Patent Application
20170159064
The field of the present disclosure relates generally to the field of molecular biology, more particularly relating to small RNA-directed regulation of gene expression. In particular, it relates to methods for down-regulating the expression of one or more target sequences in vivo. The disclosure also provides polynucleotide constructs and compositions useful in such methods, as well as cells, plants and seeds comprising the polynucleotides.
Reduction of the activity of specific genes (also known as gene silencing or gene suppression) is critical for normal cellular function in a variety of eukaryotes. Important to regulating gene expression, controlling integration of mobile genetic elements and defending against pathogens or pests, RNA-directed gene silencing is a conserved biological process that involves small RNA molecules. Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. The consequence of these events, regardless of the specific mechanism, is that gene expression is modulated. In recent years, gene silencing technology involving small RNAs has been used as an important tool to study and manipulate gene expression.
microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) are two distinct classes of plant small RNAs that act in post-transcriptional RNA silencing pathways to silence target RNA transcripts with sequence complementary (Chapman and Carrington, 2007; Martinez de Alba et al., 2013). Target repression can occur through direct endonucleolytic cleavage, or through other mechanisms such as target destabilization or translational repression (Huntzinger and Izaurralde, 2011). MicroRNAs and tasiRNAs differ in their biogenesis pathway. While miRNAs originate from transcripts with imperfect self-complementary foldback structures that are usually processed by DICER-LIKE1 (DCL1), tasiRNAs are formed through a refined RNA silencing pathway. TAS transcripts are initially targeted and sliced by a specific miRNA/AGO complex, and one of the cleavage products is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE6 (RDR6). The resulting dsRNA is sequentially processed by DCL4 into 21-nt siRNA duplexes in register with the miRNA-guided cleavage site (Allen et al., 2005; Dunoyer et al., 2005; Gasciolli et al., 2005; Xie et al., 2005; Yoshikawa et al., 2005; Axtell et al., 2006; Montgomery et al., 2008; Montgomery et al., 2008). For both miRNA and tasiRNA intermediate duplexes, usually one strand is selectively sorted to an ARGONAUTE (AGO) protein according to the identity of the 5′ nucleotide or to other sequence/structural elements of the small RNA or small RNA duplex (Mi et al., 2008; Montgomery et al., 2008; Takeda et al., 2008; Zhu et al., 2011).
Small RNA-directed gene silencing has been used extensively to selectively regulate plant gene expression. Artificial miRNA (amiRNA), synthetic tasiRNA (syn-tasiRNA), hairpin-based RNA interference (hpRNAi), virus-induced gene silencing (VIGS) or transcriptional silencing (TGS) methods have been developed (Ossowski et al., 2008; Baykal and Zhang, 2010). Since their initial application (Alvarez et al., 2006; Schwab et al., 2006), amiRNAs produced from different MIRNA precursors have been used to silence reporter genes (Parizotto et al., 2004), endogenous plant genes (Alvarez et al., 2006; Schwab et al., 2006), viruses (Niu et al., 2006) and non-coding RNAs (Eamens et al., 2011). Syn-tasiRNAs have been shown to target RNAs in Arabidopsis when produced from TAS1a (Felippes and Weigel, 2009), TAS1c (de la Luz Gutierrez-Nava et al., 2008; Montgomery et al., 2008) and TAS3a (Montgomery et al., 2008; Felippes and Weigel, 2009) transcripts, or from gene fragments fused to an upstream miR173 target site (Felippes et al., 2012). Current methods to generate amiRNA or syn-tasiRNA constructs, however, can be tedious and cost- and time-ineffective for high-throughput applications.
Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production, or for multiplexing to specifically suppress multiple targets. Here we describe simple, fast and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis and other plant species. AmiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB (B/c′)-based expression vector. B/c vectors for amiRNA and syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that a) exhibited the expected phenotypes predicted by loss of target gene function, b) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and c) had reduced levels of the corresponding target RNAs.
However, current methods for generating small RNAs for targeting specific sequences are tedious and cost- and time-ineffective. Therefore, there is an unfulfilled need for efficient constructs and methods for inducing inhibition or suppression of one or more target genes or RNAs. It is to such constructs and methods, that this disclosure is drawn.
Further scope of the applicability of the present disclosure will become apparent from the detailed description and accompanying figures provided below. However, it should be understood that the detailed description and specific examples, while indicating several embodiments, are given by way of illustration only since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The present disclosure relates to methods and constructs for modulating expression of one or more target sequences. Provided herein are methods for producing one or more sequence-specific microRNAs in vivo; also provided are constructs and compositions useful in the methods.
The methods and constructs provided in this disclosure are highly efficient methods for production of a new generation of plant MIR390a-based amiRNAs. The new methods and constructs use positive insert selection, and eliminate PCR steps, gel-based DNA purification, restriction digestions and sub-cloning of inserts between vectors, making them more suitable for high-throughput libraries.
Constructs and methods for producing specific small RNAs for inactivation or suppression of one or more target sequences or other entities, such as pathogens or pests (e.g. viruses, fungi, bacteria, nematodes, etc.) are also provided by this disclosure. Cells and organisms into which have been introduced a construct or a vector of this disclosure are also provided. Also provided are constructs and methods, where the small RNAs are produced in a tissue-specific, cell-specific or other regulated manner.
The present disclosure also relates to the production of plants with improved properties and traits using molecular techniques and genetic transformation. In particular, the invention relates to methods of modulating the expression of a target sequence in a cell using small RNAs. The disclosure also relates to cells or organisms obtained using such methods. Provided herein are plant cell and plants derived from such cells, as well as the progeny of such plants and to seeds derived from such plants. In such plant cells or plants, the modulation of the target sequence or expression of a particular gene is more effective, selective and more predictable than the modulation of the gene expression of a particular gene obtained using current methods known in the art.
The invention can be more fully understood form the following detailed description and the accompanying Sequence Listing, which form a part of this application.
The sequence descriptions summarize the Sequence Listing attached hereto. The Sequence Listing contains standard symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
The foregoing and other aspects, features, and advantages of the present disclosure will be better understood from the following detailed description taken in conjunction with the accompanying figures, all of which are given by way of illustration only, and are not limitative of the present specification, in which:
The following detailed description is provided to aid those skilled in the art. Even so, the following detailed description should not be construed to unduly limit, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present specification.
The contents of each of the publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure pertains. Units, prefixes and symbols may be denoted in their SI accepted form. Provision, or lack of the provision, of a definition for a particular term or phrase is not meant to signify any particular importance, or lack thereof. Rather, and unless otherwise noted, terms used and the manufacture or laboratory procedures described herein are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. The following definitions are provided to aid the reader in understanding the various aspects of the present disclosure.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants, reference to “a cell” includes one or more cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. Furthermore, the use of the term “including”, as well as other related forms, such as “includes” and “included”, is not limiting.
Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as is known to one of ordinary skill in the art and is understood as included in embodiments where it would be appropriate. Nucleotides may be referred to by their commonly accepted single-letter codes. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation, respectfully. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUM Biochemical Nomenclature Commission. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
If ranges are disclosed, the endpoints of all ranges directed to the same component or property are inclusive and independently combinable (e.g., ranges of “up to about 25 wt. %, or, more specifically, about 5 wt. % to about 20 wt. %,” is inclusive of the endpoints and all intermediate values of the ranges of “about 5 wt. % to about 25 wt. %,” etc.). Numeric ranges recited with the specification are inclusive of the numbers defining the range and include each integer within the defined range.
The term “about” as used herein is a flexible word with a meaning similar to “approximately” or “nearly”. The term “about” indicates that exactitude is not claimed, but rather a contemplated variation. Thus, as used herein, the term “about” means within 1 or 2 standard deviations from the specifically recited value, or ±a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 4%, 3%, 2%, or 1% compared to the specifically recited value.
As used herein, “altering level of production” or “altering level of expression” shall mean changing, either by increasing or decreasing, the level of production or expression of a nucleic acid sequence or an amino acid sequence (for example a polypeptide, an siRNA, a miRNA, an mRNA, a gene), as compared to a control level of production or expression.
By “amplification” when used in reference to a nucleic acid, this refers to techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of in vitro amplification can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. Methods of nucleic acid amplification can include, but are not limited to: polymerase chain reaction (PCR), strand displacement amplification (SDA), for example multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), ligase chain reaction (LCR), immuno-amplification, and a variety of transcription-based amplification procedures, including transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), and rolling circle amplification. See, e.g., Mullis, “Process for Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences,” U.S. Pat. No. 4,683,195; Walker, “Strand Displacement Amplification,” U.S. Pat. No. 5,455,166; Dean et al, “Multiple displacement amplification,” U.S. Pat. No. 6,977,148; Notomi et al, “Process for Synthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278; Landegren et al. U.S. Pat. No. 4,988,617 “Method of detecting a nucleotide change in nucleic acids”; Birkenmeyer, “Amplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat. No. 5,427,930; Cashman, “Blocked-Polymerase Polynucleotide Immunoassay Method and Kit,” U.S. Pat. No. 5,849,478; Kacian et al, “Nucleic Acid Sequence Amplification Methods,” U.S. Pat. No. 5,399,491; Malek et al, “Enhanced Nucleic Acid Amplification Process,” U.S. Pat. No. 5,130,238; Lizardi et al, BioTechnology, 6: 1197 (1988); Lizardi et al., U.S. Pat. No. 5,854,033 “Rolling circle replication reporter systems.” In some embodiments, two or more of the listed nucleic acid amplification methods are performed, for example sequentially.
“Antisense” and “Sense”: DNA has two antiparallel strands, a 5′ →3′ strand, referred to as the plus strand, and a 3′→5′ strand, referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5′ →3′ direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, an RNA transcript will have a sequence complementary to the minus strand, and identical to the plus strand (except that U is substituted for T). “Antisense” molecules are molecules that are hybridizable or sufficiently complementary to either RNA or the plus strand of DNA. “Sense” molecules are molecules that are hybridizable or sufficiently complementary to the minus strand of DNA.
As used herein “binds” or “binding” includes reference to an oligonucleotide that binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the target-oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional and physical binding assays. For instance, binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like. Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (Tm) at which 50% of the oligomer is melted from its target. A higher (Tm) means a stronger or more stable complex relative to a complex with a lower (Tm).
By “complementarity” refers to molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, or hybridize, to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide remains detectably bound to a target nucleic acid sequence under the required conditions. Complementarity is the degree to which bases in one nucleic acid strand base pair with (are complementary to) the bases in a second nucleic acid strand. Complementarity is conveniently described by the percentage, i.e., the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. “Sufficient complementarity” means that a sufficient number of base pairs exist between the oligonucleotide and the target sequence to achieve detectable binding, and disrupt or reduce expression of the gene product(s) encoded by that target sequence. When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In some embodiments, sufficient complementarity is at least about 50%, about 75% complementarity, or at least about 90% or 95% complementarity. In particular embodiments, sufficient complementarity is 98% or 100% complementarity. Likewise, “complementary” means the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.
As used herein “control” or “control level” means the level of a molecule, such as a polypeptide or nucleic acid, normally found in nature under a certain condition and/or in a specific genetic background. In certain embodiments, a control level of a molecule can be measured in a cell or specimen that has not been subjected, either directly or indirectly, to a treatment. A control level is also referred to as a wildtype or a basal level. These terms are understood by those of ordinary skill in the art. A control plant, i.e. a plant that does not contain a recombinant DNA that confers (for instance) an enhanced agronomic trait in a transgenic plant, is used as a baseline for comparison to identify an enhanced agronomic trait in the transgenic plant. A suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant DNA, or does not contain all of the recombinant DNAs in the test plant.
As used herein, “encodes” or “encoding” refers to a DNA sequence which can be processed to generate an RNA and/or polypeptide. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
As used herein, “expression” or “expressing” refers to production of a functional product, such as, the generation of an RNA transcript from an introduced construct, an endogenous DNA sequence, or a stably incorporated heterologous DNA sequence. A nucleotide encoding sequence may comprise intervening sequence (e.g. introns) or may lack such intervening non-translated sequences (e.g. as in cDNA). Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated (for example, siRNA, transfer RNA and ribosomal RNA). The term may also refer to a polypeptide produced from an mRNA generated from any of the above DNA precursors. Thus, expression of a nucleic acid fragment, such as a gene or a promoter region of a gene, may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide), or both.
The term “genome” as it applies to a plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found Within subcellular components (e.g., mitochondrial, plastid) of the cell.
As used herein, “heterologous” with respect to a sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus. For example, with respect to a nucleic acid, it can be a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form.
By “host cell” or “cell” it is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Alternatively, the host cells are monocotyledonous or dicotyledonous plant cells.
The term “hybridize” or “hybridization” as used herein means hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as base pairing. Complementary refers to the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization, though waste times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed Green and Sambrook (2012) Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, herein incorporated by reference.
The term “introduced” means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, “introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct/expression construct) into ac ell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
As used here in “interfering” or “inhibiting” with respect to expression of a target sequence): This phrase refers to the ability of a small RNA, or other molecule, to measurably reduce the expression and/or stability of molecules carrying the target sequence. “Interfering” or “inhibiting” expression contemplates reduction of the end-product of the gene or sequence, e.g., the expression or function of the encoded protein or a protein, nucleic acid, other biomolecule, or biological function influenced by the target sequence, and thus includes reduction in the amount or longevity of the miRNA transcript or other target sequence. In some embodiments, the small RNA or other molecule guides chromatin modifications which inhibit the expression of a target sequence. It is understood that the phrase is relative, and does not require absolute inhibition (suppression) of the sequence. Thus, in certain embodiments, interfering with or inhibiting expression of a target sequence requires that, following application of the small RNA or other molecule (such as a vector or other construct encoding one or more small RNAs), the target sequence is expressed at least 5% less than prior to application, at least 10% less, at least 15% less, at least 20% less, at least 25% less, or even more reduced. Thus, in some particular embodiments, application of a small RNA or other molecule reduces expression of the target sequence by about 30%, about 40%, about 50%, about 60%, or more. In specific examples, where the small RNA or other molecule is reduces expression of the target sequence by 70%, 80%, 85%, 90%, 95%, or even more.
The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment; the isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material. Nucleic acids and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
As used here “modulate” or “modulating” or “modulation” and the like are used interchangeably to denote either up-regulation or down-regulation of the expression of the product of a target sequence relative to its normal expression level in a wild type organism. Modulation includes expression that is increased or decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165% or 170% or more relative to the wild type expression level.
As used herein, “microRNA” (also referred to herein interchangeable as “miRNA” or “miR”) refers to an oligoribonucleic acid, which regulates the expression of a polynucleotide comprising the target sequence transcript. Typically, microRNAs (miRNAs) are noncoding RNAs of approximately 21 nucleotides (nt) in length that have been identified in diverse organisms, including animals and plants (Lagos-Quintana et al., Science 294:853-858 2001, Lagos-Quintana et al., Curr. Biol. 12:735-739 2002; Lau et al., Science 294:858-862 2001; Lee and Ambros, Science 294:862-864 2001; Llave et al., Plant Cell 14: 1 605-1619 2002; Mourelatos et al., Genes. Dev. 16:720-728 2002; Park et al., Curr. Biol. 12: 1484-1495 2002; Reinhart et al., Genes. Dev. 16: 1616-1626 2002). Primary transcripts of miRNA genes form hairpin structures that are processed by the multidomain RNaseIII-like nuclease DICER and DROSHA (in animals) or DICER-LIKE1 (DCL1; in plants) to yield miRNA duplexes. As used herein “pre-microRNA” refers to these miRNA duplexes, wherein the foldback includes a “distal stem-loop” or “distal SL region” of partially complementary oligonucleotides. “mature miRNA” refers to the miRNA which is incorporated into RISC complexes after duplex unwinding. In one embodiment, the miRNA is the region comprising R1 to Rn, wherein “n” corresponds to the number of nucleotides in the miRNA. In another embodiment, the miRNA is the region comprising R′i to R′n, wherein “n” corresponds to the number of nucleotides in the miRNA. In one aspect, “n” is in the range of about from 15 to about 25 nucleotides, in another aspect, “n” is about 20 or about 21 nucleotides. The term miRNA is specifically intended to cover naturally occurring polynucleotides, as well as those that are recombinantly or synthetically or artificially produced, or amiRNAs.
As used herein “operably linked” refers to a functional arrangement of elements. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked” to the coding sequence. In specific embodiments, operably linked nucleic acids as discussed herein are aligned in a linear concatamer capable of being cut into fragments, at least one of which is a small RNA molecule.
As used herein, “nucleic acid” means a polynucleotide (or oligonucleotide) and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Nucleic acids may also include fragments and modified nucleotides.
As used herein, “nucleic acid construct” or “construct” refers to an isolated polynucleotide which is introduced into a host cell. This construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides. The construct may be transcribed to form an RNA, wherein the RNA may be capable of forming a double-stranded RNA and/or hairpin structure. This construct may be expressed in the cell, or isolated or synthetically produced. The construct may further comprise a promoter, or other sequences which facilitate manipulation or expression of the construct.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and isolated plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Also included with the term “plant” is algae and generally comprises all plants of economic importance. The term “plant” also includes plants which have been modified by breeding, mutagenesis or genetic engineering (transgenic and non-transgenic plants).
As used herein the phrase “plant cell” refers to plant cells which are derived and isolated from a plant or plant cell cultures.
As used herein the phrase “plant cell culture” refers to any type of native (naturally occurring) plant cells, plant cell lines and genetically modified plant cells, which are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally, the plant cell culture of this aspect of the present invention may comprise a particular type of a plant cell or a plurality of different types of plant cells. It should be noted that optionally plant cultures featuring a particular type of plant cell may be originally derived from a plurality of different types of such plant cells.
The term “plant parts” includes differentiated and undifferentiated tissues including, but not limited to the following: roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture.
The term “plant organ” refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “polypeptide”, “peptide” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
As used herein “promoter” includes reference to an array of nucleic acid control sequences which direct transcription of a nucleic acid. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” or “regulatable” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, the presence of a specific molecule, such as C02, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. Examples of inducible promoters include Cu-sensitive promoter, Gall promoter, Lac promoter, while Trp promoter, Nitl promoter and cytochrome c6 gene (Cyc6) promoter. A “constitutive” promoter is a promoter which is active under most environmental conditions. Examples of constitutive promoters include Ubiquitin promoter, actin promoter, PsaD promoter, RbcS2 promoter, heat shock protein (hsp) promoter variants, and the like. Representative examples of promoters that can be used in the present disclosure are described herein.
A skilled person appreciates a promoter sequence can be modified to provide for a range of expression levels of an operably linked heterologous nucleic acid molecule. Less than the entire promoter region can be utilized and the ability to drive expression retained. However, it is recognized that expression levels of mRNA can be decreased with deletions of portions of the promoter sequence. Thus, the promoter can be modified to be a weak or strong promoter. A promoter is classified as strong or weak according to its affinity for RNA polymerase (and/or sigma factor); this is related to how closely the promoter sequence resembles the ideal consensus sequence for the polymerase. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a strong promoter drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts.
As used herein “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed.
As used herein, a “recombinant construct”, “expression construct”, “chimeric construct”, “construct” and “recombinant expression cassette” are used interchangeable herein. A recombinant construct comprises an artificial combination of nucleic acid fragments (e.g. regulatory and coding sequences) that are not found in nature. For example, a recombinant construct may comprise a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant construct can be incorporated into a plasmid, vector, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. This construct may comprise any combination of deoxyribonucleotides, ribonucleotides, and/or modified nucleotides. The construct may be transcribed to form an RNA, wherein the RNA may be capable of forming a double-stranded RNA and/or hairpin structure. This construct may be expressed in the cell, or isolated or synthetically produced. The construct may further comprise a promoter, or other sequences which facilitate manipulation or expression of the construct.
The term “residue” or “amino acid residue” or “amino acid” is used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
As used herein, the phrase “sequence identity” or “sequence similarity” is the similarity between two (or more) nucleic acid sequences, or two (or more) amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity or sequence homology. Sequence identity is frequently measured as the percent of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions.
One of ordinary skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant similarity could be obtained that fall outside of the ranges provided. Nucleic acid sequences that do not show a high degree of identity can nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Means for making this adjustment are well-known to those of skill in the art. When percentage of sequence identity is used in reference to amino acid sequences it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
Sequence identity (or similarity) can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997)).
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; & Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5877 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chern., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chern., 17: 191-201 (1993)) low-complexity filters can be employed alone or in combination.
The term “silencing agent” or “silencing molecule” as used herein means a specific molecule, which can exert an influence on a cell in a sequence-specific manner to reduce or silence the expression or function of a target, such as a target gene or protein. Examples of silence agents include nucleic acid molecules such as naturally occurring or synthetically generated small interfering RNAs (siRNAs), naturally occurring or synthetically generated microRNAs (miRNAs), naturally occurring or synthetically generated dsRNAs, and antisense sequences (including antisense oligonucleotides, hairpin structures, and antisense expression vectors), as well as constructs that code for any one of such molecules.
A “small interfering RNA” or “siRNA” means RNA of approximately 21-25 nucleotides that is processed from a dsRNA by a DICER enzyme (in animals) or a DCL enzyme (in plants). The initial DICER or DCL products are double-stranded, in which the two strands are typically 21-25 nucleotides in length and contain two unpaired bases at each 3′ end. The individual strands within the double stranded siRNA structure are separated, and typically one of the siRNAs then are associated with a multi-subunit complex, the RNAi-induced silencing complex (RISC). A typical function of the siRNA is to guide RISC to the target based on base-pair complementarity. The term siRNA is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
As used here “suppression” or “silencing” or “inhibition” are used interchangeably to denote the down-regulation of the expression of the product of a target sequence relative to its normal expression level in a wild type organism. Suppression includes expression that is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the wild type expression level.
As used herein, the phrases “target sequence” and “sequence of interest” are used interchangeably and encompass DNA, RNA (comprising pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, and may also refer to a polynucleotide comprising the target sequence. Target sequence is used to mean the nucleic acid sequence that is selected for suppression of expression, and is not limited to polynucleotides encoding polypeptides. Target sequences may include coding regions and non-coding regions such as promoters, enhancers, terminators, introns and the like. The target sequence may be an endogenous sequence, or may be an introduced heterologous sequence, or transgene. The specific hybridization of an oligomeric compound with its target sequence interferes with the normal function of the nucleic acid. The target sequence comprises a sequence that is substantially or completely complementary between the oligomeric compound and the target sequence. This modulation of function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as “antisense”.
The term “trans-acting siRNA” or “tasiRNA” or “ta-siRNA” refer to a subclass of siRNAs that function like miRNAs to repress expression of target genes, yet have unique biogenesis requirements. Trans-acting siRNAs form by transcription of tasiRNA-generating genes, cleavage of the transcript through a guided RISC mechanism, conversion of one of the cleavage products to dsRNA, and processing of the dsRNA by DCL enzymes. tasiRNAs are unlikely to be predicted by computational methods used to identify miRNA because they fail to form a stable foldback structure. A ta-siRNA precursor is any nucleic acid molecule, including single-stranded or double-stranded DNA or RNA, that can be transcribed and/or processed to release a tasiRNA. The term tasiRNA is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
In one embodiment, the invention relates to a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) construct comprising: (i) a microRNA and a complement thereof, and (ii) a distal SL region operably linked in between the microRNA and the complement thereof, wherein the distal SL region consists of less than about 50 nucleotides.
In another embodiment, the invention relates to a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) construct comprising: (i) a microRNA and a complement thereof, and (ii) a distal SL region operably linked in between the microRNA and the complement thereof wherein the distal SL region consists of less than about 45 nucleotides or less than about 44 nucleotides or less than about 43 nucleotides or less than about 42 nucleotides or less than about 41 nucleotides or less than about 40 nucleotides or less than about 39 nucleotides or less than about 38 nucleotides or less than about 37 nucleotides or less than about 36 nucleotides or less than about 35 nucleotides or less than about 34 nucleotides or less than about 33 nucleotides or less than about 32 nucleotides or less than about 31 nucleotides or less than about 30 nucleotides or less than about 29 nucleotides or less than about 28 nucleotides or less than about 27 nucleotides or less than about 26 nucleotides or less than about 25 nucleotides or less than about 24 nucleotides or less than about 23 nucleotides or less than about 22 nucleotides or less than about 21 nucleotides or less than about 20 nucleotides or less than about 19 nucleotides or less than about 18 nucleotides or less than about 17 nucleotides or less than about 16 nucleotides or less than about 15 nucleotides or less than about 14 nucleotides or less than about 13 nucleotides or less than about 12 nucleotides or less than about 11 nucleotides or less than about 10 nucleotides or less than about 9 nucleotides or less than about 8 nucleotides or less than about 7 nucleotides or less than about 6 nucleotides or less than about 5 nucleotides or less than about 4 nucleotides or less than about 3 nucleotides.
In another embodiment, the invention is a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof, wherein the distal SL region consists of about 3 to about 40 nucleotides.
In accordance with another embodiment of the invention, the distal SL region can consists of between about 3 to about 50 nucleotides, between about 3 to about 45 nucleotides, between about 3 to about 40 nucleotides, between about 3 to about 35 nucleotides, between about 3 to about 30 nucleotides, between about 3 to about 20 nucleotides, between about 3 to about 15 nucleotides, between about 3 to about 10 nucleotides, between about 5 to about 50 nucleotides, between about 5 to about 50 nucleotides, between about 5 to about 45 nucleotides, between about 5 to about 40 nucleotides, between about 5 to about 35 nucleotides, between about 5 to about 30 nucleotides, between about 5 to about 20 nucleotides, between about 5 to about 15 nucleotides, between about 5 to about 10 nucleotides, between about 10 to about 50 nucleotides, between about 10 to about 45 nucleotides, between about 10 to about 40 nucleotides, between about 10 to about 35 nucleotides, between about 10 to about 30 nucleotides, between about 10 to about 20 nucleotides, between about 10 to about 15 nucleotides, between about 15 to about 50 nucleotides, between about 15 to about 45 nucleotides, between about 15 to about 40 nucleotides, between about 15 to about 35 nucleotides, between about 15 to about 30 nucleotides, between about 15 to about 20.
As used herein, the region that folds back between the micro-RNA and the complement thereof is referred to as the “distal stem-loop region” or “distal SL region.” In an aspect of the invention, the region in between the microRNA and complement thereof could adopt a stem-loop structure or just a loop structure. In one embodiment of the invention, the region in between the micro RNA and the complement thereof is folded to form a symmetric stem-loop structure. In another embodiment, the region in between the micro RNA and the complement thereof is folded to form an asymmetric stem-loop structure.
In one embodiment of invention, the stem-loop is distal or downstream or 3′ of the miRNA. In another embodiment, the stem-loop is proximal or upstream or 5′ of the miRNA.
In another embodiment, the invention is a heterologous or synthetic or artificial single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof, wherein the nucleotide sequence of the distal SL region is at least 75% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.
In accordance with another embodiment of the invention, the nucleotide sequence identity of the distal SL region is at least 70%, is at least 75%, is at least 80%, is at least 85%, is at least 90%, is at least 95%, is at least 97%, is at least 99%. In accordance with another embodiment of the invention, the nucleotide sequence identity of the distal SL region is identical or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.
In one embodiment of the invention, the RNA construct is operably linked between complementary nucleotide sequences. In another embodiment, the complementary nucleotide sequences are at least 75% identical to SEQ ID NO: 3 and SEQ ID NO: 4, or complements thereof. In another embodiment the complementary nucleotide sequences are at least 75% identical to SEQ ID NO: 5 and SEQ ID NO: 6, or complements thereof. In yet another embodiment the complementary nucleotide sequences are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical. In accordance with another embodiment of the invention, the complementary nucleotide sequences are identical or have 100% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4, or complements thereof; or the complementary nucleotide sequences are identical or have 100% sequence identity to SEQ ID NO: 5 and SEQ ID NO: 6, or complements thereof.
In one embodiment of the invention, the RNA construct is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates the expression of a target sequence. In another embodiment of the invention, the RNA is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates or suppresses or reduces the expression of a target sequence. In accordance with another embodiment of the invention, the microRNA is an artificial microRNA. In yet another embodiment of the invention, the target sequence is a promoter, or an enhancer, or a terminator or an intron. In another embodiment, the target sequence is an endogenous sequence, in another embodiment the target sequence is a heterologous sequence. In one embodiment of the invention, the microRNA is substantially complementary to the target sequence. In another embodiment, the microRNA is sufficiently complementary to the target sequence. In another embodiment, the microRNA is completely complementary to the target sequence.
In one embodiment of the invention, the pre-microRNA has at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10; and wherein the region comprising R1 to Rn and the region comprising R′1 to R′n represent the microRNA or the complement thereof; and wherein “n” corresponds to the number of nucleotides in the miRNA. In one aspect, “n” is in the range of from about 15 to about 25 nucleotides, in another aspect, “n” is from about 20, or “n” is from about 21 nucleotides.
In another embodiment of the invention, the pre-microRNA has a nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10. In accordance with another embodiment of the invention, the pre-microRNA has a nucleotide sequence is identical or has 100% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10.
Also provided herein, is a heterologous or synthetic or an artificial deoxyribonucleic acid (DNA) comprising a polynucleotide or nucleotide sequence encoding an artificial or synthetic or heterologous single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof.
In one embodiment, the invention relates to a vector comprising DNA encoding an artificial or synthetic or heterologous single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof. In one embodiment, the vector further comprises a promoter or regulatory sequence. In another embodiment, the vector comprises a tissue-specific, cell-specific or other regulated manner. In another embodiment, the vector comprises a selectable marker or resistance gene. Typical markers and/or resistance genes are well known in the art and include antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin, the streptomycin phosphotransferase gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e. g., the bar gene), or other such genes known in the art.
In another embodiment of the invention, the vector comprises flanking nucleotide sequences; wherein the flanking nucleotide sequences are at least 75% identical to SEQ ID NO: 11 and SEQ ID NO: 12, or complements thereof; or wherein the flanking nucleotide sequences are at least 75% identical to SEQ ID NO: 13 and SEQ ID NO: 14, or complements thereof. In another embodiment, the vector comprises flanking nucleotide sequences; wherein the flanking nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to SEQ ID NO: 11 and SEQ ID NO: 12, or complements thereof; or wherein the flanking nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to SEQ ID NO: 13 and SEQ ID NO: 14, or complements thereof. In accordance with another embodiment of the invention, the vector comprises flanking nucleotide sequences; wherein the flanking nucleotide sequences are identical or 100% sequence identity to SEQ ID NO: 11 and SEQ ID NO: 12, or complements thereof; or wherein the flanking nucleotide sequences are identical or 100% sequence identity to SEQ ID NO: 13 and SEQ ID NO: 14, or complements thereof.
In one embodiment, the invention relates to a cell expressing RNA or DNA, or complements thereof; or a vector encoding an artificial or synthetic or heterologous single-stranded ribonucleic acid (RNA) comprising (i) a microRNA and a complement thereof, and (ii) a distal SL region in between the microRNA and the complement thereof. In another embodiment the invention relates to a cell, wherein the cell expresses a RNA construct which is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates the expression of a target sequence. In another embodiment of the invention, the RNA is a pre-microRNA that is processed into a microRNA, and wherein the microRNA modulates or suppresses or reduces the expression of a target sequence. Target sequences may include coding regions and non-coding regions such as promoters, enhancers, terminators, introns and the like. The target sequence may be an endogenous sequence, or may be an introduced heterologous sequence, or transgene. In one embodiment, the cell is a plant cell. In another aspect the plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.
Provided herein, is a method of modulating expression of a target sequence, comprising: transforming a cell with a vector as described herein, or expressing a vector in a cell or applying or providing or introducing a microRNA to a cell. A method of modulating expression of a target sequence in a cell, comprising: transforming a cell with the vector as described herein, wherein the cell produces the microRNA, and wherein the microRNA modulates the expression of a target sequence in the cell.
In another embodiment, the invention relates to a method of modulating expression of a target sequence in cell, comprising providing, introducing, or applying the microRNA produced by the cell to a second cell, wherein the microRNA modulates the expression of a target sequence in the second cell. In one aspect the invention relates to passive provision of the microRNA to another cell; in another aspect the microRNA is actively provided to another cell. In one embodiment the second cell is from the same organism, in another embodiment the second cell is from a different organism. As a non-limiting example, passive provision of the microRNA to a cell in a different organism involves the uptake of the microRNA by a pathogen or pest, for example a virus, a bacterium, a fungus, an insect, etc.
The following examples are provided to illustrate various aspects of the present disclosure, and should not be construed as limiting the disclosure only to these particularly disclosed embodiments.
The materials and methods employed in the examples below are for illustrative purposes only, and are not intended to limit the practice of the present embodiments thereto. Any materials and methods similar or equivalent to those described herein as would be apparent to one of ordinary skill in the art can be used in the practice or testing of the present embodiments.
Several properties of the AtMIR390a precursor make it attractive as a backbone to engineer a new generation of amiRNA vectors. First, small RNA library analyses indicate that the AtMIR390a precursor is processed accurately, as the majority of reads mapping to the AtMIR390a foldback correspond to the authentic 21-nucleotide (nt) miR390a guide strand (
Details of the zero background cloning strategy to generate AtMIR390a-based amiRNA constructs are illustrated in
A series of AtMIR390a-based cloning vectors were developed and named AtMIR390a-B/c′ vectors (from AtMIR390a-BsaI/ccdB). They contain a truncated AtMIR390a precursor sequence whose miRNA/distal stem-loop/amiRNA* region was replaced by a 1461 bp DNA cassette including the ccdB gene (Bernard and Couturier, 1992) flanked by two BsaI sites (
pMDC32B-AtMIR390a-B/c, pMDC123SB-AtMIR390a-B/c or pFK210B-AtMIR390a-B/c expression vectors were generated for direct cloning of amiRNAs and tested in different plant species (Table I,
-
A. thaliana
A. thaliana
N. benthamiana
A. thaliana
N. benthamiana
N. benthamiana
A. thaliana
N. benthamiana
indicates data missing or illegible when filed
To verify the accumulation in planta of AtMIR390a-derived amiRNAs, six different amiRNA sequences (amiR-1 to amiR-6) (
For comparative purposes, the same six amiRNA sequences were also expressed from AtMIR319a precursor, which has been most widely used to express amiRNAs in plants (Schwab et al., 2006). In this case, amiRNAs were cloned into pMDC32B-AtMIR319a-B/c (amiR-2 and amiR-3) or pMDC123SB-AtMIR319a-B/c (amiR-1, amiR-4, amiR-5 and amiR6;
In transient expression assays using N. benthamiana, each of the six amiRNAs derived from the AtMIR390a foldbacks accumulated predominantly as 21 nt species, suggesting that the amiRNA foldbacks were likely processed accurately. In each case, the amiRNA from the AtMIR390a foldbacks accumulated to significantly higher levels than did the corresponding amiRNA from the AtMIR319a or AtMIR319a-21 foldbacks (P≦0.02 for all pairwise t-test comparisons;
To test the functionality of AtMIR390a-based amiRNAs in repressing target transcripts, four different amiRNA constructs (
Twenty-three of 67 transgenic lines containing 35S:AtMIR390a-Lfy construct showed morphological defects like lfy; mutants (Schultz and Haughn, 1991; Weigel et al., 1992; Schwab et al., 2006) (Supplemental Table SI), including obvious floral defects with leaf-like organs (
The accumulation of all four amiRNAs was confirmed by RNA blot analysis in T1 transgenic lines showing amiRNA-induced phenotypes (
Accumulation of amiRNA target mRNAs in A. thaliana transgenic lines was analyzed by quantitative RT-PCR assay. The expression of all target mRNAs was significantly reduced compared to control plants (P<0.02 for all pairwise t-test comparisons,
A new generation of functional syn-tasiRNA vectors based on a modified TAS1c gene was produced with the potential to multiplex syn-tasiRNA sequences at DCL4-processing positions 3′D3[+]′ and ′3′D4[+] of AtTAS1c transcript (see (Montgomery et al., 2008). The design of AtTAS1c-based syn-tasiRNA constructs expressing two syn-tasiRNAs is shown in
Syn-tasiRNA vector construction is similar to that described for the amiRNA constructs (
To test the functionality of single and multiplexed AtTAS1c-based syn-tasiRNAs, and to compare to the efficacy of the syn-tasiRNAs with amiRNA, several syn-tasiRNA constructs were generated and introduced into Arabidopsis Col-0 plants (
Seventy-three and 62% of the transformants expressing the dual configuration syn-tasiRNA constructs 35 S:AtTAS1c-D3Ft-D4 Trich and 35 S:AtTAS1c-D3 Trich-D4Ft, respectively, showed both Trich and Ft loss-of-function phenotypes (Supplemental Table SII), which were characterized by increased clustering of trichomes in rosette leaves and a delay in flowering time compared to the 35S: GUS transformants (
Next, accumulation of syn-tasiR-Trich and syn-tasiR-Ft was compared to accumulation of amiR-Trich and amiR-Ft was analyzed by RNA blot assays using T1 transgenic plants showing obvious syn-tasiRNA- or amiRNA-induced phenotypes (
To further analyze processing and phasing of AtTAS1c-based syn-tasiRNA expressed from the dual configuration constructs (35S:AtTAS1c-D3Trich-D4Ft and 35S:AtTAS1c-D3Ft-D4Trich), small RNA libraries were produced and analyzed. Analysis of 35S:AtTAS1c-D3Trich-D4Ft small RNAs libraries confirmed that the syn-tasiRNA transcript yielded predominantly 21-nt syn-tasiR-Trich and syn-tasiR-Ft (51 and 67% of the reads within ±4 nt of 3′D3[+] and 3′D4[+], respectively), and that the corresponding tasiRNAs were in phase with miR173 cleavage site (
Finally, accumulation of target mRNAs in the 35S:AtTAS1c-D3Trich-D4Ft and 35S:AtTAS1c-D3Ft-D4Trich transgenic lines was analyzed by quantitative RT-PCR assay (
Syn-tasiRNA technology was used before to repress single targets in Arabidopsis (de la Luz Gutierrez-Nava et al., 2008; Montgomery et al., 2008; Montgomery et al., 2008; Felippes and Weigel, 2009). Here, a single AtTAS1c-based construct expressing multiple distinct syn-tasiRNAs triggered silencing of multiple target transcripts and resultant knockdown phenotypes. Theoretically, AtTAS1c-based vectors could be designed to produce more than two syn-tasiRNAs to repress a larger number of unrelated targets. Therefore, the syn-tasiRNA approach may be preferred for applications involving specific knockdown of multiple targets.
Arabidopsis thaliana Col-0 and Nicotiana benthamiana plants were grown in a chamber under long day conditions (16/8 hr photoperiod at 200 μmol m−2 s−1) and 22° C. constant temperature. Plants were transformed using the floral dip method with Agrobacterium tumefaciens GV3101 strain (Clough and Bent, 1998). Transgenic plants were grown on plates containing Murashige and Skoog medium and Basta (50 mg/ml) or hygromycin (50 mg/ml) for 10 days before being transferred to soil. Plant photographs were taken with a Canon Rebel XT/EOS 350D digital camera and EF-S18-55 mm f/3.5-5.6 II or EF-100 mm f/2.8 Macro USM lenses.
The cassette containing the AtMIR390a sequence lacking the distal stem-loop region, and including two BsaI sites, was generated as follows. A first round of PCR was done to amplify AtMIR390a-5′ or AtMIR390a-3′ regions using primers AtMIR390a-F and BsaI-AtMIR390a-5′-R, or BsaI-AtMIR390a-3′-F and AtMIR390a-R, respectively. A second round of PCR was done using as template a mixture of the products of the first PCR round and primers AtMIR390a-F and AtMIR390a-R. The PCR product was cloned into pENTR-D-TOPO (Life Technologies) to generate pENTR-AtM/R390a-BsaI. A similar strategy was used to generate pENTR-AtTAS1c-BsaI containing the AtTAS1c cassette for syn-tasiRNA cloning: oligo pairs AtTAS1c-F/BsaI-AtTAS1c-5′-R and BsaI-AtTAS1c-3′-F/AtTAS1c-R were used for the first round of PCR, and oligo pair AtTAS1c-F/AtTAS1c-R was used for the second PCR.
A 2×35S promoter cassette including the Gateway attR sites ofpMDC32 (Curtis and Grossniklaus, 2003) was transferred into pMDC123 (Curtis and Grossniklaus, 2003) to make pMDC123S. An undesired BsaI site contained in pMDC32, pMDC123S and pFK210 (de Felippes and Weigel, 2010) was disrupted to generate pMDC32B, pMDC123SB and pFK210B, respectively. pMDC32B-AtMIR390a-BsaI, pMDC123SB-AtMIR390BsaI and pFK210B-AtMIR390a-BsaI intermediate plasmids were obtained by LR recombination using pENTR-AtMIR390a-BsaI as the donor plasmid and pMDC32B, pMDC123SB and pFK210B as destination vectors, respectively. Similarly, pMDC32B-AtTAS1c-BsaI and pMDC123SB-AtTAS1c-Bs& intermediate plasmids were obtained by LR recombination using pENTR-AtTAS1c-Bs& as the donor plasmid and pMDC32B and pMDC123SB as destination vectors, respectively.
To generate zero background cloning vectors, a ccdB cassette was inserted in between the BsaI sites of plasmids containing the AtMIR390a-BsaI or AtTAS1c-BsaI cassettes. ccdB cassettes flanked with BsaI sites and with AtMIR390a or AtTAS1c specific sequences were amplified from pFK210 using primers AtMIR390a-B/c-F and AtMIR390a-B/c-R or AtTAS1c-B/c-F and AtTAS1c-Bc-R, respectively, with an overlapping PCR to disrupt an undesired BsaI site from the original ccdB sequence. These modified ccdB cassettes were then inserted between the BsaI sites into pENTR-AtMIR390a-BsaI, pENTR-AtTAS1c-BsaI, pMDC32B-AtMIR390a-BsaI, pMDC32B-AtTAS1c-BsaI, pMDC123SB-AtMIR390-BsaI, pMDC123SB-AtTAS1c-BsaI and pFK210B-AtMIR390-BsaI to generate pENTR-AtMIR390a-B/c, pENTR-AtTAS1c-B/c, pMDC32B-AtMIR390a-B/c, pMDC32B-AtTAS1c-B/c, pMDC123SB-AtMIR390a-B/c, pMDC123SB-AtTAS1c-B/c and pFK210B-AtMIR390a-B/c, respectively.
AtMIR319a-based amiRNA constructs (pMDC32-AtMIR319a-amiR-1, pMDC32-AtMIR319a-amiR-2, pMDC32-AtMIR319a-amiR-3, pMDC32-AtMIR319a-21-amiR-4, pMDC32-AtMIR319a-21-amiR-5 and pMDC32-AtMIR319-21-amiR-6) were generated as previously described (Schwab et al., 2006) using the WMD3 tool (wmd3.weigelworld.org). The CACC sequence was added to the 5′ end of the PCR fragments for pENTR-D-TOPO cloning (Life Technologies) and to allow LR recombination to pMDC32B or pMDC123SB. amiR-1, amiR-2 and amiR-3 were inserted in the AtMIR319a foldback, while amiR-4, amiR-5, amiR-6, were inserted in the AtMIR319a-21 foldback.
The rest of the amiRNA and syn-tasiRNA constructs (pMDC32B-AtMIR390a-amiR-1, pMDC32B-AtMIR390a-amiR-2, pMDC32B-AtMIR390a-amiR-3, pMDC32B-AtMIR390a-21-amiR-4, pMDC32B-AtMIR390a-21-amiR-5, pMDC32B-AtMIR390a-amiR-6, pMDC32B-AtMIR390a-Ft, pMDC32B-AtMIR390a-Lfy, pMDC32B-AtMIR390a-Ch42, pMDC32B-AtMIR390a-Trich, pMDC32B-AtTAS1c-D3&D4Ft, pMDC32B-AtTAS1c-D3&D4Trich, pMDC32B-AtTAS1c-D3Trich-D4Ft, pMDC32B-AtTAS1c-D3Ft-D4Trich) were obtained as described in the next section. pMDC32-GUS construct was described previously (Montgomery et al., 2008).
All oligonucleotides used for generating the constructs described above are listed in Supplemental Table SIV. The sequences and predicted targets for all the amiRNAs and syn-tasiRNAs used in this study are listed in Supplemental Table SV. The sequences of the amiRNA and syn-tasiRNA vectors are listed in the sections tht follow. The following amiRNA and syn-tasiRNA vectors are available from Addgene at www.addgene.org/: pENTR-AtMIR390a-B/c (Addgene plasmid 51778), pMDC32B-AtMIR390a-B/c (Addgene plasmid 51776), pMDC123SB-AtMIR390a-B/c (Addgene plasmid 51775), pFK210B-AtMIR390a-B/c (Addgene plasmid 51777), pENTR-AtTAS1c-B/c (Addgene plasmid 51774), pMDC32B-AtTAS1c-B/c (Addgene plasmid 51773) and pMDC123SB-AtTAS1c-B/c (Addgene plasmid 51772).
Detailed amiRNA and syn-tasiRNA oligo design and cloning protocols are given in
For cloning amiRNA or syn-tasiRNA inserts into B/c vectors, 2 μl of each of the two overlapping oligonucleotides (100 μM stock) were annealed in 46 μl of Oligo Annealing Buffer (60 mM Tris-HCl pH7.5, 500 mM NaCl, 60 mM MgCl2 and 10 mM DTT) by heating the reaction for 5 min at 94° C. and then cooling to 20° C. (0.05° C./sec decrease). The annealed oligonucleotides were diluted in dH20 to a final concentration of 0.30 μM. A 20 μl ligation reaction was incubated for 1 h at room temperature, and included 3 ul of the annealed and diluted oligonucleotides (0.30 μM) and 1 μl (75 ng/μl) of the corresponding B/c vector previously digested with BsaI. One-μl of the ligation reaction was used to transform and E. coli strain such as DH10B or TOP10 that does not have ccdB resistance.
Transient expression assays in N. benthamiana leaves were done as described (Llave et al. 2002, Carbonell et al., 2012) using Agrobacterium tumefaciens GV3101 strain.
Total RNA from A. thaliana or N. benthamiana was extracted using TRIzol reagent (Life Technologies) as described (Cuperus et al., 2010). RNA blot assays were done as described (Montgomery et al., 2008; Cuperus et al., 2010). Oligonucleotides used as probes for small RNA blots are listed in Supplemental Table SIV.
RT-qPCR reactions were done using those RNA samples that were used for RNA blot and small RNA library analyses. Two micrograms of DNAseI-treated total RNA were used to produce first-strand cDNA using the Superscript III system (Life Technologies). RT-qPCR reactions were done in optical 96-well plates in a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using the following program: 20 seconds at 95° C., followed by 40 cycles of 95° C. for 3 seconds, 60° C. for 30 seconds, and an additional melt curve stage consisting of 15 seconds at 95° C., 1 minute at 60° C. and 15 seconds at 95° C. The 20 μl reaction mixture contained 10 μl of Fast SYBR® Green Master Mix (2×) (Applied Biosystems), 2 μl diluted cDNA (1:5), and 300 nM of each gene-specific primer. Primers used for RT-qPCR are listed in Supplemental Table SIV. Target mRNA expression levels were calculated relative to 4 reference genes (AtACT2, AtCPB20, AtSAND and AtUBQ10) using the ΔΔCt comparative Ct method (Applied Biosystems) of the StepOne Software (Applied Biosystems, version 2.2.2). Three independent biological replicates were analyzed. For each biological replicate, two technical replicates were analyzed by RT-qPCR analysis.
Small RNA libraries were produced using the same RNA samples as used for RNA blots. Fifty-100μg of Arabidopsis total RNA were treated as described (Carbonell et al. 2012), but each small RNA library was barcoded at the amplicon PCR reaction step using an indexed 3′ PCR primer (i1, i3, i4, i5 or i9) and the standard 5′PCR primer (P5) (Supplemental Table SVI). Libraries were multiplexed and submitted for sequencing using a HiSeq 2000 sequencer (Illumina).
Sequencing reads were parsed to identify library-specific barcodes and remove the 3′ adaptor sequence, and were collapsed to a unique set with read counts. Unique sequences were aligned to a database containing the sequences of AtMIR390a-based amiRNA, AtTAS1c-based syn-tasiRNA and the control constructs using BOWTIE version 0.12.8 (Langmead et al., 2009) with settings that identified only perfect matches (-f -v 0 -a -S). Small RNA alignments were saved in Sequence Alignment/Map (SAM) format and were queried using SAMTOOLS version 0.1.19+(Li et al., 2009). Processing of amiRNA foldbacks and syn-tasiRNA transcripts was assessed by quantifying the proportion of small RNA, by position and size, that mapped within ±4 nt of the 5′ end of the miRNA and miRNA* or DCL4 processing position 3′D3[+] and 3′D4[+], respectively.
syn-tasiRNA constructs differ from endogenous AtTAS1c at positions 3′D3 and 3′D4, but are otherwise the same. Therefore, reads for other syn-tasiRNA positions are indistinguishable from endogenous AtTAS1c-derived small RNAs. To assess the phasing of syn-tasiRNA constructs, small RNA reads from libraries generated from plants containing 35S: GUS, 35S:AtTAS1c-D3Trich-D4Ft or 35S:AtTAS1c-D3Ft-D4Trich were first normalized to account for library size differences (reads per million total sample reads). Next, normalized reads for 21-nt small RNA that mapped to AtTAS1c in the 35S:GUS plants were subtracted from the corresponding small RNA reads in plants containing syn-tasiRNA constructs to correct for endogenous background tasiRNA expression. Phasing register tables were constructed by calculating the proportion of reads in each register relative to the miR173 cleavage site for all 21-nt positions downstream of the cleavage site.
A summary of high-throughput small RNA sequencing libraries from Arabidopsis transgenic lines is provided in Supplemental Table SVI.
Arabidopsis gene and locus identifiers are as follows: CH42 (AT4G18480), CPC (AT2G46410), ETC2 (AT2G30420), LFY (AT5G61850), FT (AT1G65480), TRY (AT5G53200). The miRBase (mirbase.org) locus identifiers of the conserved Arabidopsis MIRNA precursors (
High-throughput sequencing data from this article can be found in the Sequence Read Archive (ncbi.nlm.nih.gov/sra) under accession number SRP036134.
A. thaliana Col-0 T1 transgenic plants
aThe Ft phenotype was defined as a higher ‘days to flowering’ value when compared to the average ‘days to flowering’ value of the 35S:GUS control set.
aThe Ft Phenotype was defined as a higher ‘days to flowering’ value when compared to the average ‘days to flowering’ value of the 35S:GUS control set.
a80-100 individuals for each T2 independent line were analyzed.
bThe Trich phenotype was defined as a higher number of trichomes when compared to transformants of the 35S:GUS control set. Plants with a Trich phenotype were considered ‘try cpc type’ if they resembled the Arabidopsis try cpc double mutant.
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indicates data missing or illegible when filed
indicates data missing or illegible when filed
indicates data missing or illegible when filed
MI0000214
MI0000989
MI0000990
MI0000215
MI0000216
MI0000991
MI0001089
MI0000217
MI0000544
MI0000545
MI0001001
MI0001002
MI0001003
MI0001018
MI0001023
MI0001024
MI0005383
MI0000214
MI0000989
MI0000990
MI0000215
MI0000216
MI0000991
MI0001089
MI0000217
MI0000544
MI0000545
MI0001001
MI0001002
MI0001003
MI0001018
MI0001023
MI0001024
MI0005383
Arabidopsis thaliana
MI0000189
Arabidopsis thaliana
MI0000218
Arabidopsis thaliana
MI0000197
Arabidopsis thaliana
MI0000198
Arabidopsis thaliana
MI0000978
Arabidopsis thaliana
MI0000214
Arabidopsis thaliana
MI0000215
Arabidopsis thaliana
MI0000544
Arabidopsis thaliana
MI0001000
Arabidopsis thaliana
MI0001007
Chlamydomonas reinhardtii
MI0006219
Chlamydomonas reinhardtii
MI0006123
Gossypium herbaceum
MI0005645
Oryza sativa
MI0003201
Populus trichocarpa
MI0002352
Solanum lycopersicum
MI0009974
Solanum lycopersicum
We generated Brachypodium distachyon transgenic plants expressing artificial miRNAs against Brachypodium distachyon BRI1, CAD, CAO1 or SPL11 genes. In all cases, these artificial miRNAs were expressed them from two different foldbacks: OsMIR390 (the wild-type) and OsMIR390a (the chimeric foldback with rice OsMIR390 stem sequence but with Arabidopsis MIR390a distal stem-loop sequence).
Rice MIR390 foldback (OsMIR390) has a very short distal stem-loop, making expensive oligos unnecessary for cloning the amiRNAs (
Artificial microRNA target mRNAs were significantly reduced in transgenic plants regardless the MIRNA foldback the amiRNA was expressed from (
We suspect that because we are expressing the artificial microRNAs through an extremely potent promoter (called 35S, that leads to very high levels of artificial microRNA) we may be ‘saturating’ the system and that may explain why we do not see significant differences in phenotypes or in target mRNA accumulation in plants expressing the wild-type (OsMIR390) or the chimeric (OsMIR390-AtL) foldbacks.
However, we can predict that by expressing the artificial microRNAs to lower levels (without ‘saturating’ the system) we might see then a higher RNA silencing effect (stronger phenotypes, stronger reduction in target mRNAs) of artificial microRNAs expressed from the chimeric foldback compared to artificial microRNAs expressed from the wild-type foldback. This hypothesis is being tested by expressing the artificial microRNAs from a vector (pH7GW2) that contains a rice Ubiquitin promoter (called UBI) that is less strong than 35S.
We generated Arabidopsis thaliana transgenic plants expressing artificial microRNAs against Arabidopsis FT and CH42 gens. In both cases these artificial miRNAs were expressed from two different foldbacks: AtMIR390a (wild-type) and AtMIR390a-OsL (a MIRNA foldback with Arabidopsis MIR390a stem and shorter rice MIR390 distal stem-loop).
A very high proportion of transgenic plants showed the expected amiRNA-induced phenotype, regardless the MIRNA foldback (AtMIR390 or AtMIR390-OsL) the amiRNA was expressed from (
Therefore, we can use the chimeric MIRNA foldback AtMIR390a-OsL to express efficient artificial microRNAs in Arabidopsis and saving money in the oligos needed for cloning (the length of the oligos for the AtMIR390a wild-type is 75 nt, and the length of the oligos for the chimeric AtMIR390a-OsL is 60 bp) (
Nicotiana benthamiana
Brachypodium distachyon
Nicotiana benthamiana
Brachypodium distachyon
Brachypodium distachyon
This example provides further information for designing and cloning amiRNAs or syn-tasiRNAs in BsaI/ccdB-based (B/c′) vectors containing AtMIR390a or AtTAS1c precursors, respectively.
1. Selection of the amiRNA or Syn-tasiRNA(s) Sequence(s)
A link to a web tool for automated design of the amiRNA or syn-tasiRNA sequence(s) will be available at http://p-sams.carringtonlab.org/2.
2. Design of amiRNA or syn-tasiRNA oligonucleotides
A link to a web tool for automated design of the amiRNA or syn-tasiRNA oligonucleotide sequences will be available at http://p-sams.carringtonlab.org/2.1
2.1 Design of amiRNA Oligonucleotides
2.1.1 Sequence of the AtMIR390a Cassette Containing the amiRNA
The following FASTA sequence includes the amiRNA sequence inserted in the AtMIR390a precursor sequence:
>amiRNA in AtMIR390a precursor
C
TGTA
X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21AT
Where:
X is a DNA base of the amiRNA sequence, and the subscript number is the base position in the amiRNA 21-mer
X is a DNA base of the amiRNA* sequence, and the subscript number is the base position in the amiRNA* 21-mer
X is a DNA base of the AtMIR390a foldback
X is a DNA base of the AtMIR390a foldback included in the oligonucleotides required to clone the amiRNA insert in B/c vectors
X is a DNA base of the AtMIR390a foldback that may be modified to preserve the authentic AtMIR390a duplex structure
X is a DNA base of the AtMIR390a precursor.
In the sequence above:
Insert the amiRNA sequence where you see
Insert the amiRNA* sequence that has to verify the following base-pairing:
Note that:—In general, X=T for amiRNA association with AGO1. SEQ ID NO:372
In this case, X19=A SEQ ID NO:373
Bases X11 and X9 DO NOT base-pair to preserve the central bulge of the authentic AtMIR390a duplex. The following base-pair rule applies:
The sequences of the two amiRNA oligonucleotides are:
3Y2Y1
X1X2=AtMIR390a sequence that may be modified to preserve authentic AtMIR390a duplex structure.
Y2Y2=reverse-complement of X1X2
The sequences of the two oligonucleotides to clone the amiRNA ‘amiR-Trich’
Note: The 75 b long oligonucleotides can be ordered PAGE-purified, although oligonucleotides of ‘Standard Desalting’ quality worked well.
2.2 Design of Syn-tasiRNA Oligonucleotides
2.2.1 Sequence of the AtTAS1c Cassette Containing the syntasiRNA(s)
The following FASTA sequence includes two syn-tasiRNA sequences inserted in the AtTAS1c precursor sequence:
Where:
X is a DNA base of the syn-tasiRNA-1 sequence, and the subscript number is the base position in the syn-tasiRNA-1 21-mer
X is a DNA base of the syn-tasiRNA-2 sequence, and the subscript number is the base position in the syn-tasiRNA-2 21-mer
X is a DNA base of the AtTAS1c precursor included in the oligonucleotides required to clone the syn-tasiRNA insert in B/c vectors
X is a DNA base of the AtTAS1c precursor
Note that in general, X1=T and X1=T for syn-tasiRNA association with AGO1. SEQ ID NO:388
In the sequence above, replace the sequences
The sequences of the two syn-tasiRNA oligonucleotides are:
Where:
The sequences of the two oligonucleotides to clone syn-tasiRNAs ‘syn-tasiR-Trich’
of AtTAS1c, respectively, are:
3. Cloning of the amiRNA/Syn-tasiRNA Sequences in BsaI ccdB (B/c) Vectors
Notes:—Available B/c vectors are listed in Table I at the end of the section.
Alternatively, BsaI digestion of the B/c vector and subsequent ligation of the amiRNA oligonucleotide insert can be done in separate reactions
Dilute sense oligonucleotide and antisense oligonucleotide in sterile H2O to a final concentration of 100 μM.
Prepare Oligo Annealing Buffer:
60 mM Tris-HCl (pH 7.5), 500 mM NaCl, 60 mM MgCl2, 10 mM DTT
Note: Prepare 1 ml aliquots of Oligo Annealing Buffer and store at −20° C.
Assemble the annealing reaction in a PCR tube as described below:
The final concentration of each oligonucleotide is 4 μM.
Use a thermocycler to heat the annealing reaction 5 min at 94° C. and then cool down (0.05° C./sec) to 20° C.
Dilute the annealed oligonucleotides just prior to assembling the digestion-ligation reaction as described below:
The final concentration of each oligonucleotide is 0.15 μM.
Note: Do not store the diluted oligonucleotides.
Mix the reactions by pipetting. Incubate the reactions at room temperature for 5 minutes at 37° C.
Transform 1-5 ul of the digestion-ligation reaction into an E. coli strain that doesn't have ccdB resistance (e.g. DH10B, TOP10, . . . ) to do counter-selection.
Pick two colonies/construct, grow LB-Kan (100 mg/ml) cultures and purify plasmids.
A. thaliana
A. thaliana
A. thaliana
A. thaliana
indicates data missing or illegible when filed
DNA sequence of 13/c vectors used for direct cloning of amiRNAs in zero-background vectors containing the OsMIR390 sequence.
Index:
cagctttcttgtacaaagttggcattataagaaagcattgcttatcaatt
tgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgCCA
TCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACA
PURPLE/UPPERCASE: M13-forward binding site
orange/lowercase: attL1
BLUE/UPPERCASE: OsMIR390a5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390a 3′ region
orange/lowercase/underlines: attL2
PURPLE/UPPERCASE/UNDERLINED: M13-reverse binding site
brown/lowercase: kanamycin resistance gene
GTGGTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCCACCGCGGTGG
ATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAG
CATGTAATAATTAACATGTAATGCATGACGTTTTTATGAGATGGGTTTTTATGA
TTAGAGTCCCGCAATTATACATITAATACGCGATATTAAAACAAAATATAGCGCG
CAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTGAATTCGTAATC
tccataataatgtgtgagtagttcccagataagggaattagggttcctatagggtttcgctcatgtgttgagcatataagaaacccttagtat
gtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtactaaaatccagatcCCCCGAATTA
brown/lowercase: kanamycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector'sBsaI site
cyan/lowercase: T-DNA right border
GREEN/UPPERCASE: 2×35S CaMV promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: OsMIR390 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Nos terminator
green/lowercase: CaMV promoter
BROWN/UPPERCASE: hygromycin resistance gene
green/lowercase/underlined: CaMV terminator
CYAN/UPPERCASE: T-DNA left border
GTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCT
TGTTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATG
TAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAG
AGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAA
CTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTA
GCCGGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTT
CCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCG
GGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGC
CTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGAC
CGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACC
CACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCCGTTGTCATC
GGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCrCGGATATGCCCCC
CGCGGCATGCGTCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGT
TTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCA
brown/lowercase: kanamycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
cyan/lowercase: T-DNA right border
GREEN/UPPERCASE: 2×35S CaMV promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: OsMIR390 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Nos terminator
green/lowercase: CaMV promoter
BROWN/UPPERCASE: hygromycin resistance gene
green/lowercase/underlined: CaMV terminator
CYAN/UPPERCASE: T-DNA left border
GGTGATATCCCCcggccatgctagagtccgcaaaaatcaccagtctctctctacaaatctatctctctctatttttctccagaat
aatgtgtgagtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgagcatataagaaaccttagtatgtatttgt
atttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtgacctGCAGGCATGCGACGTCGGGC
cyan/lowercase: T-DNA right border
grey/lowercase: OsUbi promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: OsMIR390 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
green/lowercase: CaMV promoter
GREY/UPPERCASE: ZmUbi promoter
BROWN/UPPERCASE: hygromycin resistance gene
CYAN/UPPERCASE: T-DNA left border
brown/lowercase: spectinomycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
DNA sequence of BsaI-ccdB-based (B/c) vectors used for direct cloning of amiRNAs or syn-tasiRNAs.
1. amiRNA vectors
AAGAAAGCATTGCTTTATCAATTTGTTTTCAACGAACACTTTCACTATCAGTCAAAAT
AAAATCATTATTTGCTTCCAGCTGATATCCCCTATAGTGAGTCGTATTACATGG
TCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATT
PURPLE/UPPERCASE: M13-F binding site
orange/lowercase: attL1
BLUE/UPPERCASE: AtMIR390a 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: AtMIR390a 3′ region
orange/lowercase/underlined: attL2
PURPLE/UPPERCASE/UNDERLINED: M13-Reverse binding site
AATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATA
ATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTA
TTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTAATACGCGA
TAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCAT
CTATGTTACTGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT
tcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaattctaagttcctaaaaccaaatccgt
actaaaatccagatcCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCA
brown/lowercase: kanamycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector'BsaI site
cyan/lowercase: T-DNA right border
GREEN/UPPERCASE: 2×35S CaMV promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: AtMIR390a 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Nos terminator
green/lowercase: CaMV promoter
BROWN/UPPERCASE: hygromycin resistance gene
green/lowercase/underlined: CaMV terminator
CYAN/UPPERCASE: T-DNA left border
CCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCG
GTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATT
AACATCTAATTTCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGC
AATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATA
AATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCATGGT
CCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCGGCG
GTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTACCG
AGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCT
ATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGG
GCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTACG
TCTCCCCCCGCCACCAGCGGAGGGGGACTGGGTTTTCACGGTTCTACACCCACCTGCT
GAAGTCCCTGGAGGCACAGGGCTTTCAAGATTCGTGGTCGCTTGTCATCGGGCTGCC
CAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGGCAT
GCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCA
GCTGGACTTCAGCCCTGCCGCTACCGCCCCCGTCCGGTCCTGCCCGTCACCGAGATT
tataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtactaaaatccagat
gCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTA
brown/lowercase: kanamycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->transversion to block vector's BsaI site
cyan/lowercase: T-DNA right border
GREEN/UPPERCASE: 2×35S CaMV promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: AtMIR390a 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: AtMIR390a 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Nos terminator
green/lowercase: CaMV promoter
BROWN/UPPERCASE: hygromycin resistance gene
green/lowercase/underlined: CaMV terminator
CYAN/UPPERCASE: T-DNA left border
TAGTAACATAGATGACACCGCGCGCGATAATTTATCCTAGTTTGCGCGCTATATT
TTGTTTTCTATCGCGTATTAAATGTATAATTGCGGGACTCTAATCATAAAAACCC
ATCTCATAAATAACGTCATGCATTACATGTTAATTATTACATGCTTAACGTAATTC
AACAGAAATTATATGATAATCATCGCAAGACCGGCAACAGGATTCAATCTTAAG
AAACTTTTATTGTAAATGTTTGAACTTTCTGCTTGACTCTAGGGGTCATCAGAT
brown/lowercase: spectinomycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
CYAN/UPPERCASE: T-DNA left border
GREY/UPPERCASE/UNDERLINED: Nos terminator
BROWN/UPPERCASE/UNDERLINED: BASTA resistance gene
GREY/UPPERCASE: Nos promoter
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
GREEN/UPPERCASE: 35S CaMV promoter
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
GREEN/UPPERCASE: 35S promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: AtMIR390a 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: AtMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Pea rbcs terminator
cyan/lowercase: T-DNA right border
2. syn-tasiRNA vectors
CCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTT
GCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGCCATCCAGCTGA
PURPLE/UPPERCASE: M13-F binding site
orange/lowercase: attL1
BLUE/UPPERCASE: AtTAS1c 5′ region
RED/UPPERCASE: BsaI site
red/lowercase: inverted BsaI site
magenta/lowercase: Chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
blue/lowercase: AtTAS1c 3′ region
orange/lowercase/underlined: attL2
PURPLE/UPPERCASE/UNDERLINED: M13-R binding site
brown/lowercase: Kanamycin resistance gene
ACAAAGTGGTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCCACCGC
ATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACG
TTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTT
TATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATA
GCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTGAATTCG
tagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtactaaaatccagatcCCCGAA
brown/lowercase: kanamycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
cyan/lowercase: T-DNA right border
GREEN/UPPERCASE: 2×35S CaMV promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: AtTAS1c 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: AtTAS1c 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Nos terminator
green/lowercase: CaMV promoter
BROWN/UPPERCASE: hygromycin resistance gene
green/lowercase/underlined: CaMV terminator
CYAN/UPPERCASE: T-DNA left border
TTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGC
CGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATA
ATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCC
CGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGG
ATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCAT
AGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCG
GCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTA
CCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGC
GCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACG
CGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGT
ACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCACCT
GCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCT
GCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGG
CATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTG
GCAGCTGGACTTCAGCCTGCCGGTACCGCCCCTCCGGTCCTGCCCGTCACCGAG
agcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtactaaatc
cagatcCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTG
a A transformant shows the Ft phenotype when its ‘days to flowering’ value is higher than the ‘days of flowering’ average of the 35S:GUS control set.
Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to generate amiRNA constructs for silencing transcripts in monocot species are not well adapted for simple, cost-effective and large-scale production. Here, a new series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants exhibited the expected phenotypes predicted by loss of target gene function, and accumulated high levels of amiRNAs and reduced levels of the corresponding target RNAs. Genome-wide transcriptome profiling combined with 5′-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.
A new generation of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale production of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.
MicroRNAs (miRNAs) are a class of ≈21 nt long endogenous small RNAs that posttranscriptionally regulate gene expression in eukaryotes (Bartel, 2004). In plants, DICER-LIKE1 processes MIRNA precursors with imperfect self-complementary foldback structures into miRNA/miRNA* duplexes (Bologna and Voinnet, 2014). Typically, one strand of the miRNA duplex is sorted into an ARGONAUTE (AGO) protein according to the identity of the 5′-terminal nucleotide (nt) of the miRNA (Mi et al., 2008; Montgomery et al., 2008; Takeda et al., 2008) and/or to other sequence or structural properties of the miRNA duplex (Zhu et al., 2011; Endo et al., 2013; Zhang et al., 2014). Plant miRNAs target transcripts with highly complementary sequence through direct AGO-mediated endonucleolytic cleavage, or through other cleavage-independent mechanisms such as target destabilization or translational repression (Axtell, 2013).
Artificial miRNAs (amiRNAs) can be produced accurately by modifying the miRNA/miRNA* sequence within a functional MIRNA precursor (Alvarez et al., 2006; Schwab et al., 2006). AmiRNAs have been used in plants to selectively and effectively knockdown reporter and endogenous genes, non-coding RNAs and viruses (Ossowski et al., 2008; Tiwari et al., 2014). Recently, cost- and time-effective methods to generate large numbers of amiRNA constructs were developed and validated for eudicot species (Carbonell et al., 2014). These included a new generation of eudicot amiRNA vectors based on Arabidopsis thaliana MIR390a (AtMIR390a) precursor, whose relatively short distal stem-loop allows the cost-effective synthesis and cloning of the amiRNA inserts into “B/c” expression vectors (Carbonell et al., 2014). In monocots, OsMIR528 precursor has been used successfully to express amiRNAs for silencing endogenous genes in rice (Warthmann et al., 2008; Butardo et al., 2011; Chen et al., 2012a; Chen et al., 2012b). However, OsMIR528-based cloning methods have not been optimized for efficient generation of monocot amiRNA constructs.
A new series of amiRNA expression vectors for high-throughput cloning and high-level expression in monocot species are described and tested. The new vectors contain a truncated sequence from Oryza sativa MIR390 (OsMIR390) precursor in a configuration that allows the direct cloning of amiRNAs. OsMIR390-based amiRNAs were generally more accurately processed and accumulated to higher levels in transgenic Brachypodium distachyon (Brachypodium) when processed from chimeric precursors (OsMIR390-AtL) containing Arabidopsis thaliana (Arabidopsis) MIR390a (AtMIR390a) distal stem-loop sequences. Functionality of OsMIR390-AtL-based amiRNAs was confirmed in Brachypodium transgenic plants that exhibited the phenotypes expected from loss of target gene function, accumulated high levels of amiRNAs and reduced levels of the corresponding target RNAs. Moreover, genome-wide transcriptome profiling in combination with 5′-RLM RACE analysis confirmed that the amiRNAs were highly specific. We also describe a cost-optimized alternative to generate amiRNA constructs for eudicots, as amiRNAs produced from chimeric AtMIR390a-based precursors including AtMIR390a basal stem and OsMIR390 short distal stem-loop sequences are highly expressed, accurately processed, and effective in target gene knockdown in A. thaliana.
AmiRNA vectors based on the OsMIR390 precursor
Previously, the short AtMIR390a precursor was selected as the backbone for high-throughput cloning of amiRNAs in a new generation of vectors for eudicot species (Carbonell et al., 2014). These vectors allow a zero-background, oligonucleotide cloning strategy that requires no enzymatic modifications, PCR steps, restriction digestions, or DNA fragment isolation (Carbonell et al., 2014). The short distal stem-loop (
Publicly available small RNA data sets from rice (Heisel et al., 2008; Zhu et al., 2008; Johnson et al., 2009; Zhou et al., 2009; He et al., 2010) were analyzed to assess the OsMIR390 precursor processing accuracy. Approximately 70% of reads mapping to the OsMIR390 foldback correspond to the authentic 21-nt miR390 guide strand (
A series of OsMIR390-based cloning vectors named ‘OsMIR390-B/c’ (from OsMIR390-BsaI/ccdB) were developed for direct cloning of amiRNAs (Figure S1, Table I). OsMIR390-B/c vectors contain a truncated OsMIR390 precursor sequence whose miRNA/distal stem-loop/amiRNA* region was replaced by a DNA cassette containing the counter-selectable ccdB gene (Bernard and Couturier, 1992) flanked by two BsaI sites. AmiRNA inserts corresponding to amiRNA/OsMIR390-distal-stem-loop/amiRNA* sequences are synthesized using two overlapping and partially complementary 60-base oligonucleotides (Figure S2). Forward and reverse oligonucleotides must have 5′-CTTG and 5′-CATG overhangs, respectively, for direct cloning into OsMIR390-based vectors (Figure S2).
OsMIR390-B/c vectors include pMDC32B-OsMIR390-B/c, pMDC123SB-OsMIR390-B/c and pH7WG2B-OsMIR390-B/c plant expression vectors, each of which contains a unique combination of bacterial and plant antibiotic resistance genes and regulatory sequences (Figure S1, Table I). Additionally, a pENTR-OsMIR390-B/c GATEWAY-compatible entry vector was generated for direct cloning of the amiRNA insert and subsequent recombination into a preferred GATEWAY expression vector containing a promoter, terminator or other features of choice (Figure S1, Table I).
High Accumulation of amiRNAs Derived from Chimeric Precursors in Brachypodium calli
To test amiRNA expression from OsMIR390 precursors, transformed B. distachyon calli containing amiRNA constructs expressing miR390 or modified versions of several miRNAs from Arabidopsis (amiR173-21, amiR472-21 or amiR828-21) (Cuperus et al., 2010) were analyzed (
Surprisingly, miR390 accumulated to highest levels when expressed from the chimeric OsMIR390-AtL precursor compared to each of the other three precursors (P≦0.001 for all pairwise t-test comparisons;
To assess the accuracy of precursor processing, small RNA libraries from samples expressing OsMIR390-AtL-based amiRNAs were prepared and sequenced (
Gene Silencing in Brachypodium and Arabidopsis by amiRNAs Derived from Chimeric Precursors
To test the functionality of OsMIR390-AtL-derived amiRNAs in repressing target transcripts in Brachypodium, BRASSINOSTEROID-INSENSITIVE 1 (BdBRI1), CINNAMYL ALCOHOL DEHYDROGENASE 1 (BdCAD1), CHLOROPHYLLIDE A OXYGENASE (BdCAO) and SPOTTED LEAF 11 (BdSPL11) gene transcripts were targeted by amiRNAs expressed from the chimeric OsMIR390-AtL and from authentic OsMIR390 precursors (
Sixteen out of 20 and 11 out of 17 transgenic lines containing 35S:OsMIR390-AtL-Bri1 or 35S:OsMIR390-Bri1, respectively, which were predicted to have brassinosteroid signaling defects, had reduced height and altered architecture (
Each of 27 35S:OsMIR390-AtL-Cao-expressing plants, and 12 of 12 of 35S:OsMIR390-Cao-expressing plants exhibited light green color compared to control plants (
Accumulation of amiRNA target mRNAs in Brachypodium transgenic lines expressing OsMIR390-AtL- or OsMIR390-based amiRNAs was analyzed by quantitative real time RT-PCR (RT-qPCR) assay. The expression of all target mRNAs was significantly reduced compared to control plants (P<0.005 for all pairwise t-test comparisons,
AmiR-BdBri1, amiR-BdCao and amiR-BdSpl11 produced from chimeric OsMIR390-AtL precursors were also expressed using pH7WG2B-based constructs that contain the rice ubiquitin (UBI) regulatory sequences. Each of the three UBI promoter-driven amiRNAs induced the expected phenotypes in a relatively high proportion of Brachypodium T0 lines (Table S3), and in the one case tested (amiR-BdSpl11), phenotypes were heritable in the T1 generation (Table S4).
Finally, we tested if the reciprocal chimeric AtMIR390a-OsL precursor could be used to express amiRNAs efficiently in eudicots. The synthesis of AtMIR390a-OsL-based constructs requires shorter oligonucleotides than the generation of AtMIR390a-based constructs, and therefore would be a further cost-optimized alternative. As shown in Nicotiana benthamiana and Arabidopsis assays, AtMIR390-OsL precursors are accurately processed (Appendix S1, Figures S8-S10). Indeed, amiRNAs produced from chimeric AtMIR390a-OsL precursors are highly expressed, accurately processed and highly effective in target gene knockdown in T1 Arabidopsis transgenic plants (Appendix S1, Figures S9-S11, Table S5). Moreover, amiRNA induced phenotypes were still obvious in T2 plants confirming the heritability of the effects (Table S6). Therefore, the use of AtMIR390a-OsL precursors may be an attractive alternative to express effective amiRNAs in eudicots in a cost-optimized manner.
Accuracy of Processing of OsMIR390 and OsMIR390-AtL Chimeric Precursors in Brachypodium
The accumulation of each amiRNA from chimeric and OsMIR390 precursors was analyzed by RNA blot analysis in T0 transgenic lines showing amiRNA-induced phenotypes (
To more accurately assess processing and accumulation of the amiRNA populations, small RNA libraries from transgenic lines expressing amiRNAs from chimeric OsMIR390-AtL or authentic OsMIR390 precursors were prepared (
The reasons explaining the accumulation of OsMIR390a-AtL-based amiRNAs that are 1 nt-shorter than expected are not clear. AmiRNAs shorter than expected and differing on their 3′ end were also described using AtMIR319a precursors in Arabidopsis (Schwab et al., 2006). Importantly, a recent study has shown that amiRNA efficacy is not affected by the loss of the base-pairing at the 5′ end of the target site (Liu et al., 2014). Regardless, the inaccurate processing of an amiRNA precursor leading to the accumulation of diverse small RNA populations could conceivably induce undesired off-target effects. This potential complication argues against using authentic OsMIR390 precursors to express amiRNAs in Brachypodium and possibly other monocot species.
Reads from the amiRNA* strands from each of the OsMIR390 and OsMIR390-AtL-derived precursors were under-represented, relative to the amiRNA strands (
High Specificity of amiRNA Derived from Chimeric Precursors in Brachypodium
To assess amiRNA target specificity at a genome-wide level, transcript libraries from control (35S: GUS) and amiRNA-expressing lines were generated and analyzed. Only lines expressing amiRNAs from the more accurately processed OsMIR390-AtL precursors were analyzed. Differential gene expression analyses were done by comparing, in each case, the transcript libraries obtained from four independent control lines with those obtained from four independent amiRNA-expressing lines exhibiting the expected phenotypes. Four hundred and ninety four, 1847 and 818 genes were differentially expressed in plants expressing amiR-BdBri1, amiR-BdCao and amiR-BdSpl11, respectively (
To assess potential off-target effects of the amiRNAs, TargetFinder (Fahlgren and Carrington, 2010) was used to generate a genome-wide list of potential candidate targets that share relatively high sequence complementarity with each amiRNA. TargetFinder ranks the potential amiRNA targets based on a Target Prediction Score (TPS) assigned to each amiRNA-target interaction. Scores range from 1 to 11, that is, from highest to lowest levels of sequence complementarity between the small RNA and putative target RNA. Indeed, when designing amiRNAs with the “P-SAMS amiRNA Designer” tool, “optimal” amiRNAs are selected when i) their interaction with the desired target has a TPS=1, and ii) no other amiRNA-target interactions have a TPS<4 (Fahlgren et al., in preparation). Therefore, direct off-target effects with amiRNAs described here can only occur through amiRNA-target RNA interactions with a TPS in the [4, 11] interval. It was hypothesized that off-target effects, if due to base-pairing between amiRNAs and the affected transcripts, would be reflected by the presence of differentially underexpressed genes corresponding to target RNAs with lower TPS scores in the [4, 11] interval. Therefore, we next analyzed for all TargetFinder-predicted targets for each amiRNA if their corresponding genes were differentially underexpressed in amiRNA-expressing lines versus controls.
As expected from P-SAMS design, BdCad1, BdCao and BdSpl11 were the only genes differentially underexpressed in the [1,4[TPS interval in plants expressing amiR-BdCad1, amiR-BdCao and amiR-BdSpl11, respectively (
Next, we used 5′-RLM-RACE to test for amiRNA-directed off-target cleavage of underrepresented transcripts. This analysis detects 3′ cleavage products expected from small RNA-guided cleavage events. Only TargetFinder predicted targets with a TPS≦7 were included in the analysis, as targets with higher score are not considered likely to be cleaved, according to previous studies (Addo-Quaye et al., 2008). For all specific targets, 3′ cleavage products of the expected size were detected in samples expressing the corresponding amiRNA, but not in control samples expressing 35S:GUS (
High amiRNA specificity was previously indicated for AtMIR319a-derived amiRNAs in Arabidopsis based on genome-wide expression profiling (Schwab et al., 2006). However, a recent and systematic processing analysis of AtMIR319a-based amiRNA precursors in petunia (Guo et al., 2014) showed that multiple small RNA variants are generated from different regions of the precursor, and that many of these small RNAs meet the required criteria for amiRNA design (Schwab et al., 2006). Here, the fact that chimeric OsMIR390-AtL precursors produce high levels of accurately processed amiRNAs not only in Brachypodium (
We have developed and validated a new generation of expression vectors based on the OsMIR390 precursor for high-throughput cloning and high expression of amiRNAs in monocots. OsMIR390-B/c-based vectors allow the direct cloning of amiRNAs in a zero-background strategy that requires no oligonucleotide enzymatic modifications, PCR steps, restriction digestions, or DNA fragment isolation. Thus, OsMIR390-B/c-based vectors are particularly attractive for generating large-scale amiRNA construct libraries for silencing genes in monocots.
“P-SAMS amiRNA Designer” tool was used to design four different amiRNAs, each of which was aimed to target specifically one Brachypodium gene transcript. We show that chimeric OsMIR390-AtL precursors including OsMIR390 basal stem and AtMIR390a distal stem-loop were processed more accurately, and the resulting amiRNAs generally accumulated to higher levels than amiRNAs derived from authentic OsMIR390 precursors in Brachypodium transgenic plants. Each P-SAMS-designed amiRNA induced the expected phenotypes predicted by loss of target gene function, and specifically decreased expression of the expected target gene. Chimeric OsMIR390-AtL precursors designed using P-SAMS, therefore, are likely to be highly effective and specific in silencing genes in monocot species.
Plant Materials and Growth Conditions
Arabidopsis thaliana Col-0 and N. benthamiana plants were grown as described (Carbonell et al., 2014). Brachypodium distachyon 21-3 plants were grown in a chamber under long day conditions (16/8 hr photoperiod at 200 μmol m−2 s−1) and 24° C./18° C. temperature cycle.
Arabidopsis thaliana plants were transformed using the floral dip method with Agrobacterium tumefaciens GV3101 strain (Clough and Bent, 1998). A. thaliana transgenic plants were grown on plates containing Murashige and Skoog medium hygromycin (50 mg/ml) for 10 days before being transferred to soil. Embryogenic calli from B. distachyon 21-3 plants were transformed as described (Vogel and Hill, 2008). Photographs of plants were taken as described (Carbonell et al., 2014).
DNA Constructs
pENTR-OsMIR390-BsaI construct was generated by ligating into pENTR (Life Technologies) the DNA insert resulting from the annealing of oligonucleotides BsaI-OsMIR390-F and BsaI-OsMIR390-R. Rice ubiquitin 2 promoter and maize ubiquitin promoter-hygromycin cassettes were transferred into the GATEWAY binary destination vector pH7WG2 (Karimi et al 2002) to generate pH7WG2-OsUbi. pH7WG2-OsMIR390-BsaI, pMDC123SB-OsMIR390-BsaI and pMDC32-OsMIR390-BsaI were obtained by LR recombination using pENTR-OsMIR390-BsaI as the donor plasmid and pH7WG2-OsUbi, pMDC32B (Carbonell et al., 2014) and pMDC123SB (Carbonell et al., 2014) as destination vectors, respectively. A modified ccdB cassette (Carbonell et al., 2014) was inserted between the BsaI sites of pENTR-OsMIR390-BsaI, pMDC123SB-OsMIR390-BsaI, pMDC32B-OsMIR390-BsaI and pH7WG2-OsMIR390-BsaI to generate pENTR-OsMIR390-B/c, pMDC123SB-OsMIR390-B/c, pMDC32B-OsMIR390-B/c and pH7WG2-OsMIR390-B/c, respectively. Finally, an undesired BsaI site was disrupted in pH7WG2-OsMIR390-B/c to generate pH7WG2B-OsMIR390-B/c. The sequences of the OsMIR390-B/c-based amiRNA vectors are listed in Appendix S2. The following amiRNA vectors for monocots are available from Addgene (http://www.addgene.org/): pENTR-OsMIR390-B/c (Addgene plasmid 61468), pMDC32B-OsMIR390-B/c (Addgene plasmid 61467) pMDC123SB-OsMIR390-B/c (Addgene plasmid 61466) and pH7WG2B-OsMIR390-B/c (Addgene plasmid 61465). pMDC32B-AtMIR390a-B/c (Addgene plasmid 51776) was described before (Carbonell et al., 2014).
The rest of the amiRNA constructs (pMDC32B-AtMIR390a-OsL-173-21, pMDC32B-AtMIR390a-OsL-472-21, pMDC32B-AtMIR390a-OsL-828-21, pMDC32B-AtMIR390a-OsL-Ch42, pMDC32B-AtMIR390a-OsL-Ft, pMDC32B-AtMIR390a-OsL-Trich, pMDC32B-OsMIR390, pMDC32B-OsMIR390-AtL, pMDC32B-OsMIR390-173-21, pMDC32B-OsMIR390-173-21-AtL, pMDC32B-OsMIR390-472-21, pMDC32B-OsMIR390-AtL-472-21, pMDC32B-OsMIR390-828-21, pMDC32B-OsMIR390-AtL-828-21, pMDC32B-OsMIR390-Bri1, pMDC32B-OsMIR390-AtL-Bri1, pMDC32B-OsMIR390-Cao, pMDC32B-OsMIR390-AtL-Cao, pMDC32B-OsMIR390-Cad1, pMDC32B-OsMIR390-AtL-Cad1, pMDC32B-OsMIR390-Spl11, pMDC32B-OsMIR390-AtL-Spl11, pH7WG2B-OsMIR390-Bri1-AtL, pH7WG2B-OsMIR390-Cao-AtL, and pH7WG2B-OsMIR390-Spl11-AtL) were obtained as described in the next section. Control construct pH7WG2-GUS was obtained by LR recombination using pENTR-GUS (Life technologies) as the donor plasmid and pH7GW2-OsUbi as the destination vector. pMDC32-GUS construct was described previously (Montgomery et al., 2008). The sequence of all amiRNA precursors used in this study are listed in Appendix S3. All oligonucleotides used for generating the constructs described above are listed in Table S7.
amiRNA Oligonucleotide Design and Cloning
Sequences of the amiRNAs expressed in A. thaliana were described previously (Schwab et al., 2006; Felippes and Weigel, 2009; Liang et al., 2012; Carbonell et al., 2014). Sequences of the amiRNAs expressed in Brachypodium, and their corresponding oligonucleotides for cloning in OsMIR390-B/c vectors, were designed with the “P-SAMS amiRNA Designer” tool (http://p-sams.carringtonlab.org) (Fahlgren et al., in preparation). The sequences and predicted targets for all the amiRNAs used in this study are listed in Table S8.
The generation of constructs to express amiRNAs from authentic AtMIR390a precursors was described before (Carbonell et al., 2014). Detailed oligonucleotide design for amiRNA cloning in OsMIR390, OsMIR390-AtL and AtMIR390a-OsL precursors is given in Figures S2, S3 and S4, respectively. The amiRNA cloning procedure is described in Appendix S4. All oligonucleotides used in this study for cloning amiRNA sequences are listed in Table S7.
Transient Expression Assays in N benthamiana
Transient expression assays in N. benthamiana leaves were done as described (Carbonell et al., 2014) with A. tumefaciens GV3101 strain.
RNA-Blot Assays
Total RNA from Arabidopsis, Brachypodium or N. benthamiana was extracted using TRIzol® reagent (Life Technologies) as described (Cuperus et al., 2010). RNA blot assays were done as described (Cuperus et al., 2010). Oligonucleotides used as probes for small RNA blots are listed in Table S7.
Quantitative Real-Time RT-qPCR
RT-qPCR reactions and analyses were done as described (Carbonell et al., 2014). Primers used for RT-qPCR are listed in Table S7 (and are named with the prefix ‘q’). Target mRNA expression levels were calculated relative to four A. thaliana (AtACT2, AtCPB20, AtSAND and AtUBQ10) or B. distachyon (BdSAMDC, BdUBC18, BdUBI4 and BdUBI10) reference genes as described (Carbonell et al., 2014).
5′-RLM-RACE
5′ RNA ligase-mediated rapid amplification of cDNA ends (5′-RLM-RACE) was done using the GeneRacer™ kit (Life Technologies) but omitting the dephosphorylation and decapping steps. Total RNA (2 μg) was ligated to the GeneRacer RNA Oligo Adapter. The GeneRacer Oligo dT primer was then used to prime first strand cDNA synthesis in reverse transcription reaction. An initial PCR was done by using the GeneRacer 5′ and 3′ primers. The 5′ end of cDNA specific to each mRNA was amplified with the GeneRacer 5′ Nested primer and a gene specific reverse primer. For each gene, control PCR reactions were done using gene specific forward and reverse primers. Oligonucleotides used are listed in Table S7. 5′-RLM-RACE products were gel purified using MinElute gel extraction kit (Qiagen), cloned using the Zero Blunt® TOPO® PCR cloning kit (Life Technologies), introduced into Escherichia coli DH10B, screened for inserts, and sequenced.
Chlorophyll and Carotenoid Extraction and Analysis
Pigments from Brachypodium leaf tissue (40 mg of fresh weight) were extracted with 5 ml 80% (v/v) acetone in the dark at room temperature for 24 hours, and centrifuged at 4000 rpm during two minutes. One hundred μl of supernatant was diluted 1:2 with 80% (v/v) acetone and loaded to flat bottom 96-well plates. Absorbance was measured from 400 to 750 nm wavelengths in a SpectrMax M2 microplate reader (Molecular Devices, Sunnyvale, Calif.) using the software SoftMax Pro 5 (Molecular Devices, Sunnyvale, Calif.). Content in chlorophyll a, chlorophyll b, and carotenoids was calculated with the following formulas: Chlorophyll a (mg/L in extract)=12.21*Absorbance663 nm−2.81*Absorbance647 nm; Chlorophyll b (mg/L in extract)=20.13*Absorbance647 nm−5.03*Absorbance663 nm; Carotenoid (mg/L in extract)=[1000*Absorbance470 nm−3.27*Chlorophyll a (mg/L)−104*Chlorophyll b (mg/L)]/227.
Preparation of Small RNA Libraries
Fifty to 100 μs of Arabidopsis, Brachypodium or Nicotiana total RNA were treated as described (Carbonell et al., 2012; Gilbert et al., 2014), but each small RNA library was barcoded at the amplicon PCR reaction step using an indexed 3′ PCR primer (i1-i8, i10 or ill) and the standard 5′PCR primer (P5) (Table S7). Libraries were multiplexed and subjected to sequencing analysis using a HiSeq 2000 sequencer (Illumina).
Small RNA Sequencing Analysis
Small RNA sequencing analysis was done as described (Carbonell et al., 2014). Custom scripts to process small RNA data sets are available at https://github.com/carringtonlab/srtools. A summary of high-throughput small RNA sequencing libraries from transgenic Arabidopsis inflorescences and Brachypodium calli or leaves, and from N. benthamiana agroinfiltrated leaves, is provided in Table S9. O. sativa small RNA data sets used in the processing analysis of authentic OsMIR390 presented in
Preparation of Strand-Specific Transcript Libraries
Ten μg of total RNA extracted from four independent lines per construct were treated with TURBO DNAse I DNA-free (Life Technologies). Samples were depleted of ribosomal RNAs by treatment with Ribo-Zero Magnetic Kit “Plant Leaf” (Epicentre) according to manufacturer's instructions. cDNA synthesis and strand-specific transcript libraries were made as described (Wang et al., 2011; Carbonell et al., 2012), with the following modifications. Ribo-Zero treated RNAs were fragmented with metal ions during 4 minutes at 95° C. prior to library construction, and 14 cycles were used in the linear PCR reaction. DNA adaptors 1 and 2 were annealed to generate the Y-shape adaptors, and PE-F oligonucleotide was combined with one indexed oligonucleotide (PE-R-N701 to PE-R-N710) in the linear PCR (see Table S7). DNA amplicons were analyzed with a Bioanalyzer (DNA HS kit; Agilent), quantified using the Qubit HS Assay Kit (Invitrogen), and sequenced on a HiSeq 2000 sequencer (Illumina).
Transcriptome Analysis
FASTQ files were de-multiplexed with the parseFastq.pl perl script (https://github.com/carringtonlab/srtools). Sequencing reads from each de-multiplexed transcript library were mapped to B. distachyon transcriptome (v2.1, Phytozome 10) using Butter (Axtell, 2014) and allowing one mismatch. Differential gene expression analysis was done using DESeq2 (Love et al., 2014) with a false discovery rate of 1%. For each 35S:GUS versus 35S:OsMIR390-AtL pairwise comparison, genes having no expression (0 gene counts) in at least five of the eight samples were removed from the analysis. Differential gene expression analysis results are shown in Data S1.
TargetFinder v1.7 (https://github.com/carringtonlab/TargetFinder) (Fahlgren and Carrington, 2010) was used to obtain a ranked list of potential off-targets for each amiRNA.
A summary of high-throughput RNA-Seq libraries from transgenic Brachypodium leaves is provided in Table S10.
Accession Numbers
A. thaliana gene and locus identifiers are as follows: AtACT2 (AT3G18780), AtCBP20 (AT5G44200), AtCH42 (AT4G18480), AtCPC (AT2G46410), AtETC2 (AT2G30420), AtFT (AT1G65480), AtSAND (AT2G28390), AtTRY (AT5G53200) and AtUBQ10 (AT4G05320). B. distachyon gene and locus identifiers are as follows: BdBRI1 (Bradi2g48280), BdCAD1 (Bradi3g06480), BdCAO (Bradi2g61500), BdSAMDC (Bradi5g14640), BdSPL11 (Bradi4g04270), BdUBC18 (Bradi4g00660), BdUBI4 (Bradi3g04730) and BdUBI10 (Bradi1g32860). The miRBase (http://mirbase.org) (Kozomara and Griffiths-Jones, 2014) locus identifiers of the conserved rice MIRNA precursors and plant MIR390 precursors (
High-throughput sequencing data from this article can be found in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under accession number SRP052754.
Nicotiana benthamiana
Nicotiana benthamiana
Brachypodium distachyon
Brachypodium distachyon
MI0000654
MI0000655
MI0000656
MI0000657
MI0000658
MI0000659
MI0000660
MI0000661
MI0000662
MI0001090
MI0001091
MIMAT0001022
MI0001093
MI0001094
MI0001095
MI0001096
MI0001097
MI0000663
MI0000664
MI0000665
MI0000666
MI0001100
MI0001101
MI0000667
MI0001102
MI0000668
MI0000669
MI0001103
MI0001104
MI0001105
MI0001159
MI0000670
MI0000671
MI0000672
MI0000673
MI0000674
MI0000675
MI0001142
MI0001143
MI0001144
MI0001158
MI0001107
MI0001108
MI0001157
MIMAT0001088
MI0000676
MI0000677
MI0000678
MI0001109
MI0001110
MI0001111
MI0001112
MI0001113
MI0001114
MI0001156
MI0001115
MI0000679
MI0001117
MI0001118
MI0001119
MI0001120
MI0001121
MI0001122
MI0001123
MI0001124
MI0001125
MI0001126
MI0001127
MI0001128
MI0001129
MI0001130
MI0001131
MI0001132
MI0000680
MI0001133
MI0001134
MI0001135
MI0001136
MI0001137
MI0001138
MI0001147
MI0001155
MI0001139
MI0001140
MI0001141
MI0001154
MI0001098
MI0001099
MI0001690
MI0001026
MI0001148
MI0001027
MI0001042
MI0001028
MI0001041
MI0001029
MI0001030
MI0001043
MI0001031
MI0001032
MI0001033
MI0001034
MI0001035
MI0001036
MI0005084
MI0005085
MI0005086
MI0005087
MI0005088
MI0005092
MI0001037
MI0001038
MI0001044
MI0005090
MI0005091
MI0001046
MI0001047
MI0001048
MI0013049
MI0001703
MI0010563
MI0013048
MI0001049
MI0001050
MI0001051
MI0001052
MI0001053
MI0001054
MI0001055
MI0001056
MI0001057
MI0001058
MI0001059
MI0001060
MI0001061
MI0001062
MI0001063
MI0001149
MI0003201
MI0010490
MI0014569
MI0014570
MI0001000
MI0001001
MI0006447
MI0006448
MI0006449
MI0021077
MI0023238
MI0018164
MI0023239
MI0023237
MI0013317
MI0005647
MI0005648
MI0005649
MI0007214
MI0007215
MI0007845
MI0021700
MI0021701
MI0021702
MI0021703
MI0022249
MI0022250
MI0023073
MI0023074
MI0023075
MI0023076
MI0023077
MI0023078
MI0005586
MI0021391
MI0021392
MI0021393
MI0022095
MI0005787
MI0002305
MI0002306
MI0002307
MI0002308
MI0013410
MI0013411
MI0017503
MI0017504
MI0006552
aThe Bri1 phenotype was defined as a shorter height and presence of splindly leaves in amiR-Bri1 transformants when compared to transformants of the 35S:GUS control set.
aThe Bri1 phenotype was defined as a shorter height and presence of splindly leaves in amiR-Bri1 transformants when compared to transformants of the 35S:GUS control set.
aThe Ft phenotype was defined as a higher ‘days to flowering’ value when compared to the average ‘days to flowering’ value of the 35S:GUS control set.
aThe Ft phenotype was defined as a higher ‘days to flowering’ value when compared to the average ‘days to flowering’ value of the 35S:GUS control set.
Arabidopis
thaliana
Arabidopis
thaliana
Arabidopis
thaliana
Arabidopis
thaliana
Arabidopis
thaliana
Brachypodium
distachyon
Brachypodium
distachyon
Brachypodium
distachyon
Brachypodium
distachyon
Brachypodium
distachyon
benthamiana plants.
N. benthamiana
N. benthamiana
N. benthamiana
N. benthamiana
N. benthamiana
N. benthamiana
B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
A. thaliana
A. thaliana
A. thaliana
B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
B. distachyon
Characterization of AtMIR390a-OsL-Based amiRNAs in Eudicots
Accumulation and Processing of amiRNAs Produced from AtMIR390a- or OsMIR390-Based Precursors in Nicotiana benthamiana
A key feature of the AtMIR390a-B/c-based cloning system to produce amiRNA constructs for eudicots is that the amiRNA insert can be synthesized by annealing two relatively short 75 bases-long oligonucleotides (Carbonell et al., 2014). Because the oligonucleotides containing OsMIR390 distal stem-loop sequences are even shorter (60 bases), we first tested if amiRNAs derived from precursors including OsMIR390 distal stem-loop sequences could be expressed efficiently in eudicot species. This would reduce the synthesis cost of the oligonucleotides required for generating AtMIR390a-based amiRNA constructs, and benefit the generation of large amiRNA construct libraries for gene knockdown in eudicots such as those reported recently (Hauser et al., 2013; JoverGil et al., 2014).
To test the functionality of authentic OsMIR390 precursors to produce high levels of accurately processed small RNAs, miR390 and three different amiRNA sequences (amiR173-21, amiR472-21 and amiR828-21) (Cuperus et al., 2010) were directly cloned into pMDC32B-OsMIR390-B/e (Figure S1, Table I) and expressed transiently in N. benthamiana leaves (Figure S5). The same small RNA sequences were also expressed from the chimeric AtMIR390a-OsL precursor including AtMIR390a basal stem and OsMIR390 distal stem-loop sequences (Figure S4, Figure S8a). For comparative purposes, the same small RNA sequences were expressed from the authentic AtMIR390a precursor or from a chimeric precursor including OsMIR390 basal stem and AtMIR390a stem-loop sequences (OsMIR390-AtL) (Figure S3, Figure S8a). Samples expressing the B-glucuronidase transcript from the 35S: GUS construct were used as negative controls.
MiR390 accumulated to similar levels when expressed from each of the different precursors (Figure S8b). In each case, amiRNAs expressed from AtMIR390a-OsL precursors did not accumulate to significantly different levels than did the corresponding amiRNAs produced from authentic AtMIR390a precursors (P>0.11 for all pairwise t-test comparisons) (Figure S8b). AtMIR390a-OsL-derived amiRNAs accumulated predominantly to 21 nt species, suggesting that the chimeric amiRNA precursors were likely processed accurately (Figure S8b). Finally, amiRNAs produced from either authentic OsMIR390 or chimeric OsMIR390-Ath precursors did not always accumulated as 21 nt species (e g miR828-21 and amiR472-21 from OsMIR390 or OsMIR390-AtL precursors, respectively) (Figure S8b). Therefore, further analyses focused on characterizing AtM1R390a-OsL-based amiRNAs.
To more accurately assess processing of the amiRNA populations produced from AtMIR390a-OsL precursors, small RNA libraries were prepared and sequenced. For comparative purposes, small RNA libraries from samples containing AtMIR390a-derived amiRNAs were also analyzed. In each case, the majority of reads from either the chimeric AtMIR390a-OsL or authentic AtMIR390a precursors corresponded to correctly processed, 21 nt amiRNA (Figure S8c).
Gene Silencing in Arabidopsis by amiRNAs Derived from Chimeric Precursors
To test the functionality of AtMIR390a-OsL based amiRNAs in repressing target transcripts, three different amiRNA constructs were introduced into A. thaliana Col-Oplants. For comparative purposes, the same three amiRNA sequences were also expressed from authentic AtMIR390a precursors as reported before (Carbonell et aL, 2014). In particular, amiR-AtFt, and amiR-AtCh42 each targeted a single gene transcript [FLOWERING LOCUS T (FT) and CHLORINA 42 (CH42), respectively], and amiRAtTrich targeted three MYB transcripts [TRIPTYCHON (TRY), CAPRICE (CPC) and ENHANCER OF TRIPTYCHON AND CAPRICE2 (ETC2)] (Figure S9). Plants including 35S: GUS were used as negative controls. Plant phenotypes, amiRNA accumulation, mapping of amiRNA reads in AtMIR390a-OsL precursors and target mRNA accumulation were measured in Arabidopsis Ti transgenic lines.
Each of the 44 transformants containing 35S:AtMIR390a-OsL-Ft was significantly delayed in flowering time compared to control plants not expressing the amiRNA (P<0.01 two sample t-test, Figure S 1 Ob, Figure S11, Table S5), as previously observed in amiRNA knockdown lines (Schwab et al., 2006; Liang et al., 2012; Carbonell et al., 2014) and ft mutants (Koornneef et aL, 1991). Two hundred and sixty-six out of 267 transgenic lines containing 35S:AtMIR390a-OsL-Ch42 were smaller than controls and had bleached leaves and cotyledons (Figure SlOc, Figure S11, Table S5), as consequence of defective chlorophyll biosynthesis and loss of Ch42 magnesium chelatase (Koncz et al., 1990; Felippes and Weigel, 2009). One hundred and seventy of these plants had a severe bleached phenotype with a lack of visible true leaves at 14 days after plating (Figure S 10c, Figure S11, Table S5). Finally, 68 out of 69 lines containing 35S:AtMIR390a-OsL-Trick had increased number of trichomes in rosette leaves; six lines had highly clustered trichomes on leaf blades like try cpc double mutants (Schellmann et al., 2002) or other amiR-Trich overexpressor transgenic lines (Schwab et al., 2006; Liang et al., 2012; Carbonell et al., 2014) (Figure SlOd, Table S5). The delayed flowering and trichome phenotypes were maintained in the Arabidopsis T2 progeny expressing amiR-Ft and amiR-Trich, respectively, from chimeric AtMIR390a-OsL precursors (Table S6). No obvious phenotypic differences were observed between plants expressing the amiRNAs from the AtMIR390a-OsL or AtMIR390a precursors in either T1 or T2 generations (Figure S 10b-d, Figure S11, Tables S5 and S6). In summary, AtMIR390-OsL-based amiRNAs conferred a high proportion of expected and heritable target-knockdown phenotypes in transgenic plants.
The accumulation of all three amiRNAs produced from chimeric Ati111R390-OsL or authentic Atl11IR390a precursors was confirmed by RNA blot analysis in T1 transgenic lines showing amiRNA-induced phenotypes (Figure S10e). In all cases, AtM[R390-OsL and AtMIR390a-derived amiRNAs accumulated to similarly high levels and as a single species of 21 nt (Figure S10e), suggesting that AtMIR390a-OsL-based amiRNAs were as accurately processed as AtMIR390a-based amiRNAs. To more precisely assess processing and accumulation of the AtMIR390a-OsL-based amiRNA populations, small RNA libraries from samples containing each of the AtMIR390a-OsL-based constructs were prepared. In each case, the majority of reads from AtMIR390a-OsL precursors corresponded to correctly processed, 21 nt amiRNA while reads from the amiRNA* strands were always relatively under-represented (Figure SlOg) as observed before with the same amiRNAs expressed from AtMIR390a precursors (Carbonell et al., 2014).
Finally, accumulation of target mRNAs in A. thaliana transgenic lines expressing AtMIR390a-OsL- or AtMIR390a-based amiRNAs was analyzed by quantitative real time RT-PCR assay. The expression of all target mRNAs was significantly reduced compared to control plants (P<0.023 for all pairwise t-test comparisons, Figure SlOf) when the specific amiRNA was expressed. No significant differences were observed in target mRNA expression between lines expressing AtMIR390a-OsL- or Ati111R390a-based amiRNAs.
Collectively, all these results indicate that amiRNAs produced from chimeric AtIVER390a-OsL precursors are highly expressed, accurately processed and highly effective in target gene knockdown. Therefore, the use of chimeric AtM1R390a-OsL precursors is an attractive alternative to express effective amiRNAs in eudicots in a cost-optimized manner.
DNA sequence of B/c vectors used for direct cloning of amiRNAs in zero-background vectors containing the OsMIR390 sequence.
gctttcttgtacaaagttggcattataagaaaccattgcttatcgatttg
ttgcaacgaacaggtcactatcagtcaaaataaaatcattatttaCCATC
TGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGA
PURPLE/UPPERCASE: M13-forward binding site
orange/lowercase: attL1
BLUE/UPPERCASE: OsMIR390a5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390a 3′ region
orange/lowercase/underlines: attL2
PURPLE/UPPERCASE/UNDERLINED: M13-reverse binding site
brown/lowercase: kanamycin resistance gene
GTGGTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGCGCGCCCACCGCGGTGG
ATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAG
CATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGA
TTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCG
CAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTGAATTCGTAATC
tccataataatgtgtgagtagttcccagataagggaattagggttcctatagggtttcagctcatgtgttgagcatataagaaacccttagtat
gtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtactaaaatccagatcCCCCGAATTA
brown/lowercase: kanamycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
cyan/lowercase: T-DNA right border
GREEN/UPPERCASE: 2×35S CaMV promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: OsMIR390 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Nos terminator
green/lowercase: CaMV promoter
BROWN/UPPERCASE: hygromycin resistance gene
green/lowercase/underlined: CaMV terminator
CYAN/UPPERCASE: T-DNA left border
GTTCGATAATTCCTTAATTAACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCT
TGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATG
TAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAG
AGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAA
CTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTA
GCCGGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTT
CCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCG
GGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGC
CTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGAC
CGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACC
CACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTQTCATC
GGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCC
CGCGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGT
TTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCA
catgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaaataaaattctaattcctaaaaccaaaatccagta
ctaaaatccagatcCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCA
brown/lowercase: kanamycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
cyan/lowercase: T-DNA right border
GREEN/UPPERCASE: 2×35S CaMV promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: OsMIR390 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
GREY/UPPERCASE/UNDERLINED: Nos terminator
green/lowercase: CaMV promoter
BROWN/UPPERCASE: hygromycin resistance gene
green/lowercase/underlined: CaMV terminator
CYAN/UPPERCASE: T-DNA left border
TTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGA
ACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACG
CTCTTTTCTCTTAGGtttacccgccaatatatcctgtcaAACACTGATAG
TTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTaagct
tattcgggtcaaggcggaagccagcgcgccaccccacgtcagcaaatacg
gaggcgcggggttgacggcgtcacccggtcctaacggcgaccaacaaacc
agccagaagaaattacagtaaaaaaaaagtaaattgcactttgatccacc
ttttattacctaagtctcaatttggatcacccttaaacctatcttttcaa
tttgggccgggttgtggtttggactaccatgaacaacttttcgtcatgtc
taacttccctttcagcaaacatatgaaccatatatagaggagatcggccg
tatactagagctgatgtgtttaaggtcgttgattgcacgagaaaaaaaaa
tccaaatcgcaacaatagcaaatttatctggttcaaagtgaaaagatatg
tttaaaggtagtccaaagtaaaacttatagataataaaatgtggtccaaa
gcgtaattcactcaaaaaaaatcaacgagacgtgtaccaaacggagacaa
acggcatcttctcgaaatttcccaaccgctcgctcgcccgcctcgtcttc
ccggaaaccgcggtggtttcagcgtggcggattctccaagcagacggaga
cgtcacggcacgggactcctcccaccacccaaccgccataaataccagcc
ccctcatctcctctcctcgcatcagctccacccccgaaaaatttctcccc
aatctcgcgaggctctcgtcgtcgaatcgaatcctctcgcgtcctcaagg
tacgctgcttctcctctcctcgcttcgtttcgattcgatttcggacgggt
gaggttgttttgttgctagatccgattggtggttagggttgtcgatgtga
ttatcgtgagatgtttaggggttgtagatctgatggttgtgatttgggca
cggttggttcgataggtggaatcgtggttaggttttgggattggatgttg
gttctgatgattggggggaatttttacggttagatgaattgttggatgat
tcgattggggaaatcggtgtagatctgttggggaattgtggaactagtca
tgcctgagtgattggtgcgatttgtagcgtgttccatcttgtaggccttg
ttgcgagcatgttcagatctactgttccgctcttgattgagttattggtg
cggttggtgcaaacacaggctttaatatgttatatctgttttgtgtttga
tgtagatctgtagggtagttcttcttagacatggttcaattatgtagctt
gtgcgtttcgatttgatttcatatgttcacagattagataatgatgaact
cttttaattaattgtcaatggtaaataggaagtcttgtcgctatatctgt
cataatgatctcatgttactatctgccagtaatttatgctaagaactata
ttagaatatcatgttacaatctgtagtaatatcatgttacaatctgtagt
tcatctatataatctattgtggtaatttctttttactatctgtgtgaaga
ttattgccactagttcattctacttatttctgaagttcaggatacgtgtg
ctgttactacctatctgaatacatgtgtgatgtgcctgttactatctttt
tgaatacatgtatgttctgttggaatatgtttgctgtttgatccgttgtt
gtgtccttaatcttgtgctagttcttaccctatctgtaggtgattatact
tgcagattcagatcgggcccAAGCTTGACTAGTGATATCACAAGTTTGTA
CAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCGAGCTCGAGATGTTTT
GAGGAAGGGTATGGAACAATCCTTGAGAGACCATTAGGCACCCCAGGCTT
TACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGAGC
CGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAatggagaaaaaaatcact
ggatataccaccgttgatatatcccaatggcatcgtaaagaacattagag
gcatttcagtcagttgctcaatgtacctataaccagaccgttcagctgga
tattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatc
cggcctttattcacattcttgcccgcctgatgaatgctcatccggagttc
cgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcaccc
ttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctgga
gtgaataccacgacgatttccggcagtttctacacatatattcgcaagat
gtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattga
gaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttg
atttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatg
ggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattca
ggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatg
aattacaacagtactgcgatgagtggcagggcggggcgtaaACGCGTGGA
GCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTT
TTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAA
GAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCT
ATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCA
CAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCG
GAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGG
CTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTAC
ACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGA
TATTATTGACACGCCCGGCCGACGGATGGTGATCCCCCTGGCCAGTGCAC
GTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATC
GGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGT
TTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACA
TCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCC
CTTATACACAGCCAGTCTGCACCTCGACggtctcAcatggtttgttctta
ccacacgaccaattaaatcGAGCTCAAGGGTGGGCGCGCCG
ACCCAGCTT
TCTTGTACAAAGTGGT
GATATCCCG
cggccatgctagagtccgcaaaaat
caccagtctctctctacaaatctatctctctctatttttctccagaataa
tgtgtgagtagttcccagataagggaattagggttcttatagggtttcgc
tcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaa
atacttctatcaataaaatttctaattcctaaaaccaaaatccagtgacc
t
GCAGGCATGCGACGTCGGGCCCTCTAGAGGATCCCCGGGTACCGTGCAG
CGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTCGCTGCC
TCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGT
CGGCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCA
GGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTT
CCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGACAC
CCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACAC
AACACAACCAGATCTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAG
GTACGCCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCTAGATCGG
CGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTG
TGTTAGATCCGTGTTTGTGTTAGATCCGTGCTGCTAGCGTTCGTACACGG
ATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTC
TCTTTGGGGAATCCTGGGTGGCTCTAGCCGTTCCGCAGACGGGATCGATT
TCATGATTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTT
TATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATCTTTTCATGCTT
TTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCTAGA
TCGGAGTAGAAATCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGA
TCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGG
ATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTG
ATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGT
GGTTGGGCGGTCGTTCATTCGTTCTAGATCGGAGTAGAATACTGTTTCAA
ACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACAT
CTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGT
ATACATGTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCA
GCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACA
AGTATGTTTTATAATTATTTTGATCTTGATATACTTGGATGATGGCATAT
GCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTAT
TTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTA
CTTCTGCAGGTCGACTCTAGAGGATCCATGAAAAAGCCTGAACTCACCGC
GACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACC
TGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTA
GGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTA
CAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTC
CGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATC
TCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACT
GCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGG
CCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATC
GGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCA
TGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCG
CGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTC
CGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAA
TGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATT
CCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGT
ATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGG
ATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCT
ATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGT
CGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACA
AATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTAC
TCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGAAA
TAGGAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC
GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCT
GGGGTGCCTAATGAGTGAGCTAACTCACATTACTTAAGATTGAATCCTGT
TGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGC
ATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTT
ATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAAT
ATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTAC
TAGATCGACCGGCATGCAAGCTGATAATTCAATTCGGCGTTAATTCAGTA
CATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGT
TTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGG
CAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGA
CGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTAC
CGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGA
TGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCT
GCCTGTGATCAATTCGggcacgaacccagtggacataagcctcgttcggt
tcgtaagctgtaatgcaagtagcgtaactgccgtcacgcaactggtccag
aaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatgg
cttcttgttatgacatgtttttttggggtacagtctatgcctcgggcatc
caagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagca
gcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaaacatc
atgggggaagcggtgatcgccgaagtatcgactcaactatcagaggtagt
tggcgtcatcgagcgccatctcgaaccgacgttgctggccgtacatttgt
acggctccgcagtggatggcggcctgaagccacacagtgatattgatttg
ctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgat
caacgaccttttggaaacttcggcttcccctggagagagcgagattctcc
gcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgt
tatccagctaagcgcgaactgcaatttggagaatggcagcgcaatgacat
tcttgcaggtatcttcgagccagccacgatcgacattgatctggctatct
tgctgacaaaagcaagagaacatagcgttgccttggtaggtccagcggcg
gaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaa
tgaaaccttaacgctatggaactcgccgcccgactgggctggcgatgagc
gaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggc
aaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgcc
ggcccagtatcagcccgtcatacttgaagctagacaggcttatcttggac
aagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtc
cactacgtgaaaggcgagatcaccaaggtagtcggcaaataatgtctagc
tagaaattcgttcaagccgacgccgcttcgccggcgttaactcaagcgat
tagatgcactaagcacataattgctcacagccaaactatcaggtcaagtc
tgcttttattatttttaagcgtgcataataagccctacacaaattgggag
atatatcatgcatgacCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA
GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT
TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG
TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT
GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTA
GTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC
TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT
ACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGG
CTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA
CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCC
GAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG
AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC
CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG
TCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACG
GTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTAT
CCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC
GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC
GGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTT
CACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG
TTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCG
CCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT
CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGT
GTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTG
ATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTG
GTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCG
ATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGT
AGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCAC
AGGCCAGGCGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCG
GAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCG
GTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAA
TGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAGAGAACT
TTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGT
ACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCA
TGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCC
GGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTT
GAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATC
TGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGG
CCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTC
CGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGA
TCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGG
CTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGC
CTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGG
TTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAAC
TTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCC
CTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCA
TCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCG
GAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGC
TCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGC
GACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGT
TGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGG
CGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATT
CACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCG
GCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTAC
CGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTT
CCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCA
ACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTT
GTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTC
ATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATA
GCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTG
GTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGT
GTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGAC
GGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCAT
TAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCG
CGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGC
GGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGG
GCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTG
CCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGT
GACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATA
TGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTG
ATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTAC
GAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGT
CGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCG
CGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGC
GAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACC
CGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTA
TTTGTTTATTTACTCATCGCATCATATACGCAGCGACCGCATGACGCAAG
CTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTC
TTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAAC
CGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCG
AACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAA
AAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTG
GCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCT
CACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTG
CGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGC
CGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCG
CGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGG
GCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACG
CTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCGGCCG
GCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCG
GCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGC
CAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGT
CAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTC
TCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTT
GGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAG
CGGCGGCGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTA
TTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGCCAGGAAAAGGG
CAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTT
CAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGG
AGGGGTTGGATCAAAGTAC
cyan/lowercase: T-DNA right border
grey/lowercase: OsUbi promoter
ORANGE/UPPERCASE: attB1
BLUE/UPPERCASE: OsMIR390 5′ region
RED/UPPERCASE: BsaI site
magenta/lowercase: chloramphenicol resistance gene
MAGENTA/UPPERCASE: ccdB gene
red/lowercase: inverted BsaI site
blue/lowercase: OsMIR390 3′ region
ORANGE/UPPERCASE/UNDERLINED: attB2
green/lowercase/underlined: CaMV terminator
GREY/UPPERCASE: ZmUbi promoter
BROWN/UPPERCASE: hygromycin resistance gene
CYAN/UPPERCASE: T-DNA left border
brown/lowercase: spectinomycin resistance gene
CYAN/UPPERCASE/UNDERLINED: C->A transversion to block vector's BsaI site
DNA sequence in FASTA format of all the MIRNA precursors used in this study to express and analyze amiRNAs.
(a) Sequences of OsMIR390-Based amiRNA Precursors
Sequences unique to the pri-miRNA, pre-miRNA, miRNA/amiRNA guide strand and miRNA*/amiRNA* strand sequences are highlighted in grey, white, blue and green, respectively. Bases of the pre-OsMIR390 that had to be modified to preserve the authentic OsMIR390 precursor structure are highlighted in red.
(b) Sequences of AtMIR390a-Based amiRNA Precursors
Sequence unique to the pri-AtMIR390a sequence is highlighted in black. Bases of the pre-AtMIR390a that had to be modified to preserve the authentic AtMIR390a precursor structure are highlighted in red. Other details as in (a).
Protocol to clone amiRNAs in BsaI/ccdB-based (‘B/c’) vectors containing the OsMIR390 precursor.
Notes: Available OsMIR390 B/c vectors are listed in Table I at the end of this protocol.
OsMIR3 90-B/c-based vectors must be propagated in a ccdB resistant E. coli strain such as DB3.1.
Alternatively, BsaI digestion of the B/c vector and subsequent ligation of the amiRNA oligonucleotide insert can be done in separate reactions
3.1. Oligonucleotide Annealing
Dilute sense oligonucleotide and antisense oligonucleotide in sterile H2O to a final concentration of 100 μM.
Prepare Oligo Annealing Buffer:
60 mM Tris-HCl (pH 7.5)
500 mM NaCl
60 mM MgCl2
10 mM DTT
Note: Prepare 1 ml aliquots of Oligo Annealing Buffer and store at −20° C.
Assemble the annealing reaction in a PCR tube as described below:
The final concentration of each oligonucleotide is 4 μM.
Use a thermocycler to heat the annealing reaction 5 min at 94° C. and then cool down (0.05° C./sec) to 20° C.
Dilute the annealed oligonucleotides just prior to assembling the digestion-ligation reaction as described below:
The final concentration of each oligonucleotide is 0.15 μM.
Note: Do not store the diluted oligonucleotides.
3.2. Digestion-Ligation Reaction
Assemble the digestion-ligation reaction as described below:
Prepare a negative control reaction lacking BsaI.
Mix the reactions by pipetting. Incubate the reactions for 5 minutes at 37° C.
3.3. E. coli Transformation and Analysis of Transformants
Transform 1-5 ul of the digestion-ligation reaction into an E. coli strain that doesn't have ccdB resistance (e.g. DH10B, TOP10, . . . ) to do counter-selection.
Pick two colonies/construct, grow LB-Kan (100 mg/ml) cultures and purify plasmids.
vectors for direct cloning of
indicates data missing or illegible when filed
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure specifically described herein. Such equivalents are intended to be encompassed within the scope of the following claims.
Montgomery, T. A., Howell, M. D., Cuperus, J. T., Li, D., Hansen, J. E., Alexander, A. L., Chapman, E. J., Fahlgren, N., Allen, E. and Carrington, J. C. (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128-141.
Ossowski, S., Schwab, R. and Weigel, D. (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J. 53, 674-690.
The present application claims priority to U.S. Provisional Application No. 62/947,732, filed Mar. 4, 2014, entitled “New Generation of Artificial MicroRNAs, which is herein incorporated by reference. The present application also claims priority to U.S. Provisional Application No. 62/950,588, filed Mar. 10, 2014, entitled “New Generation of Artificial MicroRNAs, which also is herein incorporated by reference. The present application is a continuation of PCT/US2015/018529, filed Mar. 3, 2015 entitled “New Generation of Artificial MicroRNAs,” which is also herein incorporated by reference.
The development of this invention was partially funded by the government under grants from the National Science Foundation (MCB-0956526, MCB-1231726), National Institutes of Health (AI043288), National Institute of Food and Agriculture (MOW-2012-01361). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61947732 | Mar 2014 | US | |
| 61950588 | Mar 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2015/018529 | Mar 2015 | US |
| Child | 15256578 | US |
