Patent Application
20100192261
The material on the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing was created on Jul. 9, 2006. The accompanying sequence listing is a text file and is 203 KB. The file can be accessed using Microsoft Word on a computer that uses Windows OS.
1. Technical Field
This document relates to methods and materials involved in plant UV-B tolerance. For example, this document provides seeds and plants having cells comprising an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity.
2. Background Information
Levels of terrestrial UV-B (280-320 nm) irradiation have increased as a result of alterations in the ozone layer. Elevated terrestrial UV-B irradiation can have detrimental effects, such as causing DNA damage and protein damage, on living organisms, including plants. Plant responses to UV-B irradiation include reduced growth rates, changes in plant form, and altered nutrient distribution. Plant responses to UV-B irradiation can cause reduced crop yields.
This document provides methods and materials related to plants having increased or decreased levels of UV-B tolerance. For example, this document provides seeds and plants having cells comprising an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity. Such seeds can be used to grow plants having cells comprising an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity. In some cases, plants having an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity can exhibit increased tolerance to UV-B light exposure. For example, a plant having cells comprising an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity can have a hypocotyl length, when exposed to UV-B light (e.g., light having a wavelength of 280-320 nm at a fluence of 5 watts/m2), that is greater than the hypocotyl length of a control plant lacking the exogenous nucleic acid that is grown under similar conditions.
Plants having cells comprising an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity can produce a higher crop yield than control plants lacking the exogenous nucleic acid when grown in conditions having excess UV-B light exposure.
In one aspect a method for producing a plant is provided. The method comprises growing a plant cell comprising an exogenous nucleic acid, where the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide, and where a plant produced from the plant cell has a difference in UV-B tolerance as compared to the corresponding control plant that does not comprise the exogenous nucleic acid. The nucleic acid can encode a polypeptide having an amino acid sequence with an HMM bit score greater than 50, where the HMM is based on the amino acid sequences depicted in one of
The nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding SEQ ID NO:87. The nucleotide sequence can encode a polypeptide comprising an amino acid sequence corresponding SEQ ID NO:97.
In another aspect a method for producing a plant is provided. The method comprises introducing into a plant cell an exogenous nucleic acid, where the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide, and where a plant produced from the plant cell has a difference in UV-B tolerance as compared to the corresponding control plant that does not comprise the exogenous nucleic acid. The nucleic acid can encode a polypeptide having an amino acid sequence with an HMM bit score greater than 50, where the HMM is based on the amino acid sequences depicted in one of
The methods can further comprise the step of producing a plurality of plants from said plant cell. The methods can further comprise the step of selecting one or more plants from the plurality of plants that have the difference in UV-B tolerance. The introducing step can comprise introducing the nucleic acid into a plurality of plant cells. The methods can further comprise selecting a plurality of plants from the plurality of plant cells. The regulatory region can be a tissue-preferential, broadly expressing, or inducible promoter.
The plant can be a dicot or a monocot. The plant can be a member of the genus Anacardium, Arachis, Azadirachta, Brassica, Cannabis, Carthamus, Corylus, Crambe, Cucurbita, Glycine, Gossypium, Helianthus, Jatropha, Juglans, Linum, Olea, Papaver, Persea, Prunus, Ricinus, Sesamum, Simmondsia, or Vitis. The plant can be a member of the genus Cocos, Elaeis, Oryza, Panicum, or Zea. The plant can be a species selected from the group consisting of Miscanthus hybrid (Miscanthus x giganteus), Miscanthus sinensis, Miscanthus sacchariflorus, Panicum virgatum, Populus balsamifera, Sorghum bicolor, and Saccharum spp.
In another aspect, a plant cell is provided. The plant cell comprises an exogenous nucleic acid, the exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide, where a plant produced from the plant cell has a difference in UV-B tolerance as compared to the corresponding control plant that does not comprise the nucleic acid. The nucleic acid can encode a polypeptide having an amino acid sequence with an HMM bit score greater than 50, where the HMM is based on the amino acid sequences depicted in one of
A transgenic plant is also provided. The transgenic plant comprises a plant cell comprising an exogenous nucleic acid, the exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide, where the plant has a difference in UV-B tolerance as compared to the corresponding control plant that does not comprise the nucleic acid. The nucleic acid can encode a polypeptide having an amino acid sequence with an HMM bit score greater than 50, where the HMM is based on the amino acid sequences depicted in one of
In another aspect, an isolated nucleic acid is provided. The nucleic acid comprises a nucleotide sequence having 95% or greater sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:88, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:119.
Also provided is an isolated nucleic acid. The nucleic acid comprises a nucleotide sequence encoding a polypeptide having 80% or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:103, and SEQ ID NO:107.
In another aspect, an article of manufacture is provided. The article of manufacture comprises packaging material and a plurality of seeds within the packaging material, where the seeds comprise an exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having UV-B tolerance activity. The article of manufacture can further comprise a regulatory region operably linked to the nucleotide sequence encoding the polypeptide.
The plants grown from the seeds can express the polypeptide. The polypeptide can comprise an amino acid sequence having 80 percent or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:87. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:87. The polypeptide can comprise an amino acid sequence having 80 percent or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:97. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:97.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials related to UV-B tolerance in plants, plant cells, and seeds. For example, this document provides seeds and plants containing cells having an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity. The term “polypeptide having UV-B tolerance activity” as used herein refers to a polypeptide having the ability to increase a plant's tolerance of UV-B light when that polypeptide is expressed by cells of that plant. In general, an increase in a plant's tolerance to UV-B light refers to the plant's ability to experience a negative effect of UV-B light to a degree less than that experienced by a similar plant (e.g., a comparable plant such as a plant lacking an exogenous nucleic acid described herein) when grown under similar conditions. Negative effects of UV-B light include, without limitation, reduced hypocotyl length, reduced silique size, delayed maturation, reduced biomass, reduced crop yield, reduced seed yield, and combinations thereof.
The cells of any type of plant or plant seed can be designed to contain an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity. For example, plants such as corn, wheat, soybean, sunflower, tobacco, cotton, and rice plants can be designed to contain cells having an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity.
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and encompass both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Polynucleotides can have any three-dimensional structure. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
An isolated nucleic acid can be, for example, a naturally-occurring DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid. Nucleic acids described herein include nucleic acids encoding polypeptides having UV-B tolerance activity. Nucleic acids encoding polypeptides having UV-B tolerance activity can be effective to modulate UV-B tolerance when transcribed in a plant or plant cell.
Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to 5′ direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids also can be obtained by mutagenesis of, e.g., a naturally occurring DNA.
The term “exogenous” as used herein with respect to a nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration. For example, a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant. Such progeny are considered to contain the exogenous nucleic acid.
A polypeptide having UV-B tolerance activity can include, without limitation, a nodulin MtN3 polypeptide and can have an amino acid sequence set forth in SEQ ID NO:87. Alternatively, a polypeptide having UV-B tolerance activity can be a homolog, ortholog, or variant of the polypeptide having the amino acid set forth in SEQ ID NO:87. For example, a polypeptide having UV-B tolerance activity can have an amino acid sequence with at least 45 percent sequence identity (e.g., at least about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent identity) to the amino acid sequence set forth in SEQ ID NO:87.
The term “polypeptide” as used herein refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post-translational modification, e.g., phosphorylation or glycosylation. The subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds. The term “amino acid” refers to natural and/or unnatural or synthetic amino acids, including D/L optical isomers. Full-length proteins, analogs, mutants, and fragments thereof are encompassed by this definition.
Amino acid sequences of homologs and/or orthologs of the polypeptide having the amino acid sequence set forth in SEQ ID NO:87 are provided in
In some cases, a polypeptide having UV-B tolerance activity includes a polypeptide having at least 80 percent sequence identity (e.g., 80, 85, 90, 95, 97, 98, or 99 percent sequence identity), to an amino acid sequence corresponding to SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, or SEQ ID NO:113.
A polypeptide having UV-B tolerance activity can be a SET domain polypeptide and can have an amino acid sequence set forth in SEQ ID NO:97. Alternatively, a polypeptide having UV-B tolerance activity can be a homolog, ortholog, or variant of the polypeptide having the amino acid set forth in SEQ ID NO:97. For example, a polypeptide having UV-B tolerance activity can have an amino acid sequence with at least 45 percent sequence identity (e.g., at least about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100 percent identity) to the amino acid sequence set forth in SEQ ID NO:97.
Amino acid sequences of homologs and/or orthologs of the polypeptide having the amino acid sequence set forth in SEQ ID NO:97 are provided in
In some cases, a polypeptide having UV-B tolerance activity includes a polypeptide having at least 80 percent sequence identity (e.g., 80, 85, 90, 95, 97, 98, or 99 percent sequence identity), to an amino acid sequence corresponding to SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:112, or SEQ ID NO:114.
As used herein, the term “percent sequence identity” refers to the degree of identity between any given query sequence, e.g., SEQ ID NO:94, and a subject sequence. A subject sequence typically has a length that is from 80 percent to 200 percent, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200 percent, of the length of the query sequence. A query nucleic acid or amino acid sequence is aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chema et al., Nucleic Acids Res., 31(13):3497-500 (2003).
ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more nucleotides or amino acid residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair-wise alignments of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For alignments of multiple nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pair-wise alignment of amino acid sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For alignments of multiple amino acid sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher internet site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
To determine percent identity of a subject nucleic acid or amino acid sequence to a query sequence, the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
Known methods, such as PCR, can be used to obtain a nucleic acid encoding a polypeptide having UV-B tolerance activity. In addition, known methods can be used to identify polypeptide having UV-B tolerance activity. For example, the methods provided in the Example section can be used to identify a polypeptide having UV-B tolerance activity. In addition, polypeptides having UV-B tolerance activity can be identified using nucleic acid or amino acid sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs and/or orthologs of polypeptides having UV-B tolerance activity. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using known amino acid sequences of polypeptides having UV-B tolerance activity. Those polypeptides in the database that have greater than, for example, 40 percent sequence identity can be identified as candidates for further evaluation for suitability as polypeptides having UV-B tolerance activity. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains suspected of being present in polypeptides having UV-B tolerance activity, e.g., conserved functional domains.
The identification of conserved regions in a template or subject polypeptide can facilitate production of variants of wild type polypeptides having UV-B tolerance activity. Conserved regions can be identified by locating a region within the primary amino acid sequence of a template polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains at sanger.ac.uk/Pfam and genome.wust1.edu/Pfam. A description of the information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). Amino acid residues corresponding to Pfam domains included in polypeptides having UV-B tolerance activity provided herein are set forth in the sequence listing. For example, amino acid residues 9 to 98 and amino acid residues 134 to 221 of the amino acid sequence set forth in SEQ ID NO:87 correspond to MtN3/saliva domains, as indicated in fields <222> and <223> for SEQ ID NO:87 in the sequence listing. In another example, amino acid residues 83 to 338 of the amino acid sequence set forth in SEQ ID NO:97 correspond to a SET domain, as indicated in the fields <222> and <223> for SEQ ID NO:97 in the sequence listing.
Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate. For example, sequences from Arabidopsis and Zea mays can be used to identify one or more conserved regions.
Typically, polypeptides that exhibit at least about 40 percent amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides can exhibit at least 45 percent amino acid sequence identity (e.g., at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, or at least 90 percent amino acid sequence identity). In some embodiments, a conserved region of target and template polypeptides exhibit at least 92, 94, 96, 98, or 99 percent amino acid sequence identity. Amino acid sequence identity can be deduced from amino acid or nucleotide sequences. In certain cases, highly conserved domains have been identified within polypeptides having UV-B tolerance activity. These conserved regions can be useful in identifying functionally similar (orthologous) polypeptides having UV-B tolerance activity.
In some instances, suitable polypeptides having UV-B tolerance activity can be synthesized on the basis of consensus functional domains and/or conserved regions in polypeptides that are homologous polypeptides having UV-B tolerance activity. Domains are groups of substantially contiguous amino acids in a polypeptide that can be used to characterize protein families and/or parts of proteins. Such domains have a “fingerprint” or “signature” that can comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three-dimensional conformation. Generally, domains are correlated with specific in vitro and/or in vivo activities. A domain can have a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino acids.
Representative homologs and/or orthologs of polypeptides having UV-B tolerance activity are shown in
Useful polypeptides can be constructed based on the conserved regions in
Conserved regions can be identified by homologous polypeptide sequence analysis as described above. The suitability of polypeptides for use as polypeptides having UV-B tolerance activity can be evaluated by functional complementation studies.
Useful polypeptides can also be identified based on the polypeptides set forth in any of
The default parameters for building an HMM (hmmbuild) are as follows: the default “architecture prior” (archpri) used by MAP architecture construction is 0.85, and the default cutoff threshold (idlevel) used to determine the effective sequence number is 0.62. The HMMER 2.3.2 package was released Oct. 3, 2003 under a GNU general public license, and is available from various sources on the World Wide Web such as hmmerjanelia.org, hmmer.wust1.edu, and fr.com/hmmer232. Hmmbuild outputs the model as a text file.
The HMM for a group of homologous and/or orthologous polypeptides can be used to determine the likelihood that a subject polypeptide sequence is a better fit to that particular HMM than to a null HMM generated using a group of sequences that are not homologous and/or orthologous. The likelihood that a subject polypeptide sequence is a better fit to an HMM than to a null HMM is indicated by the HMM bit score, a number generated when the subject sequence is fitted to the HMM profile using the HMMER hmmsearch program. The following default parameters are used when running hmmsearch: the default E-value cutoff (E) is 10.0, the default bit score cutoff (T) is negative infinity, the default number of sequences in a database (Z) is the real number of sequences in the database, the default E-value cutoff for the per-domain ranked hit list (domE) is infinity, and the default bit score cutoff for the per-domain ranked hit list (domT) is negative infinity. A high HMM bit score indicates a greater likelihood that the subject sequence carries out one or more of the biochemical or physiological function(s) of the polypeptides used to generate the HMM. A high HMM bit score is at least 20, and often is higher.
A polypeptide having UV-B tolerance activity can fit an HMM provided herein with an HMM bit score greater than 20 (e.g., greater than 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500). In some cases, a polypeptide having UV-B tolerance activity can fit an HMM provided herein with an HMM bit score that is about 50, 60, 70, 80, 90, or 95 percent of the HMM bit score of any homologous and/or orthologous polypeptide provided in either of Tables 3 and 4. In some cases, a polypeptide having UV-B tolerance activity can fit an HMM described herein with an HMM bit score greater than 20, and can have a conserved domain, e.g., a PFAM domain, or a conserved region having 70 percent or greater sequence identity (e.g., 75, 80, 85, 90, 95, or 100 percent sequence identity) to a conserved domain or region present in a polypeptide having UV-B tolerance activity disclosed herein.
For example, a polypeptide having UV-B tolerance activity can fit an HMM generated using the amino acid sequences set forth in
It will be appreciated that a number of different nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Given the genetic code degeneracy, any of the nucleic acids provided herein can be modified so as to have a different nucleic acid sequence that encodes the same amino acid sequence. In some cases, a nucleic acid provided herein can be modified such that expression of the encoded polypeptide is optimized for a particular plant species. Such codon optimization can be achieved using an appropriate codon bias table for the desired species.
A polypeptide having UV-B tolerance activity can be designed to contain additional amino acid residues. For example, a polypeptide having UV-B tolerance activity can be designed to include an amino acid sequence that functions as a reporter. Such a polypeptide having UV-B tolerance activity can be a fusion polypeptide to which a green fluorescent protein (GFP) polypeptide is fused or to which a yellow fluorescent protein (YFP) polypeptide is fused. In some cases, a polypeptide having UV-B tolerance activity can contain a purification tag, a chloroplast transit polypeptide, a mitochondrial transit polypeptide, or a leader sequence. Any additional amino acid residues can be located at the amino terminus, at the carboxy terminus, within the polypeptide, or combinations thereof. For example, a polypeptide having UV-B tolerance activity can be designed to contain an amino terminus leader sequence and an internal epitope tag (e.g., a FLAG™ tag or myc tag).
The following can be used to determine whether or not a particular polypeptide is a polypeptide having UV-B tolerance activity. A vector designed to express a nucleic acid encoding a test polypeptide and a control vector (e.g., vector lacking the nucleic acid encoding the test polypeptide) are introduced into plants (e.g., corn, wheat, soybean, or Arabidopsis plants) to generate plants containing the vector expressing the polypeptide to be tested (test plants) and plants containing the control vector (control plants). A population of test plant seedlings and control plant seedlings are grown under normal laboratory growth conditions for that species with the exception that the seedlings are exposed to the following sequence of light conditions: a 23 hour period of darkness, a 30 minute to 3 hour pulse of 280-320 nm light at a fluence of 5 watts/m2, a 23 hour period of darkness, another 30 minute to 3 hour pulse of 280-320 nm light at a fluence of 5 watts/m2, and a 23 hour period of darkness. These two populations are referred to as UV-B light-treated test plants and UV-B light-treated control plants. Another population of control plants is grown under growth conditions identical to the UV-B light-treated test plants and UV-B light-treated control plants with the exception that the plants of this population are not exposed to UV-B. This population of plants can be referred to as dark grown plants.
Growth characteristics of the UV-B light-treated control plants and the dark grown plants are compared to determine the level of an effect (e.g., a negative effect) of UV-B light exposure. For example, hypocotyl length, silique size, time to maturation, biomass, crop yield, seed yield, leaf senescence or a combination thereof can be assessed to determine the effect of UV-B light exposure. The ability of the test polypeptide to reduce an identified effect of UV-B light exposure is assessed by comparing UV-B light-treated test plants and UV-B light-treated control plants for that identified growth characteristic. If the level of a UV-B light exposure effect is reduced in the UV-B light-treated test plants as compared to the UV-B-treated control plants, then the test polypeptide is considered a polypeptide having UV-B tolerance activity.
A plant grown under elevated UV-B light conditions and having (1) cells containing an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity and (2) increased UV-B tolerance can have a greater hypocotyl length, an increased silique size, an earlier maturation, a greater seed yield, an increased biomass, or a combination thereof when compared to a comparable plant grown under similar conditions and lacking cells containing an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity. For example, a plant provided herein can have hypocotyl lengths that are at least 2 percent (e.g., at least 2, 3, 4, 5, 10, 25, 50, 75, 100, or more percent) greater than the average hypocotyl length of similar plants that lack cells containing an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity. In some cases, a plant provided herein can have siliques that are at least 2 percent (e.g., at least 2, 3, 4, 5, 10, 25, 50, 75, 100, or more percent) greater than the average silique size of similar plants that lack cells containing an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity. Typically, a difference (e.g., an increase) in the hypocotyl length or silique length in a plant provided herein (e.g., a plant containing cells having an exogenous nucleic acid encoding a polypeptide having UV-B tolerance activity) relative to a control plant is considered statistically significant at p<0.05 with an appropriate parametric or non-parametric statistic, e.g., Chi-square test, Student's t-test, Mann-Whitney test, or F-test.
In general, a recombinant nucleic acid construct can be used to introduce an exogenous nucleic acid into plant cells. Such a recombinant nucleic acid construct can include a nucleic acid sequence encoding a polypeptide having UV-B tolerance activity operably linked to a regulatory region suitable for expressing the polypeptide having UV-B tolerance activity in a plant, plant tissue, plant seed, or plant cell. For example, a recombinant nucleic acid construct can contain a nucleic acid sequence that encodes an amino acid sequence set forth in
Examples of nucleic acids encoding polypeptides having UV-B tolerance activity are set forth in SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, and SEQ ID NO:119.
In some cases, a recombinant nucleic acid construct can include a nucleic acid sequence having less than the full-length coding sequence of a polypeptide having UV-B tolerance activity. Typically, such a construct also includes a regulatory region operably linked to the nucleic acid encoding a polypeptide having UV-B tolerance activity.
Vectors containing nucleic acids such as those described herein also are provided. A vector is a replicon, such as a plasmid, phage, or cosmid into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).
The vectors provided herein can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers. A marker gene can confer a selectable phenotype on a plant cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or an herbicide (e.g., chlorosulfuron or phosphinothricin). In some cases, a recombinant nucleic acid construct provided herein can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
The term “regulatory region” refers to a nucleotide sequence that influences transcription initiation, transcription rate, translation initiation, translation rate, transcription product stability, transcription product mobility, translation product stability, translation product mobility, or combinations thereof. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, tolerance elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
As used herein, the term “operably linked” refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site. A promoter typically contains at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). For example, a suitable enhancer can be a cis-regulatory element (−212 to −154) from the upstream region of the octopine synthase (ocs) gene (Fromm et al., The Plant Cell, 1:977-984 (1989)). The choice of promoters to be included depends upon several factors, including, without limitation, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. The expression of a coding sequence can be modulated by selecting a desired regulatory region or altering the position of a regulatory region relative to the coding sequence.
Some suitable promoters initiate transcription only, or predominantly, in certain cell types. For example, a promoter that is active predominantly in a reproductive tissue (e.g., fruit, ovule, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo, zygote, endosperm, integument, or seed coat) can be used. A cell type- or tissue-preferential promoter can be a promoter that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano et al., Plant Cell, 1:855-866 (1989); Bustos et al., Plant Cell, 1:839-854 (1989); Green et al., EMBO J., 7:4035-4044 (1988); Meier et al., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology, 110:1069-1079 (1996).
Examples of various classes of promoters are provided below. Some of the promoters indicated below, as well as additional promoters, are described in more detail in U.S. Patent Application Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569; 11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891; 11/097,589; 11/233,726; 10/950,321; PCT/U505/011105; PCT/U505/034308; PCT/U505/23639; 11/408,791; 11/414,142; 11/360,017; PCT/US05/034343; PCT/US06/038236; PCT/US06/040572; and PCT/US07/62762. Nucleotide sequences of promoters are set forth in SEQ ID NOS:1-85. It will be appreciated that a regulatory region (e.g., a promoter) may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a different classification based on its activity in another plant species.
In some cases, a broadly expressing promoter can be used to drive expression of a polypeptide provided herein. A promoter can be said to be “broadly expressing” when it promotes transcription in many, but not necessarily all, plant tissues. For example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems. As another example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds. Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326 (SEQ ID NO:75), YP0144 (SEQ ID NO:54), YP0190 (SEQ ID NO:58), p13879 (SEQ ID NO:74), YP0050 (SEQ ID NO:34), p32449 (SEQ ID NO:76), 21876 (SEQ ID NO:1), YP0158 (SEQ ID NO:56), YP0214 (SEQ ID NO:60), YP0380 (SEQ ID NO:69), PT0848 (SEQ ID NO:26), and PT0633 (SEQ ID NO:7) promoters. Additional examples include the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the l′ or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34S promoter, actin promoters such as the rice actin promoter, and ubiquitin promoters such as the maize ubiquitin-1 promoter. In some cases, the CaMV 35S promoter is excluded from the category of broadly expressing promoters.
Promoters active in photosynthetic tissue confer transcription in green tissues such as leaves and stems. Most suitable are promoters that drive expression only or predominantly in such tissues. Examples of such promoters include the ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricina), the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778 (1994)), the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol., 15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et al., Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from rice (Luan et al., Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al., Proc. Natl. Acad. Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcb1*2 promoter (Cerdan et al., Plant Mol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al., Planta, 196:564-570 (1995)), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissue promoters include PT0535 (SEQ ID NO:3), PT0668 (SEQ ID NO:2), PT0886 (SEQ ID NO:29), YP0144 (SEQ ID NO:54), YP0380 (SEQ ID NO:69), and PT0585 (SEQ ID NO:4).
Examples of promoters that have high or preferential activity in vascular bundles include YP0087 (SEQ ID NO:82), YP0093 (SEQ ID NO:83), YP0108 (SEQ ID NO:84), YP0022 (SEQ ID NO:80), and YP0080 (SEQ ID NO:81). Other vascular tissue-preferential promoters include the glycine-rich cell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell, 3(10):1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV) promoter (Medberry et al., Plant Cell, 4(2):185-192 (1992)), and the rice tungro bacilliform virus (RTBV) promoter (Dai et al., Proc. Natl. Acad. Sci. USA, 101(2):687-692 (2004)).
Inducible promoters confer transcription in response to external stimuli such as chemical agents or environmental stimuli. For example, inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought. Examples of drought-inducible promoters include YP0380 (SEQ ID NO:69), PT0848 (SEQ ID NO:26), YP0381 (SEQ ID NO:70), YP0337 (SEQ ID NO:65), PT0633 (SEQ ID NO:7), YP0374 (SEQ ID NO:67), PT0710 (SEQ ID NO:18), YP0356 (SEQ ID NO:66), YP0385 (SEQ ID NO:72), YP0396 (SEQ ID NO:73), YP0388 (SEQ ID NO:85), YP0384 (SEQ ID NO:71), PT0688 (SEQ ID NO:15), YP0286 (SEQ ID NO:64), YP0377 (SEQ ID NO:68), PD1367 (SEQ ID NO:77), PD0901 (SEQ ID NO:79), and PD0898 (SEQ ID NO:78).
Nitrogen-inducible promoters include PT0863 (SEQ ID NO:27), PT0829 (SEQ ID NO:23), PT0665 (SEQ ID NO:10), and PT0886 (SEQ ID NO:29).
A basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation. Basal promoters frequently include a “TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation. Basal promoters also may include a “CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
Other classes of promoters include, but are not limited to, leaf-preferential, stem/shoot-preferential, callus-preferential, guard cell-preferential, such as PT0678 (SEQ ID NO:13), and senescence-preferential promoters. Promoters designated YP0086 (SEQ ID NO:35), YP0188 (SEQ ID NO:57), YP0263 (SEQ ID NO:61), PT0758 (SEQ ID NO:22), PT0743 (SEQ ID NO:21), PT0829 (SEQ ID NO:23), YP0119 (SEQ ID NO:48), and YP0096 (SEQ ID NO:38), as described in the above-referenced patent applications, may also be useful.
A 5′ untranslated region (UTR) can be included in nucleic acid constructs described herein. A 5′ UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide. A 3′ UTR can be positioned between the translation termination codon and the end of the transcript. UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3′ UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.
The nucleic acids and recombinant nucleic acid constructs provided herein can contain one or more than one regulatory region. For example, a recombinant nucleic acid constructs provided herein can contain multiple introns, enhancers, upstream activation regions, transcription terminators, and inducible elements. Typically, each included regulatory element is operably linked to the sequence encoding a polypeptide having UV-B tolerance activity. Regulatory regions, such as promoters for endogenous genes, can be obtained by chemical synthesis or by subcloning from a genomic DNA that includes such a regulatory region. A nucleic acid comprising such a regulatory region can also include flanking sequences that contain restriction enzyme sites that facilitate subsequent manipulation.
The invention also features transgenic plant cells and plants comprising at least one recombinant nucleic acid construct described herein. A plant or plant cell can be transformed by having a construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid with each cell division. A plant or plant cell can also be transiently transformed such that the construct is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant. Progeny include descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F1, F2, F3, F4, F5, F6 and subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants, or seeds formed on F1BC1, F1BC2, F1BC3, and subsequent generation plants. The designation F1 refers to the progeny of a cross between two parents that are genetically distinct. The designations F2, F3, F4, F5 and F6 refer to subsequent generations of self- or sib-pollinated progeny of an F1 plant. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct.
Transgenic plants can be grown in suspension culture, or tissue or organ culture. For the purposes of this invention, solid and/or liquid tissue culture techniques can be used. When using solid medium, transgenic plant cells can be placed directly onto the medium or can be placed onto a filter that is then placed in contact with the medium. When using liquid medium, transgenic plant cells can be placed onto a flotation device, e.g., a porous membrane that contacts the liquid medium. Solid medium typically is made from liquid medium by adding agar. For example, a solid medium can be Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g., kinetin.
When transiently transformed plant cells are used, a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation. A suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days. The use of transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous protein-modulating polypeptide whose expression has not previously been confirmed in particular recipient cells.
A transformed cell, callus, tissue, or plant can be identified and isolated by selecting or screening engineered plant material for particular traits or activities (e.g., expression of a selectable marker gene or expression of a UV-B tolerance polypeptide). Such screening and selection methodologies are well known to those having ordinary skill in the art. In some cases, physical and biochemical methods can be used to identify transformants. These include, without limitation, Southern analysis or PCR amplification for detecting a nucleotide sequence; Northern blots, S1 RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polynucleotides or polypeptides. Methods for performing all of the referenced techniques are well known.
A population of transgenic plants can be screened and/or selected for those members of the population that have a desired trait or phenotype conferred by expression of the transgene. Selection and/or screening can be carried out over one or more generations, which can be useful to identify those plants that have a desired trait, such as a modulated level of UV-B tolerance. Selection and/or screening can also be carried out in more than one geographic location. In some cases, transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant. In addition, selection and/or screening can be carried out during a particular developmental stage in which the phenotype is exhibited by the plant.
Plants grown from transgenic seeds can have an altered phenotype as compared to a corresponding control plant that either lacks the transgene or does not express the transgene. Expression of an introduced polypeptide at the appropriate time(s), in the appropriate tissue(s), or at the appropriate expression level can affect the phenotype of a plant. Phenotypic effects can be evaluated relative to a control plant that does not express the exogenous nucleic acid, such as a corresponding wild-type plant, a corresponding plant that is not transgenic for the exogenous nucleic acid but otherwise is of the same genetic background as the transgenic plant of interest, or a corresponding plant of the same genetic background in which expression of the polypeptide is suppressed, inhibited, or not induced (e.g., where expression is under the control of an inducible promoter). A plant can be said “not to express” a polypeptide when the plant exhibits less than 10 percent, e.g., less than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.01, or 0.001 percent, of the amount of polypeptide or mRNA encoding the polypeptide exhibited by the plant of interest. Expression can be evaluated using methods including, for example, RT-PCR, Northern blots, S1 RNase protection, primer extensions, Western blots, protein gel electrophoresis, immunoprecipitation, enzyme-linked immunoassays, chip assays, and mass spectrometry. It should be noted that if a polypeptide is expressed under the control of a tissue-preferential or broadly expressing promoter, expression can be evaluated in the entire plant or in a selected tissue. Similarly, if a polypeptide is expressed at a particular time, e.g., at a particular time in development or upon induction, expression can be evaluated selectively at a desired time period.
In some embodiments, a plant in which expression of a polypeptide having UV-B tolerance activity is modulated can have increased UV-B tolerance. For example, a polypeptide having UV-B tolerance activity described herein can be expressed in a transgenic plant, resulting in increased UV-B tolerance. For example, the hypocotyl length can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 25, 50, 75, 100, or more percent, as compared to the hypocotyl length in a corresponding control plant that does not express the transgene. In some embodiments, a plant in which expression of a polypeptide having UV-B tolerance activity is modulated can have decreased UV-B tolerance. For example, the hypocotyl length can be decreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35 percent, as compared to the hypocotyl length in a corresponding control plant that does not express the transgene.
Techniques for introducing nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium-mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
The plants, plant cells, or seeds provided herein can contain one or more exogenous nucleic acids that encode one or more polypeptides having UV-B tolerance activity. For example, a plant provided herein can have one exogenous nucleic acid encoding a first polypeptide having UV-B tolerance activity and another exogenous nucleic acid encoding a second polypeptide having UV-B tolerance activity. As another example, coding sequences for two polypeptides having UV-B tolerance activity can be present on the same exogenous nucleic acid.
The polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, including dicots such as alfalfa, almond, amaranth, apple, apricot, avocado, beans (including kidney beans, lima beans, dry beans, green beans), broccoli, cabbage, canola, carrot, cashew, castor bean, cherry, chick peas, chicory, clover, cocoa, coffee, cotton, crambe, flax, grape, grapefruit, hazelnut, hemp, jatropha, jojoba, lemon, lentils, lettuce, linseed, mango, melon (e.g., watermelon, cantaloupe), mustard, neem, olive, orange, peach, peanut, pear, peas, pepper, plum, poppy, potato, pumpkin, oilseed rape, rapeseed (high erucic acid and canola), safflower, sesame, soybean, spinach, strawberry, sugar beet, sunflower, sweet potatoes, tea, tomato, walnut, and yams, as well as monocots such as banana, barley, bluegrass, coconut, date palm, fescue, field corn, garlic, millet, oat, oil palm, onion, palm kernel oil, pineapple, popcorn, rice, rye, ryegrass, sorghum, sudangrass, sugarcane, sweet corn, switchgrass, timothy, and wheat.
Thus, the methods and compositions described herein can be used with dicotyledonous plants belonging, for example, to the orders Apiales, Arecales, Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Cucurbitales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Illiciales, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Linales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papaverales, Piperales, Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales, Polygonales, Populus, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Solanales, Trochodendrales, Theales, Umbellales, Urticales, and Violales. The methods and compositions described herein also can be utilized with monocotyledonous plants such as those belonging to the orders Alismatales, Arales, Arecales, Asparagales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Liliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, Zingiberales, and with plants belonging to Gymnospermae, e.g., Cycadales, Ginkgoales, Gnetales, and Pinales.
The methods and compositions can be used over a broad range of plant species, including species from the dicot genera Amaranthus, Anacardium, Arachis, Azadirachta, Brassica, Calendula, Camellia, Canarium, Cannabis, Capsicum, Carthamus, Cicer, Cichorium, Cinnamomum, Citrus, Citrullus, Coffea, Corylus, Crambe, Cucumis, Cucurbita, Daucus, Dioscorea, Fragaria, Glycine, Gossypium, Helianthus, Jatropha, Juglans, Lactuca, Lens, Linum, Lycopersicon, Malus, Mangifera, Medicago, Mentha, Nicotiana, Ocimum, Olea, Papaver, Persea, Phaseolus, Pistacia, Pisum, Prunus, Pyrus, Ricinus, Rosmarinus, Salvia, Sesamum, Simmondsia, Solanum, Spinacia, Theobroma, Thymus, Trifolium, Vaccinium, Vigna, and Vitis; and the monocot genera Allium, Ananas, Asparagus, Avena, Cocos, Curcuma, Elaeis, Festuca, Festulolium, Hordeum, Lemna, Lolium, Miscanthus, Musa, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale, Sorghum, Triticosecale, Triticum, and Zea; and the gymnosperm genera Abies, Cunninghamia, Picea, Pinus, Populus, and Pseudotsuga.
In some embodiments, a plant is a member of the species Arachis hypogea, Brassica spp., Carthamus tinctorius, Elaeis oleifera, Glycine max, Gossypium spp., Helianthus annuus, Linum usitatissimum, Miscanthus hybrid (Miscanthus x giganteus), Miscanthus sinensis, Miscanthus sacchariflorus, Oryza sativa, Panicum virgatum, Populus balsamifera, Saccharum spp., Sorghum bicolor, Triticum aestivum, or Zea mays.
The polynucleotides and recombinant vectors described herein can be used to express or inhibit expression of a polypeptide having UV-B tolerance activity in a plant species of interest. “Up-regulation” or “activation” refers to regulation that increases the production of expression products (mRNA, polypeptide, or both) relative to basal or native states, while “down-regulation” or “repression” refers to regulation that decreases production of expression products (mRNA, polypeptide, or both) relative to basal or native states.
A number of nucleic-acid based methods, including antisense RNA, co-suppression, ribozyme directed RNA cleavage, and RNA interference (RNAi) can be used to inhibit protein expression in plants. Antisense technology is one well-known method. In this method, a nucleic acid segment from a gene to be repressed is cloned and operably linked to a promoter so that the antisense strand of RNA is transcribed. The recombinant vector is then transformed into plants, as described above, and the antisense strand of RNA is produced. The nucleic acid segment need not be the entire sequence of the gene to be repressed, but typically will be substantially complementary to at least a portion of the sense strand of the gene to be repressed. Generally, higher homology can be used to compensate for the use of a shorter sequence. Typically, a sequence of at least 30 nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more.
Thus, for example, an isolated nucleic acid provided herein can be an antisense nucleic acid to any of the aforementioned nucleic acids encoding a polypeptide having UV-B tolerance activity set forth in SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:113, or SEQ ID NO:114. A nucleic acid that decreases the level of a transcription or translation product of a gene encoding a polypeptide having UV-B tolerance activity is transcribed into an antisense nucleic acid that anneals to the sense coding sequence of the polypeptide having UV-B tolerance activity.
Constructs containing operably linked nucleic acid molecules in the sense orientation can also be used to inhibit the expression of a gene. The transcription product can be similar or identical to the sense coding sequence of a polypeptide having UV-B tolerance activity. The transcription product can also be unpolyadenylated, lack a 5′ cap structure, or contain an unsplicable intron. Methods of co-suppression using a full-length cDNA as well as a partial cDNA sequence are known in the art. See, e.g., U.S. Pat. No. 5,231,020.
In another method, a nucleic acid can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA. (See, U.S. Pat. No. 6,423,885). Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide. Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contain a 5′-UG-3′ nucleotide sequence. The construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Pat. No. 5,254,678 and WO 02/46449 and references cited therein. Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo. Perriman et al., Proc. Natl. Acad. Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes in Plants,” Edited by Turner, P.C., Humana Press Inc., Totowa, N.J. RNA endoribonucleases which have been described, such as the one that occurs naturally in Tetrahymena thermophile, can be useful. See, for example, U.S. Pat. Nos. 4,987,071 and 6,423,885.
RNAi can also be used to inhibit the expression of a gene. For example, a construct can be prepared that includes a sequence that is transcribed into an interfering RNA. Such an RNA can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure. One strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of the polypeptide of interest, and that is from about 10 nucleotides to about 2,500 nucleotides in length. The length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides. The other strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the antisense strand of the coding sequence of the polypeptide of interest, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence. The loop portion of a double stranded RNA can be from 10 nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides. The loop portion of the RNA can include an intron. A construct including a sequence that is transcribed into an interfering RNA is transformed into plants as described above. Methods for using RNAi to inhibit the expression of a gene are known to those of skill in the art. See, e.g., U.S. Pat. Nos. 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S. Patent Publications 20030175965, 20030175783, 20040214330, and 20030180945.
In some nucleic-acid based methods for inhibition of gene expression in plants, a suitable nucleic acid can be a nucleic acid analog. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et al., Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
Information that the polypeptides disclosed herein can modulate UV-B tolerance can be useful in breeding of crop plants. Based on the effect of disclosed polypeptides on protein content, one can search for and identify polymorphisms linked to genetic loci for such polypeptides. Polymorphisms that can be identified include simple sequence repeats (SSRs), rapid amplification of polymorphic DNA (RAPDs), amplified fragment length polymorphisms (AFLPs) and restriction fragment length polymorphisms (RFLPs).
If a polymorphism is identified, its presence and frequency in populations is analyzed to determine if it is statistically significantly correlated to an alteration in protein content. Those polymorphisms that are correlated with an alteration in protein content can be incorporated into a marker assisted breeding program to facilitate the development of lines that have a desired alteration in UV-B tolerance. Typically, a polymorphism identified in such a manner is used with polymorphisms at other loci that are also correlated with a desired alteration in UV-B tolerance.
A plurality of seeds of a transgenic plant described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture. Packaging material such as paper and cloth are well known in the art. Such an article of manufacture typically has a package label accompanying the bag, e.g., a tag or label secured to the packaging material, a label printed on the packaging material or a label inserted within the packaging material. The package label may indicate that the seeds therein incorporate one or more transgenes, e.g., a transgene that encodes a polypeptide having UV-B tolerance activity. The plurality of seeds in such an article of manufacture can be at least 25, 500, 1,000, 2,500, 10,000, or 80,000 seeds.
Transgenic plants provided herein have particular uses in the agricultural and nutritional industries. For example, transgenic plants described herein can be used to maintain growth and development of such plants under conditions of increased incident UV-B light, relative to non-transgenic control plants. Such a trait can increase plant survival and result in increased yields of grain or biomass under increased UV-B light conditions.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
The following symbols are used in the Examples: T1: first generation transformant; T2: second generation, progeny of self-pollinated T1 plants; T3: third generation, progeny of self-pollinated T2 plants. Independent transformations are referred to as events.
The following nucleic acids were isolated from Arabidopsis thaliana plants: SEQ ID NO:86 (Ceres Clone 11922, At5g40260) and SEQ ID NO: 96 (Ceres Clone 41610, At5g14260). The sequence set forth in SEQ ID NO:86 is a cDNA predicted to encode a polypeptide having nodulin MtN3 activity. The polypeptide sequence is set forth in SEQ ID NO:87 and is 239 amino acid residues in length. The sequence set forth in SEQ ID NO: 96 is a cDNA predicted to encode a polypeptide SET domain activity. The polypeptide sequence is set forth in SEQ ID NO:97 and is 516 amino acid residues in length.
Ceres Clones 11922 and 41610 were individually cloned into separate Ti plasmid vectors, each containing a phosphinothricin acetyltransferase gene (bar gene), which confers Finale™ resistance to transformed plants, such that the Ceres Clone 11922 and Ceres Clone 41610 coding sequences were operably linked to a CaMV 35S promoter.
Wild-type Arabidopsis thaliana ecotype Wassilewskija (WS) plants were transformed separately with each construct. The transformations were performed using methods similar to those described elsewhere (Bechtold and Pelletier, Methods in Mol. Biol., 82:259-66 (1998)).
Transgenic Arabidopsis lines containing Ceres Clone 11922 or Ceres Clone 41610 were designated ME04971 or ME01765, respectively. Finale™ resistance, polymerase chain reaction (PCR) amplification, and sequencing of PCR products were used to confirm the presence of each vector containing a Ceres clone in the respective transgenic Arabidopsis lines.
To analyze inhibition of hypocotyl elongation by exposure to UV-B light, 40 T2 seeds were cold treated, plated onto standard MS medium (0.5% sucrose, 0.5×MS) plates, and allowed to germinate in 16 hours light at 22° C. After 24 hours growth in complete darkness, seedlings were treated with 1 hour of UV-B at a fluence of 5 watts/m2, and then kept in darkness for 23 hours, after which, seedlings received a second round of UV-B illumination. The seedlings were kept in darkness for another 23 hours. Seedling hypocotyl length was observed on day 4 post germination. The hypocotyls of individual seedlings were determined to be “long” or “short” based on qualitative observation (see, for example,
Chi-square analysis of these segregating T2 seed lines indicated that the bar-gene co-segregates with the trans-gene in a 3:1 ratio indicating a single insertion. Homozygous T3 seeds from self-pollinated T2 plants were allowed to germinate and were exposed to UV-B as above. Hypocotyl lengths of T3 seedlings and wild-type seedlings were measured, and results subjected to a standard student's T-test to determine statistical significance.
T2 and T3 seeds from two events of ME04971 containing Ceres Clone 11922 were analyzed for hypocotyl length as described in Example 2. Significantly more Finale™ resistant T2 and T3 seedlings had long hypocotyls than Finale® sensitive seedlings (-segregants). See Table 1.
There were no observable or statistically significant differences between T2 ME04971 plants and control plants in germination, onset of flowering, rosette area, fertility, and general morphology/architecture.
T2 seeds and T3 seeds from two events of ME01765 containing Ceres Clone 41610 were analyzed for hypocotyl length as described in Example 2. Significantly more Finale™ resistant T2 and T3 seedlings had long hypocotyls than Finale® sensitive seedlings (-segregants). See Table 2.
There were no observable or statistically significant differences between T2 ME01765 plants and control plants in germination, onset of flowering, rosette area, fertility, and general morphology/architecture.
A process known as Reciprocal BLAST (Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244 (1998)) was used to identify potential functional homolog and/or ortholog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.
Before starting a Reciprocal BLAST process, a specific query polypeptide was searched against all peptides from its source species using BLAST in order to identify polypeptides having sequence identity of 80% or greater to the query polypeptide and an alignment length of 85% or greater along the shorter sequence in the alignment. The query polypeptide and any of the aforementioned identified polypeptides were designated as a cluster.
The BLASTP version 2.0 program from Washington University at Saint Louis, Mo., USA was used to determine BLAST sequence identity and E-value.
The BLASTP version 2.0 program includes the following parameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3) the -postsw option. The BLAST sequence identity was calculated based on the alignment of the first BLAST HSP (High-scoring Segment Pairs) of the identified potential functional homolog and/or ortholog sequence with a specific query polypeptide. The number of identically matched residues in the BLAST HSP alignment was divided by the HSP length, and then multiplied by 100 to get the BLAST sequence identity. The HSP length typically included gaps in the alignment, but in some cases gaps were excluded.
The main Reciprocal BLAST process consisted of two rounds of BLAST searches; forward search and reverse search. In the forward search step, a query polypeptide sequence, “polypeptide A,” from source species SA was BLASTed against all protein sequences from a species of interest. Top hits were determined using an E-value cutoff of 10−5 and a sequence identity cutoff of 35%. Among the top hits, the sequence having the lowest E-value was designated as the best hit, and considered a potential functional homolog or ortholog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original query polypeptide was considered a potential functional homolog or ortholog as well. This process was repeated for all species of interest.
In the reverse search round, the top hits identified in the forward search from all species were BLASTed against all protein sequences from the source species SA. A top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit was also considered as a potential functional homolog or ortholog.
Functional homologs and/or orthologs were identified by manual inspection of potential functional homolog and/or ortholog sequences. Representative functional homologs and/or orthologs for SEQ ID NO:87 and SEQ ID NO:97 are shown in
Hidden Markov Models (HMMs) were generated by the program HMMER 2.3.2 using groups of sequences as input that are homologous and/or orthologous to each of SEQ ID NO:87 and SEQ ID NO:97. To generate each HMM, the default HMMER 2.3.2 program parameters configured for glocal alignments were used.
An HMM was generated using the sequences set forth in SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:105, and SEQ ID NO:107, which are aligned in
An HMM was generated using the sequences set forth in SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, and SEQ ID NO:114, which are aligned in
Arabidopsis thaliana
Populus balsamifera
Zea mays
Oryza sativa subsp.
japonica
Panicum virgatum
Triticum aestivum
Lycopersicon
esculentum
Glycine max
Glycine max
Gossypium hirsutum
Panicum virgatum
Oryza sativa subsp.
japonica
Oryza sativa subsp.
japonica
Oryza sativa subsp.
indica
Oryza sativa subsp.
indica
Oryza sativa subsp.
japonica
Arabidopsis thaliana
Populus balsamifera
Glycine max
Oryza sativa subsp.
japonica
Oryza sativa subsp.
japonica
Medicago truncatula
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims priority to U.S. Patent Application No. 60/819,763 filed on Jul. 10, 2006 and entitled “Increasing UV-B Tolerance in Plants,” the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/73154 | 7/10/2007 | WO | 00 | 4/6/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60819763 | Jul 2006 | US | |
| 60819763 | Jul 2006 | US |
