Patent Grant
9834778
The present invention relates to methods for the design and production of synthetic promoters with a defined specificity and promoters produced with these methods.
Manipulation of plants to alter and/or improve phenotypic characteristics such as productivity or quality requires expression of heterologous genes in plant tissues. Such genetic manipulation relies on the availability of a means to drive and to control gene expression as required. For example, genetic manipulation relies on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant.
Advanced traits often require the coordinated expression of more than one gene in a transgenic plant. For example, to achieve the production of polyunsaturated fatty acids such as archachidonic acid in a plant requires expression of at least 5 genes. There is also increasing demand of trait stacking which requires the combination of more than one gene in transgenic plants.
The availability of suitable promoters for such coordinated expression is limited. Promoters would often need to have the same tissue and/or developmental specificity and preferably comparable expression strength. One solution has been to use the same promoter for the expression of several genes. Expression constructs comprising more than one expression cassette with tandem or inverted sequence repeats of for example a promoter cause various problems. When located on one vector, handling of the vector in bacteria for cloning, amplification and transformation is difficult due to recombination events which lead to the loss and/or rearrangement of part of the expression construct. Moreover, sequence verification of constructs comprising repeated sequences is difficult and sometimes impossible. A further problem of such expression constructs comprising repeats of the same promoter sequence is that recombination may also occur after introduction into the genome of the target organism such as a plant.
Additionally it is well known that repeated promoter sequences in the genome of organisms such as a plant may induce silencing of expression derived from these promoters, for example by methylation of the promoter or increase of chromatin density at the site of the promoters which makes the promoter inaccessible for transcription factors.
The use of different promoters in expression constructs comprising more than one expression cassette is one possibility to circumvent these problems. Isolation and analysis of promoters is laborious and time consuming. It is unpredictable what expression pattern and expression strength an isolated promoter will have and hence a high number of promoters need to be tested in order to find at least two promoters with comparable expression pattern and optionally comparable expression strength.
There is, therefore, a great need in the art for the availability of new sequences that may be used for expression of selected transgenes in economically important plants. It is thus an objective of the present invention to provide new methods for the production of synthetic promoters with identical and/or overlapping expression pattern or expression specificity and optionally similar expression strength. This objective is solved by the present invention.
A first embodiment of the invention is a method for the production of one or more synthetic regulatory nucleic acid molecules of a defined specificity comprising the steps of
In one embodiment of the invention, additional preferably associated motives may be introduced into the sequence of the synthetic nucleic acid molecule.
Production of the nucleic acid molecule comprising the mutated sequence could for example be done by chemical synthesis or by oligo ligation whereby smaller oligos comprising parts of the sequence of the invention are stepwise annealed and ligated to form the nucleic acid molecule of the invention.
In a preferred embodiment of the invention, the synthetic regulatory nucleic acid molecule is a synthetic promoter, in a more preferred embodiment the synthetic regulatory nucleic acid molecule is a synthetic promoter functional in a plant, plant tissue or plant cell.
The at least one starting molecule comprising the starting sequence may for example be identified by searches in literature or internet resources such as sequence and/or gene expression data bases. The at least one starting molecule comprising the starting sequence may in another example be identified by isolation and characterization of a natural occurring promoter from the respective organism, for example plants, algae, fungi, animals and the like. Such methods are well known to a person skilled in the art and for example described in Back et al., 1991, Keddie et al., 1992, Keddie et al., 1994.
Motives in a series of nucleic acid molecules may be identified by a variety of bioinformatic tools available in the art. For example see Hehl and Wingender, 2001, Hehl and Bulow, 2002, Cartharius et al., 2005, Kaplan et al., 2006, Dare at al., 2008.
In addition, there are various databases available specialized in promoter analysis and motif prediction in any given sequence. For example as reviewed in Hehl and Wingender, 2001.
It is also possible to identify motives necessary for regulation of expression of the defined specificity with experimental methods known to a skilled person. Such methods are for example deletion or mutation analysis of the respective starting sequence as for example described in Montgomery et al., 1993.
Essential motives known to be involved in transcription initiation for example by being bound by general initiation factors and/or RNA polymerases as described above under ii) are for example the TATA box, the CCAAT box, the GC box or other functional similar motives as for example identified in Roeder (1996, Trends in Biochemical Science, 21(9)) or Baek et al. (2006, Journal of Biological Chemistry, 281). These motives allow a certain degree of degeneration or variation of their sequence without changing or destroying their functionality in initiation of transcription. The skilled person is aware of such sequence variations that leave the respective motives functional. Such variations are for example given in the Transfac database as described by Matys et al, ((2003) NAR 31 (1)) and literature given therein. The Transfac database may for example be accessed via ftp://ftp.ebi.ac.uk/pub/databases/transfac/transfac32.tar.Z. Hence it is to be understood that the term “leaving motives unaltered involved in transcription initiation” means that the respective motives may be mutated, hence altered in their sequence as long as their respective function which is enabling initiation of transcription is not altered, hence as long as the essential motives are functional. In another embodiment of the invention the first 49, preferably 44, more preferably 39, even more preferably 34, most preferably 29 bp directly upstream of the transcription initiation site are kept unaltered.
The term “keeping the arrangement of the motives unchanged” as used above under iv) means, that the order of the motives and/or the distance between the motives are kept substantially unchanged, preferably unchanged. Substantially unchanged means, that the distance between two motives in the starting sequence does not differ from the distance between these motives in the synthetic regulatory nucleic acid sequence, hence the distance between said motives is not longer or shorter, by more than 100%, for example 90%, 80% or 70%, preferably 60%, 50% or 40%, more preferably not more than 30% or 20%, most preferably not more than 10% in the synthetic regulatory nucleic acid sequence as compared to the starting sequence. Preferably the distance between two motives in the starting sequence differs by not more than 10, preferably 9, more preferably 8 or 7 or 6 or 5 or 4, even more preferably not more than 3 or 2, most preferably not more than 1 basepairs from the distance in the permutated sequence.
Inverted and/or direct stretches of repeated sequences may lead to the formation of secondary structures in plasmids or genomic DNA. Repeated sequences may lead to recombination, deletion and/or rearrangement in the plasmid both in E. coli and Agrobacterium. In eukaryotic organisms, for example plants, repeated sequences also tend to be silenced by methylation. Recombination events which lead to deletions or rearrangements of one or more expression cassettes and/or T-DNAs are likely to lead to loss of function for example loss of expression of such constructs in the transgenic plant (Que and Jorgensen, 1998, Hamilton et al., 1998). It is therefore a critical feature of the invention at hand to avoid identical stretches of 50 basepairs, preferably 45 basepairs, more preferably 40 basepairs, most preferably 35 basepairs, for example 30 basepairs between each of the starting sequence and the one or more permutated sequences. In case of the production of more than one permutated sequences said identical stretches must be avoided between the starting sequence and each of the permutated sequences in a pair wise comparison. In another embodiment, such identical stretches must be avoided between all permutated sequences and the starting sequence; hence none of the permutated and starting sequences shares such identical stretches with any of the other sequences.
The skilled person is aware that regulatory nucleic acids may comprise promoters and functionally linked to said promoters 5′UTR the latter may comprise at least one intron. It has been shown, that introns may be lead to increased expression levels derived from the promoter to which the 5′UTR comprising the intron is functionally linked. The 5′ UTR and the intron may be altered in their sequence as described, wherein the splice sites and putative branching point are not altered in order to ensure correct splicing of the intron after permutation. No nucleotide exchanges are introduced into sequences at least 2, preferably at least 3, more preferably at least 5, even more preferably at least 10 bases up- and downstream of the splice sites (5′ GT; 3′ CAG) are kept unchanged. In addition, “CURAY” and “TNA” sequence elements being potential branching points of the intron are kept unchanged within the last 200 base pairs, preferably the last 150 base pairs, more preferably the last 100 base pairs, even more preferably the last 75 base pairs of the respective intron.
The 5′UTR may be permutated according to the rules as defined above, wherein preferably at least 25, more preferably at least 20, even more preferably at least 15, for example at least 10, most preferably at least 5 base pairs up- and downstream of the transcription start are kept unchanged. The AT content of both the 5′ UTR and the intron is not changed by more than 20%, preferably not more than 15%, for example 10% or 5% compared to the AT content of the starting sequence.
A further embodiment of the invention is a synthetic regulatory nucleic acid molecule produced according to the method of the invention.
An expression construct comprising the said synthetic regulatory nucleic acid molecule is another embodiment of the invention.
A vector comprising the regulatory nucleic acid molecule or the expression construct of the invention is also comprised in this invention, as well as microorganisms, plant cells or animal cells comprising the regulatory nucleic acid molecule, the expression construct and/or the vector of the invention.
A further embodiment of the invention is a plant, plant seed, plant cell or part of a plant comprising the regulatory nucleic acid molecule, the expression construct and/or the vector of the invention.
A further embodiment of the invention are exemplary recombinant seed specific or seed preferential synthetic regulatory nucleic acid molecules produced according to the method of the invention wherein the regulatory nucleic acid molecule is comprised in the group consisting of
Another embodiment of the invention are exemplary recombinant seed specific or seed preferential synthetic regulatory nucleic acid molecules produced according to the method of the invention wherein the regulatory nucleic acid molecule is comprised in the group consisting of
Further embodiments of the invention are exemplary recombinant constitutive regulatory nucleic acid molecules produced according to the method of the invention wherein the regulatory nucleic acid molecule is comprised in the group consisting of
Another embodiment of the invention are exemplary recombinant constitutive synthetic regulatory nucleic acid molecules produced according to the method of the invention wherein the regulatory nucleic acid molecule is comprised in the group consisting of
It is to be understood, that the group of exemplary recombinant seed specific or seed preferential or constitutive synthetic regulatory nucleic acid molecules produced according to the method of the invention as defined above under I) to V) and i) to vi) does not comprise the starting molecules as defined by SEQ ID NO: 1, 3, 5 and 13 or a complement thereof or a nucleic acid molecule having at least 250 consecutive base pairs of a sequence described by SEQ ID NO: 1, 3, 5 or 13 or a complement thereof or any other nucleic acid molecule occurring in a wild type plant as such nucleic acid molecules are molecules that are not produced according to the invention but are naturally present in wild type plants.
An expression construct comprising any of said synthetic regulatory nucleic acid molecules as defined above under I) to VI) and i) to vi) is another embodiment of the invention.
A vector comprising the regulatory nucleic acid molecule or the expression construct of the invention is also comprised in this invention, as well as microorganisms, plant cells or animal cells comprising the regulatory nucleic acid molecule, the expression construct and/or the vector of the invention.
A further embodiment of the invention is a plant, plant seed, plant cell or part of a plant comprising the regulatory nucleic acid molecule, the expression construct and/or the vector of the invention.
Abbreviations: GFP—green fluorescence protein, GUS—beta-Glucuronidase, BAP—6-benzylaminopurine; 2,4-D—2,4-dichlorophenoxyacetic acid; MS—Murashige and Skoog mediurn; NAA—1-naphtaleneacetic acid; MES, 2-(N-morpholino-ethanesulfonic acid, IAA indole acetic acid; Kan: Kanamycin sulfate; GA3—Gibberellic acid; Timentin™: ticarcillin disodium/clavulanate potassium.
It is to be understood that this invention is not limited to the particular methodology or protocols. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a vector” is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and so forth. The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower). As used herein, the word “or” means any one member of a particular list and also includes any combination of members of that list. The words “comprise,” “comprising,” “include,” “including,” and “includes” when used in this specification and in the following claims are intended to specify the presence of one or more stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. For clarity, certain terms used in the specification are defined and used as follows:
Antiparallel: “Antiparallel” refers herein to two nucleotide sequences paired through hydrogen bonds between complementary base residues with phosphodiester bonds running in the 5′-3′ direction in one nucleotide sequence and in the 3′-5′ direction in the other nucleotide sequence.
Antisense: The term “antisense” refers to a nucleotide sequence that is inverted relative to its normal orientation for transcription or function and so expresses an RNA transcript that is complementary to a target gene mRNA molecule expressed within the host cell (e.g., it can hybridize to the target gene mRNA molecule or single stranded genomic DNA through Watson-Crick base pairing) or that is complementary to a target DNA molecule such as, for example genomic DNA present in the host cell.
“Box” or as synonymously used herein “motif” or “cis-element” of a promoter means a transcription factor binding sequence defined by a highly conserved core sequence of approximately 4 to 6 nucleotides surrounded by a conserved matrix sequence of in total up to 20 nucleotides within the plus or minus strand of the promoter, which is able of interacting with the DNA binding domain of a transcription factor protein. The conserved matrix sequence allows some variability in the sequence without loosing its ability to be bound by the DNA binding domain of a transcription factor protein.
One way to describe transcription factor binding sites (TFBS) is by nucleotide or position weight matrices (NWM or PWM) (for review see Stormo, 2000). A weight matrix pattern definition is superior to a simple IUPAC consensus sequence as it represents the complete nucleotide distribution for each single position. It also allows the quantification of the similarity between the weight matrix and a potential TFBS detected in the sequence (Cartharius et al. 2005).
Coding region: As used herein the term “coding region” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5′-side by the nucleotide triplet “ATG” which encodes the initiator methionine and on the 3′-side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA). In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5′- and 3′-end of the sequences which are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5′ or 3′ to the non-translated sequences present on the mRNA transcript). The 5′-flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3′-flanking region may contain sequences which direct the termination of transcription, post-transcriptional cleavage and polyadenylation.
Complementary: “Complementary” or “complementarity” refers to two nucleotide sequences which comprise antiparallel nucleotide sequences capable of pairing with one another (by the base-pairing rules) upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences. For example, the sequence 5′-AGT-3′ is complementary to the sequence 5′-ACT-3′. Complementarity can be “partial” or “total.” “Partial” complementarity is where one or more nucleic acid bases are not matched according to the base pairing rules. “Total” or “complete” complementarity between nucleic acid molecules is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid molecule strands has significant effects on the efficiency and strength of hybridization between nucleic acid molecule strands. A “complement” of a nucleic acid sequence as used herein refers to a nucleotide sequence whose nucleic acid molecules show total complementarity to the nucleic acid molecules of the nucleic acid sequence.
Conserved motives: A conserved motif as used herein means a sequence motif or box found in various promoters having the same or overlapping specificity. Overlapping specificity means the specificity of at least two promoters wherein the expression derived from one promoter is in part or completely in the same for example tissue as the other promoter, wherein the latter one may drive expression in additional tissues in which the first promoter may not drive expression. Motives may be grouped in three classes:
Essential: motives present in the promoters of most genes that are transcribed by RNA Polymerase II and which are preferentially localized close to the transcription start side. Such motives must not be made dysfunctional by mutations according to the method of the invention. Hence they must not be altered in a way that prevents them from being bound by the respective DNA binding domain of the transcription factor protein that would have bound to the unaltered sequence.
non exclusively associated: motives present in the promoters of genes that are associated with certain tissues/physiological states/treatments but not exclusively, they may be expressed also in other tissues/physiological states/treatments. According to the method of the invention, such motives should preferably not be made dysfunctional by mutations or at least only a certain percentage of such motives present in one particular promoter or starting sequence. Hence they should preferably not be altered in a way that prevents them from being bound by the respective DNA binding domain of the transcription factor protein that would have bound to the unaltered sequence.
preferentially associated: motives present in the promoters of genes that are expressed preferentially in specific tissues/physiological states/treatments. The vast majority of such motives identified in a starting sequence must not be made dysfunctional by mutations according to the method of the invention. Hence they must not be altered in a way that prevents them from being bound by the respective DNA binding domain of the transcription factor protein that would have bound to the unaltered sequence.
Defined specificity: the term “defined specificity” means any expression specificity of a promoter, preferably a plant specific promoter, which is beneficial for the expression of a distinct coding sequence or RNA. A defined specificity may for example be a tissue or developmental specificity or the expression specificity could be defined by induction or repression of expression by biotic or abiotic stimuli or a combination of any of these.
Double-stranded RNA: A “double-stranded RNA” molecule or “dsRNA” molecule comprises a sense RNA fragment of a nucleotide sequence and an antisense RNA fragment of the nucleotide sequence, which both comprise nucleotide sequences complementary to one another, thereby allowing the sense and antisense RNA fragments to pair and form a double-stranded RNA molecule.
Endogenous: An “endogenous” nucleotide sequence refers to a nucleotide sequence, which is present in the genome of the untransformed plant cell.
Expression: “Expression” refers to the biosynthesis of a gene product, preferably to the transcription and/or translation of a nucleotide sequence, for example an endogenous gene or a heterologous gene, in a cell. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and—optionally—the subsequent translation of mRNA into one or more polypeptides. In other cases, expression may refer only to the transcription of the DNA harboring an RNA molecule. Expression may also refer to the change of the steady state level of the respective RNA in a plant or part thereof due to change of the stability of the respective RNA.
Similar expression strength: Two or more regulatory nucleic acid molecules have a similar expression strength when the expression derived from any of the regulatory nucleic acid molecule in a distinct cell, tissue or plant organ does not deviate by more than factor 2.
Expression construct: “Expression construct” as used herein mean a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate part of a plant or plant cell, comprising a promoter functional in said part of a plant or plant cell into which it will be introduced, operatively linked to the nucleotide sequence of interest which is—optionally—operatively linked to termination signals. If translation is required, it also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region may code for a protein of interest but may also code for a functional RNA of interest, for example RNAa, siRNA, snoRNA, snRNA, microRNA, ta-siRNA or any other noncoding regulatory RNA, in the sense or antisense direction. The expression construct comprising the nucleotide sequence of interest may be chimeric, meaning that one or more of its components is heterologous with respect to one or more of its other components. The expression construct may also be one, which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host, i.e., the particular DNA sequence of the expression construct does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression construct may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a plant, the promoter can also be specific to a particular tissue or organ or stage of development.
Expression pattern or expression specificity of a regulatory nucleic acid molecule as used herein defines the tissue and/or developmental and/or environmentally modulated expression of a coding sequence or RNA under the control of a distinct regulatory nucleic acid molecule.
Foreign: The term “foreign” refers to any nucleic acid molecule (e.g., gene sequence) which is introduced into the genome of a cell by experimental manipulations and may include sequences found in that cell so long as the introduced sequence contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) and is therefore distinct relative to the naturally-occurring sequence.
Functional linkage: The term “functional linkage” or “functionally linked” is to be understood as meaning, for example, the sequential arrangement of a regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator or an enhancer) in such a way that each of the regulatory elements can fulfill its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. As a synonym the wording “operable linkage” or “operably linked” may be used. The expression may result depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. In a preferred embodiment, the nucleic acid sequence to be transcribed is located behind the promoter in such a way that the transcription start is identical with the desired beginning of the chimeric RNA of the invention. Functional linkage, and an expression construct, can be generated by means of customary recombination and cloning techniques as described (e.g., in Maniatis T, Fritsch E F and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience; Gelvin et al. (Eds) (1990) Plant Molecular Biology Manual; Kluwer Academic Publisher, Dordrecht, The Netherlands). However, further sequences, which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences. The insertion of sequences may also lead to the expression of fusion proteins. Preferably, the expression construct, consisting of a linkage of a regulatory region for example a promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a plant genome, for example by transformation.
Gene: The term “gene” refers to a region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term “structural gene” as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
Genome and genomic DNA: The terms “genome” or “genomic DNA” is referring to the heritable genetic information of a host organism. Said genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also the DNA of the plastids (e.g., chloroplasts) and other cellular organelles (e.g., mitochondria). Preferably the terms genome or genomic DNA is referring to the chromosomal DNA of the nucleus.
Heterologous: The term “heterologous” with respect to a nucleic acid molecule or DNA refers to a nucleic acid molecule which is operably linked to, or is manipulated to become operably linked to, a second nucleic acid molecule to which it is not operably linked in nature, or to which it is operably linked at a different location in nature. A heterologous expression construct comprising a nucleic acid molecule and one or more regulatory nucleic acid molecule (such as a promoter or a transcription termination signal) linked thereto for example is a constructs originating by experimental manipulations in which either a) said nucleic acid molecule, or b) said regulatory nucleic acid molecule or c) both (i.e. (a) and (b)) is not located in its natural (native) genetic environment or has been modified by experimental manipulations, an example of a modification being a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the sequence of the nucleic acid molecule is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1,000 bp, very especially preferably at least 5,000 bp, in length. A naturally occurring expression construct—for example the naturally occurring combination of a promoter with the corresponding gene—becomes a transgenic expression construct when it is modified by non-natural, synthetic “artificial” methods such as, for example, mutagenization. Such methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815). For example a protein encoding nucleic acid molecule operably linked to a promoter, which is not the native promoter of this molecule, is considered to be heterologous with respect to the promoter. Preferably, heterologous DNA is not endogenous to or not naturally associated with the cell into which it is introduced, but has been obtained from another cell or has been synthesized. Heterologous DNA also includes an endogenous DNA sequence, which contains some modification, non-naturally occurring, multiple copies of an endogenous DNA sequence, or a DNA sequence which is not naturally associated with another DNA sequence physically linked thereto. Generally, although not necessarily, heterologous DNA encodes RNA or proteins that are not normally produced by the cell into which it is expressed.
Hybridization: The term “hybridization” as used herein includes “any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing.” (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules. As used herein, the term “Tm” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acid molecules is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41 (% G+C), when a nucleic acid molecule is in aqueous solution at 1 M NaCl [see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. Stringent conditions, are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
Medium stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/L NaCl, 6.9 g/L NaH2PO4.H2O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 1% SDS, 5× Denhardt's reagent [50× Denhardt's contains the following per 500 mL 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mL denatured salmon sperm DNA followed by washing (preferably for one times 15 minutes, more preferably two times 15 minutes, more preferably three time 15 minutes) in a solution comprising 1×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS at room temperature or—preferably 37° C.—when a DNA probe of preferably about 100 to about 500 nucleotides in length is employed.
High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/L NaCl, 6.9 g/L NaH2PO4.H2O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 1% SDS, 5× Denhardt's reagent [50× Denhardt's contains the following per 500 mL 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mL denatured salmon sperm DNA followed by washing (preferably for one times 15 minutes, more preferably two times 15 minutes, more preferably three time 15 minutes) in a solution comprising 0.1×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 1% SDS at room temperature or—preferably 37° C.—when a DNA probe of preferably about 100 to about 500 nucleotides in length is employed.
Very high stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5× Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing (preferably for one times 15 minutes, more preferably two times 15 minutes, more preferably three time 15 minutes) in a solution comprising 0.1×SSC, and 1% SDS at 68° C., when a probe of preferably about 100 to about 500 nucleotides in length is employed.
“Identity”: “Identity” when used in respect to the comparison of two or more nucleic acid or amino acid molecules means that the sequences of said molecules share a certain degree of sequence similarity, the sequences being partially identical.
To determine the percentage identity (homology is herein used interchangeably) of two amino acid sequences or of two nucleic acid molecules, the sequences are written one underneath the other for an optimal comparison (for example gaps may be inserted into the sequence of a protein or of a nucleic acid in order to generate an optimal alignment with the other protein or the other nucleic acid).
The amino acid residues or nucleic acid molecules at the corresponding amino acid positions or nucleotide positions are then compared. If a position in one sequence is occupied by the same amino acid residue or the same nucleic acid molecule as the corresponding position in the other sequence, the molecules are homologous at this position (i.e. amino acid or nucleic acid “homology” as used in the present context corresponds to amino acid or nucleic acid “identity”. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology=number of identical positions/total number of positions×100). The terms “homology” and “identity” are thus to be considered as synonyms.
For the determination of the percentage identity of two or more amino acids or of two or more nucleotide sequences several computer software programs have been developed. The identity of two or more sequences can be calculated with for example the software fasta, which presently has been used in the version fasta 3 (W. R. Pearson and D. J. Lipman, PNAS 85, 2444 (1988); W. R. Pearson, Methods in Enzymology 183, 63 (1990); W. R. Pearson and D. J. Lipman, PNAS 85, 2444 (1988); W. R. Pearson, Enzymology 183, 63 (1990)). Another useful program for the calculation of identities of different sequences is the standard blast program, which is included in the Biomax pedant software (Biomax, Munich, Federal Republic of Germany). This leads unfortunately sometimes to suboptimal results since blast does not always include complete sequences of the subject and the query. Nevertheless as this program is very efficient it can be used for the comparison of a huge number of sequences. The following settings are typically used for such a comparisons of sequences:
-p Program Name [String]; -d Database [String]; default=nr; -i Query File [File In]; default=stdin; -e Expectation value (E) [Real]; default=10.0; -m alignment view options: 0=pairwise; 1=query-anchored showing identities; 2=query-anchored no identities; 3=flat query-anchored, show identities; 4=flat query-anchored, no identities; 5=query-anchored no identities and blunt ends; 6=flat query-anchored, no identities and blunt ends; 7=XML Blast output; 8=tabular; 9 tabular with comment lines [Integer]; default=0; -o BLAST report Output File [File Out] Optional; default=stdout; -F Filter query sequence (DUST with blastn, SEG with others) [String]; default=T; -G Cost to open a gap (zero invokes default behavior) [Integer]; default=0; -E Cost to extend a gap (zero invokes default behavior) [Integer]; default=0; -X X dropoff value for gapped alignment (in bits) (zero invokes default behavior); blastn 30, megablast 20, tblastx 0, all others 15 [Integer]; default=0; -I Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotide mismatch (blastn only) [Integer]; default=−3; -r Reward for a nucleotide match (blastn only) [Integer]; default=1; -v Number of database sequences to show oneline descriptions for (V) [Integer]; default=500; -b Number of database sequence to show alignments for (B) [Integer]; default=250; -f Threshold for extending hits, default if zero; blastp 11, blastn 0, blastx 12, tblastn 13; tblastx 13, megablast 0 [Integer]; default=0; -g Perfom gapped alignment (not available with tblastx) [T/F]; default=T; -Q Query Genetic code to use [Integer]; default=1; -D DB Genetic code (for tblast[nx] only) [Integer]; default=1; -a Number of processors to use [Integer]; default=1; -O SeqAlign file [File Out] Optional; -J Believe the query defline [T/F]; default=F; -M Matrix [String]; default=BLOSUM62; —W Word size, default if zero (blastn 11, megablast 28, all others 3) [Integer]; default=0; -z Effective length of the database (use zero for the real size) [Real]; default=0; -K Number of best hits from a region to keep (off by default, if used a value of 100 is recommended) [Integer]; default=0; —P 0 for multiple hit, 1 for single hit [Integer]; default=0; —Y Effective length of the search space (use zero for the real size) [Real]; default=0; —S Query strands to search against database (for blast[nx], and tblastx); 3 is both, 1 is top, 2 is bottom [Integer]; default=3; -T Produce HTML output [T/F]; default=F; —I Restrict search of database to list of GI's [String] Optional; -U Use lower case filtering of FASTA sequence [T/F] Optional; default=F; -y X dropoff value for ungapped extensions in bits (0.0 invokes default behavior); blastn 20, megablast 10, all others 7 [Real]; default=0.0; -Z X dropoff value for final gapped alignment in bits (0.0 invokes default behavior); blastn/megablast 50, tblastx 0, all others 25 [Integer]; default=0; —R PSI-TBLASTN checkpoint file [File In] Optional; -n MegaBlast search [T/F]; default=F; -L Location on query sequence [String] Optional; -A Multiple Hits window size, default if zero (blastn/megablast 0, all others 40 [Integer]; default=0; -w Frame shift penalty (OOF algorithm for blastx) [Integer]; default=0; -t Length of the largest intron allowed in tblastn for linking HSPs (0 disables linking) [Integer]; default=0.
Results of high quality are reached by using the algorithm of Needleman and Wunsch or Smith and Waterman. Therefore programs based on said algorithms are preferred. Advantageously the comparisons of sequences can be done with the program PileUp (J. Mol. Evolution., 25, 351 (1987), Higgins et al., CABIOS 5, 151 (1989)) or preferably with the programs “Gap” and “Needle”, which are both based on the algorithms of Needleman and Wunsch (J. Mol. Biol. 48; 443 (1970)), and “BestFit”, which is based on the algorithm of Smith and Waterman (Adv. Appl. Math. 2; 482 (1981)). “Gap” and “BestFit” are part of the GCG software-package (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991); Altschul et al., (Nucleic Acids Res. 25, 3389 (1997)), “Needle” is part of the The European Molecular Biology Open Software Suite (EMBOSS) (Trends in Genetics 16 (6), 276 (2000)). Therefore preferably the calculations to determine the percentages of sequence identity are done with the programs “Gap” or “Needle” over the whole range of the sequences. The following standard adjustments for the comparison of nucleic acid sequences were used for “Needle”: matrix: EDNAFULL, Gap_penalty: 10.0, Extend_penalty: 0.5. The following standard adjustments for the comparison of nucleic acid sequences were used for “Gap”: gap weight: 50, length weight: 3, average match: 10.000, average mismatch: 0.000.
For example a sequence, which is said to have 80% identity with sequence SEQ ID NO: 1 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence represented by SEQ ID NO: 1 by the above program “Needle” with the above parameter set, has a 80% identity. The identity is calculated on the complete length of the query sequence, for example SEQ ID NO:1.
Isogenic: organisms (e.g., plants), which are genetically identical, except that they may differ by the presence or absence of a heterologous DNA sequence.
Isolated: The term “isolated” as used herein means that a material has been removed by the hand of man and exists apart from its original, native environment and is therefore not a product of nature. An isolated material or molecule (such as a DNA molecule or enzyme) may exist in a purified form or may exist in a non-native environment such as, for example, in a transgenic host cell. For example, a naturally occurring polynucleotide or polypeptide present in a living plant is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides can be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and would be isolated in that such a vector or composition is not part of its original environment. Preferably, the term “isolated” when used in relation to a nucleic acid molecule, as in “an isolated nucleic acid sequence” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its natural source. Isolated nucleic acid molecule is nucleic acid molecule present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acid molecules are nucleic acid molecules such as DNA and RNA, which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs, which encode a multitude of proteins. However, an isolated nucleic acid sequence comprising for example SEQ ID NO: 1 includes, by way of example, such nucleic acid sequences in cells which ordinarily contain SEQ ID NO:1 where the nucleic acid sequence is in a chromosomal or extrachromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the nucleic acid sequence may be double-stranded).
Minimal Promoter: promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
Naturally occurring as used herein means a cell or molecule, for example a plant cell or nucleic acid molecule that occurs in a plant or organism which is not manipulated by man, hence which is for example neither mutated nor genetically engineered by man.
Non-coding: The term “non-coding” refers to sequences of nucleic acid molecules that do not encode part or all of an expressed protein. Non-coding sequences include but are not limited to introns, enhancers, promoter regions, 3′ untranslated regions, and 5′ untranslated regions.
Nucleic acids and nucleotides: The terms “Nucleic Acids” and “Nucleotides” refer to naturally occurring or synthetic or artificial nucleic acid or nucleotides. The terms “nucleic acids” and “nucleotides” comprise deoxyribonucleotides or ribonucleotides or any nucleotide analogue and polymers or hybrids thereof in either single- or double-stranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term “nucleic acid” is used interchangeably herein with “gene”, “cDNA, “mRNA”, “oligonucleotide,” and “polynucleotide”. Nucleotide analogues include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil, and the like; and 2′-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2′-OH is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN. Short hairpin RNAs (shRNAs) also can comprise non-natural elements such as non-natural bases, e.g., ionosin and xanthine, non-natural sugars, e.g., 2′-methoxy ribose, or non-natural phosphodiester linkages, e.g., methylphosphonates, phosphorothioates and peptides.
Nucleic acid sequence: The phrase “nucleic acid sequence” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′- to the 3′-end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role. “Nucleic acid sequence” also refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides. In one embodiment, a nucleic acid can be a “probe” which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length. A “target region” of a nucleic acid is a portion of a nucleic acid that is identified to be of interest. A “coding region” of a nucleic acid is the portion of the nucleic acid, which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein.
Oligonucleotide: The term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. An oligonucleotide preferably includes two or more nucleomonomers covalently coupled to each other by linkages (e.g., phosphodiesters) or substitute linkages.
Overhang: An “overhang” is a relatively short single-stranded nucleotide sequence on the 5′- or 3′-hydroxyl end of a double-stranded oligonucleotide molecule (also referred to as an “extension,” “protruding end,” or “sticky end”).
Overlapping specificity: The term “overlapping specificity” when used herein related to expression specificity of two or more promoters means that the expression regulated by these promoters occur partly in the same plant tissues, developmental stages or conditions. For example, a promoter expressed in leaves and a promoter expressed in root and leaves have an overlap in expression specificity in the leaves of a plant.
Plant: is generally understood as meaning any eukaryotic single- or multi-celled organism or a cell, tissue, organ, part or propagation material (such as seeds or fruit) of same which is capable of photosynthesis. Included for the purpose of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred. The term includes the mature plants, seed, shoots and seedlings and their derived parts, propagation material (such as seeds or microspores), plant organs, tissue, protoplasts, callus and other cultures, for example cell cultures, and any other type of plant cell grouping to give functional or structural units. Mature plants refer to plants at any desired developmental stage beyond that of the seedling. Seedling refers to a young immature plant at an early developmental stage. Annual, biennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the generation of transgenic plants. The expression of genes is furthermore advantageous in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawns. Plants which may be mentioned by way of example but not by limitation are angiosperms, bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses); Pteridophytes such as ferns, horsetail and club mosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms), and Euglenophyceae. Preferred are plants which are used for food or feed purpose such as the families of the Leguminosae such as pea, alfalfa and soya; Gramineae such as rice, maize, wheat, barley, sorghum, millet, rye, triticale, or oats; the family of the Umbelliferae, especially the genus Daucus, very especially the species carota (carrot) and Apium, very especially the species Graveolens dulce (celery) and many others; the family of the Solanaceae, especially the genus Lycopersicon, very especially the species esculentum (tomato) and the genus Solanum, very especially the species tuberosum (potato) and melongena (egg plant), and many others (such as tobacco); and the genus Capsicum, very especially the species annuum (peppers) and many others; the family of the Leguminosae, especially the genus Glycine, very especially the species max (soybean), alfalfa, pea, lucerne, beans or peanut and many others; and the family of the Cruciferae (Brassicacae), especially the genus Brassica, very especially the species napus (oil seed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and of the genus Arabidopsis, very especially the species thaliana and many others; the family of the Compositae, especially the genus Lactuca, very especially the species sativa (lettuce) and many others; the family of the Asteraceae such as sunflower, Tagetes, lettuce or Calendula and many other; the family of the Cucurbitaceae such as melon, pumpkin/squash or zucchini, and linseed. Further preferred are cotton, sugar cane, hemp, flax, chillies, and the various tree, nut and wine species.
Polypeptide: The terms “polypeptide”, “peptide”, “oligopeptide”, “polypeptide”, “gene product”, “expression product” and “protein” are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.
Pre-protein: Protein, which is normally targeted to a cellular organelle, such as a chloroplast, and still comprising its transit peptide.
Primary transcript: The term “primary transcript” as used herein refers to a premature RNA transcript of a gene. A “primary transcript” for example still comprises introns and/or is not yet comprising a polyA tail or a cap structure and/or is missing other modifications necessary for its correct function as transcript such as for example trimming or editing.
Promoter: The terms “promoter”, or “promoter sequence” are equivalents and as used herein, refer to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into RNA. Such promoters can for example be found in the following public databases www.grassius.org/grasspromdb.html, mendel.cs.rhul.ac.uk/mendel.php?topic=plantprom, ppdb.gene.nagoya-u.ac.jp/cgi-bin/index.cgi. Promoters listed there may be addressed with the methods of the invention and are herewith included by reference. A promoter is located 5′ (i.e., upstream), proximal to the transcriptional start site of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. Said promoter comprises for example the at least 10 kb, for example 5 kb or 2 kb proximal to the transcription start site. It may also comprise the at least 1500 bp proximal to the transcriptional start site, preferably the at least 1000 bp, more preferably the at least 500 bp, even more preferably the at least 400 bp, the at least 300 bp, the at least 200 bp or the at least 100 bp. In a further preferred embodiment, the promoter comprises the at least 50 bp proximal to the transcription start site, for example, at least 25 bp. The promoter does not comprise exon and/or intron regions or 5′ untranslated regions. The promoter may for example be heterologous or homologous to the respective plant. A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety). Suitable promoters can be derived from genes of the host cells where expression should occur or from pathogens for this host cells (e.g., plants or plant pathogens like plant viruses). A plant specific promoter is a promoter suitable for regulating expression in a plant. It may be derived from a plant but also from plant pathogens or it might be a synthetic promoter designed by man. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. Also, the promoter may be regulated in a tissue-specific or tissue preferred manner such that it is only or predominantly active in transcribing the associated coding region in a specific tissue type(s) such as leaves, roots or meristem. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., petals) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., roots). Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected. The term “cell type specific” as applied to a promoter refers to a promoter, which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., GUS activity staining, GFP protein or immunohistochemical staining. The term “constitutive” when made in reference to a promoter or the expression derived from a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid molecule in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.) in the majority of plant tissues and cells throughout substantially the entire lifespan of a plant or part of a plant. Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue.
Promoter specificity: The term “specificity” when referring to a promoter means the pattern of expression conferred by the respective promoter. The specificity describes the tissues and/or developmental status of a plant or part thereof, in which the promoter is conferring expression of the nucleic acid molecule under the control of the respective promoter. Specificity of a promoter may also comprise the environmental conditions, under which the promoter may be activated or down-regulated such as induction or repression by biological or environmental stresses such as cold, drought, wounding or infection.
Purified: As used herein, the term “purified” refers to molecules, either nucleic or amino acid sequences that are removed from their natural environment, isolated or separated. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. A purified nucleic acid sequence may be an isolated nucleic acid sequence.
Recombinant: The term “recombinant” with respect to nucleic acid molecules refers to nucleic acid molecules produced by recombinant DNA techniques. Recombinant nucleic acid molecules as such do not exist in nature but are modified, changed, mutated or otherwise manipulated by man. A “recombinant nucleic acid molecule” is a non-naturally occurring nucleic acid molecule that differs in sequence from a naturally occurring nucleic acid molecule by at least one nucleic acid. The term “recombinant nucleic acid molecule” may also comprise a “recombinant construct” which comprises, preferably operably linked, a sequence of nucleic acid molecules, which are not naturally occurring in that order wherein each of the nucleic acid molecules may or may not be a recombinant nucleic acid molecule. Preferred methods for producing said recombinant nucleic acid molecule may comprise cloning techniques, directed or non-directed mutagenesis, synthesis or recombination techniques.
Sense: The term “sense” is understood to mean a nucleic acid molecule having a sequence which is complementary or identical to a target sequence, for example a sequence which binds to a protein transcription factor and which is involved in the expression of a given gene. According to a preferred embodiment, the nucleic acid molecule comprises a gene of interest and elements allowing the expression of the said gene of interest.
Starting sequence: The term “starting sequence” when used herein defines the sequence of a promoter of a defined specificity which is used as a reference sequence for analysis of the presence of motives. The starting sequence is referred to for the definition of the degree of identity to the sequences of the promoters of the invention. The starting sequence could be any wild-type, naturally occurring promoter sequence or any artificial promoter sequence. The sequence of a synthetic promoter sequence produced with the method of the invention may also be used as a starting sequence.
Substantially complementary: In its broadest sense, the term “substantially complementary”, when used herein with respect to a nucleotide sequence in relation to a reference or target nucleotide sequence, means a nucleotide sequence having a percentage of identity between the substantially complementary nucleotide sequence and the exact complementary sequence of said reference or target nucleotide sequence of at least 60%, more desirably at least 70%, more desirably at least 80% or 85%, preferably at least 90%, more preferably at least 93%, still more preferably at least 95% or 96%, yet still more preferably at least 97% or 98%, yet still more preferably at least 99% or most preferably 100% (the later being equivalent to the term “identical”in this context). Preferably identity is assessed over a length of at least 19 nucleotides, preferably at least 50 nucleotides, more preferably the entire length of the nucleic acid sequence to said reference sequence (if not specified otherwise below). Sequence comparisons are carried out using default GAP analysis with the University of Wisconsin GCG, SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; as defined above). A nucleotide sequence “substantially complementary” to a reference nucleotide sequence hybridizes to the reference nucleotide sequence under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions (as defined above).
Transgene: The term “transgene” as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell by experimental manipulations. A transgene may be an “endogenous DNA sequence,” or a “heterologous DNA sequence” (i.e., “foreign DNA”). The term “endogenous DNA sequence” refers to a nucleotide sequence, which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence.
Transgenic: The term transgenic when referring to an organism means transformed, preferably stably transformed, with a recombinant DNA molecule that preferably comprises a suitable promoter operatively linked to a DNA sequence of interest.
Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector”, which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. In the present specification, “plasmid” and “vector” are used interchangeably unless otherwise clear from the context. Expression vectors designed to produce RNAs as described herein in vitro or in vivo may contain sequences recognized by any RNA polymerase, including mitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III. These vectors can be used to transcribe the desired RNA molecule in the cell according to this invention. A plant transformation vector is to be understood as a vector suitable in the process of plant transformation.
Wild-type: The term “wild-type”, “natural” or “natural origin” means with respect to an organism, polypeptide, or nucleic acid sequence, that said organism is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.
Unless indicated otherwise, cloning procedures carried out for the purposes of the present invention including restriction digest, agarose gel electrophoresis, purification of nucleic acids, Ligation of nucleic acids, transformation, selection and cultivation of bacterial cells were performed as described (Sambrook et al., 1989). Sequence analyses of recombinant DNA were performed with a laser fluorescence DNA sequencer (Applied Biosystems, Foster City, Calif., USA) using the Sanger technology (Sanger et al., 1977). Unless described otherwise, chemicals and reagents were obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, USA), from Promega (Madison, Wis., USA), Duchefa (Haarlem, The Netherlands) or Invitrogen (Carlsbad, Calif., USA). Restriction endonucleases were from New England Biolabs (Ipswich, Mass., USA) or Roche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides were synthesized by Eurofins MWG Operon (Ebersberg, Germany).
Using publicly available data, two promoters showing seed specific expression in plants were selected for analyzing the effects of sequence permutation in periodic intervals throughout the full length of the promoter DNA sequence (WO2009016202, WO2009133145). The wildtype or starting sequences of the Phaseolus vulgaris p-PvARC5 (SEQ ID NO 1) (with the prefix p-denoting promoter) and the Vicia faba p-VfSBP (SEQ ID NO 3) promoters were analyzed and annotated for the occurrence of motives, boxes, cis-regulatory elements using e.g. the GEMS Launcher Software (www.genomatix.de) with default parameters (Core similarity 0.75, matrix similarity 0.75)
The “core sequence” of a matrix is defined as the usually 4 consecutive highest conserved positions of the matrix.
The core similarity is calculated as described here and in the papers related to MatInspector (Cartharius K, et al. (2005) Bioinformatics 21; Cartharius K (2005), DNA Press; Quandt K, et al (1995) Nucleic Acids Res. 23.
The maximum core similarity of 1.0 is only reached when the highest conserved bases of a matrix match exactly in the sequence. More important than the core similarity is the matrix similarity which takes into account all bases over the whole matrix length. The matrix similarity is calculated as described here and in the MatInspector paper. A perfect match to the matrix gets a score of 1.00 (each sequence position corresponds to the highest conserved nucleotide at that position in the matrix), a “good” match to the matrix has a similarity of >0.80.
Mismatches in highly conserved positions of the matrix decrease the matrix similarity more than mismatches in less conserved regions.
Opt. gives the Optimized matrix threshold: This matrix similarity is the optimized value defined in a way that a minimum number of matches is found in non-regulatory test sequences (i.e. with this matrix similarity the number of false positive matches is minimized). This matrix similarity is used when the user checks “Optimized” as the matrix similarity threshold for MatInspector. In the following, the DNA sequences of the promoters were permutated according to the method of the invention to yield p-PvArc5_perm (SEQ ID NO 2) and p-VfSBP_perm (SEQ ID NO 4). In case of the p-PvArc5 promoter 6.6% of the motives not associated with seed specific/preferential expression and transcription initiation have been altered, in case of the p-VfSBP 7.8%. DNA permutation was conducted in a way to not affect cis regulatory elements which have been associated previously with seed specific gene expression or initiation of transcription and permutations were distributed periodically over the full promoter DNA sequence with less than 46 nucleotides between permutated nucleotide positions and within a stretch of 5 nucleotides having at least one nucleotide permutated. Permutations were carried out with the aim to keep most of the cis regulatory elements, boxes, motives present in the native promoter and to avoid creating new putative cis regulatory elements, boxes, motives.
The list of motives, boxes, cis regulatory elements in the PvARC5 promoters before and after the permutation are shown in Table 1 and 2.
The list of motives, boxes, cis regulatory elements in the VfSBP promoters before and after the permutation are shown in Table 3 and 4.
Empty lines resemble motives, boxes, cis regulatory elements not found in one sequence but present in the corresponding sequence, hence, motives, boxes, cis regulatory elements that were deleted from the starting sequence or that were introduced into the permutated sequence.
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Agrobacterium
tumefaciens T-
Using the Multisite Gateway System (Invitrogen, Carlsbad, Calif., USA), promoter::reporter-gene cassettes were assembled into binary constructs for plant transformation. beta-Glucuronidase (GUS) or uidA gene which encodes an enzyme for which various chromogenic substrates are known, was utilized as reporter protein for determining the expression features of the permutated p-PvArc5_perm (SEQ ID NO2) and p-VfSBP_perm (SEQ ID NO4) promoter sequences.
The DNA fragments representing promoters p-PvArc5_perm (SEQ ID NO2) and p-VfSBP_perm (SEQ ID NO4) were generated by gene synthesis. Endonucleolytic restriction sites suitable for cloning the promoter fragments into beta-Glucuronidase reporter gene cassettes were included in the synthesis. The p-PvArc5_perm (SEQ ID NO2) promoter was cloned into a pENTR/A vector harboring the beta-Glucuronidase reporter gene c-GUS (with the prefix c-denoting coding sequence) followed by the t-PvArc (with the prefix t- denoting terminator) transcription terminafor sequence using restriction endonucleases FseI and NcoI, yielding construct LJB2012. Similarly, the p-VfSBP_perm (SEQ ID NO4) promoter was cloned into a pENTR/B vector harboring the beta-Glucuronidase reporter gene c-GUS followed by the t-StCatpA transcriptional terminator sequence using restriction endonucleases FseI and NcoI, yielding construct LJB2007.
The complementary pENTR vectors without any expression cassettes were constructed by introduction of a multiple cloning site via KpnI and HindIII restriction sites. By performing a site specific recombination (LR-reaction), the created pENTR/A, pENTR/B and pENTR/C were combined with the pSUN destination vector (pSUN derivative) according to the manufacturers (Invitrogen, Carlsbad, Calif., USA) Multisite Gateway manual. The reactions yielded a binary vector with the p-PvArc5_perm (SEQ ID NO2) promoter, the beta-Glucuronidase coding sequence c-GUS and the t-PvArc terminator, for which the full construct sequence is given (SEQ ID NO7). Accordingly, a binary vector with the p-VfSBP_perm (SEQ ID NO4) promoter, the beta-Glucuronidase reporter gene and the t-StCatpA terminator for which the full construct sequence is given (SEQ ID NO8). The resulting plant transformation vectors are summarized in table 5:
In preparation for the generation of transgenic rapeseed plants, the binary vectors were transformed into Agrobacterium tumefaciens C58C1:pGV2260 (Deblaere et al., 1985, Nucl. Acids. Res. 13: 4777-4788). A 1:50 dilution of an overnight culture of Agrobacteria harboring the respective binary construct was grown in Murashige-Skoog Medium (Murashige and Skoog, 1962, Physiol. Plant 15, 473) supplemented with 3% saccharose (3MS-Medium). For the transformation of rapeseed plants, petioles or hypocotyledons of sterile plants were incubated with a 1:50 Agrobacterium solution for 5-10 minutes followed by a three-day co-incubation in darkness at 25° C. on 3 MS. Medium supplemented with 0.8% bacto-agar. After three days, the explants were transferred to MS-medium containing 500 mg/l Claforan (Cefotaxime-Sodium), 100 nM lmazetapyr, 20 microM Benzylaminopurin (BAP) and 1.6 g/l Glucose in a 16 h light/8 h darkness light regime, which was repeated in weekly periods. Growing shoots were transferred to MS-Medium containing 2% saccharose, 250 mg/l Claforan and 0.8% Bacto-agar. After 3 weeks, the growth hormone 2-Indolbutyl acid was added to the medium to promote root formation. Shoots were transferred to soil following root development, grown for two weeks in a growth chamber and grown to maturity in greenhouse conditions.
To demonstrate and analyze the transcription regulating properties of a promoter, it is useful to operably link the promoter or its fragments to a reporter gene, which can be employed to monitor its expression both qualitatively and quantitatively. Preferably bacterial β-glucuronidase is used (Jefferson 1987). β-glucuronidase activity can be monitored in planta with chromogenic substrates such as 5-bromo-4-Chloro-3-indolyl-β-D-glucuronic acid during corresponding activity assays (Jefferson 1987). For determination of promoter activity and tissue specificity, plant tissue is dissected, stained and analyzed as described (e.g., Bäumlein 1991).
The regenerated transgenic T0 rapeseed plants harboring single or double insertions of the transgene deriving from constructs LJB2043 or LJB2045 were used for reporter gene analysis.
Table 6 summarizes the reporter gene activity observed in plants harboring transgenes containing SEQ ID NO2 and SEQ ID NO4 in constructs LJB2043 and LJB2045, respectively:
The gene expression activity conferred by p-PvArc5 perm and p-VfSBP_perm is shown exemplary in
General results for SEQ ID NO2: Strong GUS expression was detected in all stages of embryo development and in seed shells. No activity was found in other tissues analyzed.
General results for SEQ ID NO4: Weak GUS expression was detected in early and young embryo stages, strong GUS expression could be observed in medium and mature embryos. Weak expression was monitored in seed shells. No activity was found in other tissues investigated.
Using publicly available data, a promoter showing seed specific expression in plants was selected for analyzing the effects of sequence permutation in periodic intervals throughout the full length of the promoter DNA sequence. The wild type sequences of the Brassica napus p-BnNapin promoter was analyzed and annotated for the occurrence of cis-regulatory elements using available literature data (Ellerström et al., Ericson et al., Ezcurra et al.). In the following, the DNA sequence of the promoter was permutated in the region of −1000 to +1 nucleotides with the following criteria to yield p-BnNapin_perm (SEQ ID NO6): DNA permutation was conducted in a way to not affect cis regulatory elements which have been proven previously to be essential for seed specific gene expression and motives essential for gene expression. The remaining promoter sequence was randomly permutated resulting in a promoter sequence with an overall nucleotide homology of 75% to the initial p-BnNapin sequence
Using the Multisite Gateway System (Invitrogen, Carlsbad, Calif., USA), promoter::reporter-gene cassettes were assembled into binary constructs for plant transformation. Beta-Glucuronidase (GUS) or uidA gene which encodes an enzyme for which various chromogenic substrates are known, was utilized as reporter protein for determining the expression features of the permutated p-BnNapin_perm (SEQ ID NO6) promoter sequences.
The DNA fragments representing promoter p-BnNapin_perm was generated by gene synthesis. Endonucleolytic restriction sites suitable for cloning the promoter fragment into a beta-Glucuronidase reporter gene cassette was included in the synthesis. p-BnNapin_perm (SEQ ID NO6) promoter was cloned into a pENTR/A vector harboring the beta-Glucuronidase reporter gene c-GUS (with the prefix c-denoting coding sequence) followed by the t-nos transcription terminator sequence using restriction endonucleases BamHI and NcoI, yielding pENTR/A LLL1168.
A 1138 bp DNA fragment representing the native promoter p-BnNapin (SEQ ID NO5) was generated by PCR with the following primers.
Endonucleolytic restriction sites suitable for cloning the promoter fragment into a beta-Glucuronidase reporter gene cassette were included in the primers. p-BnNapin (SEQ ID NO5) promoter was cloned into a pENTR/A vector harboring the beta-Glucuronidase reporter gene c-GUS (with the prefix c- denoting coding sequence) followed by the t-nos transcription terminator sequence using restriction endonucleases KpnI and NcoI, yielding pENTR/A LLL1166.
By performing a site specific recombination (LR-reaction), the newly created pENTRs/A LLL1168 and LLL1166, were combined with pENTR/B and pENTR/C and the pSUN destination vector (pSUN derivative) according to the manufacturers (Invitrogen, Carlsbad, Calif., USA) Multi-site Gateway manual. The reaction yielded binary vector LLL 1184 with the p-BnNapin_perm (SEQ ID NO6) promoter, the beta-Glucuronidase coding sequence c-GUS and the t-nos terminator, and binary vector LLL 1176 with the native p-BnNapin (SEQ ID NO5) promoter, the beta-Glucuronidase coding sequence c-GUS and the t-nos terminator. For both vectors the full construct sequence is given (SEQ ID NO9 and 10). The resulting plant transformation vectors are shown in table 7:
A. thaliana plants were grown in soil until they flowered. Agrobacterium tumefaciens (strain C58C1 [pMP90]) transformed with the construct of interest was grown in 500 mL in liquid YEB medium (5 g/L Beef extract, 1 g/L Yeast Extract (Duchefa), 5 g/L Peptone (Duchefa), 5 g/L sucrose (Duchefa), 0.49 g/L MgSO4 (Merck)) until the culture reached an OD600 0.8-1.0. The bacterial cells were harvested by centrifugation (15 minutes, 5,000 rpm) and resuspended in 500 mL infiltration solution (5% sucrose, 0.05% SILWET L-77 [distributed by Lehle seeds, Cat. No. VIS-02]). Flowering plants were dipped for 10-20 seconds into the Agrobacterium solution. Afterwards the plants were kept in the dark for one day and then in the greenhouse until seeds could be harvested. Transgenic seeds were selected on soil by spraying the seeds directly after sowing with a solution of 0.016 g/l Imazamox. After 12 to 14 days surviving plants were transferred to pots and grown in the greenhouse.
To demonstrate and analyze the transcription regulating properties of a promoter, it is useful to operably link the promoter or its fragments to a reporter gene, which can be employed to monitor its expression both qualitatively and quantitatively. Preferably bacterial β-glucuronidase is used (Jefferson 1987). β-glucuronidase activity can be monitored in planta with chromogenic substrates such as 5-bromo-4-Chloro-3-indolyl-β-D-glucuronic acid during corresponding activity assays (Jefferson 1987). For determination of promoter activity and tissue specificity, plant tissue is dissected, stained and analyzed as described (e.g., Bäumlein 1991).
The regenerated transgenic T0 Arabidopsis plants harboring single or double insertions of the transgene deriving from constructs LLL1184 (SEQ ID NO9) and constructs LLL1176 (SEQ ID NO10) were used for reporter gene analysis. Table 8 summarizes the reporter gene activity observed in plants harboring transgenes containing SEQ ID NO9 and SEQ ID NO10 in constructs LLL1184 and LLL1176, respectively:
The gene expression activity conferred by pBn-Napin and p-BNapin_perm is shown exemplary in
General results for SEQ ID NO5 and 6: For both promoters pBn-Napin and p-BNapin_perm strong GUS expression was detected in medium to mature stages of embryo development. Weak expression was monitored in seed shells and in siliques. No activity was found in other tissues analyzed.
Using publicly available data, one promoters showing constitutive expression in plants was selected (de Pater, B. S., van der Mark, F., Rueb, S., Katagiri, F., Chua, N. H., Schilperoort, R. A. and Hensgens, L. A. (1992) The promoter of the rice gene GOS2 is active in various different monocot tissues and binds rice nuclear factor ASF-1 Plant J. 2 (6)) for analyzing the effects of sequence permutation in periodic intervals throughout the full length of the promoter DNA sequence. The wildtype or starting sequence of the Oryza sativa p-GOS2 (SEQ ID NO 13) (with the prefix p- denoting promoter) promoter was analyzed and annotated for the occurrence of motives, boxes, cis-regulatory elements using e.g. the GEMS Launcher Software (www.genomatix.de) as described above in example 1.
The promoter p-Gos2 encompasses a 5′UTR sequence with an internal intron. To ensure correct splicing of the intron after permutation, splice sites and putative branching point were not altered. No nucleotide exchanges were introduced into sequences 10 bp up- and downstream of the splice site (5′ GT; 3′ CAG) and “TNA” sequence elements within the last 100 base pairs of the original p-Gos2 were preserved after permutation.
In the following, the DNA sequence of the promoter was permutated according to the method of the invention to yield p-GOS2_perm1 and p-GOS2_perm2 respectively (SEQ ID NO 14 and 15).
The list of motives, boxes, cis regulatory elements in the p-GOS2 promoters before and after the permutation are shown in Table 9 for the starting sequence of p-GOS2, Table 10 for the p-GOS2_perm1 (SEQ ID NO 14) and Table 11 for the p-GOS2_perm2 sequence (SEQ ID NO 15).
Empty lines resemble motives, boxes, cis regulatory elements not found in one sequence but present in the corresponding sequence, hence, motives, boxes, cis regulatory elements that were deleted from the starting sequence or that were introduced into the permutated sequence.
Arabidopsis homeobox
Arabidopsis homeobox
tumefaciens T-DNA
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
tumefaciens T-DNA
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
tumefaciens T-DNA
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox protein
Arabidopsis homeobox protein
tumefaciens T-DNA
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
tumefaciens T-DNA
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
tumefaciens T-DNA
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox protein
Arabidopsis homeobox
Arabidopsis homeobox
tumefaciens T-DNA
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
tumefaciens T-DNA
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
tumefaciens T-DNA
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
Arabidopsis homeobox
The DNA fragments representing promoter p-GOS2_perm1 (SEQ ID NO14) and p-GOS2_perm2 (SEQ ID NO15), respectively, were generated by gene synthesis. Endonucleolytic restriction sites suitable for cloning the promoter fragments were included in the synthesis. The p-GOS2_perm1 (SEQ ID NO14) and p-GOS2_perm2 (SEQ ID NO15) promoters are cloned into destination vectors compatible with the Multisite Gateway System upstream of an attachment site and a terminator using Swa1 restriction endonuclease.
beta-Glucuronidase (GUS) or uidA gene which encodes an enzyme for which various chromogenic substrates are known, is utilized as reporter protein for determining the expression features of the permutated p-GOS2_perm (SEQ ID NO14) and p-GOS2_perm2 (SEQ ID NO15) promoter sequences.
A pENTR/A vector harboring the beta-Glucuronidase reporter gene c-GUS (with the prefix c-denoting coding sequence) is constructed using site specific recombination (BP-reaction).
By performing a site specific recombination (LR-reaction), the created pENTR/A is combined with the destination vector according to the manufacturers (Invitrogen, Carlsbad, Calif., USA) Multisite Gateway manual. The reaction yields a binary vector with the p-GOS2_perm1 promoter (SEQ ID NO14) or the p-Gos2_perm2 promoter (SEQ ID NO 15), respectively, the beta-Glucuronidase coding sequence c-GUS and a terminator.
The Agrobacterium containing the respective expression vector is used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare are dehusked. Sterilization is carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds are then germinated on a medium containing 2.4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli are excised and propagated on the same medium. After two weeks, the calli are multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces are sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
Agrobacterium strain LBA4404 containing the respective expression vector is used for co-cultivation. Agrobacterium is inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria are then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension is then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues are then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli are grown on 2.4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential is released and shoots developed in the next four to five weeks. Shoots are excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they are transferred to soil. Hardened shoots are grown under high humidity and short days in a greenhouse.
The primary transformants are transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent are kept for harvest of T1 seed. Seeds are then harvested three to five months after transplanting. The method yields single locus transformants at a rate of over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
To demonstrate and analyze the transcription regulating properties of a promoter, it is useful to operably link the promoter or its fragments to a reporter gene, which can be employed to monitor its expression both qualitatively and quantitatively. Preferably bacterial β-glucuronidase is used (Jefferson 1987). β-glucuronidase activity can be monitored in planta with chromogenic substrates such as 5-bromo-4-Chloro-3-indolyl-β-D-glucuronic acid during corresponding activity assays (Jefferson 1987). For determination of promoter activity and tissue specificity, plant tissue is dissected, stained and analyzed as described (e.g., Bäumlein 1991).
The regenerated transgenic T0 rice plants are used for reporter gene analysis.
General results for SEQ ID NO14: Medium-strong GUS expression is detected in all plant tissues analyzed.
General results for SEQ ID NO15: Medium-strong GUS expression is detected in all plant tissues analyzed.
General results for SEQ ID NO13: Medium-strong GUS expression is detected in all plant tissues analyzed.
| Number | Date | Country | Kind |
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| 10193800 | Dec 2010 | EP | regional |
This application is a National Stage application of International Application No. PCT/IB2011/055412, filed Dec. 1, 2011, which claims the benefit of U.S. Provisional Application No. 61/419,895, filed Dec. 6, 2010. This application also claims priority under 35 U.S.C. §119 to European Patent Application No. 10193800.9, filed Dec. 6, 2010.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2011/055412 | 12/1/2011 | WO | 00 | 6/4/2013 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2012/077020 | 6/14/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6927321 | Wang et al. | Aug 2005 | B2 |
| 20100199365 | Senger et al. | Aug 2010 | A1 |
| Number | Date | Country |
|---|---|---|
| 2721879 | Oct 2009 | CA |
| 1489631 | Apr 2004 | CN |
| WO 0250295 | Jun 2002 | WO |
| WO 2006131490 | Dec 2006 | WO |
| WO 2009016202 | Feb 2009 | WO |
| WO 2009117417 | Sep 2009 | WO |
| WO 2009133145 | Nov 2009 | WO |
| WO 2010127969 | Nov 2010 | WO |
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| 20130263330 A1 | Oct 2013 | US |
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| 61419895 | Dec 2010 | US |
