Nucleotide sequences regulating gene expression and constructs and methods utilizing same

Information

  • Patent Grant
  • 7695968
  • Patent Number
    7,695,968
  • Date Filed
    Thursday, March 11, 2004
    20 years ago
  • Date Issued
    Tuesday, April 13, 2010
    14 years ago
Abstract
Novel plant derived regulatory sequences and constructs and methods of using such sequences for directing expression of exogenous polynucleotide sequences in plants are provided.
Description
FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to isolated polynucleotides which are capable of regulating gene expression in an organism and more specifically, to novel nucleic acid sequences which include constitutive, inducible, tissue-specific and developmental stage-specific promoters which are capable of directing gene expression in plants.


A promoter is a nucleic acid sequence approximately 200-1500 base pairs (bp) in length which is typically located upstream of coding sequences. A promoter functions in directing transcription of an adjacent coding sequence and thus acts as a switch for gene expression in an organism. Thus, all cellular processes are ultimately governed by the activity of promoters, making such regulatory elements important research and commercial tools.


Promoters are routinely utilized for heterologous gene expression in commercial expression systems, gene therapy and a variety of research applications.


The choice of the promoter sequence determines when, where and how strongly the heterologous gene of choice is expressed. Accordingly, when a constitutive expression throughout an organism is desired, a constitutive promoter is preferably utilized. On the other hand, when triggered gene expression is desired, an inductive promoter is preferred. Likewise, when an expression is to be confined to a particular tissue, or a particular physiological or developmental stage, a tissue specific or a stage specific promoter is respectively preferred.


Constitutive promoters are active throughout the cell cycle and have been utilized to express heterologous genes in transgenic plants, such that the expression of traits encoded by the heterologous genes is effected throughout the plant at all time. Examples of known constitutive promoters often used for plant transformation include the cauliflower heat shock protein 80 (hsp80) promoter, 35S cauliflower mosaic virus promoter, nopaline synthase (nos) promoter, octopine (ocs) Agrobacterium promoter and the mannopine synthase (mas) Agrobacterium promoter.


Inducible promoters can be switched on by an inducing agent and are typically active as long as they are exposed to the inducing agent. The inducing agent can be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, or through the action of a microbial pathogen or an insecticidal pest. Accordingly, inducible promoters can be utilized to regulate expression of desired traits, such as genes that control insect pests or microbial pathogens, whereby the protein is only produced shortly upon infection or first bites of the insect and transiently so as to decrease selective pressure for resistant insects. For example, plants can be transformed to express insecticidal or fungicidal traits such as the Bacillus thuringiensis (Bt) toxins, viruses coat proteins, glucanases, chitinases or phytoalexins. In another example, plants can be transformed to tolerate herbicides by overexpressing, upon exposure to a herbicide, the acetohydroxy acid synthease enzyme, which neutralizes multiple types of herbicides [Hattori, J. et al., Mol. General. Genet. 246: 419 (1995)].


Several fruit-specific promoters have been described, including an apple-isolated Thi promoter (U.S. Pat. No. 6,392,122); a strawberry-isolated promoter (U.S. Pat. No. 6,080,914); tomato-isolated E4 and E8 promoters (U.S. Pat. No. 5,859,330); a polygalacturonase promoter (U.S. Pat. No. 4,943,674); and the 2AII tomato gene promoter [Van Haaren et al., Plant Mol. Biol. 21: 625-640 (1993)]. Such fruit specific promoters can be utilized, for example, to modify fruit ripening by regulating expression of ACC deaminase which inhibits biosynthesis of ethylene. Other gene products which may be desired to express in fruit tissue include genes encoding flavor or color traits, such as thaumatin, cyclase or sucrose phosphate synthase.


Seed specific promoters have been described in U.S. Pat. Nos. 6,403,862, 5,608,152 and 5,504,200; and in U.S. patent application Ser. Nos. 09/998,059 and 10/137,964. Such seed specific promoters can be utilized, for example, to alter the levels of saturated or unsaturated fatty acids; to increase levels of lysine- or sulfur-containing amino acids, or to modify the amount of starch contained in seeds.


Several promoters which regulate gene expression specifically during germination stage have been described, including the α-glucoronidase and the cystatin-1 barely-isolated promoters (U.S. Pat. No. 6,359,196), and the hydrolase promoter [Skriver et al., Proc. Natl. Acad. Sci. USA, 88:7266-7270 (1991)].


While reducing the present invention to practice, the present inventors have uncovered several regulatory sequences which exhibit a wide range of promoter activities in plants, as is further described hereinunder, such regulatory sequences can be used in a variety of commercial and research applications.


SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 211, 210 and 213, wherein the isolated polynucleotide is capable of regulating expression of at least one polynucleotide sequence operably linked thereto.


According to another aspect of the present invention there is provided a nucleic acid construct which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to yet another aspect of the present invention there is provided a transgenic cell which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to still another aspect of the present invention there is provided a transgenic cell comprising the nucleic acid construct which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to an additional aspect of the present invention there is provided a transgenic organism which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to yet an additional aspect of the present invention there is provided a transgenic organism comprising a nucleic acid construct which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to still an additional aspect of the present invention there is provided a transgenic plant which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to a further aspect of the present invention there is provided a transgenic plant comprising a nucleic acid construct which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41,46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to yet a further aspect of the present invention there is provided a method of producing a transgenic plant comprising transforming a plant with an isolated polynucleotide which includes a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to still a further aspect of the present invention there is provided a method of producing a transgenic plant comprising transforming a plant with a nucleic acid construct which includes the isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213.


According to still a further aspect of the present invention there is provided a method of expressing a polypeptide of interest in a cell comprising transforming the cell with a nucleic acid construct including a polynucleotide sequence encoding the polypeptide of interest operably linked to a regulatory nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213 thereby expressing the polypeptide of interest in the cell.


According to still a further aspect of the present invention there is provided a method of co-expressing two polypeptides of interest in a cell comprising transforming the cell with a nucleic acid construct including two polynucleotide sequences encoding the two polypeptides of interest operably linked to a regulatory nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202, 203, 210 and 213 such that said two polynucleotide sequences flank said regulatory nucleic acid sequence, thereby expressing the two polypeptides of interest in the cell.


According to further features in preferred embodiments of the invention described below, the isolated polynucleotide includes at least one promoter region.


According to still further features in the described preferred embodiments the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 1, 6, 41, 46, 51, 61, 86, 121, 136, 171, 181 and 202, and whereas the at least one promoter region is capable of directing transcription of said at least one polynucleotide sequence in a constitutive manner.


According to still further features in the described preferred embodiments the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 1, 11, 16, 21, 26, 31, 36, 56, 66, 71, 76, 81, 91, 96, 101, 116, 126, 141, 146, 151, 156, 161, 166, 176, 186, 191, 196, 201, 203, 210 and 213, and whereas the at least one promoter region is capable of directing transcription of said at least one polynucleotide sequence in an inductive manner.


According to still further features in the described preferred embodiments the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 1, 11, 16, 21, 26, 31, 36, 56, 61, 66, 71, 76, 91, 116, 126, 141, 146, 151, 156, 161, 166, 176, 186, 191, 196, 201, 203, 210 and 213, and whereas the at least one promoter region is capable of directing transcription of said at least one polynucleotide sequence in a tissue specific manner.


According to still further features in the described preferred embodiments the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 81, 96, 101, 106 and 131, and whereas the at least one promoter region is capable of directing transcription of said at least one polynucleotide sequence in a developmental stage specific manner.


According to still further features in the described preferred embodiments the nucleic acid construct further includes at least one heterologous polynucleotide operably linked to the isolated polynucleotide.


According to still further features in the described preferred embodiments the at least one heterologous polynucleotide is a reporter gene.


According to still further features in the described preferred embodiments the nucleic acid construct further includes two heterologous polynucleotides each being operably linked to an end of the isolated polynucleotide such that the two heterologous polynucleotides flank the isolated polynucleotide.


The present invention successfully addresses the shortcomings of the presently known configurations by providing a plurality of isolated polynucleotide sequences which exhibit a wide spectrum of promoter function patterns. These polynucleotides can be used to generate nucleic acid constructs, such as expression vectors suitable for transforming an organism. Such nucleic acid constructs can be used to promote expression of desired traits or expression products in transgenic organisms, such as plants, in a constitutive, induced, tissue specific, or a developmental stage specific manner.





BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.


In the drawings:



FIGS. 1
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 11 operably linked to a luciferase encoding sequence. FIG. 1a shows the transgenic plant under normal light. FIG. 1b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression luciferase in flower tissue.



FIGS. 2
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 21 operably linked to a luciferase encoding sequence. FIG. 2a shows the transgenic plant under normal light. FIG. 2b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in root tissue.



FIGS. 3
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 36 operably linked to a luciferase encoding sequence. FIG. 3a shows the transgenic plant under normal light. FIG. 3b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in root and flower tissue.



FIGS. 4
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 61 operably linked to a luciferase encoding sequence. FIG. 4a shows the transgenic plant under normal light. FIG. 4b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in young tissue.



FIGS. 5
a-b are photographs showing an Arabidopsis thaliana seedling transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 66 operably linked to a luciferase encoding sequence. FIG. 5a shows the transgenic plant under normal light. FIG. 5b is an ultra-low light photograph of the same plant in the dark, illustrating an expression of luciferase in leaf tissue.



FIGS. 6
a-b are photographs showing an Arabidopsis thaliana mature plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 66 operably linked to a luciferase encoding sequence. FIG. 6a shows the transgenic plant under normal light. FIG. 6b is an ultra-low light photograph of the same plant in the dark, illustrating an expression of luciferase in stem tissue.



FIGS. 7
a-b are photographs showing an Arabidopsis thaliana plant seedlings transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 81 operably linked to a luciferase encoding sequence. FIG. 7a shows the transgenic plant under normal light. FIG. 7b is an ultra-low light photograph of the same plant in the dark, illustrating an expression of luciferase in above ground tissue.



FIGS. 8
a-b are photographs showing an Arabidopsis thaliana mature plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 81 operably linked to a luciferase encoding sequence. FIG. 8a shows the transgenic plant under normal light. FIG. 8b is an ultra-low light photograph of the same plant in the dark, illustrating an expression of luciferase in flower tissue.



FIGS. 9
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 91 operably linked to a luciferase encoding sequence. FIG. 9a shows the transgenic plant under normal light. FIG. 9b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in root and flower tissue.



FIGS. 10
a-b are photographs showing an Arabidopsis thaliana seedling transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 96 operably linked to a luciferase encoding sequence. FIG. 10a shows the transgenic plant under normal light. FIG. 10b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in above ground tissue.



FIGS. 11
a-b are photographs showing an Arabidopsis thaliana mature plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 96 operably linked to a luciferase encoding sequence. FIG. 11a shows the transgenic plant under normal light. FIG. 11b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in above ground tissue.



FIGS. 12
a-b are photographs showing seeds of an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 111 operably linked to a luciferase encoding sequence. FIG. 12a shows the seeds under normal light. FIG. 12b is an ultra-low light photograph of the same seeds in the dark, illustrating a specific expression of luciferase in seeds.



FIGS. 13
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 111 operably linked to a luciferase encoding sequence. FIG. 13a shows the transgenic plant under normal light. FIG. 13b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in roots.



FIGS. 14
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 121 operably linked to a luciferase encoding sequence. FIG. 14a shows the transgenic plant under normal light. FIG. 14b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in meristematic tissue.



FIGS. 15
a-b are photographs showing an Arabidopsis thaliana seedling transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 126 operably linked to a luciferase encoding sequence. FIG. 15a shows the transgenic plant under normal light. FIG. 15b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in root meristematic tissue.



FIGS. 16
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 126 operably linked to a luciferase encoding sequence. FIG. 16a shows the transgenic plant under normal light. FIG. 16b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in flower meristematic tissue.



FIGS. 17
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 131 operably linked to a luciferase encoding sequence. FIG. 17a shows the transgenic plant under normal light. FIG. 17b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in leaf tissue.



FIGS. 18
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 136 operably linked to a luciferase encoding sequence. FIG. 18a shows the transgenic plant under normal light. FIG. 18b is an ultra-low light photograph of the same plant in the dark, illustrating a non-specific constitutive expression of luciferase.



FIGS. 19
a-b are photographs showing an Arabidopsis thaliana seedling transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 156 operably linked to a luciferase encoding sequence. FIG. 19a shows the transgenic plant under normal light. FIG. 19b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in above ground tissue.



FIGS. 20
a-b are photographs showing an Arabidopsis thaliana mature plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 156 operably linked to a luciferase encoding sequence. FIG. 20a shows the transgenic plant under normal light. FIG. 20b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in above ground tissue.



FIGS. 21
a-b are photographs showing seeds of an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 161 operably linked to a luciferase encoding sequence. FIG. 21a shows the seeds under normal light. FIG. 21b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in seed tissue.



FIGS. 22
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 186 operably linked to a luciferase encoding sequence. FIG. 22a shows the transgenic plant under normal light. FIG. 22b is an ultra-low light photograph of the same plant in the dark, illustrating an expression of luciferase in stalk and stem tissue.



FIGS. 23
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 191 operably linked to a luciferase encoding sequence. FIG. 23a shows the transgenic plant under normal light. FIG. 23b is an ultra-low light photograph of the same plant in the dark, illustrating a weak expression of luciferase in vegetative tissue.



FIGS. 24
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 201 operably linked to a luciferase encoding sequence. FIG. 24a shows the transgenic plant under normal light. FIG. 24b is an ultra-low light photograph of the same plant in the dark, illustrating an above ground tissue specific expression of luciferase.



FIGS. 25
a-b are photographs showing an Arabidopsis thaliana plant transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 176 operably linked to a luciferase encoding sequence. FIG. 25a shows the transgenic plant under normal light. FIG. 25b is an ultra-low light photograph of the same plant in the dark, illustrating a specific expression of luciferase in flower tissue.



FIGS. 26
a-b are photographs showing transformed Arabidopsis thaliana plants transformed with nucleic acid constructs including partial DREs operably each linked to a GUS encoding sequence. FIG. 26a shows a plant transformed with a nucleic acid construct including the nucleic acid sequence set forth in SEQ ID NO: 210 operably linked to a GUS encoding sequence. FIG. 26b shows root tips of a plant, transformed with a nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 213 operably linked to a GUS encoding sequence.



FIG. 27 is a nucleic acid sequence alignment between DRE 6669 (SEQ ID NO: 61, QUERY) and a prior art sequence (SEQ ID NO: 214, SBJCT), revealing a different 5′ sequence which is important for constitutive expression, as is exemplified in the Examples section hereinbelow.





DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated polynucleotides capable of regulating the expression of operably linked heterologous polynucleotides, and more specifically, novel nucleic acid sequences which are capable of promoting gene expression in a constitutive, inductive, tissue specific and/or developmental stage specific manner. The present invention also provides nucleic acid constructs, as well transgenic organisms which carry the polynucleotides of the present invention and methods of producing thereof.


The principles and operation of the present invention may be better understood with reference to the accompanying descriptions.


Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following descriptions or illustrated in the Examples section. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.


The term “polynucleotide” or the phrase “nucleic acid sequence” are used herein interchangeably and refer to a polymer of deoxyrebonucleotide (DNA) or ribonucleotide (RNA).


The phrase “heterologous polynucleotide” refers to a polynucleotide sequence which originates from a heterologous organism or to a polynucleotide sequence which is linked to a regulatory sequence of the same organism which does not normally regulate expression of the polynucleotide sequence in the organism.


PCT Publication WO 02/07989 describes a unique approach developed by the present inventors in order to uncover novel regulatory sequences in organisms such as plants. This approach combines molecular and bioinformatics techniques for high throughput isolation of DNA regulating elements (DREs), located within the non-transcribed (non-coding) regions of the genome and which include, for example, promoters, enhancers, suppressors, silencers, locus control regions and the like.


Utilizing this approach, the present inventors have uncovered several novel polynucleotide sequences which, as illustrated in the Examples section which follows, exhibit regulatory activity in plants.


Thus, according to one aspect of the present invention, there is provided isolated polynucleotides which are capable of regulating the expression of at least one polynucleotide operably linked thereto. As is further described in the Examples section which follows, these isolated polynucleotides are as set forth in SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202 and 203, or fragments (e.g., SEQ ID NOS: 210 and 213), variants or derivatives thereof.


A coding nucleic acid sequence is “operably linked” to a regulatory sequence if it is capable of exerting a regulatory effect on the coding sequence linked thereto. Preferably, the regulatory sequence is positioned 1-500 bp upstream of the ATG codon of the coding nucleic acid sequence, although it will be appreciated that regulatory sequences can also exert their effect when positioned elsewhere with respect to the coding nucleic acid sequence (e.g., within an intron).


As is clearly illustrated in the Examples section which follows, the isolated polynucleotides of the present invention are capable of regulating expression of a coding nucleic acid sequence (e.g., luciferase) operably linked thereto (see FIGS. 1-25).


The isolated polynucleotides of the present invention range in length from 174 to 3, 348 nucleotides and include one or more sequence regions which are capable of recognizing and binding RNA polymerase II and other proteins (trans-acting transcription factors) involved in transcription.


Although most of the isolated polynucleotides described herein include one promoter region, some include two distinct promoter regions each positioned on a different strand of the same genomic sequence. Such bidirectional DREs are further described in the Examples section which follows (see for example, Tables 3-17).


As is further illustrated by the Examples section which follows, the isolated polynucleotides of the present invention exhibit a range of activities and tissue specificities.


Thus for example, the nucleic acid sequences set forth in SEQ ID NOS: 1, 6, 41, 46, 51, 61, 86, 121, 136, 171, 181 and 202 or fragment, variants or derivatives thereof, are capable of directing transcription of coding nucleic acid sequences operably linked thereto in a constitutive manner and thus include a constitutive promoter region.


In another example, the nucleic acid sequences set forth in SEQ ID NOS: 1, 11, 16, 21, 26, 31, 36, 56, 66, 71, 76, 81, 91, 96, 101, 116, 126, 141, 146, 151, 156, 161, 166, 176, 186, 191, 196, 201 and 203, or fragments (e.g., SEQ ID NOS: 210 and 213), variants or derivatives thereof, are capable of directing transcription of coding nucleic acid sequences operably linked thereto in an inductive manner and thus include an inductive promoter region.


In yet another example, the nucleic acid sequences set forth in SEQ ID NOS: 1, 11, 16, 21, 26, 31, 36, 56, 61, 66, 71, 76, 91, 116, 126, 141, 146, 151, 156, 161, 166, 176, 186, 191, 196, 201 and 203, or fragments (e.g., SEQ ID NOS: 210 and 213), variants or derivatives thereof, are capable of directing transcription of coding nucleic acid sequences operably linked thereto in a tissue specific manner and thus include a tissue specific promoter region.


In further yet another example, the nucleic acid sequences set forth in SEQ ID NOS: 81, 96, 101, 106 and 131, or fragment, variants or derivatives thereof, are capable of directing transcription of coding nucleic acid sequences operably linked thereto in a developmental stage specific manner and thus include a developmental stage specific promoter region.


Preferably, the polynucleotide of the present invention are modified to create variations in the molecule sequences such as to enhance their promoting activities, using methods known in the art, such as PCR-based DNA modification, or standard DNA mutagenesis techniques, or by chemically synthesizing the modified polynucleotides.


Accordingly, the sequences set forth in SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 11, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202 and 203 may be truncated or deleted and still retain the capacity of directing the transcription of an operably linked DNA sequence (e.g., SEQ ID NOS: 210 and 213). The minimal length of a promoter region can be determined by systematically removing sequences from the 5′ and 3′-ends of the isolated polynucleotide by standard techniques known in the art, including but not limited to removal of restriction enzyme fragments or digestion with nucleases. Consequently, any sequence fragments, portions, or regions of the disclosed polypeptide sequences of the present invention can be used as regulatory sequences. It will be appreciated that modified sequences (mutated, truncated and the like) can acquire different transcriptional properties such as the direction of different pattern of gene expression as compared to the unmodified element (e.g., SEQ ID NO: 61 as compared to SEQ ID NO: 213, see the Examples section which follows).


Optionally, the sequences set forth in SEQ ID NOS: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 202 and 203 may be modified, for example for expression in a range of plant systems. In another approach, novel hybrid promoters can be designed or engineered by a number of methods. Many promoters contain upstream sequences which activate, enhance or define the strength and/or specificity of the promoter, such as described, for example, by Atchison [Ann. Rev. Cell Biol. 4:127 (1988)]. T-DNA genes, for example contain “TATA” boxes defining the site of transcription initiation and other upstream elements located upstream of the transcription initiation site modulate transcription levels [Gelvin In: Transgenic Plants (Kung, S.-D. and Us,R., eds, San Diego: Academic Press, pp.49-87, (1988)]. Another chimeric promoter combined a trimer of the octopine synthase (ocs) activator to the mannopine synthase (mas) activator plus promoter and reported an increase in expression of a reporter gene [Mm Ni et al., The Plant Journal 7:661 (1995)]. The upstream regulatory sequences of the polynucleotide sequences of present invention can be used for the construction of such chimeric or hybrid promoters. Methods for construction of variant promoters include, but are not limited to, combining control elements of different promoters or duplicating portions or regions of a promoter (see for example, U.S. Pat. Nos. 5,110,732 and 5,097,025). Those of skill in the art are familiar with the specific conditions and procedures for the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.), generation of recombinant organisms and the screening and isolation of genes, [see for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, (1989); Mailga ei al., Methods in Plant Molecular Biology, Cold Spring Harbor Press, (1995); Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997); volume 2, Detecting Genes, (1998); volume 3, Cloning Systems, (1999); and volume 4, Mapping Genomes, (1999), Cold Spring Harbor, N.Y].


The polynucleotides of the present invention, or fragment, variants or derivatives thereof, can be incorporated into nucleic acid constructs, preferably expression constructs (i.e., expression vectors) which can be introduced and replicate in a host cell.


Thus, according to another aspect of the present invention there is a provided a nucleic acid construct which includes at least one of the polynucleotides of the present invention, or fragments, variants or derivatives thereof.


Preferably, the nucleic acid construct of the present invention includes at least one operably linked heterologous polynucleotide. More preferably, at least one operably linked reporter gene.


The phrase “reporter gene” used herein refers to a gene encoding a selectable, screenable or detectable phenotype.


Reporter genes which may be utilized in the present invention may include, but not limited to, LUX or LUC coding for luciferase, GUS coding for β-glucoronidase, GFP coding for green-fluorescent protein, or antibiotic or herbicide tolerance genes. A general review of suitable markers is found in Wilmink and Dons, Plant Mol. Biol. Reprt. 11:165-185 (1993).


Further preferably, the nucleic acid construct of the present invention includes at least one heterologous polynucleotide encoding a desirable trait or an expression product.


A desirable trait which may be utilized in this invention may include, but not limited to, any phenotype associated with organism's morphology, physiology, growth and development, yield, produce quality, nutritional enhancement, disease or pest resistance, or stress tolerance.


Alternatively, the heterologous polynucleotide can encode any naturally occurring or man-made recombinant protein, such as pharmaceutical proteins [e.g., growth factors and antibodies Schillberg Naturwissenschaften. (2003) April; 90(4):145-55] and food additives. It will be appreciated that molecular farming is a well-proven way of producing a range of recombinant proteins, as described in details in Ma Nat Rev Genet. 2003 Oct.; 4(10):794-805; Twyman Trends Biotechnol. 2003 Dec.; 21(12):570-8.


An expression product which may be utilized in this invention may include, but not limited to, pharmaceutical polypeptides, industrial enzymes, oils, dyes, flavors, biofuels, or industrial biopolymers.


In cases of bidirectional DREs, the nucleic acid construct of this invention may include two heterologous polynucleotides each being operably linked to an end of the isolated polynucleotide of this invention, such that the two heterologous polynucleotides flank the isolated polynucleotide of this invention.


The nucleic acid construct can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome. Preferably, the nucleic acid construct of the present invention is a plasmid vector, more preferably a binary vector.


The phrase “binary vector” refers to an expression vector which carries a modified T-region from Ti plasmid, enable to be multiplied both in E. coli and in Agrobacterium cells, and usually comprising reporter gene(s) for plant transformation between the two boarder regions. A binary vector suitable for the present invention includes pBI2113, pBI121, pGA482, pGAH, pBIG, pBI101 (Clonetech), or a modification thereof such as pVER1 which is a modified pBI101 plasmid, where the GUS gene was replaced by the LucII gene from pGL3-Basic (Promega).


The nucleic acid construct of the present invention can be utilized to transform a host cell. Thus, according to another aspect of the present invention there is provided a transgenic cell, a transgenic organism or a transgenic plant which is transformed with an isolated polynucleotide of the present invention. Preferably the transgenic cell, the transgenic organism or the transgenic plant is transformed with the nucleic acid construct of the present invention.


As used herein, the terms “transgenic” or “transformed” are used interchangeably referring to a cell or an organism into which cloned genetic material has been transferred.


Methods of introducing nucleic acid constructs into a cell, an organism or a plant are well known in the art. Accordingly, suitable methods for introducing nucleic acid sequences into plants include, but are not limited to, bacterial infection, direct delivery of DNA (e.g., via PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and acceleration of DNA coated particles, such as described by Potrykus Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225 (1991).


Methods for specifically transforming dicots primarily use Agrobacterium tumefaciens. For example, transgenic plants reported include but are not limited to cotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908; and WO 97/43430), soybean [U.S. Pat. Nos. 5,569,834, 5,416,011; McCabe et al., Bio/Technology, 6:923 (1988); and Christou et al., Plant Physiol., 87:671, (1988)]; Brassica (U.S. Pat. No. 5,463,174), and peanut [Cheng et al., Plant Cell Rep., 15: 653, (1996)].


Similar methods have been reported in the transformation of monocots. Transformation and plant regeneration using these methods have been described for a number of crops including but not limited to asparagus [Asparagus officinalis; Bytebier et al., Proc. Natl. Acad. Sci. U.S.A., 84: 5345, (1987); barley (Hordeum vulgarae; Wan and Lemaux, Plant Physiol., 104: 37, (1994)]; maize [Zea mays; Rhodes, C. A., et al., Science, 240: 204, (1988); Gordon-Kamm, et al., Plant Cell, 2: 603, (1990); Fromm, et al., Bio/Technology, 8: 833, (1990); Koziel, et al., Bio/Technology, 11: 194, (1993)]; oats [Avena sativa; Somers, et al., Bio/Technology, 10: 1589, (1992)]; orchardgrass [Dactylis glomerata; Horn, et al., Plant Cell Rep., 7: 469, (1988); rice [Oryza sativa, including indica and japonica varieties, Toriyama, et al., Bio/Technology, 6: 10, (1988); Zhang, et al., Plant Cell Rep., 7: 379, (1988); Luo and Wu, Plant Mol. Biol. Rep., 6: 165, (1988); Zhang and Wu, Theor. Appl. Genet., 76: 835, (1988); Christou, et al., Bio/Technology, 9: 957, (1991); sorghum [Sorghum bicolor; Casas, A. M., et al., Proc. Natl. Acad. Sci. U.S.A., 90: 11212, (1993)]; sugar cane [Saccharum spp.; Bower and Birch, Plant J., 2: 409, (1992)]; tall fescue [Festuca arundinacea; Wang, Z. Y. et al., Bio/Technology, 10: 691, (1992)]; turfgrass [Agrostis palustris; Zhong et al., Plant Cell Rep., 13: 1, (1993)]; wheat [Triticum aestivum; Vasil et al., Bio/Technology, 10: 667, (1992); Weeks T., et al., Plant Physiol., 102: 1077, (1993); Becker, et al., Plant, J. 5: 299, (1994)], and alfalfa [Masoud, S. A., et al., Transgen. Res., 5: 313, (1996)]. It is apparent to those of skill in the art that a number of transformation methodologies can be used and modified for production of stable transgenic plants from any number of target crops of interest.


The transformed plants can be analyzed for the expression features conferred by the polynucleotides of the present invention, using methods known in the art for the analysis of transformed plants. A variety of methods are used to assess gene expression and determine if the introduced gene(s) is integrated, functioning properly, and inherited as expected. Preferably, the promoters can are evaluated by determining the expression levels and the expression features of genes to which the promoters are operatively linked. A preliminary assessment of promoter function can be determined by a transient assay method using reporter genes, but a more definitive promoter assessment can be determined from the analysis of stable plants. Methods for plant analysis include but are not limited to Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field evaluations, and immunodiagnostic assays.


Preferably, the capacity of isolated polynucleotides of this invention to promote gene expression in plants is evaluated according to phenotypic expression of reporter genes using procedures as described in the Examples section that follows. Briefly, the expression of luciferase in transgenic Arabidopsis is determined and consistently classified by quantitatively scoring certain features of expression, such as the intensity, specificity, development stage and positioning of expression. Accordingly, a luciferase gene that is expressed in a constitutive manner would indicate a putative constitutive promoter activity of the isolated polynucleotide. Likewise, a luciferase gene that is expressed in an inductive, tissue specific or a development-stage specific manner, would respectively indicate a putative inductive, a tissue specific or a stage specific promoter activity.


Hence, the present invention provides a plurality of isolated polynucleotide sequences which exhibit a wide spectrum of promoter function patterns. These polynucleotides can be used to generate nucleic acid constructs, such as expression vectors suitable for transforming an organism. Such nucleic acid constructs can be used to promote expression of desired traits or expression products in transgenic organisms, such as plants, in a constitutive, induced, tissue specific, or a developmental stage specific manner.


Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.


EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.


Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” RL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.


Identification, Isolation and Characterization of DNA Regulating elements (DREs)

Novel DREs were identified by luciferase expression assay driven by bioinformatically identified DNA fragments from Arabidopsis thaliana genomic DNA. Positive DREs were fused upstream a reporter gene in a vector which was used to transform Arabidopsis thaliana plants. The reporter gene expression driven by these DREs was characterized.


Materials and Experimental Methods

Isolation of DREs: A high throughput method of cloning DNA regulating elements (DREs) using a single reaction tube, referred to herein as the “one-tube” method, was utilized in order to enable large scale production of DRE transformed plants. Accordingly, genomic DNA (gDNA) was extracted from leaves of Arabidopsis thaliana Col1 using DNAeasy Plant Mini Kit (Qiagen, Germany). Primers for PCR amplification of DREs were designed using PRIMER3© software and modified to contain restriction sites absent from the DRE sequence, for PCR product insertion into pVER1 binary plasmid, which is a pBI101 (clontech) modified plasmid, where the GUS reporter gene was replaced by LucII gene from pGL3-Basic (promega). Briefly, GUS gene was cut out of pBI101 using the blunt restriction enzymes Ecl136II and SmaI. The pGL-Basic plasmid [after eliminating the HindIII and BamHI sites, by digestion, fill-in using klenow fragment (Roche) and self ligating the plasmid, using T4 DNA ligase (Roche)] was cut SacI and XbaI and the LucII gene insert was inserted into pBluescript, digested with the same enzymes. The new plasmid was digested SmaI, as a result a blunt ends LucII gene was cut out. The LucII gene was inserted into The pBI plasmid instead of the GUS gene. To eliminate all possible read-through of the Nos-promoter, which regulates Kanamycin resistance gene on pBI101, a poly-A signal was added between the Nos-terminator and the LucII gene. Poly-A signal was amplified from pGL3-Basic using proof reading Taq polymerase PFU (Promega) and using primers 5′-aggtacttggagcggccgca-3′ and 5′-tagagaaatgttctggcacctg-3′. The Product was inserted into HindIII site on pVerI after filling the overhang 5′ ends, using Klenow fragment (Roche).


Polymerase chain reaction analyses were performed using Taq Expand Long Template PCR kit (Roche), according to the manufacturer's instructions, using as thermal cycle: 92° C./2 min→10×[94° C./10 min→55° C./30 sec→68° C./5 min]→18×[94° C./10 min 55° C./30 sec→68° C./5 min (+20 sec each cycle)]→68° C./7 min. PCR products were double-digested with restriction endonucleases according to the protocols described in Table 1.









TABLE 1







DRE double digestion protocols


















Digest
Heat


Digest
Heat


Enzyme
First
Buffer
time
inactivation
Second

time
inactivation


combination
digest
(Roche)
(min)
conditions
digest
Buffer
(min)
conditions





HindIII, SalI
HindIII
M
90
20 min,
SalI
M + NaCl + Tris
60
20 min,






70° C.



70° C.


HindIII,
HindIII
B
30
No
BamHI
B
60
20 min,


BamHI







70° C.


SalI, BamHI
BamHI
M
60
20 min,
SalI
M + NaCl + Tris
60
20 min,






80° C.



70° C.


HindIII,
HindIII
B
30
No
EcoRV
B
60
20 min,


EcoRV







70° C.


SalI, ScaI
SalI, ScaI
H
60
20 min,






80° C.


BamHI, SmaI
SmaI
A
60
20 min,
BamHI
A
60
20 min,





(30° C.)
70° C.



80° C.


SalI, PvuII
PvuII
M
60
20 min,
SalI
M + NaCl + Tris
60
20 min






80° C.


HindIII,
HindIII
M
30
No
PvuII
M
60
20 min,


PvuII







80° C.


HindIII, StuI
HindIII,
B
90
20 min,



StuI


80° C.


BamHI, StuI
StuI
B
30
No
BamHI
B
60
20 min,










80° C.









Cloning of DREs in luciferase reporter gene expression: PCR amplified DREs were cloned into a luciferase reporter gene expression vector pVER1, derived from the binary vector pBI101 (Clontech), was double-digested using the same restriction endonucleases used to excise cloned DREs from vector, purified using PCR Purification Kit (Qiagen, Germany), treated with alkaline-phophatase (Roche) according to the manufacturer's instructions and re-purified using PCR Purification Kit (Qiagen, Germany). Insertion of DRE into vector pVER1 was performed by adding to DRE digests: 500 ng of double digested pVer1 plasmid, 1 μl of T4 DNA ligase (40 U/μl; Roche) and 6 μl of T4 buffer (Roche). Following overnight incubation of ligation mixes at 4° C., Agrobacterium tumefaciens GV303 competent cells were transformed using 1-2 μl of ligation reaction mixture by electroporation, using a MicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program (Biorad). Agrobacterium cells were grown on LB at 28° C. for 3 h and plated on LB-agar plates supplemented with the antibiotics gentamycin 50 mg/L (Sigma) and kanamycin 50 mg/L (Sigma). Plates were then incubated at 28° C. for 48 h. Cloned DREs were identified by PCR analysis of bacterial colony DNA using the vector specific, insert flanking upstream and downstream primers 5′-AGGTACTTGGAGCGGCCGCA-3′ and 5′-CGAACACCACGGTAGGCTG-3′, respectively and the thermal cycle: 92° C./3 min→31×[94° C./30 sec→54° C./30 sec→72° C./X min (X=length (kb) of longest PCR product expected)] 72° C./10 min. Positive Agrobacterium colonies were subsequently used for Arabidopsis plant transformation.


Plant transformation and cultivation: Arabidopsis thaliana Columbia (T0 plants) were transformed using the Floral Dip procedure described by Clough S J and Bent A F [The Plant J. 16:735-743 (1998)] and by Desfeux et al. [Plant Physiology 123:895-904 (2000)] with minor modifications. Briefly, T0 Plants were sown in 250 ml pots filled with wet peat-based growth mix. The pots were covered with aluminum foil and a plastic dome, kept at 4° C. for 3-4 days, then uncovered and incubated in a growth chamber at 18-24° C. under 16/8 hr light/dark cycle. The T0 plants were ready for transformation six days before anthesis.


Single colonies of Agrobacterium carrying plant DREs were cultured in LB medium supplemented with kanamycin (50 mg/L) and gentamycin (50 mg/L). The cultures were incubated at 28° C. for 48 hours under vigorous shaking and centrifuged at 4000 rpm for 5 minutes. The pellets comprising Agrobacterium cells were resuspended in a transformation medium which contained half-strength (2.15 g/L) Murashig-Skoog (Duchefa); 0.044 μM benzylamino purine (Sigma); 112 μg/L B5 Gambourg vitamins (Sigma); 5% sucrose; and 0.2 ml/L Silwet L-77 (OSI Specialists, CT) in double-distilled water, at pH of 5.7.


Transformation of T0 plants was effected by inverting each plant into an Agrobacterium suspension such that the above ground plant tissue was submerged for 3-5 seconds. Each inoculated T0 plant was immediately placed in a plastic tray, then covered with clear plastic dome to maintain humidity and kept in the dark at room temperature for eighteen hours to facilitate infection and transformation. Transformed (transgenic) plants were then uncovered and transferred to a greenhouse for recovery and maturation. The transgenic T0 plants were grown in the greenhouse for 3-5 weeks until siliques were brown and dry then seeds were harvested from plants and kept at room temperature until sowing


Generating T1 and T2 transgenic plants harboring DREs: Seeds collected from transgenic T0 plants were surface-sterilized by soaking in 70% ethanol for 1 minute, followed by soaking in 5% sodium hypochloride and 0.05% triton for 5 minutes. The surface-sterilized seeds were thoroughly washed in sterile distilled water then placed on culture plates containing half-strength Murashig-Skoog (Duchefa); 2% sucrose; 0.8% plant agar; 50 mM kanamycin; and 200 mM carbenicylin (Duchefa). The culture plates were incubated at 4° C. for 48 hours then transferred to a growth room at 25° C. for an additional week of incubation. Vital T1 Arabidopsis plants were transferred to a fresh culture plates for another week of incubation. Following incubation the T1 plants were removed from culture plates and planted in growth mix contained in 250 ml pots. The transgenic were allowed to grow in a greenhouse to maturity. Seeds harvested from T1 plants were cultured and grown to maturity as T2 plants under the same conditions as used for culturing and growing the T1 plants.


Evaluating DRE gene-promoting activity in transgenic plants: The ability of DREs to promote gene expression in plants was determined based on the expression of luciferase reporter gene. Accordingly, transgenic Arabidopsis plantlets at a development stage of 2-3 true leaves were subjected to luminescence assays using the procedure described by Messinner R. [Plant. J. 22:265 (2000)]. The imaging of luciferase was performed in a darkroom using ultra-low light detection camera (Princeton Instruments Inc., USA). Using the procedure described by Messinner R. [Plant. J. 22:265 (2000)].


Scoring promoter activity in transgenic plants: DREs promoting gene expression was characterized based luciferase expression in transgenic plants using quantitative values such as to enable consistent evaluations of a large volume of transgenic plants, as follows:


Scoring distribution and intensity of expression: The distribution of reporter genes' expression in transgenic plants was presented in a three variables functions, as follows: (i) plant ID (X axis), (ii) plant organ (Y axis), and (iii) development stage (Z axis). The intensity of expression, relevant to any of these three variables, was measured by a distribution function value (DF), referred hereinbelow as fx,y,z (Promoter). The DF received a value ranging from 0 to 5, representing no expression and the highest expression intensity, respectively.


Scoring specificity of expression: The specificity of reporter genes' expression in transgenic plants was calculated by summing two independent addends:


(a) the zero value/nonzero values ratio, as described in table 2 below and which further referred to as the Binary Function B( ); and (b) the variance of the nonzero values only.












TABLE 2









No. of non zero
No. of zero values














values
0
1
2
3
4


















0

0
0
0
0



1
0
0.7
1.5
2



2
0
0.6
1



3
0
0.5



4
0










The Organ Specificity expression value (SpOr) was calculated according to the following equation:

SpOr(promoter)=Vary(Avx,z(fx,y,z(promoter))|y>0)+B(Avx,z(fx,y,z(promoter)))

Whereas Var is the variance, Av is the average and B is the Binary Function.


The development Stage Specificity expression value (SpDs) was calculated according to the following equation:

SpDS(promoter)=Varz(Avx,y(fx,y,z(promoter))|z>0)+B(Avx,y(fx,y,z(promoter)))

Whereas Var is the variance, Av is the average and B is the Binary Function.


Scoring position effect: Similarly to the Binary Factor approach described above, position values were also classified as either zero or nonzero values. Accordingly, the reporter genes' expression in a given organ in a given development stage was measured by a Local Position Effect value (LoPoEf). The Position Effect value (PoEf) was the average of all the Local Position Effects, calculate in three steps as follows:








1
)








h

x
,
y
,
z




(
promoter
)



=



{




0








f

x
,
y
,
z




(
promoter
)


=
0






1






f

x
,
y
,
z




(
promoter
)


=
1

,
2
,
3
,
4
,
5









2

)






LoPoEf






(

promoter
,
organ
,
development_stage

)


=
min












(






no_of

_

0

s_in


(


h

x
,

y
=
Y

,

z
=
Z





(
promoter
)


)



no_of

_non

_

0

s_in


(


h

x
,

y
=
Y

,

z
=
Z





(
promoter
)


)



,







no_of

_non

_

0

s_in


(


h

x
,

y
=
Y

,

z
=
Z





(
promoter
)


)



no_of

_

0

s_in


(


h

x
,

y
=
Y

,

z
=
Z





(
promoter
)


)






)






3) PoEf(promoter)=Av(LoPoEf(promoter,Y,Z)).


Scoring expression level: The average expression level value (ExLe) and the ExLe variance (VrExLe) were calculated per each DRE promoter×plant organ×plant development stage combination, according the following equations:

ExLe(promoter,organ,development_stage)=Avx(fx,y,z(promoter))
VrExLe(promoter,organ,development_stage)=varx(fx,y,z(promoter))


Scoring evaluation reliability: The General Reliability value (Grel) was the number of independent plants that were used for evaluating a specific DRE promoter activity. Hence, GRel(promoter)=Countx(fx,y,z(promoter)). The Development Stage Reliability value (Rel(DS)) was the number of independent plants that were used for evaluating a specific DRE promoter activity in any given plant developing stage. Rel(promoter, development_stage)=Countx|z=developmentstage(fx,y,z(promoter)).


Creation of partial fragments from vDREs 4209 and 6669: Genomic DNA derived from Arabidopsis thaliana var Col0 was extracted and PCR-amplified using oligonucleotide primers complementary to sequences within vDRE 4209 (SEQ ID NO:36) [sense primer 5′-GTTGGTTCGTCGACTAGAGAAGGT-3′ (SEQ ID NO: 208), antisense primer 5′-TTGGATCCGGGAGGCAATGATGCTTTAG-3′ (SEQ ID NO: 209)], and vDRE 6669 (SEQ ID NO:61) [sense primer 5′-TTGTAAGCTTGCAGGGATACGGATGGGTAG-3′ (SEQ ID NO: 211), antisense primer 5′-AAATATTGGATCCTTTGGGGTTCTC-3′ (SEQ ID NO: 212)].


The above PCR amplifications resulted in a 470 bp fragment, containing bp 76-548 of the original vDRE 4209 (SEQ ID NO:210) and a 1569 bp fragment, containing bp 748-2316 of the original vDRE 6669 (SEQ ID NO:213), respectively.


PCR products were digested with HindIII and BamHI and ligated into the binary vector, pBI121 (Clontech, accession number: AF485783) upstream to the GUS gene, generating plasmids p4209short-GUS, and p6669short-GUS, respectively. Arabidopsis plants (var col0) were transformed with the binary constructs generated (p4209short-GUS and p6669short-GUS), and GUS activity was analyzed on 10 independent T1 transformed plants using standard GUS staining protocol [Jefferson R A, Kavanagh T A, Bevan M W. 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6(13): 3901-7]. Genomic DNA extraction, PCR amplification, DNA restriction, ligation and transformation of Arabidopsis plant were preformed according to the protocols described above.


Experimental Results

Characterization of DREs:


Various features of the isolated DREs of the present invention are described in Tables 3-17 which follow. As is clearly evident from the Table provided data, the DREs of the present invention exhibit a wide range of gene-promoting activities including: constitutive, inductive, tissue specific, and stage specific activities.












TABLE 3







DRE number1
1345
1495
2176


Cluster reference2
Z18125
Z17428
ATBIBBI


Cluster position2
Upstream
Upstream
Upstream


DRE regulatory direction3
Bidirectional
Unidirectional
Unidirectional


DRE length (bp)
1611
901
2192


DRE sequence
SEQ ID NO: 1
SEQ ID NO: 6
SEQ ID NO: 11


Internal forward primer
SEQ ID NO: 2
SEQ ID NO: 7
SEQ ID NO: 12


sequence4


External forward primer
SEQ ID NO: 3
SEQ ID NO: 8
SEQ ID NO: 13


sequence4


Internal reverse primer
SEQ ID NO: 4
SEQ ID NO: 9
SEQ ID NO: 14


sequence4


External reverse primer
SEQ ID NO: 5
SEQ ID NO: 10
SEQ ID NO: 15


sequence4


Position Effect Value5
0.37
0.21
8.33


Development Stage
1.09
0.32
0.62


Specificity Value5


Organ Specificity Value5
1.56
0.38
2.60


Number of transgenic
11
10
7


plants


Young roots score (No.,
10, 12, 3.36
6, 2.333, 1.555
4, 0, 0


Ave., Var)6


Mature roots score (No.,
7, 1.571, 3.387
7, 3, 2.285
5, 0, 0


Ave., Var)6


Young above-ground
10, 3.3, 2.21
6, 4.16, 0.13
4, 0, 0


Tissue (No., Ave., Var)6


Mature above-ground
7, 3, 2
7, 3.28, 1.06
5, 0, 0


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
3, 4.33, 0.88
3, 2, 2
3, 1.67, 5.56


Var)6


Flowers (No., Ave., Var)6
7, 1.42, 3.10
7, 3.14, 4.40
5, 4.2, 2.56


Description
Constitutive.
Constitutive.
Specific to flower tissue.



Strong in seeds.

Strong in flower buds.





Lower expression in open





flowers.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 4







DRE number1
2524
3560
3583


Cluster reference2
Z17778
Z17937
av558751


Cluster position2
Upstream
Upstream
Upstream


DRE regulatory direction
Bidirectional
Bidirectional
Bidirectional


DRE length (bp)
1975
3126
2501


DRE sequence
SEQ ID NO: 16
SEQ ID NO: 21
SEQ ID NO: 26


Internal forward primer
SEQ ID NO: 17
SEQ ID NO: 22
SEQ ID NO: 27


sequence4


External forward primer
SEQ ID NO: 18
SEQ ID NO: 23
SEQ ID NO: 28


sequence4


Internal reverse primer
SEQ ID NO: 19
SEQ ID NO: 24
SEQ ID NO: 29


sequence4


External reverse primer
SEQ ID NO: 20
SEQ ID NO: 25
SEQ ID NO: 30


sequence4


Position Effect Value5
0.15
0.3
5.555


Development Stage
0.69
0.77
1.5


Specificity Value5


Organ Specificity Value5
1.16
1.14
2


Number of transgenic
8
11
6


plants


Young roots score (No.,
5, 0, 0
6, 3.5, 1.92
5, 0, 0


Ave., Var)6


Mature roots score (No.,
5, 0, 0
7, 3.71, 1.63
4, 0, 0


Ave., Var)6


Young above-ground
5, 0.4, 0.24
6, 1.83, 2.14
5, 0, 0


Tissue (No., Ave., Var)6


Mature above-ground
5, 2, 0.8
7, 1.43, 1.10
4, 0.25, 0.19


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
3, 0, 0
3, 0.67, 0.89
3, 0, 0


Var)6


Flowers (No., Ave., Var)6
5, 0.4, 0.64
7, 1.86, 1.55
4, 0, 0


Description
Specific to above ground
Specific to root tissue.
Weak in above ground



tissue.
Strong expression,
tissue.




mainly in root meristems.




Weak expression in above




ground tissues.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 5







DRE number1
3714
4209
5095


Cluster reference2
Z25961
Z29176
AI996150


Cluster position2
Upstream
Downstream
Downstream


DRE regulatory direction
Unidirectional
Bidirectional
Bidirectional


DRE length (bp)
513
1022
1056


DRE sequence
SEQ ID NO: 31
SEQ ID NO: 36
SEQ ID NO: 41


Internal forward primer
SEQ ID NO: 32
SEQ ID NO: 37
SEQ ID NO: 42


sequence4


External forward primer
SEQ ID NO: 33
SEQ ID NO: 38
SEQ ID NO: 43


sequence4


Internal reverse primer
SEQ ID NO: 34
SEQ ID NO: 39
SEQ ID NO: 44


sequence4


External reverse primer
SEQ ID NO: 35
SEQ ID NO: 40
SEQ ID NO: 45


sequence4


Position Effect Value5
0.3625
0.40
0.6


Development Stage
0.11241
0.57
0


Specificity Value5


Organ Specificity Value5
0.377
0.40
0.85


Number of transgenic
11
18
3


plants


Young roots score (No.,
9, 0.611, 0.987
14, 3.46, 2.87
2, 0.5, 0.25


Ave., Var)6


Mature roots score (No.,
3, 0, 0
9, 2.11, 3.65
2, 1, 1


Ave., Var)6


Young above-ground
9, 2.38, 1.20
14, 2.89, 2.36
2, 1.5, 1.25


Tissue (No., Ave., Var)6


Mature above-ground
3, 1, 2
9, 2.44, 1.80
2, 2, 0


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
3, 1.66, 0.22
3, 1.33, 1.56
Not available


Var)6


Flowers (No., Ave., Var)6
3, 1.33, 3.55
9, 2, 3.78
2, 0, 0


Description
Weak in above ground tissue
Strong in roots, mainly roo
Constitutive, weak.




tips, and flower buds.




Lower expression in veins.




Very low expression in




seeds.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 6







DRE number1
5311
5532
5587


Cluster reference2
ATHCOL2A
ATASCO
Z26363


Cluster position2
Upstream
Upstream
Upstream


DRE regulatory direction
Bidirectional
Unidirectional
Unidirectional


DRE length (bp)
435
3348
1331


DRE sequence
SEQ ID NO: 46
SEQ ID NO: 51
SEQ ID NO: 56


Internal forward primer
SEQ ID NO: 47
SEQ ID NO: 52
SEQ ID NO: 57


sequence4


External forward primer
SEQ ID NO: (none)
SEQ ID NO: 53
SEQ ID NO: 58


sequence4


Internal reverse primer
SEQ ID NO: 49
SEQ ID NO: 54
SEQ ID NO: 59


sequence4


External reverse primer
SEQ ID NO: (none)
SEQ ID NO: 55
SEQ ID NO: 60


sequence4


Position Effect Value5
0.36
0.25
8.33


Development Stage
1.15
1.30932
1.5


Specificity Value5


Organ Specificity Value5
0.332
1.246
2


Number of transgenic
8
6
4


plants


Young roots score (No.,
7, 0.36, 0.48
5, 2.2, 1.36
4, 0, 0


Ave., Var)6


Mature roots score (No.,
4, 0.5, 0.75
4, 4.25, 0.187
3, 0, 0


Ave., Var)6


Young above-ground
7, 1.57, 1.74
5, 3.6, 1.84
4, 0, 0


Tissue (No., Ave., Var)6


Mature above-ground
4, 1.5, 2.25
4, 3.5, 0.25
3, 0, 0


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
Not available
3, 0.67, 0.22
3, 1.33, 3.55


Var)6


Flowers (No. Ave., Var)6
4, 0.25, 0.18
4, 2, 4.5
3, 0, 0


Description
Constitutive, weak.
Constitutive, mainly in
Siliques specific.




vegetative tissue.
High position effect.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 7







DRE number1
6669
6762
7357


Cluster reference2
Z26440
Z17588
F13952


Cluster position2
Upstream
Upstream
Upstream


DRE regulatory direction
Unidirectional
Bidirectional
Unidirectional


DRE length (bp)
2316
379
979


DRE sequence
SEQ ID NO: 61
SEQ ID NO: 66
SEQ ID NO: 71


Internal forward primer
SEQ ID NO: 62
SEQ ID NO: 67
SEQ ID NO: 72


sequence4


External forward primer
SEQ ID NO: 63
SEQ ID NO: 68
SEQ ID NO: 73


sequence4


Internal reverse primer
SEQ ID NO: 64
SEQ ID NO: 69
SEQ ID NO: 74


sequence4


External reverse primer
SEQ ID NO: 65
SEQ ID NO: 70
SEQ ID NO: 75


sequence4


Position Effect Value5
0.28
9.72
0.32


Development Stage
1.18
0.16
0.6


Specificity Value5


Organ Specificity Value5
1.32
1.42
0.64


Number of transgenic
4
11
7


plants


Young roots score (No.,
3, 2.67, 0.22
8, 1.25, 4.69
6, 0.5, 0.58


Ave., Var)6


Mature roots score (No.,
4, 4.75, 0.19
9, 0.33, 0.89
5, 0, 0


Ave., Var)6


Young above-ground
3, 3.67, 3.56
8, 4.19, 0.87
6, 0.43, 0.47


Tissue (No., Ave., Var)6


Mature above-ground
4, 1, 3
9, 3.61, 1.10
5, 0.8, 0.16


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
4, 0.75, 0.69
3, 3.33, 1.56
3, 0, 0


Var)6


Flowers (No., Ave., Var)6
4, 3, 4.5
9, 3.11, 2.57
5, 1.2, 1.36


Description
Specific to young and
Strong in above ground
Weak in above ground



meristematic tissue.
tissue.
tissue.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 8







DRE number1
7617
8463
9136


Cluster reference2
Z17636
Z26728
F15462


Cluster position2
Upstream
Downstream
Downstream


DRE regulatory direction
Bidirectional
Unidirectional
Unidirectional


DRE length (bp)
665
2834
486


DRE sequence
SEQ ID NO: 76
SEQ ID NO: 81
SEQ ID NO: 86


Internal forward primer
SEQ ID NO: 77
SEQ ID NO: 82
SEQ ID NO: 87


sequence4


External forward primer
SEQ ID NO: 78
SEQ ID NO: 83
SEQ ID NO: 88


sequence4


Internal reverse primer
SEQ ID NO: 79
SEQ ID NO: 84
SEQ ID NO: 89


sequence4


External reverse primer
SEQ ID NO: 80
SEQ ID NO: 85
SEQ ID NO: 90


sequence4


Position Effect Value5
0.42
0.16
0.48


Development Stage
0.16
0.68
0.60


Specificity Value5


Organ Specificity Value5
0.41
2.02
0.53


Number of transgenic
3
12
13


plants


Young roots score (No.,
3, 0, 0
6, 0, 0
9, 0.778, 2.617


Ave., Var)6


Mature roots score (No.,
3, 0, 0
7, 1.14, 3.55
11, 0.73, 1.107


Ave., Var)6


Young above-ground
3, 0.17, 5.56
6, 3.33, 3.32
9, 0.778, 0.84


Tissue (No., Ave., Var)6


Mature above-ground
3, 0.33, 0.22
7, 2, 2.57
11, 1.18, 2.51


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
2, 1, 1
4, 0, 0
3, 0, 0


Var)6


Flowers (No., Ave., Var)6
3, 0.33, 0.22
7, 3.57, 3.96
11, 0.55, 0.98


Description
Very weak in above ground.
Strong in above ground
Constitutive, weak.



tissue.
tissue of seedlings.




Strong in flower tissue of




mature plants.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 9







DRE number1
10826
12582
13257


Cluster reference2
Z30896
Z33953
Z17541


Cluster position2
Upstream
Downstream
Upstream


DRE regulatory direction
Bidirectional
Unidirectional
Bidirectional


DRE length (bp)
1840
1665
807


DRE sequence
SEQ ID NO: 91
SEQ ID NO: 96
SEQ ID NO: 101


Internal forward primer
SEQ ID NO: 92
SEQ ID NO: 97
SEQ ID NO: 102


sequence4


External forward primer
SEQ ID NO: 93
SEQ ID NO: 98
SEQ ID NO: 103


sequence4


Internal reverse primer
SEQ ID NO: 94
SEQ ID NO: 99
SEQ ID NO: 104


sequence4


External reverse primer
SEQ ID NO: 95
SEQ ID NO: 100
SEQ ID NO: 105


sequence4


Position Effect Value5
0.27
0.19
0


Development Stage
0.50
0.32
1.5


Specificity Value5


Organ Specificity Value5
8.19
1.38
1.14


Number of transgenic
5
20
2


plants


Young roots score (No.,
3, 1.67, 2.89
18, 0.56, 1.36
2, 0, 0


Ave., Var)6


Mature roots score (No.,
4, 3.38, 4.17
10, 0.5, 1.05
2, 0, 0


Ave., Var)6


Young above-ground
3, 2, 2.67
18, 2.39, 3.90
2, 0, 0


Tissue (No., Ave., Var)6


Mature above-ground
4, 3.12, 1.55
10, 3.2, 0.36
2, 2.5, 0.25


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
Not available
3, 1, 0
2, 0, 0


Var)6


Flowers (No., Ave., Var)6
4, 3.25, 3.06
10, 4.4, 1.84
2, 2, 1


Description
Strong in root and flower
Strong in above ground
Specific to above ground



tissue.
tissue of seedlings.
tissue of mature plants.




Lower expression in mature




plants.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 10







DRE number1
13277
15980
16665


Cluster reference2
Z18392
BE522497
T04806


Cluster position2
Upstream
Downstream
Downstream


DRE regulatory direction
Bidirectional
Unidirectional
Bidirectional


DRE length (bp)
3297
2183
1358


DRE sequence
SEQ ID NO: 106
SEQ ID NO: 111
SEQ ID NO: 116


Internal forward primer
SEQ ID NO: 107
SEQ ID NO: 112
SEQ ID NO: 117


sequence4


External forward primer
SEQ ID NO: 108
SEQ ID NO: 113
SEQ ID NO: 118


sequence4


Internal reverse primer
SEQ ID NO: 109
SEQ ID NO: 114
SEQ ID NO: 119


sequence4


External reverse primer
SEQ ID NO: 110
SEQ ID NO: 115
SEQ ID NO: 120


sequence4


Position Effect Value5
0.22
0.38
0.33


Development Stage
1.5
1.18
4.44


Specificity Value5


Organ Specificity Value5
1
1.45
1.5


Number of transgenic
5
16
5


plants


Young roots score (No.,
5, 0.6, 0.24
10, 2.1, 1.49
5, 0, 0


Ave., Var)6


Mature roots score (No.,
3, 0, 0
13, 2.46, 0.86
2, 0, 0


Ave., Var)6


Young above-ground
5, 0.4, 0.24
10, 12, 1.76
5, 0.6, 0.34


Tissue (No., Ave., Var)6


Mature above-ground
3, 0, 0
13, 0.46, 0.86
2, 0.5, 0.25


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
3, 0, 0
4, 3.75, 1.69
Not available


Var)6


Flowers (No., Ave., Var)6
3, 0, 0
13, 0.92, 1.76
2, 0, 0


Description
Weak in seedlings.
Root tissue, mainly root
Above ground vegetative




tips; and seeds.
tissue of mature plants.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 11







DRE number1
16900
17109
17809


Cluster reference2
Z25996
Z17897
Z18103


Cluster position2
Upstream
Upstream
Upstream


DRE regulatory direction
Bidirectional
Bidirectional
Bidirectional


DRE length (bp)
824
2927
3165


DRE sequence
SEQ ID NO: 121
SEQ ID NO: 126
SEQ ID NO: 131


Internal forward primer
SEQ ID NO: 122
SEQ ID NO: 127
SEQ ID NO: 132


sequence4


External forward primer
SEQ ID NO: 123
SEQ ID NO: 128
SEQ ID NO: 133


sequence4


Internal reverse primer
SEQ ID NO: 124
SEQ ID NO: 129
SEQ ID NO: 134


sequence4


External reverse primer
SEQ ID NO: 125
SEQ ID NO: 130
SEQ ID NO: 135


sequence4


Position Effect Value5
4.17
0.26
0.21


Development Stage
0.21
0.63
0.60


Specificity Value5


Organ Specificity Value5
0.22
1.85
1.38


Number of transgenic
5
10
10


plants


Young roots score (No.,
4, 3.5, 0.75
6, 4, 1.25
5, 0.8, 0.56


Ave., Var)6


Mature roots score (No.,
5, 3.2, 0.56
7, 3.07, 2.60
7, 1.5, 1.5


Ave., Var)6


Young above-ground
4, 4, 0
6, 0.42, 0.37
5, 4, 0.8


Tissue (No., Ave., Var)6


Mature above-ground
5, 3.8, 0.56
7, 1.05, 1.07
7, 3.07, 1.03


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
3, 4.67, 0.22
3, 0, 0
3, 0, 0


Var)6


Flowers (No., Ave., Var)6
5, 4, 4
7, 1.07, 2.89
7, 2.71, 2.99


Description
Constitutive pattern.
Strong in root, flower and
Strong in leaf tissue of



Strong in meristematic tissue
meristematic tissue.
seedlings.



and seeds.

Variable in mature plants.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 12







DRE number1
19672
19678
19827


Cluster reference2
Z25683
BE523552
Z17577


Cluster position2
Upstream
Upstream
Upstream


DRE regulatory direction
Unidirectional
Bidirectional
Bidirectional


DRE length (bp)
1155
2877
578


DRE sequence
SEQ ID NO: 136
SEQ ID NO: 141
SEQ ID NO: 146


Internal forward primer
SEQ ID NO: 137
SEQ ID NO: 142
SEQ ID NO: 147


sequence4


External forward primer
SEQ ID NO: 138
SEQ ID NO: 143
SEQ ID NO: 148


sequence4


Internal reverse primer
SEQ ID NO: 139
SEQ ID NO: 144
SEQ ID NO: 149


sequence4


External reverse primer
SEQ ID NO: 140
SEQ ID NO: 145
SEQ ID NO: 150


sequence4


Position Effect Value5
0.03
5.55
0.37


Development Stage
9.78
1.5
0.60


Specificity Value5


Organ Specificity Value5
3.99
2
0.64


Number of transgenic
17
5
12


plants


Young roots score (No.,
15, 4.33, 0.76
5, 0, 0
10, 0, 0


Ave., Var)6


Mature roots score (No.,
17, 4, 1.76
4, 0, 0
5, 0.1, 0.04


Ave., Var)6


Young above-ground
15, 4.53, 0.38
5, 0, 0
10, 2.9, 2.29


Tissue (No., Ave., Var)6


Mature above-ground
17, 3.12, 2.22
4, 0.25, 0.18
5, 0.8, 1.36


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
3, 4.33, 0.22
3, 0, 0
3, 0, 0


Var)6


Flowers (No., Ave., Var)6
17, 4.06, 2.17
4, 0, 0
5, 0.8, 1.36


Description
Strong, constitutive.
Very weak.
Above ground tissue.



Lower expression in mature
High position effect.
Weak.



leaf tissue.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 13







DRE number1
20607
22397
22604


Cluster reference2
AI998130
ATHD12A
ATHFEDAA


Cluster position2
Upstream
Upstream
Downstream


DRE regulatory direction
Bidirectional
Unidirectional
Bidirectional


DRE length (bp)
2819
1313
2080


DRE sequence
SEQ ID NO: 151
SEQ ID NO: 156
SEQ ID NO: 161


Internal forward primer
SEQ ID NO: 152
SEQ ID NO: 157
SEQ ID NO: 162


sequence4


External forward primer
SEQ ID NO: 153
SEQ ID NO: 158
SEQ ID NO: 163


sequence4


Internal reverse primer
SEQ ID NO: 154
SEQ ID NO: 159
SEQ ID NO: 164


sequence4


External reverse primer
SEQ ID NO: 155
SEQ ID NO: 160
SEQ ID NO: 165


sequence4


Position Effect Value5
0.25
0.38
9.72


Development Stage
2.50
0.89
0.71


Specificity Value5


Organ Specificity Value5
0.916
1.33
1.10


Number of transgenic
5
12
17


plants


Young roots score (No.,
5, 0, 0
12, 1.13, 2.09
15, 0, 0


Ave., Var)6


Mature roots score (No.,
3, 0, 0
12, 1.67, 4.22
13, 0, 0


Ave., Var)6


Young above-ground
5, 2.2, 2.16
12, 3.33, 0.89
15, 0.2, 0.16


Tissue (No., Ave., Var)6


Mature above-ground
3, 2, 2
12, 2.63, 2.69
13, 0, 0


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
Not available
3, 4, 0.67
4, 0.75, 1.69


Var)6


Flowers (No., Ave., Var)6
3, 1, 2
12, 1.21, 3.06
13, 0, 0


Description
Above ground tissue.
Above ground tissue and
Above ground tissue and




seed.
seed.





High position effect.





Very weak.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 14







DRE number1
24136
24291
24728


Cluster reference2
Z34788
Z17960
AV530349


Cluster position2
Downstream
Upstream
Upstream


DRE regulatory direction
Unidirectional
Bidirectional
Unidirectional


DRE length (bp)
174
2096
1617


DRE sequence
SEQ ID NO: 166
SEQ ID NO: 171
SEQ ID NO: 176


Internal forward primer
SEQ ID NO: 167
SEQ ID NO: 172
SEQ ID NO: 177


sequence4


External forward primer
SEQ ID NO: none
SEQ ID NO: 173
SEQ ID NO: 178


sequence4


Internal reverse primer
SEQ ID NO: 169
SEQ ID NO: 174
SEQ ID NO: 179


sequence4


External reverse primer
SEQ ID NO: none
SEQ ID NO: 175
SEQ ID NO: 180


sequence4


Position Effect Value5
0.17
0.56
5.71


Development Stage
1.5
7.76
0.17


Specificity Value5


Organ Specificity Value5
2
6.93
1.75


Number of transgenic
5
12
9


plants


Young roots score (No.,
1, 0, 0
9, 1.56, 3.14
8, 0, 0


Ave., Var)6


Mature roots score (No.,
2, 0, 0
8, 2.37, 1.48
8, 0.5, 1.75


Ave., Var)6


Young above-ground
1, 0, 0
9, 2.33, 3.11
8, 2.63, 0.48


Tissue (No., Ave., Var)6


Mature above-ground
2, 0.25, 0.06
8, 1.62, 2.33
8, 2.5, 1.5


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
3, 0, 0
4, 2, 1.5
Not available


Var)6


Flowers (No., Ave., Var)6
2, 0, 0
8, 1.37, 1.98
8, 3.38, 2.73


Description
Above ground tissue.
Constitutive.
Strong in flower tissue.



Very weak.

Low expression in veins.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 15







DRE number1
24811
4209
5095


Cluster reference2
H36200
H36237
Z29720


Cluster position2
Upstream
Upstream
Upstream


DRE regulatory direction
Bidirectional
Bidirectional
Bidirectional


DRE length (bp)
428
1022
1056


DRE sequence
SEQ ID NO: 181
SEQ ID NO: 186
SEQ ID NO: 191


Internal forward primer
SEQ ID NO: 182
SEQ ID NO: 187
SEQ ID NO: 192


sequence4


External forward primer
SEQ ID NO: none
SEQ ID NO: 188
SEQ ID NO: 193


sequence4


Internal reverse primer
SEQ ID NO: 184
SEQ ID NO: 189
SEQ ID NO: 194


sequence4


External reverse primer
SEQ ID NO: none
SEQ ID NO: 190
SEQ ID NO: 195


sequence4


Position Effect Value5
0.53
0.60
0.33


Development Stage
8.11
0.28
0.61


Specificity Value5


Organ Specificity Value5
0.23
0.49
1.35


Number of transgenic
4
5
3


plants


Young roots score (No.,
4, 0.75, 1.69
5, 0.4, 0.34
3, 0.33, 0.22


Ave., Var)6


Mature roots score (No.,
3, 1, 2
4, 1.2, 2.16
2, 1, 1


Ave., Var)6


Young above-ground
4, 1, 3
5, 2.4, 1.84
3, 1.33, 1.56


Tissue (No., Ave., Var)6


Mature above-ground
3, 1.33, 3.56
5, 2, 2.8
2, 2, 0


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
Not available
5, 0.4, 0.24
2, 0, 0


Var)6


Flowers (No., Ave., Var)6
3, 2, 4
5, 1.6, 3.44
2, 0, 0


Description
Constitutive, weak.
Leaf-stalk and stem tissue.
Vegetative tissue, weak.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 16







DRE number1
17109
20607
24811


Cluster reference2
R29912
R90407
T22055


Cluster position2
Downstream
Downstream
Downstream


DRE regulatory direction
Bidirectional
Bidirectional
Bidirectional


DRE length (bp)
2027
2834
428


DRE sequence
SEQ ID NO: 196
SEQ ID NO: 201
SEQ ID NO: 202


Internal forward primer
SEQ ID NO: 197
SEQ ID NO: 168
SEQ ID NO: 48


sequence4


External forward primer
SEQ ID NO: 198
SEQ ID NO: 170
SEQ ID NO: none


sequence4


Internal reverse primer
SEQ ID NO: 199
SEQ ID NO: 183
SEQ ID NO: 50


sequence4


External reverse primer
SEQ ID NO: 200
SEQ ID NO: 185
SEQ ID NO: none


sequence4


Position Effect Value5
0.46
0.26
0.24


Development Stage
0
0.60
0.61


Specificity Value5


Organ Specificity Value5
0.49
1.16
0.51


Number of transgenic
5
5
5


plants


Young roots score (No.,
5, 0.6, 0.64
5, 0, 0
4, 0.75, 1.69


Ave., Var)6


Mature roots score (No.,
Not available
5, 0, 0
5, 0.6, 1.44


Ave., Var)6


Young above-ground
5, 2, 2
5, 2.2, 2.16
4, 1, 3


Tissue (No., Ave., Var)6


Mature above-ground
Not available
5, 1.8, 2.16
5, 0.8, 2.56


tissue (No., Ave., Var)6


Siliques/Seed (No., Ave.,
Not available
4, 0, 0
3, 0, 0


Var)6


Flowers (No., Ave., Var)6
Not available
5, 1.2, 1.36
5, 0.8, 2.56


Description
Above ground tissue, weak
Above ground tissue,
Constitutive, weak




mainly in leaves.






1ID number of the DRE as assigned by the present inventors.




2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.




3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.




4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.




5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.




6No. = number; Ave. = average; Var. = variance.

















TABLE 17









DRE number1
16665



Cluster reference2
Z26101



Cluster position2
Upstream



DRE regulatory direction
Bidirectional



DRE length (bp)
1358



DRE sequence
SEQ ID NO: 203



Internal forward primer
SEQ ID NO: 204



sequence4



External forward primer
SEQ ID NO: 206



sequence4



Internal reverse primer
SEQ ID NO: 206



sequence4



External reverse primer
SEQ ID NO: 207



sequence4



Position Effect Value5
0.51



Development Stage
8.82



Specificity Value5



Organ Specificity Value5
0.403



Number of transgenic
12



plants



Young roots score (No.,
10, 0, 0



Ave., Var)6



Mature roots score (No.,
5, 0.6, 0.64



Ave., Var)6



Young above-ground
10, 1.5, 3.05



Tissue (No., Ave., Var)6



Mature above-ground
5, 2, 2.08



tissue (No., Ave., Var)6



Siliques/Seed (No., Ave.,
3, 1, 0.66



Var)6



Flowers (No., Ave., Var)6
5, 1.8, 3.76



Description
Above ground tissue








1ID number of the DRE as assigned by the present inventors.





2Internal reference assigned by the present inventors to a cluster of Arabidopsis genes (contig) downstream or upstream of the DRE.





3Unidirectional implies that only the sense strand of the DRE is capable of regulating gene expression; Bidirectional implies that both the sense and antisense strands of the DRE are capable of regulating gene expression.





4A PCR primer for isolating the DRE from Arabidopsis genomic DNA.





5Position Effect Values (PoEf), Development Stage-Specificity Values (SpDs) and Organ Specificity Values (SpOr) were calculated as described in the materials and methods section hereinabove.





6No. = number; Ave. = average; Var. = variance.








Deletion analysis of DREs 4209 and 6669:


The ability of partial DRE sequences to modify in vivo gene expression pattern, was tested by comparing reporter gene expression driven by unmodified DREs (SEQ ID NO:36 and 61) with that of deletion mutants thereof (SEQ ID NO:210 and 213, respectively).


GUS expression pattern in p4209short-GUS (including the DRE 4209 partial sequence set forth in SEQ ID NO:210) transformed plants was similar to that driven by the full length promoter sequence, DRE 4209 (SEQ ID NO:36). As is shown in FIG. 26a, expression was strong in roots, mainly root tips, as well as in flower buds. Insterstingly, p4209short-GUS transformed plants exhibited lower reporter gene expression in veins, while leaves exhibited higher expression. Note, expression in seeds was not examined.


GUS expression pattern in the p6669short-GUS (comprising the DRE 6669 partial sequence set forth in SEQ ID NO:213) transformed plants was restricted to the root tips (FIG. 26b) while expression in other young or meristematic tissues, as was obtained by the full length DRE 6669 promoter (SEQ ID NO:61), was lost.


These results demonstrate that the 5′ nucleic acid sequence of SEQ ID NO: 61 (e.g., nucleotide coordinates 1-747), is important for constitutive gene expression. Indeed, a DNA sequence (SEQ ID NO: 214, see FIG. 27 WO 02/16655) which does not include the 5′ first 400 nucleotides of SEQ ID NO: 61 has been implicated in stress regulated gene expression.


These results indicate that the promoters of the present invention may be modified by partial deletions, to generate inductive or tissue specific expression pattern as demonstrated for DRE 6669 (SEQ ID NO:61).


As is clearly illustrated by Tables 3-17 and FIGS. 1-26, the DREs isolated according to the teachings of the present invention exhibit a wide range of activities as well as a wide range of tissue and developmental stage specificities. The DREs of the present invention were classified according to function as determined using the Arabidopsis assay described hereinabove.


The luciferase gene was expressed in a constitutive manner in Arabidopsis when functionally linked to SEQ ID NOS: 1, 6, 41, 46, 51, 61, 86, 121, 136, 171, 181 and 202 (illustrated in FIG. 18), thus the promoters of these DREs are putatively classified herein as constitutive promoters.


The luciferase gene was expressed in an inductive manner in Arabidopsis when functionally linked to SEQ ID NOS: 1, 11, 16, 21, 26, 31, 36, 56, 66, 71, 76, 81, 91, 96, 101, 116, 126, 141, 146, 151, 156, 161, 166, 176 and 203, thus the promoters of these DREs are putatively classified herein as inductive promoters.


The luciferase gene was expressed in a young or meristematic, tissue-specific manner in Arabidopsis when functionally linked to SEQ ID NOS: 61, 121, 126, 213 (illustrated in FIGS. 4, 14, 15, 16 and 26b), thus the promoters of these DREs are putatively classified herein as young or meristematic, tissue-specific promoters.


The luciferase gene was expressed in root tissue specific manner in Arabidopsis when functionally linked to SEQ ID NOS: 21, 36, 91, 111, and 126 (illustrated in FIGS. 2, 3, 9, and 13), thus the promoters of these DREs are putatively classified herein as root tissue-specific promoters


The luciferase gene was expressed in an above ground tissue-specific manner in Arabidopsis when functionally linked to SEQ ID NOS: 16, 26, 31, 66, 71, 76, 81, 96, 106, 101, 116, 131, 146, 151, 156, 161, 166, 196, 201 and 203 (illustrate in FIGS. 10, 11, 17, 19 and 20), thus the promoters of these DREs are putatively classified herein as above ground tissue-specific promoters.


The luciferase gene was expressed in a stem tissue specific manner in Arabidopsis when functionally linked to SEQ ID NO: 186 (illustrated in FIG. 22), thus the promoter(s) of this DRE are putatively classified herein as stem tissue specific promoter(s).


The luciferase gene was expressed in a flower tissue specific manner in Arabidopsis when functionally linked to SEQ ID NOS: 11, 36, 81, 91, 126, 176 and 210 (illustrated in FIGS. 1, 3, 9 and 26a), thus the promoters of these DREs are putatively classified herein as flower tissue-specific promoters.


The luciferase gene was expressed in a fruit (silique) tissue specific manner in Arabidopsis when functionally linked to SEQ ID NO: 56, thus the promoter(s) of this DRE are putatively classified herein as fruit (silique) tissue specific promoter(s).


The luciferase gene was expressed in a seed tissue specific manner in Arabidopsis when functionally linked to SEQ ID NOS: 1, 156, and 161 (illustrated in FIGS. 12 and 21), thus the promoters of these DREs are putatively classified herein as seed tissue specific promoters.


The luciferase gene was expressed in a developmental stage specific manner in Arabidopsis when functionally linked to SEQ ID NOS: 81, 96, 101, 106, and 131 (illustrated comparatively in FIGS. 5-6, 7-8, 10-11 and 15-16), thus the promoters of these DREs are putatively classified herein as developmental stage specific promoters.


The GUS gene was expressed in an inductive manner in Arabidopsis when functionally linked to SEQ ID NOS: 210 and 213 (illustrated in FIG. 26b), thus the promoters of these partial DREs sequences are putatively classified herein as inductive promoters.


The GUS gene was expressed in a root, as well as in a flower bud tissue specific manner in Arabidopsis when functionally linked to SEQ ID NO: 210 (illustrated in FIG. 26a), thus the promoter of this partial DRE sequence is putatively classified herein as a root as well as a flower tissue-specific promoter.


The GUS gene was expressed in a root-tip tissue specific manner in Arabidopsis when functionally linked to SEQ ID NO: 213 (illustrated in FIG. 26b), thus this promoter is putatively classified herein as a root tissue-specific promoter.


Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by their accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, patent application or sequence identified by their accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims
  • 1. An isolated polynucleotide consisting of the nucleic acid sequence set forth in SEQ ID NO: 61.
  • 2. A nucleic acid construct, comprising an expressed nucleic acid sequence operably linked to a regulatory sequence consisting of SEQ ID NO :61.
  • 3. The nucleic acid construct of claim 2, wherein said expressed nucleic acid sequence is a reporter gene.
  • 4. A transgenic cell comprising the isolated polynucleotide of claim 1.
  • 5. A transgenic cell comprising the nucleic acid construct of claim 2.
  • 6. A transgenic non-human organism comprising the isolated polynucleotide of claim 1.
  • 7. A transgenic non-human organism comprising the nucleic acid construct of claim 2.
  • 8. A transgenic plant comprising the isolated polynucleotide of claim 1.
  • 9. A transgenic plant comprising the nucleic acid construct of claim 2.
  • 10. A method of producing a transgenic plant, comprising transforming a plant with the polynucleotide of claim 1.
  • 11. A method of producing a transgenic plant, comprising transforming a plant with the nucleic acid construct of claim 2.
  • 12. A method of expressing a polypeptide of interest in a cell comprising transforming the cell with a nucleic acid construct including a polynucleotide sequence encoding the polypeptide of interest operably linked to the regulatory nucleic acid sequence set forth in SEQ ID NO: 61 thereby expressing the polypeptide of interest in the cell.
RELATED PATENT APPLICATION

This application is a National Phase Application of PCT/IL2004/000235 having International Filing Date of 11 Mar. 2004, which claims the benefit of U.S. Provisional Patent Application No. 60/453,843 filed 12 Mar. 2003. The contents of the above Application are all incorporated herein by reference.

PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/IL2004/000235 3/11/2004 WO 00 9/12/2005
Publishing Document Publishing Date Country Kind
WO2004/081173 9/23/2004 WO A
US Referenced Citations (9)
Number Name Date Kind
4761367 Edgell et al. Aug 1988 A
6177611 Rice Jan 2001 B1
6359196 Lok et al. Mar 2002 B1
6363399 Maslyn et al. Mar 2002 B1
6403862 Jiao et al. Jun 2002 B1
7034204 Conner et al. Apr 2006 B2
20020029394 Allen et al. Mar 2002 A1
20040121360 Karchi et al. Jun 2004 A1
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Number Date Country
WO 9504830 Feb 1995 WO
WO 0029594 May 2000 WO
WO 0120024 Mar 2001 WO
WO 0155371 Aug 2001 WO
WO 0216655 Feb 2002 WO
WO0216655 Feb 2002 WO
WO 02079487 Oct 2002 WO
WO 2004081173 Sep 2004 WO
Related Publications (1)
Number Date Country
20070022503 A1 Jan 2007 US
Provisional Applications (1)
Number Date Country
60453843 Mar 2003 US