Abstract:
To develop a promoter inducible in response to injury, this invention provides an injury-inducible element, which may be added to a promoter. 
     The invention provides an injury-inducible element that is a base sequence referred to as nucleotide numbers from −611 to −629 SEQ ID No: 1.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a cis-element inducible in response to injury and derived from a promoter region of FAD7 of  Arabidopsis thaliana  Heynh. 
     2. Description of Related Art 
     Studies have confirmed the significant relationship between cold-resistance of a plant and the degree of unsaturation of fatty acids constructing biomembrane thereof. The inventors have experimentally indicated that a transformed-tobacco plant acquires higher-resistance against low temperatures by expressing a fatty acid desaturase, (unsaturating enzyme) gene FAD7, derived from  Arabidopsis thaliana,  in a high degree in the plant. 
     SUMMARY OF THE INVENTION 
     On the other hand, to produce certain protein in plant cells, a promoter, which exhibits constitutive expression and strong promoter activity, have been used. Such a promoter functions regardless of its external environmental conditions, sometimes providing economical losses in breeding a plant. For example, to improve the resistance of a plant against physical environmental stresses such insect damage, the use of various genes with resistant properties to the stresses is now investigated. That is, the resistance against injury might be improved by expressing ω-3 fatty acid desaturase enzyme by means of its constitutive promoter functioning in response to insect damage or physical damage. 
     Such particular expressed protein, however, is unnecessary for a plant under normal condition without injury. However, in the conventional gene expression system using a constitutive promoter, a plant is forced to express a particular protein, such as ω-3 fatty acid desaturase enzyme, unnecessary under normal condition. 
     Based on such background, it has been demanded to develop a promoter inducible in response to injury. For example, it is required to produce a breeding intermediate mother body, wherein the expression of a gene, which encodes ω-3 fatty acid desaturase enzyme or other proteins contributing to improving resistance of a plant against insect damage, may be induced in response to injury. 
     An object of the invention is to provide an element inducible in response to injury, which may be added to a promoter for developing a promoter inducible in response to injury. 
     Another object of the invention is to provide a means for developing a breed intermediate mother body with improved resistance against injury, by using a promoter provided with such element inducible in response to injury. 
     The invention provides an element inducible in response to injury, the element comprising 
     (a) a base sequence referred to as nucleotide numbers from −430 to −363 in a sequence number 1 in a sequence list, or 
     (b) a base sequence hybridizes with the base sequence (a) under stringent condition, the base sequence (b) being inducible in response to injury, or 
     (b)′ a base sequence (a), a part of which is deleted or substituted by another base sequence, or to which another base sequence is added, the base sequence (b)′ being inducible in response to injury. 
     The invention provides an element inducible in response to injury, the element comprising 
     (c) a base sequence referred to as nucleotide numbers from −242 to −223 in a sequence number 1 in a sequence list, or 
     (d) a base sequence (d) hybridizes with the base sequence (c) under stringent condition, the base sequence (d) being inducible in response to injury, or 
     (d)′ a base sequence (c), a part of which is deleted or substituted by another base sequence, or to which another base sequence is added, the base sequence (d)′ being inducible in response to injury. 
     The invention also provides an injury-inducible promoter characterized by containing each of the elements described above, and a transgenic plant comprising a recombinant DNA containing this injury-inductible promoter. 
     An ω-3 fatty acid desaturase enzyme catalyzes the final step of production of triene-fatty acids in a plant, which are main components of plant lipids. FAD7 derived from  Arabidopsis thaliana  is a gene encoding ω-3 fatty acid desaturase enzyme localized in chloroplast. FAD7 is specifically expressed in chloroplast organs and its expression is shown to be light-inducible. 
     Moreover, it is shown that localized damage on a plant body causes highly expression of FAD7, not only in chloroplast organs but also in non-chloroplast organs such as stalk or root. The invention identified a cis-element, in a promoter region of FAD7 gene, involved in organ-specific induction in response to injury, and therefore has an inventive step. 
    
    
     BRIEF DESCRIPTION OF DRAWINGS 
     The invention will be described with reference to the accompanying drawings, wherein: 
     FIG. 1 is a genome sequence of the promoter region derived from  Arabidopsis thaliana,  and 
     FIG. 2 is the experimental results on injury-inductivity of each element, which is a promoter region of FAD7 gene with a part of which being deleted. A: blade, B: stalk, C: root. 
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
     The base sequence of FAD7 promoter region derived from  Arabidopsis thaliana  was already published by Nishiuchi et al (Nishiuchi et al. (1995) “Plant Mol. Biol” 29: 599-609). The base sequence of the promoter region is shown in a sequence list 1 described below and FIG.  1 . 
     Parts of the promoter sequence were deleted to provide elements. The parts were selected from 5′ upstream to each predetermined nucleotide number in a stepwise manner, as described below. Each element was ligated with a GUS gene of plasmid binary vector PbI101.1 (Clonetech) to provide each construct, which was incorporated into tobacco SR1 strain using leafdisc method mediated by agrobacterium (LBA4404) (Horsch et al. (1985) Science 227:1229-1231). Each reporter (GUS) activity of the R1 generation was measured. 
     FIG. 2 shows the experimental results performed on A: blade, B: stalk and C: root. Each tobacco was planted in soil and cultivated for 3 months at 26° C. under continuous light illumination. As described above, a part of the promoter region was deleted to produce each element and construct. Each deleted part is upstream of each predetermined nucleotide number indicated by each number in the lower column in the horizontal axis of each graph in FIG.  2 . The numbers in the upper column in the horizontal axis of each graph in FIG. 2 indicate line numbers of R1 generation of tobacco used. The values in the vertical axis of each graph in FIG. 2 indicate the ratios of GUS activities measured in injured samples versus those measured in intact samples. In each line, each GUS activity is shown as an average measured in 5 plants. 
     As the result, in the case of blade and stalk (A, B), a region of nucleotide numbers from −259 to −197, and in the case of root (C), a region of nucleotide numbers from −520 to −363, are shown to be involved in injury-induced FAD7 gene expression. 
     Then, the existence of transcription factors that bind to these regions was investigated by gel-shift assay analysis (Green et al. (1989) “Plant Mol. Biol. Manual” BII, 1-22). On blade, stalk and root, gel shift assay was performed using nuclear proteins extracted from an intact tissue and an injured tissue and DNA probes corin response to various base sequences of the regions described above. As a result, on blade and stalk, a band mobility shift in response to injury was detected by electrophoresis using a base sequence of −242/−223. (TAACAATCTTATATAGTCAC) as a probe. The organ-specific injury inductivity on blade and stalk was diminished when FAD7 promoter, in which the base sequence was deleted, was ligated to a GUS gene and incorporated into tobacco plant. 
     On root, a band shifting in response to injury was detected by electrophoresis, when a base sequence of −430/−363 was used as a probe. The organ-specific injury inductivity in root was diminished, when FAD7 promoter, in which the base sequence was deleted, was ligated to a GUS gene and incorporated into a tobacco plant. 
     Accordingly, it was indicated that these sequences functioned as cis-elements. Moreover, an element of −430/−363 responded to jasmonate, proving that it exists at the end of octadecanoid intracellular signal transmission pathway. An element of −242/−223 did not respond to jasmonate, proving that it exists at the end of a signal transmission pathway different from the octadecanoid intracellular signal transmission pathways. 
     In this invention, a plasmid can be used as a vector to construct a recombinant DNA. A preferred plant to incorporate such recombinant DNA includes monocotyledon useful cultivated plants such as maize, rice, wheat, barely, oat, millet, barnyard millet and tobacco. In various injury-responsive elements, even if one or several bases of the above described element is deleted, substituted or to which another nucleotide base sequence is added, it is in the range of this invention, so far as the injury-inducible function of the element was preserved. Moreover, the injury-inductive element of this invention may induce the production of various proteins including ω-3 fatty acid desaturase enzyme and protease inhibitor II. 
     
       
         
           
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             DNA 
             Arabidopsis thaliana heynh 
           
            1
aaattcatgc ggaatcagag aacgttacca tggtgggatg aagatgaatt gcggccctgt     60
aaagttttag tcttgttcaa tagattgcac aaagaaagta aacaaacaac aacaaaaaaa    120
aagactgtag aaaagaaaaa aaaaaaggat aatcataacg gaatctttaa tttaccagcg    180
cgattaagga cctcagattg ttgttcggtg ccatgatcgg atattagggt tcgttcgctc    240
ttcttcttct ttgtctatac gcgatttgtg agaataaaaa aggtcggatc ttttgagagt    300
tctgtagttt aatgggctta tactattggg ccctagccca aatgagcgac actattgttc    360
attttgtaca aatctcttgg gctaatttat ttcaggctga ccaactaatt tggtcaacta    420
gttgggtttg gcatgtttaa tttcaatttc cacttggttc aatttttatg ttcaccgtcc    480
atgtaacttg actagtagca tgagatttgg tttgtcccta ttgaaacaat aggtataggg    540
tgtgaaacat tgaaacgtaa ttgactcaaa ttctcaaata ggtttcttca ccaaactcct    600
cttgttttgt ctaacaatct tatatagtca ctaaaataat gtgtataaat tttgctaccg    660
tcatttaaaa gttagtgtca tgaaacatat gcctcattat attttattat tttcgttcac    720
tttatttcaa aggctttaaa ctatatgaca tcataaccaa aacaagaatt aaaacgagat    780
caatcaaacc cgtgttgaaa cctcaacttg tgtctaaatt gaccgtcaca aaaaaaaatc    840
tcacatcaca ccatcactaa taaatttcct tctcctttca agttgtagct aacttatata    900
agacataagc gtgcgaacca gagacagaga tagaaattga gagacgataa gcaaagtaga    960
aaacacaagt ttctctcaca cacattatct ctttctctat taccaccact cattcataac   1020
agaaacccac caaaaaataa aaagagagac ttttcactct ggggagagag ctcaagttct   1080
aatg                                                                1084