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  • C12Y207/02011—Glutamate 5-kinase (2.7.2.11)

Definitions

• the present invention relates to a rice-derived stress-inducible promoter and a method of using the same. Background technology
• Plants have resistance mechanisms to respond to various environmental stresses in nature, such as drying, high temperature, freezing, and salt. Recently, as these stress-tolerant mechanisms have been elucidated at the molecular level, stress-tolerant plants using biotechnological techniques have also been created. For example, stress proteins such as LEA proteins, water channel proteins, and compatible solute synthases are induced in stressed cells, and protect cells from stress. Therefore, research was conducted to introduce genes such as barley LEA protein, tobacco detoxification enzyme, and osmotic pressure regulator (sugar, proline, glycine betaine, etc.) synthase genes into plants.
• stress proteins such as LEA proteins, water channel proteins, and compatible solute synthases are induced in stressed cells, and protect cells from stress. Therefore, research was conducted to introduce genes such as barley LEA protein, tobacco detoxification enzyme, and osmotic pressure regulator (sugar, proline, glycine betaine, etc.) synthase genes into plants.
• Non-Patent Document 1 a study was also conducted to enhance the stress tolerance of plants by introducing a gene for a transcription factor that simultaneously activates their expression by linking it to a constitutive promoter (Non-patent Document 2).
• Non-patent Document 2 a study was also conducted to enhance the stress tolerance of plants by introducing a gene for a transcription factor that simultaneously activates their expression by linking it to a constitutive promoter.
• the energy of the host plant is directed to the production of the gene product and intracellular metabolism caused by the gene product, so that the growth of the plant itself is reduced. There was a problem of being late or dwarfing.
• the present inventors have proposed genes that bind to stress-responsive elements from Arabidopsis thaliana and encode transcription factors that specifically activate the transcription of genes downstream of the element, DREB1A, DREB1B, DREB1C, DREB2A, and DREB2B were isolated (Patent Document 1). Then, they reported that by linking these genes to a stress-inducible rd29A promoter and introducing them into plants, a dwarf-resistant stress-tolerant plant can be produced (Patent Document 2). However, the rd29A promoter from the dicotyledon Arabidopsis thaliana functions in monocotyledonous plants but has weak activity. Therefore, a stress-inducible promoter having a strong activity in monocotyledonous plants has been desired.
• Non-Patent Document 1 S inozaki K, Yamaguchi-Shinozaki K., Plant Physiol. (1997) Oct; 115 (2) p327-334
• An object of the present invention is to find a stress-inducible promoter that can function effectively in monocotyledonous plants such as rice, and to provide a novel environmental stress-tolerant plant using the promoter.
• the present inventors have conducted intensive studies to solve the above problems, and as a result, have succeeded in isolating a strong stress-inducible promoter from a rice genome. Then, they found that it is possible to remarkably improve the environmental stress tolerance of monocotyledonous plants by using the promoter, and completed the present invention.
• the present invention relates to a rice-derived stress-inducible promoter.
• the promoter is specifically composed of the following DNA (a) or (b).
• the stress is a drought stress, a low-temperature stress, or a salt stress.
• the present invention also provides a recombinant vector containing the promoter.
• the vector may contain another structural gene or a regulatory gene under the control of the promoter of the present invention, and particularly preferably contains a structural gene for improving stress tolerance and a Z or regulatory gene.
• Preferable examples of structural genes that improve stress tolerance include the P5CS gene, a key enzyme for proline synthesis (Yoshiba Y. et al (1999) BBRC261), and the galactinol synthesis gene AtGolS3 gene (Taj i T. et al (2002). ) Plant J. 29: 417-426).
• the regulatory gene for improving stress resistance include a transcription factor DREB gene derived from Arabidopsis thaliana (Japanese Patent Application Laid-Open No. 2000-60558) and a rice transcription factor OsDREB gene (Japanese Patent Application No. 2001-358268, Dubouzet et al Plant J. in press), and the NCED gene (Iuchi S. et al (2001) Plant J. 27: 325-333) which is a key enzyme for the biosynthesis of the plant hormone ABA.
• Arabidopsis-derived transcription factor DREB gene and rice-derived transcription factor OsDREB gene are preferred, and rice-derived transcription factor OsDREB gene is most preferred.
• the present invention provides a transformant obtained by introducing the vector of the present invention into a suitable host.
• the transformant is a transgenic plant obtained by introducing the vector of the present invention into a host plant.
• monocotyledonous plants are desirable as host plants, and rice is desirable as monocotyledonous plants.
• the present invention provides a method for improving stress resistance of a plant by introducing the promoter of the present invention into the plant. Since the promoter of the present invention has an unprecedented strong stress-inducible promoter activity in monocots, it is more suitable for improving stress tolerance of monocots.
• Figure 1 shows the results of Northern analysis of a0022 (LIP9) under each stress load. You.
• FIG. 2 shows the nucleotide sequence of the promoter region of a0022 (LIP9).
• FIG. 3 shows the structure of the Gus expression construct.
• FIG. 4 is a graph showing the GUS activity under a drought stress load in transgenic Bako or rice transformed by introducing a GUS gene into various promoters.
• FIG. 5 is a photograph showing the results of GUS staining of a transformed rice plant into which a GUS gene has been linked to the LIP9 promoter and transfected with salt stress.
• Fig. 6 shows the results of comparison of the expression levels of the transgene and each of the target genes (LIP9 (a0022), WSI724 (a0066), salT (a2660)) in the transformed rice and wild type strains by the Northern method.
• b and c indicate the lines of the transformant, respectively.
• FIG. 7 shows the nucleotide sequence of the promoter region of a0066 (WSI724).
• FIG. 8 is a graph showing the GUS activity of a transformed rice plant into which the GUS gene has been linked to the WSI724 promoter and which has been subjected to drought stress (right: drought stress, left: control).
• FIG. 9 is a photograph showing the results of GUS staining of a transformed rice plant into which the GUS gene has been linked to the WSI724 promoter and which has been transfected with a drought stress.
• This description includes part or all of the contents as disclosed in the description of Japanese Patent Application No. 2003-80847, which is a priority document of the present application.
• the promoter of the present invention is a rice-derived promoter that is specifically induced against environmental stress such as low temperature, drying, and salt.
• the promoter of the present invention comprises a gene (stress-inducible gene) whose expression is significantly different between stressed and non-stressed plant individuals. It can be identified by screening, and then screening a sequence considered as a promoter of the gene from genomic information.
• mRNA for screening for a stress-inducible gene is prepared.
• the source of mRNA may be any part or whole of a plant such as leaves, stems, roots, and flowers.
• the plant may be seeded on a solid medium such as a GM medium, an MS medium, or a # 3 medium, and the plant may be grown under aseptic conditions, or may be grown under callus or under aseptic conditions.
• Cultured cells of a plant may be used.
• mRNA needs to be adjusted for each of the individual plants in order to compare the difference in the gene expression level between a plant individual subjected to stress and a plant individual not subjected to stress.
• the method of applying stress is appropriately set depending on the plant used. In general, drought stress can be applied by growing without water for 2-4 weeks.
• low temperature and freezing stress can be applied by growing at 15-10 ° C for 1-10 days.
• salt stress can be imposed by growing in 100-600 mM NaCl for 1 hour to 7 days.
• the rice grown by hydroponics is exposed to 10 to -4 t for low-temperature stress, 150 to 250 mM NaCK for salt stress, and dehydrated for dry stress.
• RNA fraction is extracted by the lithium chloride-urea method, the proteinase K-deoxyliponuclease method, or the like.
• poly (A) NA (mRNA) is purified from the crude RNA fraction by the affinity column method using oligo dT-cellulose or poly-U-Sepharose using Sepharose 2B as a carrier, or the batch method. obtain. If necessary, the mRNA may be further fractionated and used by sucrose density gradient centrifugation or the like.
• Screening for stress-inducible genes is based on This is done by comparing the difference in the gene expression level between the body and the unloaded plant individual.
• the method for comparing the gene expression levels is not particularly limited.
• the RT-PCR method, the real-time PCR method, the subtraction method, the differential-display method, the differential hybridization method, or the cross-hybridization method A known method such as a method can be used.
• methods using solid-state samples such as gene chips and cDNA microarrays are capable of qualitatively and quantitatively detecting the expression of thousands to tens of thousands of genes at once. It is suitable for performing the screening.
• the cDNA microarray used for the screening is not particularly limited as long as the cDNA of the monocotyledonous plant (for example, rice) from which the promoter is to be detected is spotted, and an existing one may be used or a known one may be used. It may be prepared according to a method (for example, see The Plant Cell (2001) 13: 61-72 Seki et al).
• the cDNA library can be prepared by a known method using the mRNA prepared according to the method (1) as type III.
• the cDNA to be spotted is not particularly limited as long as it is derived from monocotyledonous plants. However, monocotyledonous plants in which genomic analysis of rice or the like has progressed are preferable from the viewpoint of convenience of analysis of the genome database described later.
• the plant may be a normal (untreated) plant, but is preferably a plant that has been exposed to stress such as drought, salt, and low temperature.
• a cDNA library In preparing a cDNA library, first, using a commercially available kit (ZAP-cDNA Synthes is Kit (manufactured by STRATAGENE) etc.), reverse transcribe mRNA to synthesize single-stranded cDNA. Synthesize strand cDNA. Next, an adapter containing an appropriate restriction enzyme cleavage site is added to the obtained double-stranded cDNA, and then inserted into the cloning site of the lambda phage vector. This is packaged in vitro using a commercially available kit (for example, Gigapack III Gold packaging ext tract (manufactured by STRATAGENE)), infected to host Escherichia coli, and amplified to obtain the desired cDNA library. Is obtained.
• a commercially available kit for example, Gigapack III Gold packaging ext tract (manufactured by STRATAGENE)
• the gene expression level can be detected by a cDNA microarray by measuring the signal intensity when a sample mRNA (or cDNA) is labeled with an appropriate reagent and hybridized with a cDNA probe on the array. It is usually desirable to calculate the expression level of the gene as a comparison value with an appropriate control or the expression level ratio between two samples to be compared, taking into account the variation in the amount of cDNA probe spotted on the array. . In the case of this screening, the relative expression level of the mRNA derived from the stressed plant may be detected by using the mRNA derived from an untreated plant to which no stress is applied as a control.
• Detection is performed by labeling the control and sample mRNAs (or their cDNAs) with different fluorescent dyes (eg, Cy3 and Cy5) and hybridizing them with the cDNA probes on the array.
• fluorescent dyes eg, Cy3 and Cy5
• mRNA is extracted from an individual subjected to stress and reverse-transcribed in the presence of Cy5-labeled dCTP to prepare a Cy5-labeled cDNA.
• Cy3-labeled cDNA is prepared in the same manner.
• the labeling dye Cy3 may be used for the sample and Cy5 may be used for the control, or another appropriate labeling reagent may be used.
• the obtained fluorescence intensity is read by a fluorescence signal detector and quantified, this value corresponds to the ratio of the gene expression level of the sample to the control. If necessary, the fluorescence intensity read by the scanner may be adjusted for error or normalized for variation in each sample. Normalization can be performed based on genes that are commonly expressed in each sample, such as housekeeping genes. Further, A confidence limit line may be identified to exclude data with low correlation.
• the stress-inducible gene is identified as a gene whose expression level is significantly different between a plant individual that has been subjected to stress and a plant individual that has not.
• “significantly different” means that, for example, the expression levels of the two are different by at least three times or more when the intensity is 1000 or more.
• the gene selected in this manner is further confirmed by Northern analysis or the like to increase the expression of the gene in a stress-inducing manner. For example, exposing plants to various levels of salt, drought, temperature and other stresses in the manner described above. Then, RNA is extracted from the plant and separated by electrophoresis. The isolated RNA is transferred to a nitrocellulose membrane and hybridized with a labeled cDNA probe specific to the gene, whereby the expression level can be detected.
• examples of the stress-inducing gene selected from the rice cDNA library include a0022 (LIP9: SEQ ID NO: 2) and a0066 (WSI724: SEQ ID NO: 8) according to the present invention.
• A0022 and a0066 are ID numbers of cDNAs fixed on the microarray.
• the promoter sequence is selected as a region considered to be a promoter from the upstream region of the genomic gene having high homology with the stress-inducible gene (cDNA) on the genome. Devour. For example, based on the genomic information of the stress-inducible genes, the promoter region is estimated to be located at about l to 2 kb upstream from the estimated start codon of these genes.
• cis elements involved in the promoter activity are included in the sequence; for example, a drought stress responsive element (DRE), an abscisic acid responsive element (DRE), ABRE; absc is ic ac id respons ive e lement), low temperature stress responsive element
• DRE drought stress responsive element
• DRE abscisic acid responsive element
• ABRE absc is ic ac id respons ive e lement
• a stress-inducible transcription factor binds to this cis element, the promoter is activated, and a stress-resistance-imparting gene under its control is expressed. Therefore, if the cis element is contained in the searched upstream region, it can be said that the region is very likely to be a stress-inducible promoter.
• genomic information of a gene highly homologous to the aforementioned 0022 was obtained, and a putative LIP9 promoter sequence (SEQ ID NO: 1) was screened from its 1. kb upstream region.
• the putative WSI724 promoter sequence was screened from the upstream region of the gene having high homology to a0066 (WSI724: SEQ ID NO: 8).
• a primer is prepared based on the promoter sequence estimated in the previous section, PCR is performed using genomic DNA as type III, and the promoter is cloned.
• a reporter plasmid prepared by ligating a reporter gene downstream of the promoter is introduced into a plant, and expression of the reporter during stress loading of the plant (preferably its T, generation) is examined. Find out.
• reporter include j3 dalcuronidase (GUS: PBI121, Clontech, etc.), luciferase gene, green fluorescent protein gene, and the like. The activity is given numerically, and the expression is expressed by staining. GUS is preferred because it can be visually observed.
• the LIP9 promoter sequence (SEQ ID NO: 1) derived from the rice genome is a stress-inducible motor that exhibits high expression depending on stress such as drought, low temperature, and salt.
• LIP9 Promoter is a promoter specifically induced for all stresses.
• the structural and functional characteristics are listed below.
• the LIP9 promoter contains two cis-element DREs involved in the induction of drought stress in its structure (see Figure 2).
• the LIP9 promoter is a rice-derived transcription factor that binds to the cis element DRE and activates transcription of the downstream gene: OsDREBl gene (Japanese Patent Application
• the LIP9 promoter contains a DRE sequence to which the OsDREBl gene binds, it is predicted that it is the optimal promoter for overexpression of the OsDREB gene.
• the structure of WSI 724 Promo Yuichi also contains two DRE sequences in its structure, and the expression pattern of a0066 under stress load (dryness, salt, low temperature induction, low temperature induction Therefore, it was expected that the OsDREB gene would be an evening getter.
• the promoter of the present invention is not limited to ⁇ A having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10, but is complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10.
• DNAs that can hybridize under stringent conditions with DNAs having a unique base sequence are also included in the stress-inducible promoter of the present invention as long as they have a stress-inducible promoter activity.
• the stringent conditions refer to conditions of formamide concentration of 30 to 50%, 37 to 50 and 6 ⁇ SSC, preferably conditions of formamide concentration of 50%, 42 ° C. and 6 ⁇ SSC.
• the recombinant vector of the present invention is a vector containing the promoter of the present invention.
• the vector may contain another structural gene or a regulatory gene downstream of the promoter of the present invention in a manner capable of functioning. Suitable for such genes A good example is a structural gene and / or a regulatory gene that improves stress tolerance.
• the “functional aspect” means an aspect in which another structural gene or regulatory gene is appropriately expressed under the control of the promoter of the present invention.
• the structural gene that improves stress tolerance is a gene that encodes a protein that has a function of increasing plant resistance to environmental stress such as drought stress, low temperature stress, or salt stress; for example, LEA protein, Water channel protein, compatible solute synthase, tobacco detoxification enzyme, osmotic pressure regulator (sugar, proline, glycine benzoin, etc.) synthase, w-3 fatty acid desaturase of Arabidopsis thaliana, a cell membrane lipid modifying enzyme, orchid D9desaturase gene, P5CS which is a key enzyme of proline synthesis, and galactinol synthesis gene AtGolS3 gene.
• LEA protein Water channel protein
• compatible solute synthase tobacco detoxification enzyme
• osmotic pressure regulator sucgar, proline, glycine benzoin, etc.
• w-3 fatty acid desaturase of Arabidopsis thaliana a cell membrane lipid modifying enzyme
• orchid D9desaturase gene
• a regulatory gene that improves stress tolerance is a gene that enhances plant stress tolerance by regulating the activity of a stress-inducible promoter or the expression of a gene that imparts stress tolerance; Transcription factors derived from Arabidopsis thaliana: DREB1A, D-Ken A, DREB1B, and DREB1C genes (see Japanese Patent Application Laid-Open No. 2000-60558), rice-derived transcription factors: 0SDREB1A 0SMEB1B, OsDREBl OsDREBID, and 0sDREB2A gene (Japanese Patent Application 200 No. 358268), and the NCED gene, which is a key enzyme in the biosynthesis of the plant hormone ABA.
• the promoter of the present invention contains a specific cis element
• the LIP9 promoter according to the present invention contains two DRE sequences in its structure. Therefore, as a gene linked downstream of the LIP9 promoter, a DREB gene or an OsDREB gene (eg, OsDREBIA, OsDREB1B, OsDREBIC, OsDREB ID, 0sDREB2A gene, 0sDREB2B gene) is preferable, and OsDREB gene is particularly preferable.
• OsDREBIA, OsDREB1B, OsDREBIC, OsDREB ID, 0sDREB2A gene, 0sDREB2B gene is preferable, and OsDREB gene is particularly preferable.
• the gene to be linked downstream is the DREB gene or the OsDREB gene (eg, OsDREB gene). 1A, OsDREB 1B, OsDREBIC, OsDREBID, OsDREB2A gene, and 0sDREB2B gene) are preferable, and the OsDREB gene is particularly preferable.
• the vector of the present invention is constructed by ligating (inserting) the promoter of the present invention or the promoter of the present invention and another regulatory gene or structural gene into an appropriate vector.
• the vector for inserting the promoter is not particularly limited as long as it can be replicated in the host, and examples thereof include plasmid DNA and phage DNA.
• Plasmid DNA includes plasmids for Escherichia coli host such as pBR322, pBR325, pUC118 and pUC119, plasmids for Bacillus subtilis such as ⁇ 110 and ⁇ 5, plasmids for yeast host such as YEpl3, YEp24 and YCp50, and plant cell hosts such as pBI221 and PBI121 Plasmids for use.
• examples of diDNA include ⁇ phage.
• animal viruses such as retrovirus or vaccinia virus, and insect viruses such as baculovirus may be used as vectors.
• the promoter of the present invention can be inserted into a vector by cutting the purified DNA with an appropriate restriction enzyme and inserting the resulting DNA into a restriction enzyme site or a multicloning site of a vector.
• the recombinant vector of the present invention may further contain a splicing fern, a polyaddition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like, if desired.
• a selection marker for example, a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like can be used.
• the transformant of the present invention can be constructed by introducing the recombinant vector of the present invention into a host in such a manner that the promoter activity can be expressed.
• the host is not particularly limited as long as the promoter of the present invention can function, but is preferably a plant, and more preferably a monocotyledon such as rice.
• a plant or plant cell is used as a host, for example, cells established from rice, corn, wheat, Arabidopsis, tobacco, carrot, etc., or protoplasts prepared from the plant are used.
• a method for introducing a recombinant vector into a plant a method using polyethylene glycol of Abel et al. [Abel, H. et al.
• Some examples include the method of the electorotropolpoporation method. . 44 .. Sustotreress resistant totralans suzijeeninicnik plant
• a structural structural genetic gene and a gene for improving the resistance to storage stress are improved.
• the genetic genes of the control node are linked in a functionally operable manner and introduced into a plant. Improved resistance to environmental and environmental stresses, especially low- and low-temperature storages, freeze-freezing storages, dry and dry storages, etc. You will be able to create the totoranse sujieniininikku plant that has been raised. .
• the main host plant particularly preferred is a monocotyledonous cotyledon plant. .
• a method for introducing and introducing the propromotor motor and the like of the present invention to the host of the plant and plant accommodation host there is a method of faggloropapak teterium
• the indirect method such as the infectious dyeing method, the indirect method of introduction, the method of papartitikurrugagan, the method of popoliliechirirenren, the method of dadariko,
• various direct-induction and lead-in laws such as the Mum method and the law of law. .
• an appropriate linker is ligated if necessary, and Into the cloning vector to produce a recombinant vector for plant introduction.
• a binary vector-based plasmid such as pBI2113Not, PBI2113, ⁇ ⁇ ⁇ 21, pGA482, pGAH, or pBIG, or an intermediate vector-based plasmid such as pLGV23Neo or pNCAT ⁇ MON200 may be used. it can.
• Escherichia coli having a plasmid containing a target gene Escherichia coli having a helper plasmid (for example, PRK2013), and agrobacterium are mixed-cultured, and cultured on a medium containing rifampicillin and kanamycin, followed by conjugation for plant introduction.
• Body agrobacterium can be obtained.
• a plant terminator In order to express a foreign gene in a plant, it is necessary to arrange a plant terminator after the structural gene.
• the terminator sequence that can be used in the present invention includes, for example, a terminator derived from a nopaline synthase gene derived from a mosaic virus. However, the terminator is not limited to these as long as it is a terminator known to function in a plant.
• an effective selection gene in order to efficiently select a desired transformed cell.
• the selection markers used in this case are kanamycin resistance gene (NPTI I), octagromycin phosphotransferase (litp) gene that confers resistance to the antibiotic hygromycin to plants, and bialaphos (bialaphos). And the like.
• NPTI I kanamycin resistance gene
• litp octagromycin phosphotransferase
• bialaphos bialaphos (bialaphos).
• One or more genes selected from phosphinothricin acetyltransferase (bar) genes that confer resistance to) can be used.
• the promoter and the selectable marker gene of the present invention may be integrated together into a single vector, or two types of recombinant DNAs each integrated into separate vectors may be used.
• a desired transgenic plant By infecting a plant section from which the thus prepared agrobacterium has been collected, a desired transgenic plant can be produced.
• Transgenic plants are sown on a medium supplemented with an appropriate antibiotic, and individuals with the desired promoter and gene are selected. The selected individuals are replanted in pots containing Bonsol No. 1 or black clay and grown further.
• a transgene is similarly introduced into the genome of a host plant, but a phenomenon called a position effect in which the expression of the transgene varies depending on the location of the transgene is seen. Therefore, the transgene was more strongly expressed by performing a Northern blot test using the DNA fragment of the transgene as a probe. Transformants can be selected.
• the promoter of the present invention the confirmation that the structural gene for improving stress tolerance and the Z or regulatory gene have been incorporated into the transgenic plant and its next generation can be carried out from these cells and tissues using a conventional method. By extracting the introduced gene using PCR or Southern analysis or the like.
• analysis of the expression level and expression site of the transgene in the transgenic plant is performed by extracting RNA from cells and tissues of the plant according to a conventional method, and analyzing the mRNA of the transgene by RT-PCR or Northern analysis. This can be done by detecting Alternatively, the transcript of the introduced gene may be directly analyzed by Western analysis or the like using an antibody.
• the resistance to environmental stress of a transgenic plant into which the promoter of the present invention has been introduced can be determined by, for example, planting the transgenic plant in a flower pot containing soil containing vermiculite, perlite, bonsol, or the like, or cultivating the water in a water culture. It can be evaluated by examining the survival when various environmental stresses are applied. Environmental stress includes low temperature, drying, salt and the like. For example, resistance to drought stress can be assessed by examining its survival without water for 2-4 weeks. Resistance to low temperature and freezing stress can be evaluated by placing the cells at 15 to -10 for 1 to 10 days, growing them at 20 to 35 ° C for 2 to 3 weeks, and examining their survival rates. .
• salt stress can be evaluated by placing the cells in 100 to 600 mM NaCl for 1 hour to 7 days, growing them at 20 to 35 ° C for 1 to 3 weeks, and examining the survival rate.
• the use of the promoter of the present invention makes it possible to significantly improve stress tolerance of plants (particularly monocotyledonous plants) without dwarfing them.
• a preferred example of the transgenic plant according to the present invention is a transgenic plant obtained by introducing a vector in which the OsDREB gene is operably linked under the control of the LIP9 or WSI724 promoter into a monocot plant such as rice or wheat. Plants can be mentioned. Since the LIP9 promoter contains two DRE regions, the OsDREB gene can effectively exhibit its stress tolerance effect by binding to the cis element. Similarly, since the WSI724 promoter contains two DRE regions per night, the expression of the OsDREB gene can be increased to improve plant stress tolerance.
• a vector in which the OsDREB gene is operably linked under the control of the LIP9 or WSI724 promoter into a monocot plant such as rice or wheat. Plants can be mentioned. Since the LIP9 promoter contains two DRE regions, the OsDREB gene can effectively exhibit its stress tolerance effect by binding to the cis element. Similarly, since the WSI724 promoter contains two DRE regions per night, the expression of
• Rice (Nipponbare) cultivated hydroponically for 2-3 weeks was dried, salted and treated at low temperature.
• the drying treatment was air-dried at room temperature, the salt treatment was cultivated with a 250 mM NaCl solution, and the low-temperature treatment was cultivated at 4 ° C.
• the rice subjected to each stress treatment was frozen in liquid nitrogen, and total RNA was extracted by the guanidine thiosinate-cesium chloride method, and mRNA was prepared using an Oligo (dt) -cellulose column.
• the obtained mRNA was transformed into type III, cDNA was synthesized using HybriZAP-2.1 two-hybrid cDNA Gigapack cloning kit (manufactured by STRATAGENE), inserted into the EcoRI-XhoI cleavage site of HybriZAP-2.1 phagemid vector, and cloned. did.
• the phagemid layer A was packaged using Gigapack III Gold packaging extract (manufactured by STRATAGENE).
• the obtained lambda phage particles containing cDNA were infected to host Escherichia coli and amplified, and then collected as a phage suspension. Approximately 1500 independent clones were selected by sequencing the above cDNA clones.
• the selected clones were amplified by PCR, stamped on poly-L-lysine-coated micro slide glass (model S7444; Matsimami) using GTMASS System (Nippon Laser and Electronic Laboratory), and then UV-crossed.
• a rice cDNA microarray was prepared by linking (The Plant Cell (2001) 13: 61-72 Seki et al). 2.
• MRNA was purified from each of the rice plants that had been subjected to the same dry, salt, and low-temperature stress treatments as described in the previous section, or 100 xM abscisic acid treatment (5 hours or 10 hours), and untreated rice.
• CDNA microray analysis was performed using untreated rice-derived mRNA as a control, and mRNA from each stress or abscisic acid-treated rice as a sample, using the dual fluorescent labeling method with Cy3 and Cy5, respectively. .
• a gene having an intensity of 1: 1000 or more and having an expression level of 3 times or more as compared to the control was selected as a candidate for a stress-inducible gene.
• a0022 LIP9: SEQ ID NO: 2
• a0066 WSI724: SEQ ID NO: 8 were selected as stress-inducible genes.
• the expression characteristics of the gene selected in the previous section were analyzed by Northern hybridization.
• rice was exposed to abscisic acid, dry, low temperature, and saline stress, and samples were taken at 0, 1, 2, 5, and 10 hours, respectively. Abscisic acid, dryness, low temperature, and salt stress were applied in the same manner as in 1., and water stress was applied by immersion in pure water.
• Total RNA was prepared from each sample, electrophoresed, and the expression of each gene was observed by the Northern method. The results are shown in Figure 1.
• expression of a0022 is induced by the stresses of abscisic acid, drought, low temperature, and salt.
• the expression of abscisic acid, drought, and salt is increased at an early time, and is slow at a low temperature. During the period, the expression increased.
• the expression pattern of a0066 during stress loading (induced by drought, salt and low temperature, and induced by low temperature is slower than that of dry and salt) is expected to be OsDREB overnight.
• the cDNA selected as the stress-inducible gene in Example 1: a0022 was analyzed using the DDBJ rice genome database. Was used to search for homologous sites. As a result, a sequence 1 lkb upstream from the start codon of the homologous gene to the 5 'side was selected as a promoter sequence (SEQ ID NO: 1). Similar search was also performed for a0066 (WSI724: SEQ ID NO: 8), and its promoter sequence (SEQ ID NO: 10) was selected.
• FIG. 2 shows the structure of the promoter region of LIP9.
• LIP9 has two cis sequences DRE ((A / G) CCGAC) in its structure.
• Fig. 7 shows the structure of one region of the promoter of WSI724. It was also confirmed that the WSI724 promoter had two cis sequences DRE ((A / G) CCGAC) in its structure.
• a primer was designed based on the selected motor sequence, and PCR was performed using rice genomic DNA as a type II clone for cloning.
• the primer sequences and PCR conditions used are as follows.
• Reverse primer 5'-CCGGATCCTCGATCGATGGATTCAGCTA-3 '(SEQ ID NO: 4)
• Primer sequence for WSI 724 promoter 5'-CCGGATCCTCGATCGATGGATTCAGCTA-3 '(SEQ ID NO: 4)
• the G-uM plasmid generated by replacing the promoter site of pBIG29APHSNot with maize ubiquitin promoter overnight was digested with BamHI-HindIII and ligated with the similarly digested LIP9 promoter fragment.
• a plasmid incorporating the LIP9 promoter was digested with BamHI-EcoRI, and PBI 221 (Clontech) was similarly digested with BamHI-EcoRI and ligated to the excised Gus gene to prepare a Gus expression construct (G_LIP9: GUS). ( Figure 3).
• Plasmid G-LIP9 GUS was introduced into agrobacterium EHA105, which was washed with 10% glycerol after culturing, by electroporation to produce agrobacterium EHA105 (G-LIP9: GUS). The rice was infected with this agrobacterium EHA105 (G-LIP9: GUS) as follows to prepare a transformant.
• Rice seeds are immersed in 70% ethanol for 1 minute, sterilized by immersion in 2% sodium hypochlorite for 1 hour, then washed with sterile water, and N6D solid medium (per liter: CHIHN6) Basal Salt Mixture (Sigma) 3.98 g, sucrose 30 g, myo-inositol 100 mg, casamino acid 300 mg, L-proline 2878 mg, glycine 2 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, thiamine hydrochloride lmg, 2,4-D 2 mg Then, 9 seeds were inoculated on a plate of Gelrite 4g, H5.8), and cultured for 24 days to induce callus. The callus of about 20 seeds was transferred to a new N6D solid medium and cultured for another 3 days.
• N6D solid medium per CHIHN6
• Basal Salt Mixture Sigma 3.98 g, sucrose 30 g, myo-inositol 100
• a YEP medium containing 5 ml of rifampicillin 100 mg / l and kanamycin 20 mg / l containing the above-mentioned agrobacterium EHA105 (per liter: Bacto peptone 10 g, Bacto yeast extract 10 g, The cells were cultured with 5 g of NaCl, 0406 mg of MgCl 6 H 2 (pH 7.2) at 28 for 24 hours.
• AAM medium (1 liters per including Asetoshiringon of this ⁇ glow Park Teri ⁇ beam 20mg / l: MnS0 4 '5H 2 010mg, H 3 B0 3 3mg, ZnS0 4' 7H 2 02mg, NajMoO ⁇ - 2H, 0250 g, CuS0 4 -5H 2 025 g, CoCl 2 -6H 2 025 ig, KI 750 zg, CaCl 2 -2H 2 0150mg, MgS0 4 '7H 2 0250mg, Fe -.
• EDTA 40mg Na P0 4 2H 2 0150mg, lmg nicotinic acid, Thiamine hydrochloride 10 mg, pyridoxine hydrochloride lmg, myo-inositol 100 mg, L-arginine 176.7 mg, glycine 7.5 mg, L-glucamine 900 mg, aspartic acid 300 mg, KC13 g, pH 5.2) so that 0.D. becomes 0.1.
• a thin, 20 ml agrobacterium suspension was prepared.
• the agrobacterium suspension was added to the callus cultured for 3 days as described above, and mixed for 1 minute. Then placed on the callus sterile Bae one Pataoru extra ⁇ Glo Park Teri ⁇ beam suspension after removal of the, 2N6-AS solid medium (1 liters per lined with sterile filter paper: CHU [N S] Basal Salt Mixture 3.98g, sucrose 30g, glucose 10g, myo-inositol 100mg, casamino acid 300 mg, glycine 2 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, thiamine hydrochloride lmg, 2,4-D 2 mg, acetosyringone 10 mg, gellite 4 g, H5.2) at 25 ° C for 3 days, dark Cultured underneath.
• this callus was regenerated in a regeneration medium (per liter: mixed salt for Murashige-Skoog medium (Nippon Pharmaceutical) 4.6 g, sucrose 30 g, sorbitol 30 g, casamino acid 2 g, myo-inositol 100 mg, glycine 2 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, thiamine hydrochloride 0.1 mg, NAA 0.2 mg, forceinetin 2 mg, carbenicillin 250 nig, hygromycin 50 mg, agarose 8 g, pH 5.8). Every week, transplanted to a new medium, redifferentiated and the shoots grew to about lcm.
• a regeneration medium per liter: mixed salt for Murashige-Skoog medium (Nippon Pharmaceutical) 4.6 g, sucrose 30 g, sorbitol 30 g, casamino acid 2 g, myo-inositol 100 mg,
• Hormone-free medium per liter: mixed salt for Murashige-Skoog medium (Nippon Pharmaceutical) 4.6 g, The transplantation was performed on 30 g of sucrose, 2 mg of glycine, 0.5 mg of nicotinic acid, 0.5 mg of pyridoxine hydrochloride, 0.1 mg of thiamine hydrochloride, 50 mg of hygromycin, 2.5 g of gellite, pH 5.8). Plants grown to about 8 cm on a hormone free medium were transferred to a flower pot containing synthetic granular soil bonsol No. 1 (manufactured by Sumitomo Chemical Co., Ltd.) to obtain seeds of transformed plants.
• rd29A promoter overnight (Nature Bio technology (1999) 17, 287-291) or the 35S promoter and the salT promoter (SEQ ID NO: 5) were linked upstream of the GUS gene, and rice and / Or introduced into tobacco.
• the salT promoter is a stress-inducible promoter isolated from the rice genome by the same screening as the LIP9 promoter.
• the ID number of the cDNA on the microarray corresponding to the salT promoter is a2660.
• the salT promoter has no specific cis sequence in its structure, but its expression has been confirmed to be induced by abscisic acid, drought, low temperature, and salt stress (see Japanese Patent Application No. 2002-377316). ).
• the generation grows the regenerated plants in plant corn for 3 to 5 weeks, divides the grown leaves into two equal parts, and controls one of them as a control to air-dry at room temperature to dry stress. Exposed.
• Fig. 4 shows the GUS activity of various promoter-transformed plants under drought stress.
• the salT promoter and the LIP9 promoter showed stronger stress inducibility than the rd29A promoter overnight.
• LIP9 Promo overnight showed a strong activity of about 1 times that of salT.
• the LIP9 promoter also showed one stress-inducible promoter activity in tobacco, a dicotyledon, but its activity was weaker than that of rice.
• the G-ub i plasmid which was produced by substituting the ubiquitin promoter of maize overnight at the promoter site of pBIG29APHSNot, was digested with BamHI-Hind II, and ligated to the similarly digested WSI 724 promoter fragment.
• the plasmid containing the WSI 724 promoter was cut with BamHI to blunt the ends, and then ligated to the site cut with Smal of the pBIG vector to prepare a GUS expression construct (WSI724: GUS).
• the plasmid WSI724: GUS was The glycerol-washed agrobacterium EHA105 was introduced by an electroporation method to prepare an agrobacterium EHA105 (WSI724: GUS). The rice was infected with this agrobacterium EHA105 (WSI724: GUS) to prepare a transformant.
• the resulting GUS-expressing transformed rice was exposed to drought stress in the same manner as in Example 3, and the GUS activity was measured from the change in fluorescence intensity due to degradation of 4-methylumbel 1 iferyl-j3-D-glucuronide.
• higher GUS activity was observed in the transformed rice leaves subjected to the drought stress (cut and left for 24 hours) as compared to the control (cut and frozen immediately) leaves.
• GUS staining of the transformed rice plants after loading with dry stress (24 hours) showed GUS activity in both roots and leaves.
• Example 3 a transformant in which the OsDREB IA gene (SEQ ID NO: 6) or the DREB 1C gene (SEQ ID NO: 8) was introduced into rice under the control of the maize ubiquitin promoter or the 35S promoter was prepared. . Then, the mRNA levels of transgenic transgenes OsDREBIA and DREB1C and LIP9 (a0022), WSI 724 (a0066), and salT (a2660), which are considered to have altered the expression of the transgene, were examined by Northern analysis. Was.
• OsDREBIA gene SEQ ID NO: 6
• DREB 1C gene SEQ ID NO: 7
• LIP9 gene QQ22 SEQ ID NO: 2
• WSI724 gene a0066: SEQ ID NO: 8
• salT gene a2660: SEQ ID NO: 9
• rice plants transformed with only one vector were similarly prayed.
• Transformed rice was selected in a 0.1% benzilate solution containing 30 mg / ml hygromycin for 5 days, and then replanted in a pot containing Bonsol No. 1 and grown for 12 days. Wild strains were similarly raised. Prepare total RNA from each plant and run Then, the expression of each gene was observed by the Northern method in the same manner as in Example 1.
• Figure 6 shows the results. In the figure, a, b and c indicate the lines of the transformants, respectively.
• a DRE sequence is present on the LIP9 and WSI724 promoters, and the LIP9 gene and WSI724 gene are highly expressed in OsDREBlA gene overexpressors.
• LIP9 and WSI724 were considered to be target genes for OsDREB genes such as OsDREBlA. Therefore, LIP9 and WSI724 promoters were estimated to be optimal promoters for overexpressing OsDREB gene.
• G_ubi and G35S-SliA were prepared as follows.
• pBIG plasmid Nucleic Acids Research 18: 203 (1990)
• G-ubi and G35S-ShA were each digested with BamHI, and then ligated to a similarly digested OsDREBIA gene fragment encoding a rice transcription factor using ligation high (manufactured by Toyobo Co., Ltd.).
• E. coli DH5a was transformed with the product.
• plasmids pBE35S: 0sDREBlA, G-ubi: 0sDREBlA and G35S-ShA: 0sDREBlA were purified from the culture. Next, these nucleotide sequences were determined, and those having the OsDREBIA gene bound in the sense direction were selected.
• the above-mentioned plasmid pBE35S Escherichia coli DH5a having OsDREBIA, Escherichia coli HB101 having helper plasmid PRK2013, and Agrobacterium C58 were mixedly cultured at 28 ° C. for 24 hours using LB agar medium. The resulting colonies were picked up and suspended in 1 ml of LB medium. This suspension 101 was spread on an LB agar medium containing 100 mg / K of rifampicillin and 20 mg / l of kanamycin, and cultured at 28 ° C for 2 days to obtain a conjugate agrobacterium C58 (pBE35S: OsDREBlA). Was.
• the plasmids G_ubi: OsDREBIA and G35S-ShA: OsDREBIA described above were introduced into agrobacterium EHA105, which had been cultured and washed with 10% glycerol, by the electroporation method. : OsDREBIA) and Agrobacterium EHA105 (G35S-Sh ⁇ : OsDREBIA) were obtained, respectively. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial potential
• a stress-inducible promoter that can function effectively in monocotyledonous plants.
• the promoter contains a DRE sequence, and thus, under the control of the OsDREB gene or the like, can be linked to a plant to produce a strong stress-resistant transgenic plant in monocotyledonous plants such as rice. Can be. Sequence listing free text

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