CN100348723C - Reverse-tolerant concerned gene of rice and its coding protein and use - Google Patents

Abstract

The present invention discloses a reverse-resistant concerned gene of paddy rice, and a coding protein and application thereof. The present invention aims to provide a reverse-resistant concerned gene of paddy rice and a coding protein thereof, and a method for enhancing reverse resistance of plants by using the reverse-resistant concerned gene of paddy rice. The reverse-resistant concerned gene of paddy rice of the present invention is one of the following nucleotide sequences: a DNA sequence of which the protein is homologous with the DNA sequence defined in the SEQ ID No 1 in the sequence table and has the same coding function, and a polynucleotide sequence in the amino acid residue sequences in the SEQ ID No 2 in the coding sequence table. The experiment proves that when the reverse-resistant concerned gene of paddy rice is transferred to paddy rice, the drought resistance, the high salt resistance, the low temperature resistance, etc. of paddy rice can be obviously enhanced. The present invention can play an important role in cultivating plants with enhanced reverse resistance.

Description

A kind of gene of anti-retrocorrelation of paddy rice and proteins encoded and application

Technical field

The present invention relates to a kind of gene of anti-retrocorrelation the and proteins encoded and the application of paddy rice in the genetically engineered field, particularly a kind of gene of anti-retrocorrelation of paddy rice and proteins encoded thereof and utilize this gene to improve the method for plant stress tolerance.

Background technology

Under the adverse environmental factor of environment-stress such as arid, high salt and low temperature, plant can be made corresponding adjustment on molecule, cell and integral level, with the injury that reduces environment to the full extent and caused and survived.Many genes are subjected to stress-inducing to express (Shinozaki, K.and Yamaguchi-Shinozaki, K. (1994) .Molecular responses to drought and cold stress.Curr.Opin.Biotechnol 7,161-167; Thomashow, M.F. (1999) .Plant cold acclimation:freezing tolerancegenes and regulatory mechanisms.Annu Rev Plant Physiol Plant Mol Biol 50,571-599), the product of these genes not only can be participated in the stress response of plant directly, and can regulate other Expression of Related Genes or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.With coerce relevant gene product and can be divided into two big class (Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) .Improving plantdrought, salt, and freezing tolerance by gene transfer of a singlestress-inducible transcription factor.Nature biotechnol.17,287-291): the product of first kind genes encoding comprises ionophorous protein, aquaporin, the osmoregulation factor (sucrose, proline(Pro) and trimethyl-glycine etc.) synthetic enzyme etc. participates in the gene product that plant stress is replied directly; The product of second genoid coding comprises the protein factor that participates in coercing relevant signal transmission and genetic expression adjusting, as protein kinase, transcription factor etc.Wherein, transcription factor plays an important role in the gene expression regulation that plant stress is replied.

In the whole genome of plant, the gene of the encoding transcription factor has accounted for greatly, such as, the gene of the encoding transcription factor has 1500 (Riechmann at least in the Arabidopis thaliana, J.L., Heard, J., Martin, G., Reuber, L., Jiang, C.-Z., Keddie, J., Adam, L., Pineda, O., Ratcliffe, O.J., Samaha, R.R., Creelman, R., Pilgrim, M., Broun, P., Zhang, J.-Z., Ghandehari, D., Sherman, B.K., and Yu, G.-L. (2000) .Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes.Science 290,2110), account for more than 5% of whole genome.These transcription factors belong to big gene family mostly, and the gene family that has can comprise many subtribes again, and some transcription factor family is that plant is peculiar.At present known in plant with coerce relevant transcription factor and mainly contain: AP2 (APETALA2)/EREBP (the ethyleneresponsive element binding protein) transcription factor family with AP2 structural domain, bZIP (basic region/leucine zipper motif transcription factors) the class transcription factor that contains alkalescence zone and leucine zipper, the WRKY transcription factor family that contains conservative WRKY aminoacid sequence, the MYC family and MYB family of containing alkaline helix-loop-helix (bHLH) and leucine zipper with tryptophane bunch (Trp cluster).These five transcription factor families, except that WRKY family not the water of involved in plant coerce the reaction, other four families all participate in regulating the environment stress reaction of plant to arid, high salt and low temperature etc.Wherein, AP2/EREBP class transcription factor extensively exists in higher plant, and it is the peculiar class transcription factor of plant, in recent years, at Arabidopis thaliana, tobacco, corn, paddy rice, report (Elliott, R.C. are all arranged in soybean and the rape, Betzner, A.S., Huttner, E., Oakes, M.P., Tucker, W.Q., Gerentes, D., Perez, P., and Smyth, D.R. (1996) .AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic rolesin ovule development and floral organ growth.Plant Cell 8,155-168; Kevin, M.K., Leonore Reiser, and Robert, L.F. (1996) .The AINTEGUMENTA gene ofArabidopsis required for ovule and female gametophyte development is relatedto the floral homeotic gene APETALA2.Plant Cell 8,137-153), this shows AP2/EREBP class transcription factor ubiquity and have vital role in higher plant.

AP2 transcription factor family member's principal feature is to contain a DNA land high conservative, that have three βZhe Dies, be called AP2 territory (AP2 domain), sequential analysis shows, AP2 albumen except the common trait that contains an AP2 territory at least, other aminoacid sequence height difference.In the AP2 territory, two conserved sequences are arranged, one is the YRG district of alkalescence, one section sequence of the high conservative of forming by 19-22 amino acid, it contains these several conservative aminoacid sequences of YRG, and the YRG element has been proved to be combining of participation and DNA; Second conserved regions is the RAYD district, one section aminoacid sequence that the RAYD district is made up of 42-43 amino acid, its core texture is an amphiphatic α-Luo Xuanjiegou of being made up of 18 amino acid, aminoacid sequence high conservative (the Okamour of this core texture, J.K., Brian Caster, Raimundc Villarroel, Marc Van Montagu, andJofuku, K.D. (1997) .The AP2 domain of APETALA2 defines a large new familyof DNA binding proteins in Arabidopsis.Proc.Natl.Acad.Sci.U.S.A 94,7076-7081), the RAYD element may participate in albumen and proteic interaction.Simultaneously, research is also found, in YRG and RAYD conserved regions, it is being extremely conservative that the several amino acid residue is arranged, such as the 40th amino acid---glycine in the RAYD conserved regions, in all AP2 albumen, all be immovable, it brings into play its physiological function very important (Jofuku, K.D., den Boer for AP2 albumen, B.G., Van Montagu, M., and Okamuro, J.K. (1994) .Control of Arabidopsis flower and seed development by thehomeot ic gene APETALA2.Plant Cell 6,1211-1225).

According to the difference of AP2 territory number, can be divided into two subfamilies to the AP2 transcription factor family: AP2-like family and EREBP-like family.The topmost feature of AP2-like family member is to contain two AP2 territories in its protein structure, these two AP2 territories are connected by a hinge area of being made up of 25-26 amino acid, the aminoacid sequence of hinge area also is a high conservative, another feature of AP2-like family is all to contain the aminoacid sequence of the such one section high conservative of WEAR/WESH in its YRG sequence.AP2-like family mainly works in development of plants, such as arabidopsis gene AP2 (homeotic gene APETALA2) is one of member in this family, it not only participates in the foundation of floral meristem, the differentiation of floral organ, can also regulate and control simultaneously the expression of other homeotic gene relevant with flower development, in Arabidopis thaliana flower development process, play an important role, and, it also participates in the adjusting (Jofuku of Arabidopis thaliana ovary development and seed development process, K.D., den Boer, B.G., VanMontagu, M., and Okamuro, J.K. (1994) .Control of Arabidopsis flower and seeddevelopment by the homeot ic gene APETALA2.Plant Cell 6,1211-1225), another member ANT of this family plays a crucial role in the ovary development of Arabidopis thaliana and Female Gametophyte process.

The member's of EREBP-like family feature is only to contain an AP2 territory, and in the YRG conserved regions, there is not WEAR/WESH, but by 7 amino acid---the structure (Okamour of the high conservative that WAAEIRD forms, J.K., Brian Caster, Raimundc Villarroel, Marc Van Montagu, and Jofuku, K.D. (1997) .The AP2 domain of APETALA2 defines a large new family of DNA bindingproteins in Arabidopsis.Proc.Natl.Acad.Sci.U.S.A 94,7076-7081).According to estimates, in Arabidopis thaliana, there are (Riechmann, J.L. more than 124 members in EREBP-like family, Heard, J., Martin, G., Reuber, L., Jiang, C.-Z., Keddie, J., Adam, L., Pineda, O., Ratcliffe, O.J., Samaha, R.R., Creelman, R., Pilgrim, M., Broun, P., Zhang, J.-Z., Ghandehari, D., Sherman, B.K., and Yu, G.-L. (2000) .Arabidopsistranscription factors:genome-wide comparative analysis among eukaryotes.Science 290,2110), family is so huge, and perhaps the biological action widely that is participated in plant materials with the EREBP-like transcription factor is relevant.EREBP-like transcription factor involved in plant is to the stress response reaction of low temperature, arid, cause of disease infringement, mechanical wounding etc., and be subjected to the adjusting (Singh of ethene, Whitfield's ointment, jasmonic acid, K., Foley, R.C., and Onate-Sanchez, L. (2002) .Transcription factors in plantdefense and stress responses.Curr.Opin.Plant Biol 5,430-436).

The EREBP-like transcription factor can combine with GCC box (GCC box) and two kinds of cis-acting elements of DRE/CRT (dehydrationresponsive element/C-repeat).The sequence of GCC box is AGCCGCC, it is present in the promotor of the plant gene relevant with resistance, it is necessary and enough (Fujimoto for the expression of ethene inductive PR gene (pathogenesis related genes), S.Y., Ohta, M., Usui, A., Shinshi, H., and Ohme-Takagi, M. (2000) .Arabidopsisethylene-responsive element binding factors act as transcriptionalactivators or repressors of GCC box-mediated gene expression.Plant Cell 12,393-404); And the DRE/CRT sequence is present in the promotor of plant to the relevant gene of the resistance of environment-stress such as arid, high salt and low temperature, the attack that their overexpression can make plant opposing arid, high salt and damage to plants caused by sudden drop in temperature, so, the EREBP-like family member the biology of plant coerce with abiotic stress in all play important regulatory role.Simultaneously, they also find, in Arabidopis thaliana, some EREBP albumen play activation in the transcriptional control of GCC mediation, other then rise transcribes restraining effect (Fujimoto, S.Y., Ohta, M., Usui, A., Shinshi, H., and Ohme-Takagi, M. (2000) .Arabidopsis ethylene-responsive element bindingfactors act as transcriptional act ivators or repressors of GCC box-mediatedgene expression.Plant Cell 12,393-404).

Also do not report at present with AP2-like subfamily bonded cis-acting elements, but the sequence of its identification sequence with the identification of EREBP-like subfamily probably is different, because there is not (Allen in direct and GCC box bonded EREBP-like member's aminoacid sequence in AP2-like subfamily member, M.D., Yamaguchi, K., Ohme-Takagi, M., Tateno, M., and Suzuki, M. (2003) .A novel mode of DNArecognition by a b-sheet revealed by the solution structure of the GCC-boxbinding domain in complex with DNA.EMBO J.17,5484-5496).

DREB (DRE-binding protein) class transcription factor is a member in the EREBP-like subfamily in the AP2 family.According to its structural homology, the DREB class transcription factor in the Arabidopis thaliana can be divided into it DREB1 and DREB2 two big classes.DREB1 comprises CGF1/DREB1B, CBF2/DREB1C, CBF3/DREB1A, CBF4/DREB1D, six members of DRB1E and DREB1F, CBF1/DREB1B wherein, CBF2/DREB1C, CBF3/DREB1A is positioned on the tetrasome of Arabidopis thaliana, thorough (the Gilmour of the comparison of research at present, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M., and Thomashow, M.F. (1998) .Low temperature regulation of theArabidopsis CBF family ofAP2 transcriptional activators as an early step in cold-induced COR geneexpression.Plant J.16,433-442; Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) .Twotranscription factors, DREB1 and DREB2 with an EREBP/AP2 DNA binding domainseparate two cellular signal transduction pathways in drought-andlow-temperature-responsive gene expression, respectively, in Arabidopsis.Plant Cell.10,1391-1406), CBF4/DREB1D is positioned at (Nakamura, Y., Sato on the 5th karyomit(e), S., Asamizu, E., Kaneko, T., Kotani, H., Miyajima, N., and Tabata, S. (1998) .Structural analysis of Arabidopsis thaliana chromosome 5:VII.Sequence features of the regions of 1,013,767 bp covered by sixteen physicallyassigned P1 and TAG clones.DNA Res.5,297-308; Thomashow, M.F., Gilmour, S.J., Stockinger, E.J., Jaglo-Ottosen, K.R., and Zarka, D.G. (2001). Roleof Arabidopsis CBF transcriptional activators in cold acclimation.PlantPhysiol 112,171-175), DREB1E and DREB1F are positioned at (Sakuma on article one karyomit(e), Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K., and Yamaguehi-Shinozaki, K. (2002) .DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible geneexpression.Biochem Biophys Res Commun.290,998-1009).CBF1, CBF2 and CBF3 are expressed by low temperature induction, and be not subjected to arid and high salt to induce (Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) .Twotranscript ion factors, DREB1 and DREB2 with an EREBP/AP2 DNA binding domainseparate two cellular signal transduction pathways in drought-andlow-temperature-responsive gene expression, respectively, in Arabidopsis.Plant Cell.10,1391-1406; Shinwari, Z.K., Nakashima, K., Miura, S., Kasuga, M., Seki, M., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) .AnArabidopsis gene family encoding DRE/CRT binding proteins involved inlow-temperature-responsive gene expression.Biochem Biophys Res Commun.250,161-170).Different investigators is also inconsistent for the report of the expression pattern of DREBl, perhaps is that different experiment conditions causes but at present.

DREB2 class transcription factor comprises DREB2A and two member (Nakashima of DREB2B, K., Shinwari, Z.K., Sakuma, Y., Seki, M., Miura, S., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000) .Organization and expression of two Arabidopsis DREB2 genesencoding DRE-binding proteins involved in dehydration-andhigh-salinity-responsive gene expression.Plant Mol Biol.42,657-665).DREB2A is positioned on the 5th karyomit(e) of Arabidopis thaliana, and DREB2B is positioned on the 3rd karyomit(e).Except all containing AP2 territory and nuclear localization signal, they do not have tangible homology with DREB1 class transcription factor on sequence.Contain one section conserved sequence that is rich in serine/threonine in the DREB2 proteinoid, the expression pattern of DREB2 is different with DREB1, they are subjected to arid and high salt to induce (Gilmour, S.J., Zarka, D.G., Stockinger, E.J., Salazar, M.P., Houghton, J.M., and Thomashow, M.F. (1998) .Low temperatureregulation of the Arabidopsis CBF family of AP2 transcriptional activatorsas an early step in cold-induced COR gene expression.Plant J.16,433-442; Sakuma, Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K., andYamaguchi-Shinozaki, K. (2002) .DNA-binding specificity of the ERF/AP2domain of Arabidopsis DREBs, transcript ion factors involved in dehydration-and cold-inducible gene expression.Biochem Biophys Res Commun.290, not expressed by low temperature induction.So DREB1 may be different with the signal transduction path of DREB2 under the stress-inducing condition.

DREB class transcription factor can combine with the DRE/CRT cis-acting elements, the expression of gene that contains DRE/CRT cis-acting elements (TACCGACAT) in the evoked promoter, these genes all are and the resistance relevant gene of plant to environment-stress such as arid, high salt and low temperature.The core sequence of DRE/CRT is ACCGAC, be prevalent in the promotor of arid, high salt or low temperature stress response gene, such as rd29A, kin1, cor6.6, cor15a, rd17 and erd10 etc., the gene induced expression under these stress conditions is played regulating and controlling effect.So in plant was replied the molecule of arid, high salt or low temperature stress, a DREB transcription factor can be regulated and control the expression of gene relevant with stress tolerance that contains the DRE/CRT element in a group promotor.

Overexpression DREB class transcription factor can significantly strengthen plant is coerced environment to arid, high salt or low temperature etc. resistance in plant.Overexpression CBF1 in Arabidopis thaliana, transgenic plant are than there not being genetically modified plant that low temperature is had stronger patience (Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O., and Thomashow, M.F. (1998) .Arabidopsis CBF1overexpression induces COR genes and enhances freezing tolerance.Science280,104-106); Overexpression CBF3, transgenic arabidopsis has obtained the higher resistivity (Gilmour to arid, high salt and low temperature stress, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D., andThomashow, M.F. (2003) .Overexpression of the Arabidopsis CBF3transcriptional activator mimics multiple biochemical changes associatedwith cold acclimation.Plant Physiol.124,1854-1865; Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) .Improving plantdrought, salt, and freezing tolerance by gene transfer of a singlestress-inducible transcription factor.Nature biotechnol.17,287-291), but the height that also causes simultaneously transgenic plant is downgraded, and replaces constitutive promoter just can overcome this negative effect with inducible promoter such as the promotor of rd29A, can not cause the change of other proterties of plant in raising plant stress tolerance energy.

Also exist in the paddy rice and can discern DRE/CTR cis-acting elements or its core sequence and bonded DREB class transcription factor with it.2003, Dubouzet etc. separate the cDNA that has obtained with the sequence of five Arabidopis thaliana DREB class transcription factor homologs from paddy rice, and called after OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, OsDREB2A, wherein, OsDREB1A, OsDREB1B are induced by low temperature, high salt and physical abuse, OsDREB2A is expressed by arid, high salt and low temperature induction, and the transfer-gen plant of overexpression OsDREB1A and OsDREB2A has all showed higher drought-resistant, high salt and cryogenic performance in Arabidopis thaliana.And, in barley, rye, tomato and rape, obtained in recent years with Arabidopis thaliana DREB transcription factor sequence on same or analogous gene on the homology, function, these results of study show, DREB class transcription factor is extensively to exist in vegitabilia, and in different plant materialss, they are carrying out similar or identical functions.

Result to transcription factor research shows in a large number, a transcription factor may implement to regulate control to a lot of genes of a class correlated character, thereby effectively change the correlation properties of plant, simultaneously a lot of results of study also show, in plant materials, comprise Arabidopis thaliana, tobacco, barley and corn etc., the plant of overexpression DREB class transcription factor has all showed higher opposing arid, high salt and low temperature stress resistance of reverse energy.So, go to improve the resistance of reverse of crop by transgenic method, be a kind of effective way.

Paddy rice is most important in the world food crop, also is monocotyledonous model plant, and it provides food for nearly half the population in the whole world.Yet environment stresses such as arid, high salt and low temperature all can cause the large-area underproduction of paddy rice every year in worldwide, and the whole world has 40% paddy fields to be subjected in various degree environment-stress approximately.But along with the continuous increase of global population and the minimizing year by year of acreage under cultivation, improve rice yield and become the urgent challenge that the whole world is faced jointly, and reduce the production loss that environment stress caused, just can alleviate crisis in food to a great extent.

The upland rice conduct concerns the very close relative extremely ecotypic cereal crop that but has with paddy rice parent source, and research and excavation to upland rice genes involved resource have very important meaning.On the one hand, upland rice and paddy rice close source relation on evolving is very close, can make full use of the whole genome sequence information of paddy rice; On the other hand, difference on the regulatory mechanism of transgenic acceptor itself, far and near closely related with the close source relation of gene source (genetic donor material) and transgenic acceptor to a great extent, the goal gene that therefore imports nearly source crop can farthest overcome these obstacles.The gene of separating clone from upland rice will can be used for the anti-against genetically engineered of paddy rice and other cereal crop, cultivate real anti-contrary kind.

The innovation and creation content

A kind of gene of anti-retrocorrelation the and the proteins encoded thereof that the purpose of this invention is to provide paddy rice.

The paddy rice gene of anti-retrocorrelation the provided by the present invention, name is called OsCBF4, derives from Oryza paddy rice (Oryzasativa L.), is one of following nucleotide sequence:

1) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding;

2) SEQ ID № in the code sequence tabulation: the polynucleotide sequence of 2 amino acid residue sequences.

Wherein, SEQ ID № in described and the sequence table: 1 dna sequence dna that limits has 90% above homology, and coding identical function protein DNA sequence can be the SEQ worker D № in the sequence table: 1 (deriving from Brazilian upland rice) or SEQ ID №: 3 (deriving from Japanese fine); SEQ ID № in their equal code sequence tabulations: 2 amino acid residue sequence.

SFQ ID № in the sequence table: 1 and SEQ ID №: 3 cDNA sequence is by 660 based compositions, and their open reading frame is from 5 ' end the 1st to the 660th bit base.

A kind of gene coded protein of anti-retrocorrelation of paddy rice, name is called OsCBF4, derive from Oryza paddy rice (Oryzasativa L.), be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

The protein that the amino acid residue sequence of sequence 2 is made up of 219 amino-acid residues in the sequence table.

Contain expression carrier of the present invention and clone, all belong to protection scope of the present invention as recombinant vectors pMD18-T Vector-OsCBF4 and intestinal bacteria (E.coli) the DH5 α that contains this gene.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification OsCBF4.

Second purpose of the present invention provides a kind of method that improves plant stress tolerance, may further comprise the steps:

(a) make up a kind of dna fragmentation, make it comprise the OsCBF4 gene;

(b) with constructed dna fragmentation or contain the plasmid transformed plant cells of described dna fragmentation;

(c) filter out by the plant transformed cell;

(d) make by plant transformed cell regeneration and go out whole plant, obtain the transgenic plant that resistance of reverse improves.

Make in the step (d) gone out whole plant by plant transformed cell regeneration after, can expand this transfer-gen plant numerous, but the resistance of reverse of render transgenic plant (drought-enduring or salt tolerant alkali) is further improved.The expansion of described transgenic plant is numerous to comprise vegetative propagation and/or seminal propagation.Described resistance of reverse improves improving of performances such as comprising drought resisting or salt tolerant, low temperature resistant, disease and insect resistance.

In aforesaid method, the described dna fragmentation of step (a) also comprises the promoter sequence of composition or tissue specificity or abduction delivering (ABA, arid, saline and alkaline or chemical induction etc.).

In the method for the invention, described constructive expression's promotor can be CaMV 35S, corn Ubiquitin, paddy rice actinl promotor etc.; Described tissue specificity expression promoter can be that root-specific is expressed promotor, blade specific is expressed promotor or dimension pipe specific expressing promoter; Described abduction delivering promotor can be the promoter sequence of low temperature, ABA, arid, saline and alkaline or chemical induction etc.

In the method for the invention, described dna fragmentation can be the dna molecular that contains by the OsCBF4 gene of composition or tissue specific expression or abduction delivering (ABA, arid, saline and alkaline or chemical induction etc.) promotor control; The also dna fragmentation of synthetic.Described dna fragmentation can be inserted in binary vector plasmid such as the pBin 19 that is used for agrobacterium tumefaciens or Agrobacterium rhizogenes conversion plant, pCambia or pBI 101 etc., with other plasmids such as the pUC that can in prokaryotic organism, breed, in pBluescript or the PCR vector plasmid.Described dna fragmentation being inserted the method for these plasmids can be undertaken by method well known in the prior art.

The abduction delivering system of described control OsCBF4 genetic expression can be the allos inducible system, also can be plant endogenous inducible system such as heat shock, weedicide, inducible systems such as arid, salt stress or low temperature stress etc.

In the method for the present invention, described vegetable cell can derive from dicotyledonss such as tobacco, rape, cotton, soybean, willow, eucalyptus, potato, herbage, or tissue or organs such as monocotyledonous callus, stem apex, blade such as paddy rice, corn, wheat, barley, jowar, millet, turfgrass.

In the method for the present invention, described dna fragmentation can pass through protoplastis-chemical mediated method (Ca ²⁺, PEG), the particle gun mediated method, agrobacterium-mediated transformation, electrization, pollen tube imports, any method in the methods such as microinjection or the combination transformed plant cells of several method.

In the method for the present invention, describedly referred to utilize above-mentioned method for transformation to import by the plant transformed cell and prove the male vegetable cell by the molecule means with the OsCBF4 gene or with its homologous clone gene.

In the method for the present invention, describedly can be utilized microbiotic or other selection markers genes to finish, as the cell or the callus that contain neomycin phosphotransferase (NPTII) gene can be screened by kantlex or its for thing such as G 418 etc. for deriving by the screening of plant transformed cell; The cell or the callus that contain hygromix phosphotransferase (Hygromycinphosphotransferase) gene can be screened by Totomycin etc.Obtaining can to adopt Southern or PCR method behind the resistant calli cell, dot blot equimolecular detection means detects it, contains OsCBF4 or its homologous clone gene to determine it.

In the method for the present invention, make the whole plant that is gone out by plant transformed cell regeneration mainly refer to also comprise non-other stripped method regenerated plant that nourish and generate by exsomatizing by the transformant regenerated plant that includes above-mentioned DNA element.

The present invention is based on AP2 class transcription factor (comprising DREB class transcription factor) plant anti-in contrary vital role and the vital role in the anti-contrary genetically engineered of upland rice cereal crop, with Brazilian upland rice (Brazilian uplandrice) and Japan fine be material, the method that suppresses hybridization is subdued in utilization, in conjunction with the result of gene chip hybridization, separate obtaining the gene of anti-a retrocorrelation OsCBF4.Experiment showed, OsCBF4 is imported in the paddy rice, can significantly improve the tolerance that paddy rice is coerced arid, high salt and low temperature etc.The present invention will play a significant role in cultivating resistance of reverse enhanced plant.

Description of drawings

Fig. 1 is the evolutionary relationship tree of OsCBF4 and known Arabidopis thaliana DREB class transcription factor

Fig. 2 is subjected to the fine RT-PCR electrophoretogram of salt inductive Brazil upland rice and Japan

Fig. 3 is subjected to the fine RT-PCR electrophoretogram of salt inductive Japan

Fig. 4 is 9311, Japan is fine, orchid wins, Brazilian upland rice is handled 12 days photo through salt

Fig. 5 is 9311, Japan is fine, bluely win, Brazilian upland rice is through 4 days photo of subzero treatment

Fig. 6 is that the PCR of part transfer-gen plant detects electrophoretogram

Fig. 7 is that transgenosis T0 is for the photo of plant through salt processing in 60 days

Fig. 8 is the photo that fractional t1 was handled for the plant salt stress 7 days

Fig. 9 is the photo that fractional t1 was handled for the plant low temperature stress 7 days

Figure 10 is the photo that fractional t1 was handled for the plant drought stress 5 hours

Embodiment

Paddy rice (Oryza sativa L.) kind 9311, Japan is fine, and blue the victory, Brazilian upland rice (Iapar 9) etc. is used to the various experiments of following examples

The acquisition of embodiment 1, OsCBF4

1, the inhibition subtractive hybridization obtains paddy rice salt stress differential expression fragment

The fine seed of Brazil upland rice and Japan was soaked seed two days at 37 ℃ respectively, was layered on the plate that is placed with two-layer gauze after showing money or valuables one carries unintentionally and cultivated, and culture condition is 28 ℃, and continuous light intensity is 2500lux, and the photoperiod is 16/8 hour.After cultivating for two weeks, the fine seedling of Brazilian upland rice and Japan is moved to respectively in the 150mM NaCl solution and coerces processing, establish contrast simultaneously, divide different time points to get root (time point was from 15 minutes to 2 days).Extract total RNA respectively according to (2002) methods such as Wang Guanlin, then according to Clontech test kit (Clontech PCR-Select cDNASubtraction Kit, available from Clontech company) step operate, make up inhibition subtractive hybridization cDNA library.According to the steps in sequence of Clontech test kit carry out that enzyme is cut, connection, dot blot, and two-wheeled pcr amplification, obtain the fragment of differential expression.The fragment of differential expression and pMD 18-T Vector (TAKARA, Dalian) connect, and transformed into escherichia coli (Escherichia coli) DH5 α bacterial strain selects 700 positive colonies to check order at random.The differential expression fragment that is obtained compares in NCBI and BGI database, what the result showed the differential expression maximum is the cDNA fragment of a 349bp, has bigger homology with Arabidopis thaliana CBF4 gene, is single copy in paddy rice, there is not intron, called after OsCBF4.

2, the clone of OsCBF4 and checking

Design primer forward primer:5 '＞TTA AGC TTA TGG ACA CCG AGG ACA CGT C＞3 ' and reverse primer 5 '＞TTA AGC TTT CAG TCC ATC CAT AGC TTG T＞3 ', PCR method is come out this gene clone from Brazilian upland rice and Japanese fine genome respectively routinely, pMD 18-TVector packs into, obtain recombinant plasmid pMD18-T Vector-OsCBF4, pass through CaCl ₂Method transformed into escherichia coli (E.coli) DH5 α bacterial strain is selected positive bacterium colony and is joined 5ml and contain in the LB liquid nutrient medium of 50mg/L penbritin, and 37 ℃, 200rpm were cultivated 12-16 hour, extracted plasmid, after PCR and enzyme are cut evaluation, and sequencing.

The cloning and sequencing result that the inhibition subtractive hybridization is obtained shows that OsCBF4 is a fragment of the abundance difference maximum that obtains, and gained EST is long among the 349bp, has 344 Nucleotide identical.The sequencing result shows OsCBF4 total length 660bp, wherein from the OsCBF4 full length cDNA sequence of Brazilian upland rice shown in the sequence in the sequence table 1, from the fine OsCBF4 full length cDNA sequence of Japan shown in the sequence in the sequence table 3, sequence 1 only there are differences from the 402nd bit base of 5 ' end with sequence 3, the proteins encoded shown in sequence 2 that they are all encoded and are made up of 219 amino-acid residues, be positioned on article one karyomit(e) of rice genome, a typical A P2 conserved regions is arranged, have 96.7% homology with the DNA land of AP2/EREBP, have 50.6% homology with AtCBF4.The result who relatively obtains based on aminoacid sequence has set up the evolutionary tree of the transcription factor of OsCBF4 and the known DREB class of Arabidopis thaliana, as shown in Figure 1, show OsCBF4 on aminoacid sequence with Arabidopis thaliana in the homology of AtCBF1, AtCBF2, AtCBF3 and AtCBF4 be respectively 50.2%, 49.6%, 49.3%, 50.6%.

Embodiment 2, OsCBF4 are subjected to salt inductive expression pattern in the fine root of Brazilian upland rice and Japan

Get the seed of the warm and fine Brazilian upland rice of wild-type Japan, 37 ℃ of seed soaking two days, be layered on after showing money or valuables one carries unintentionally on the plate that is placed with two-layer gauze earlier, cultivate in 28 ℃ of illumination (2500lux, 16/8).After 10 days, changing 1/4 * Hogland nutritive medium continues to cultivate two days, then the seedling of the warm and fine Brazilian upland rice of Japan is moved to and carry out salt stress in the 1/4 * Hogland nutritive medium that is added with 150mM NaCl and handle, respectively 0,0.5,1,3,6,12 and 24 hours these 7 time points are got root and the blade of the warm and fine Brazilian upland rice of Japan fast, freezing in liquid nitrogen rapidly,-80 ℃ of preservations, extract total RNA of the different tissues of the warm and fine Brazilian upland rice of paddy rice Japan then according to (2002) methods such as Wang Guanlin, then according to the Reverese Transcription System (cat. of Promega company ^#3500, available from Promega company) the total RNA of root of the different time points of the step of specification sheets Japanese warm and fine Brazilian upland rice that salt is handled carries out reverse transcription, be forward and reverse primer with 5 '＞TTA AGC TTA TGG ACA CCG AGG ACA CGT C＞3 ' and 5 '＞TTAAGC TTT CAG TCC ATC CAT AGC TTG T＞3 ' respectively then, carry out 40 circulations of pcr amplification with the RT-PCR method, amplified production carries out electrophoresis with 1.2% sepharose, the result as shown in Figure 2, show that the expression amount after OsCBF4 is induced by salt is higher than the expression amount of OsCBF4 in Japan is fine under the same treatment condition far away in Brazilian upland rice, Brazilian upland rice and Japan fine in, the salt that is expressed in of OsCBF4 is induced and is reached the climax in 30 minutes rapidly, begin obvious decline in the time of 1 hour, detected fully to 6 hours less than.

Embodiment 3, the OsCBF4 expression pattern in the fine Different Organs tissue of Japan

Get the fine seed of wild-type Japan, 37 ℃ of seed soaking two days, be layered on after showing money or valuables one carries unintentionally on the plate that is placed with two-layer gauze earlier, cultivate in 28 ℃ of illumination (2500lux, 16/8).After 10 days, changing 1/4 * Hogland nutritive medium continues to cultivate two days, then the fine seedling of Japan is moved to and carry out the salt stress processing in the 1/4 * Hogland nutritive medium that is added with 150mM NaCl, get Japan fine root and blade fast at 0,0.5,1,6,12,24 and 48 hour these 7 time point respectively, freezing in liquid nitrogen rapidly,-80 ℃ of preservations, extract total RNA of the fine different tissues of paddy rice Japan then according to (2002) methods such as Wang Guanlin, then according to the Reverese TranscriptionSystem (cat. of Promega company ^#3500, available from Promega company) total RNA of the Japanese fine root that the step of specification sheets is handled salt and the different time points of blade carries out reverse transcription, be forward and reverse primer with 5 '＞TTA AGC TTA TGG ACA CCG AGGACA CGT C＞3 ' and 5 '＞TTA AGC TTT CAG TCC ATC CAT AGC TTG T＞3 ' respectively then, carry out 40 circulations of pcr amplification with the RT-PCR method, amplified production carries out electrophoresis with 1.2% sepharose, the result as shown in Figure 3, show the expression pattern difference of OsCBF4 in same kind Different Organs tissue, in fine of Japan, OsCBF4 induces the back to express rapidly at salt, promptly reached its maximum expression amount in 30 minutes, promptly began in 1 hour to descend, detected to 6 hours less than; And in leaf, 6 hours begin to express, and expression amount descends rapidly then, detect less than its expression during to 12 hours.This phenomenon is hinting after plant is subjected to high-salt stress, its root is experienced at first and is produced and coerces reaction accordingly, then, just will coerce signal and reach over-ground part, hint that also OsCBF4 may play induction and coerce the signaling molecule that information is coerced in signal or transmission in the signal conductive process that plant salt is coerced through a series of signal transduction path.Among Fig. 3, negative control is the Japanese fine plant of handling without salt, and positive control is Japanese fine genomic dna.

Embodiment 4, different rice varieties resistance of reverse can detect

Get wild-type 9311, Japan is fine, and the seed of blue victory and Brazilian upland rice 37 ℃ of seed soaking two days, is layered on after showing money or valuables one carries unintentionally on the plate that is placed with two-layer gauze earlier, cultivates in 28 ℃ of illumination (2500lux, 16/8).After 10 days, change 1/4 * Hogland nutritive medium and continue to cultivate two days, coerce processing then, that establishes wild-type simultaneously Japanese finely does contrast.Salt is handled: rice seedling is moved in the 1/4 * Hogland nutritive medium that is added with 150mM NaCl, handled 12 days under the same culture conditions; Deepfreeze: rice seedling moved in 4 ℃ of incubators handled 4 days.The result as shown in Figure 4 and Figure 5, the tolerance difference that Fig. 4 shows 9311, Japan is fine, bluely win, Brazilian upland rice is handled high salt, four different rice varieties 150mmol/L NaCl of under the same culture conditions this handled 12 days, 9311 leaf rollings, withered dead, the fine tender leaf of Japan is all curling, withered, and the Lao Ye blade tip is curling, withered, and blue victory tender leaf all curls, dries up, Lao Ye does not have curling, and Brazilian upland rice only blade tip have curling slightly.Fig. 5 shows 9311, Japan is fine, bluely win, Brazilian upland rice is to cryogenic anti-contrary ability difference, four different rice varieties of under the same culture conditions this were handled 4 days for 4 ℃, 9311 blades are all withered, fine, the blue tender leaf blade that wins of Japan dries up curling, have only nethermost a slice Lao Ye to stretch, phenotype is not coerced in Brazil's upland rice upright blade, stretching, extension.This illustrates that Brazilian upland rice is all obviously to be better than other several kinds at salt tolerant or on low temperature resistant.

Embodiment 5, agrobacterium-mediated transformation rice transformation

(OsCBF4 wherein is the SEQ ID № in the sequence table: recombinant plasmid 1 dna sequence dna) to cut the pMD18-T Vector-OsCBF4 that makes up among the embodiment 1 with the HindIII enzyme, reclaim the OsCBF4 gene fragment with glass milk, fragment behind the purifying is mended flat back and is cut through Smal I enzyme with Klenow Fragment, binary vector pCAMBIA 1300-35S-tOCS (Cambia behind the dephosphorization, Australia) connect, heat shock method transformed into escherichia coli (E.coli) DH5 α bacterial strain, selecting positive bacterium colony joins 5ml and contains in the LB liquid nutrient medium of 50mg/L penbritin, 37 ℃, 200rpm cultivated 12-16 hour, extract plasmid, carry out PCR evaluation and enzyme and cut evaluation, change Agrobacterium AGL1 subsequently over to.

The single colony inoculation of picking Agrobacterium in 20ml YEB liquid nutrient medium (containing penbritin 50mg/L, Rifampin 50mg/L), 28 ℃, 150rpm shaking culture 2-3 days.In centrifugal 3 minutes of 4 ℃ of following 5000rpm, remove supernatant, be resuspended in the AAM substratum (containing the 0.1mmol/L Syringylethanone), 28 ℃ of lucifuge shaking culture 1-2 hour are to OD ₆₀₀=0.6-0.9.After selecting the Japanese fine growth conditions of the preparation of method for plant tissue culture routinely good, shaking 20 minutes in the particulate state callus immersion Agrobacterium nutrient solution, left standstill 30 minutes, taking-up is blotted unnecessary bacterium liquid with aseptic filter paper, callus is inoculated in common substratum (NB substratum+10g/L glucose+1mg/L is the acyl syringone) goes up cultivation.After 2-3 days, callus is put into wide-necked bottle, with aseptic water washing 3-5 time, shake for several times at every turn, in water, lose thread thalline, in the sterilized water that contains the 500mg/L Pyocianil, soaked 30-60 minute then, wash again at last one time, place airing on the aseptic filter paper, change the common substratum of NB over to and cultivate altogether.

After rice callus after infecting is organized in NB altogether substratum is cultivated 3-5 days altogether, go to contain 30mg/L Totomycin and the full mycin of 400mg/L head) NB substratum (N ₆Macroelement+B ₅Trace element+B ₅Organic composition+300mg/L caseinhydrolysate+500mg/L proline(Pro)+30g/L sucrose+7-8g/L agar, pH5.8) go up 3 weeks of screening, change 4 weeks of screening on the NB substratum that contains 50mg/L Totomycin and the full mycin of 200mg/L head again over to, resistant calli changes pre-differentiation substratum (NB substratum+1mg/L naa+2mg/L 6-benzylaminopurine+5mg/L dormin) over to subsequently, 3 weeks of illumination cultivation, change on the division culture medium (NB substratum+1mg/L naa+2mg/L 6-benzylaminopurine) again and break up, regeneration plant moves in the greenhouse after taking root on strong seedling culture base (1/2MS inorganic salt+0.5mg/L naa+0.25mg/L paclobutrazol).Get plant leaf after one month, extract total DNA according to a conventional method, so that 5 '＞AGC TGC GCC GAT GGT TTC TAC AA＞3 ' and 5 '＞ATC GCC TCG CTC CAGTCA ATG＞3 ' is forward and reverse primer, carry out the pcr amplification hygromycin phosphotransferase gene, detect whether positive plant of the plant obtained, the result shows and has obtained 13 strain male transfer-gen plants that Fig. 6 provides the PCR detected result of part transfer-gen plant.Among Fig. 6, the negative contrast of N is not by genetically modified plant; The positive contrast of P (proving genetically modified positive rice plant); 151-162 is the numbering of different transgenic line.

Embodiment 6, transgenosis T0 can identify for the plant resistance of reverse

Mistake among the embodiment 5 is expressed the positive transfer-gen plant of OsCBF4 and in the greenhouse, is cultivated, treat that its generation is tillered after, the part of getting each positive transfer-gen plant is tillered and is carried out salt tolerant and arid test, establishes the fine contrast of wild-type Japan of isometric growth state simultaneously.

Salt is handled: the positive plant that is transferred in the salt pond is tillered, and recovers growth and carries out the salt processing after 15 days, irrigates 2 months continuously with 0.5%NaCl solution;

Arid is handled: the positive plant that is transferred in the culture vessel is tillered, and recovers growth and carries out arid processing 16 days after 15 days, does not rewater from this very day from handling.

The result showed the positive transgenic line of expressing OsCBF4, and its tolerance to high salt and arid obviously strengthens.Fig. 7 shows the Japanese fine contrast of handling transgenic paddy rice and wild-type in the salt pond with 0.5% NaCl, contrast death after 60 days, and the growth conditions of part transgenic line is fair.Among Fig. 7,151,154,155,157,159 is the numbering of different transgenic line shown in the arrow, and WT is the Japanese fine contrast of wild-type.

Embodiment 7, transgenosis T1 identify for the plant salt tolerance

T0 is for positive transgenic paddy rice seed for results, with the seed (T1 generation) of 151,152,153,154,155,156,157,158,159,160,161,162,163 strains of positive transfer-gen plant system earlier 37 ℃ of seed soaking two days, be layered on after showing money or valuables one carries unintentionally on the plate that is placed with two-layer gauze, cultivate in 28 ℃ of illumination (2500lux, 16/8).After 10 days, change 1/4 * Hogland nutritive medium and continue to cultivate two days, then rice seedling is moved in the 1/4 * Hogland nutritive medium that is added with 100mM NaCl, carry out salt stress under the same culture conditions and handled 7 days, that establishes wild-type simultaneously Japanese finely does contrast.Fractional t1 show the leaf rolling, withered of contrast, and three transgenic line vane extension is not coerced phenotype for the result of plant salt tolerant test as shown in Figure 8.Among Fig. 8, the strain of 151,154,157 positive transgenic lines number; WT is a wild-type, Japanese fine contrast.

Continuing above-mentioned salt stress handles to wild-type contrast death, remove stress conditions, recover under the normal culture condition growth after 15 days statistical from than, the result is as shown in table 1, show that T1 obviously improves for the tolerance of plant to salt stress, and the separation that shows 3:1 is than (survival plant number after the rehydration: dead plant number), illustrate that phenotype and transgenosis are directly chain.In the table 1,151-163 is the strain number of different positive transgenic line.

It is that T1 is for separating under the different treatment condition than analyzing that table 1. is crossed the positive strain of expression OsCBF4

Strain system	Separation is than (living: dead)
		100mM NaCl
	WT
151 152 153 154 155 156 157 158 159 160 161 162 163		0∶40 27∶10 32∶8 27∶5 27∶9 29∶9 0∶40 20∶13 25∶5 8∶32 7∶32 26∶7 24∶6 0∶40

Embodiment 8, transgenosis T1 are for the plant frigostabile evaluation

T0 is for positive transgenic paddy rice seed for results, with the seed (T1 generation) of 151,152,153,154,155,156,157,158,159,160,161,162,163 strains of positive transfer-gen plant system earlier 37 ℃ of seed soaking two days, be layered on after showing money or valuables one carries unintentionally on the plate that is placed with two-layer gauze, cultivate in 28 ℃ of illumination (2500lux, 16/8).After 10 days, changing 1/4 * Hogland nutritive medium continues to cultivate two days, rice seedling is moved in the 1/4 * Hogland nutritive medium that is added with 100mMNaCl then, will cultivate seedling and put under 10 ℃ of culture condition and carried out subzero treatment 7 days, that establishes wild-type simultaneously Japanese finely does contrast.Fractional t1 for the low temperature resistant Processing Test result of plant as shown in Figure 9, when 10 ℃ of Japanese fine contrasts (WT) that show T1 generation of positive transgenic line 151,154,157 and wild-type are handled the 7th day, the leaf rolling of contrast, withered, and three transgenic lines only tip segment have curling slightly.Test-results also shows, T1 for plant to cryogenic tolerance apparently higher than wild-type.

Embodiment 9, transgenosis T1 are for plant arid Processing Test result

T0 is for seed for results, with the seed of 151,152,153,154,155,156,157,158,159,160,161,162,163 strains of positive transfer-gen plant system earlier 37 ℃ of seed soaking two days, be layered on after showing money or valuables one carries unintentionally on the plate that is placed with two-layer gauze, cultivate in 28 ℃ of illumination (2500lux, 16/8).After 10 days, changing 1/4 * Hogland nutritive medium continues to cultivate two days later, remove the residual moisture content of its root with the gauze suction, transfer to and carry out arid processing 5 hours in the clean anhydrous plate, fractional t1 shows that the blade of contrast is withered, the cane dehydration is crooked for plant arid result as shown in figure 10, can not be upright, and the growth conditions of three transgenic lines is good, and blade is sagging slightly, and cane is upright.

Sequence table

<160>3

<210>1

<211>660

<212>DNA

＜213〉Oryza paddy rice (Oryza sativa L.)

<400>1

atggacaccg aggacacgtc gtcggcttcg tcctcgtcgg tgtcgccgcc gtcgtcgccg 60

ggcggcgggc accaccaccg gctgccgccg aagcggcggg cggggcggaa gaaattccgg 120

gagacgcggc acccggtgta ccgcggtgtg cgcgcgcggg cggaggggag caggtgggtg 180

tgcgaggtgc gcgagccgca ggcgcaggcg cgcatctggc tcggcaccta cccgacgccg 240

gagatggcgg cgcgcgcgca cgacgtcgcg gccatcgccc tccgcggcga gcgcggcgcc 300

gagctcaact tcccggactc cccctccacg ctcccgcgcg cgcgcacggg gtcgcccgag 360

gacatccgcc tcgccgccgc gcaggccgcc gagctgtacc gccgcccgcc gccgccgctg 420

gcattgccgg aggatccgca ggaaggcacg agtggcggcg gcgccaccgc cacctcgggg 480

cgtccggctg ccgtgttcgt ggacgaggac gccatcttcg acatgccggg gctgatcgac 540

gacatggcga gggggatgat gctgacgccg ccggcgattg ggagatctct cgacgactgg 600

gccgccatcg acgacgacga tgaccattac cacatggact acaagctatg gatggactga 660

<210>2

<211>219

<212>PRT

＜213〉Oryza paddy rice (Oryza sativa L.)

<400>2

Met Asp Thr Glu Asp Thr Ser Ser Ala Ser Ser Ser Ser Val Ser Pro

1 5 10 15

Pro Ser Ser Pro Gly Gly Gly His His His Arg Leu Pro Pro Lys Arg

20 25 30

Arg Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Val Tyr Arg

35 40 45

Gly Val Arg Ala Arg Ala Glu Gly Ser Arg Trp Val Cys Glu Val Arg

50 55 60

Glu Pro Gln Ala Gln Ala Arg Ile Trp Leu Gly Thr Tyr Pro Thr Pro

65 70 75 80

Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Ile Ala Leu Arg Gly

85 90 95

Glu Arg Gly Ala Glu Leu Asn Phe Pro Asp Ser Pro Ser Thr Leu Pro

100 105 110

Arg Ala Arg Thr Gly Ser Pro Glu Asp Ile Arg Leu Ala Ala Ala Gln

115 120 125

Ala Ala Glu Leu Tyr Arg Arg Pro Pro Pro Pro Leu Ala Leu Pro Glu

130 135 140

Asp Pro Gln Glu Gly Thr Ser Gly Gly Gly Ala Thr Ala Thr Ser Gly

145 150 155 160

Arg Pro Ala Ala Val Phe Val Asp Glu Asp Ala Ile Phe Asp Met Pro

165 170 175

Gly Leu Ile Asp Asp Met Ala Arg Gly Met Met Leu Thr Pro Pro Ala

180 185 190

Ile Gly Arg Ser Leu Asp Asp Trp Ala Ala Ile Asp Asp Asp Asp Asp

195 200 205

His Tyr His Met Asp Tyr Lys Leu Trp Met Asp

210 215

<210>3

<211>660

<212>DNA

＜213〉Oryza paddy rice (Oryza sativa L.)

<400>3

atggacaccg aggacacgtc gtcggcttcg tcctcgtcgg tgtcgccgcc gtcgtcgccg 60

ggcggcgggc accaccaccg gctgccgccg aagcggcggg cggggcggaa gaaattccgg 120

gagacgcggc acccggtgta ccgcggtgtg cgcgcgcggg cggaggggag caggtgggtg 180

tgcgaggtgc gcgagccgca ggcgcaggcg cgcatctggc tcggcaccta cccgacgccg 240

gagatggcgg cgcgcgcgca cgacgtcgcg gccatcgccc tccgcggcga gcgcggcgcc 300

gagctcaact tcccggactc cccctccacg ctcccgcgcg cgcgcacggg gtcgcccgag 360

gacatccgcc tcgccgccgc gcaggccgcc gagctgtacc ggcgcccgcc gccgccgctg 420

gcattgccgg aggatccgca ggaaggcacg agtggcggcg gcgccaccgc cacctcgggg 480

cgtccggctg ccgtgttcgt ggacgaggac gccatcttcg acatgccggg gctgatcgac 540

gacatggcga gggggatgat gctgacgccg ccggcgattg ggagatctct cgacgactgg 600

gccgccatcg acgacgacga tgaccattac cacatggact acaagctatg gatggactga 660

Claims (13)

1, a kind of gene of anti-retrocorrelation of paddy rice is one of following nucleotide sequence:

1) the SEQ ID № in the sequence table: 3;

2) SEQ ID № in the code sequence tabulation: the polynucleotide sequence of 2 amino acid residue sequences.

2, a kind of gene coded protein of anti-retrocorrelation of paddy rice, its aminoacid sequence are as SEQ ID №: shown in 2.

3, contain the described expression carrier of claim 1.

4, the clone that contains the described gene of claim 1.

5, a kind of method that improves plant stress tolerance may further comprise the steps:

(a) make up a kind of dna fragmentation, make it comprise the described paddy rice of claim 1 gene of anti-the retrocorrelation;

(b) with constructed dna fragmentation or contain the plasmid transformed plant cells of described dna fragmentation;

(c) filter out by the plant transformed cell;

(d) make by plant transformed cell regeneration and go out whole plant, obtain the transgenic plant that resistance of reverse improves.

6, method according to claim 5 is characterized in that: the described dna fragmentation of step (a) also comprises composition or tissue specificity or abduction delivering promoter sequence.

7, according to claim 5 or 6 described methods, it is characterized in that: described dna fragmentation is inserted in the plasmid that can breed in prokaryotic organism.

8, method according to claim 7 is characterized in that: described plasmid is pUC, pBluescript or PCR vector plasmid.

9, method according to claim 7 is characterized in that: described prokaryotic organism are agrobacterium tumefaciens or Agrobacterium rhizogenes.

10, method according to claim 9 is characterized in that: described plasmid is the binary vector plasmid.

11, method according to claim 10 is characterized in that: described binary vector plasmid is pBin 19, pCambia or pBI101.

12, according to claim 5 or 6 described methods, it is characterized in that: described vegetable cell derives from dicotyledons or monocotyledons; Described dicotyledons is tobacco, rape, cotton, soybean, willow, eucalyptus, potato or herbage; Described monocotyledons is paddy rice, wheat, barley, corn, jowar, millet or turfgrass.

13, according to claim 5 or 6 described methods, it is characterized in that: described dna fragmentation is by protoplastis-chemical mediated method, particle gun mediated method, agrobacterium-mediated transformation, electrization, the combination transformed plant cells of any or several method in pollen tube importing and the microinjection method.

Images