CN101812462B - Application of rice GT transcription factor family gene OsGT gamma-1 in controlling salt tolerance of rice - Google Patents

Abstract

The invention relates to the technical field of rice genetic engineering, in particular to the application of a GT transcription factor family gene OsGT gamma-1 in controlling the salt tolerance of rice. By functional analysis and identification, the gene OsGT gamma-1 for controlling the salt tolerance of rice is obtained, and the application of the gene in adverse resistance genetic improvement of the salt tolerance of rice is verified. The gene OsGT gamma-1 is one of the following nucleotide sequences: 1) the nucleotide sequence shown from 475th to 1707th in a sequence table SEQ NO: 1; or the nucleotide sequence of protein with the same code as that of the protein encoded by 1). The nucleotide sequence containing the gene OsGT gamma-1 is transferred to rice after being connected with an exogenous promoter, and the salt tolerance of the transgenic rice is obviously enhanced.

Description

The application of rice GT transcription factor family gene OsGT γ-1 in control paddy rice salt tolerance

Technical field

The present invention relates to paddy gene engineering field.Be specifically related to one of separating clone and can improve the rice GT transcription factor family gene OsGT γ-1 of high-salt tolerance ability, and realize its application in the improvement of paddy rice anti contravariance sex-controlled inheritance by functional verification.The present invention utilizes screening T-DNA to insert the method in rice mutant storehouse, be isolated and cloned into the gene OsGT γ-1 of control paddy rice salt tolerance, on the one hand by being divided into from this mutant of detection validation and the responsive phenotype close linkage of high salt, improved the tolerance of transgenic paddy rice by overexpression OSGT γ-1 gene on the other hand, confirmed the function of this gene high salt.

Background technology

The growth and development of plant process not only depends on its genetic background, also can be subjected to many Effect of Environmental.The environment of plant-growth is not always suitable; under the nature condition; owing to many-sided reasons such as different geographical position, weather condition and mankind's activities, usually can cause variously to plant-growth and existence hostile environment, cause plant to come to harm even dead.Concerning agriculture production, various poor environments are the most direct, the most important factors that influence crop yield and quality, also are the bottlenecks of the many regional agricultural developments of restriction.How to utilize the function and the regulatory mechanism of modern molecular biology means research anti contravariance related gene, thereby the resistance of utilizing these genes to improve crop is cultivated the crop varieties with good resistance, being the important content of plant functional genomics research, also is one of major objective of agricultural cience and farming techniques research.

Arid, low temperature and salting of soil are the main adverse circumstance factors of restriction crop growthing development and increasing both production and income always.Arid can cause the vegetable cell dehydration, causes cell turgor to completely lose, even dead; Freeze injury may cause the mechanical wounding of vegetable cell, causes the film fat phase transformation of cytolemma and dehydration; Salt damage is also poisoned with ion except that osmotic stress simultaneously, causes that injury, the plant-growth of enzyme in the vegetable cell are suppressed etc.Plant experiences the expression of regulating resistance relevant protein in the cell behind the extraneous adverse circumstance signal by the signal transduction process, and then physiological status or the form of adjusting self adapt to adverse environment, keeps basic survival activity.Plant is conducted and transcriptional control through a series of signal after sensing environment stress, start a large amount of relevant target gene expression in downstream with stress response, producing some makes cell avoid coercing the material of injury (as the LEA proteinoid, ooze accent albumen, antifreeze protein, channel protein etc.), and then tolerance (the Valliyodan B of enhancing plant to coercing, Nguyen H T.Understandingregulatory networks and engineering for enhanced drought tolerance in plants.Curr Opin Plant Biol, 2006,9:189-195).At present from plant separating clone tens of kinds of codings and the genes that improve the environment stress resistance-associated protein, function according to gene encoding production, they can be divided into two big classes: a class is that coding directly protects cell to avoid the functional gene of the functional protein of environment stress injury, comprising: the gene of some coding membranins (as aquaporin, transhipment); Some proteinase genes in tenuigenin and the chloroplast(id); The gene of coding macromole protected protein (as molecular chaperones, lea protein); The gene of coding osmoregulation material (as proline(Pro), trimethyl-glycine, sugar alcohol etc.) synthetic enzyme; The gene of coding toxicant degrading enzyme (as glutathione s-transferase, superoxide-dismutase, catalase and ascorbate peroxidase enzyme etc.).People carry out genetic transformation with the functional gene relevant with improving the environment stress resistance, and the resistance of render transgenic plant has had raising in various degree.Another kind of is signal conduction and the relevant gene of transcriptional control under the environment stress, comprising: degeneration-resistant relevant transcription factor gene (bZIP transcription factor, WRKY transcription factor, MYC, MYB class and DREB class transcription factor etc.); The protein kinase (MAPK, CDPK, S6K, receptor protein kinase, ribosomal protein kinases etc.) of signal is coerced in induction and transduction; The proteolytic enzyme (as phosphoesterase, Phospholipase C etc.) that plays an important role in the signal conduction.

Traditional breeding method practice shows, want by between kind and traditional genetic breeding means such as species hybridization improve crop the resistance of environment stress be difficult to prove effective, utilize the individual feature gene to come resistance to crop to improve and also be difficult to obtain the ideal effect.The transcription factor that some adverse circumstances of discovered in recent years are relevant can be regulated and control a plurality of adverse circumstance Expression of Related Genes, utilizes transcription factor can effectively realize improvement to stress resistance of plant.Transcription factor is meant can that special interactional DNA takes place be conjugated protein with cis-acting elements in the gene promoter region, also claim trans-acting factor, can by between them and and other associated protein between interaction on the mRNA transcriptional level, realizes expression of gene is regulated and control (.Transcription factors in rice:a genome-wide comparative analysis between monocots and eudicots.Plant Mol Biol such as Xiong, 2005,59:191-203).Facts have proved, in plant materials, some transcription factor gene is carried out overexpression and can effectively improve the tolerance of plant abiotic stress.For example the transgenic arabidopsis of overexpression DREB1A can show arid, the resistance of high salt and freeze injury, and being carried out overexpression, DREB2A can under non-stress conditions, coerce Expression of Related Genes by induced low temperature, and can improve the tolerance (Liu etc. of transfer-gen plant to low temperature stress, Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separatetwo cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis.Plant Cell, 1998,10:1391-1406).Tsil (Park etc., Overexpression of the tobacco Tsilgene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmoticstress in tobacco.Plant Cell, 2001,13:1035-1046), tomato JERF1 and the JERF3 overexpression in transgene tobacco can improve the salt tolerance (Wang etc. of tobacco, Ectopic overexpression of tomato JERF3 in tobacco activates downstream geneexpression and enhances salt tolerance.Plant Mol Biol, 2004,55:183-192; Zhang etc., The ethylene-, jasmonate-, abscisic acid-and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCCbox-containing genes and salt tolerance in tobacco.Planta, 2004,220:262-270).Overexpression SNAC1 gene has significantly improved the drought resistance and the salt tolerance (Hu etc. of transfer-gen plant in paddy rice, Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice.Proc Natl Acad Sci U S A, 2006,103:12987-12992).Because the overexpression of transcription factor can activate the relevant expression of gene of a plurality of and similar proterties, therefore compare with the traditional method that imports or improvement discrete function gene improves certain resistance of crop, the transcription factor of key is improved or strengthens its ability of regulation and control, can activate a plurality of functional genes and play a role by regulating and control a transcription factor, for the crop anti-adversity molecular breeding provides more efficiently method and approach.

As a kind of important crops and model plant, the improvement of rice quality and the raising of output are the targets that people make great efforts always.Thereby improving paddy rice anti contravariance by transcription factor relevant with adverse circumstance in the research paddy rice is an effective way of cultivating degeneration-resistant rice varieties.Be separated to one and be subjected to multiple adverse circumstance inductive gene in our early-stage Study, sequential analysis shows that it belongs to the member of plant specific one class transcription factor GT family.The existing member who studies show that this family participates in comprising some physiological responses that plant light is replied, thereby and can and corresponding GT element specific combination or and other transcription factor between take place to interact and realize multiple heterogeneic transcriptional control.Paddy rice GT-2 and Arabidopis thaliana GT-1 be activated transcription (Le etc. in vivo, Transcriptional activation by Arabidopsis GT-1 may be through interaction withTF II A-TBP-TATA complex.Plant J.1999,18:663-668; Ni etc., GT-2:in vivo transcriptional activation activityand definition of novel twin DNA binding domains with reciprocal target sequence selectivity.Plant Cell.1996,8:1041-1059), Arabidopis thaliana PTL participates in regulating the growth (Brewer etc. of floral organ, PETAL LOSS, a trihelix transcription factor gene, regulates perianth architecture in the Arabidopsis flower.Development.2004,131:4035-4045), but this family whether the adverse circumstance of involved in plant reply and still do not have report.Therefore.The gene of this family of separating clone and identify that it improving the function of being brought into play aspect the paddy rice anti contravariance, has crucial meaning for cultivating degeneration-resistant new rice variety from paddy rice.

Summary of the invention

The objective of the invention is to dna fragmentation (described in the present invention " dna fragmentation " and " nucleotide sequence " synonym that from paddy rice one of separating clone comprises this functional protein homologous gene complete coding region section, down together), utilize this gene to improve the tolerance of paddy rice, realize the application of this gene in paddy rice salt tolerance genetic improvement high-salt stress.GT-3a and GT-3b albumen that the protein sequence of this genes encoding is carried out in analysis revealed it and the Arabidopis thaliana have certain homology, the result of phylogenetic analysis shows that this gene belongs in the GT transcription factor family member among the new subfamily GT γ, therefore is named as OsGT γ-1 gene.

The present invention relates to separate and use a kind of dna fragmentation of the OsGT of comprising γ-1 gene, this fragment is given plant to high-salt stress tolerance enhanced ability.Wherein, described OsGT γ-1 gene is one of following nucleotide sequences:

1) dna sequence dna shown in the 475-1707 position among the sequence table SEQ NO:1; Or

2) the protein DNA sequence that coding and 1) encoded protein matter is identical; Or

3) 1) and 2) subfragment that comprised.

Can adopt OsGT γ-1 gene of having cloned to make probe, screening obtains gene of the present invention or homologous gene from cDNA and genomic library.Equally, also can adopt PCR (polymerase chain reaction) technology, from genome, mRNA and cDNA amplification obtain OsGT γ-1 gene of the present invention and any interested section of DNA or with its homologous section of DNA.Adopt above technology, can separate the sequence that obtains comprising OsGT γ-1 gene, this sequence is connected the back transforms plant with any carrier that can guide foreign gene to express in plant, can obtain salt tolerance enhanced transfer-gen plant.Gene of the present invention can add any strong promoter or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.Gene of the present invention also can use enhanser in being building up to plant expression vector the time, and these enhanser zones can be ATG initiator codon and neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the translation of whole sequence.

Carrying OsGT γ-1 expression carrier of the present invention can be by using Ti-plasmids, plant viral vector, directly DNA transforms, microinjection, conventional biotechnological means such as electroporation imports vegetable cell (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2 ^NdEdition).

Can use to comprise that OsGT γ-1 expression carrier host transformed of the present invention is to comprise the paddy rice various plants, cultivate the plant variety of salt tolerant.

Gene of the present invention is subjected to the adverse circumstance abduction delivering, therefore can with gene of the present invention be connected into suitable expression vector after any interested adverse circumstance evoked promoter combines, and the conversion plant host, but under adverse environmental factor the abduction delivering gene, improve the resistance of plant to high-salt stress.

The present invention will be further described below in conjunction with drawings and Examples.

Description of drawings

What sequence table SEQ ID NO:1 showed is the nucleotide sequence that includes OsGT γ-1 gene coding region of separating clone of the present invention.

Fig. 1: adopt 3.0 editions softwares of MrBayes (public use software) to carry out the result of phylogenetic analysis according to the GT factor protein sequence of having reported in GT family member in paddy rice and the Arabidopis thaliana and the part other plant species.

Fig. 2 (comprising Fig. 2 a-Fig. 2 c): with Real-time PCR detect OsGT γ-1 gene under various environment stress conditions, in the paddy rice different tissues with expression level under different HORMONE TREATMENT.4 kinds of abiotic stresses are coerced successively and are: S, high salt; D, arid; C, low temperature; W, injury.High salt, arid, low temperature stress sampling time point are 0,1,3,6 hour, and it is 0,1,10,30 minute that sampling time point is coerced in injury.13 histoorgans are successively: 1, and the root in tillering phase; 2, the stem of jointing stage; The blade of 3,4 leaf phases; The leaf sheath of 4 leaf phases; 5, fringe (0.5-1cm); 6, fringe (3-5cm); 7, fringe (greater than 10cm); 8, the fringe apical meristem before blooming; 9, stamen; 10, gynoecium; 11, the seedling that germinateed 1 day; 12, the seedling in 3 weeks of germinateing; 13; The chrysanthemum seedling in 3 weeks.7 kinds of HORMONE TREATMENT are successively: A, dormin (ABA); I, growth hormone (IAA); G, Plant hormones regulators,gibberellins (GA); K, phytokinin (KT); J, jasmonic (JA); E, ethrel (Eth); S, Whitfield's ointment (SA).Spraying hormone-treated concentration is respectively: 26.43mg/L, 10mg/L, 40mg/L, 100mg/L, 21.03mg/L, 43.35mg/L, 138.12mg/L, handle the back by the different time points sampling, ABA, IAA, GA, KT HORMONE TREATMENT sampling time point are 0,0.5,1,3 hour, and JA, Eth, SA HORMONE TREATMENT sampling time point are 0,1,2,4 hour.

Fig. 3: the proteic Subcellular Localization result of OsGT γ-1.OsGT γ-1:GFP fusion vector and pU1391GFP empty carrier be rice transformation respectively, and the root to transfer-gen plant under laser confocal microscope is observed.G, the GFP fluoroscopic image; PI is with the painted fluoroscopic image of propidium iodide; M, composograph.

Fig. 4 (comprising Fig. 4 a-Fig. 4 e): the evaluation of paddy rice osgt γ-1 mutant.A, T-DNA on position synoptic diagram in the mutant; B is according to inserting site design primer checking isolating PCR result altogether.F is illustrated in the primer that inserts the site upstream design, and R is illustrated in the primer that inserts the design of downstream, site, and VP represents the primer according to the design of T-DNA internal sequence.At T ₁In plant, the homozygous mutation body has only going out that R and VP pairing can increase, because have T-DNA insertion F and R paired product can't obtain amplified fragments too greatly, the plant of wild-type is not owing to there is the insertion of T-DNA, have only F and R pairing can obtain product, heterozygous plant then can be increased when R and VP pairing and F and R pairing and be obtained product.Wherein swimming lane 3,4, and 13,15,18 isozygoty for T-DNA; 1,2,5,6,7,9,10,11,12,14,16,17 is the T-DNA heterozygosis; 8,9 is the T-DNA feminine gender; C detects the expression level of OSGT γ-1 gene in 5 homozygous mutation body familys (M1-M5) and wild-type contrast family (CK) under normal growth condition and the high-salt stress with Real-time PCR; D, the growing state of next all back 5 homozygous mutation body family (M1-M5) of normal growth condition and 150mM NaCl high-salt stress and wild-type contrast family (CK); E, next all back 5 homozygous mutation body family (M1-M5) of normal growth condition and 150mM NaCl high-salt stress and wild-type contrast family (CK) over-ground part length statistics.

Fig. 5 (comprising Fig. 5 a-Fig. 5 d): overexpression OsGT γ-1 gene can improve the salt tolerance of transfer-gen plant.A, OsGT γ-1 gene overexpression vector construction synoptic diagram; B detects the expression level of OsGT γ-1 gene in 5 independent overexpression transgenosis familys (O1-O5) and wild-type contrast family (CK) with Real-time PCR; C, the growing state of next all back 5 overexpression family (O1-O5) of normal growth condition and 150mMNaCl high-salt stress and wild-type contrast family (CK); D, next all back 5 overexpression family (O1-O5) of normal growth condition and 150mM NaCl high-salt stress and wild-type contrast family (CK) over-ground part length statistics.

Fig. 6: overexpression is tested used pCB2004H carrier figure.

Fig. 7: Subcellular Localization is tested used pU1391cGFP carrier figure.

Embodiment

The full-length cDNA of OsGT γ-1 gene is the gained that increases in the J013022H04 plasmid from Japanese paddy rice total length database (http://cdna01.dna.affrc.go.jp).OsGT γ-1 is an anti contravariance related gene.Main according to the following aspects is arranged: (1) utilize the Real-time PCR method that it is carried out expression pattern analysis under the adverse environmental factor (Fig. 2 a), discovery is in the process of coercing processing (arid, high salt, low temperature stress and ABA handle), and the expression amount of OsGT γ-1 obviously rises.Distribution expression pattern analytical results (Fig. 2 b) shows that OsGT γ-1 all has expression to a certain degree in different tissues, and the expression amount in blade, leaf sheath and young fringe is higher.(2) predict that by bioinformatics method this gene is the member of rice GT transcription factor family, it is positioned (Fig. 3) in the nucleus the proteic Subcellular Localization analysis revealed of OsGT γ-1.(3) OsGT γ-1 expression is suppressed in the T-DNA of this gene insertion rice mutant, and the homozygous mutation body strengthens (Fig. 4) to the susceptibility of high-salt stress.(4) with its full-length gene overexpression in paddy rice, transfer-gen plant strengthens (Fig. 5) greatly to the tolerance of high-salt stress.These results show that OsGT γ-1 gene is the relevant regulatory gene of an adverse circumstance, and involved in plant is to the regulation and control of adverse circumstance.

Following examples further define the present invention, and have described the method that the present invention separating clone on above-mentioned previous work basis includes the dna fragmentation and checking OsGT γ-1 gene function of OsGT γ-1 gene complete coding section.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.

Embodiment 1: paddy rice GT family is carried out the dna fragmentation that bioinformatic analysis and separating clone include OSGT γ-1 constant gene segment C

Based on the plant GT protein sequence of having reported at NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov), TIGR (The Institute for Genomic Research, http://www.tigr.org) and TAIR (TheArabidopsis Information Research, http://www.arabidopsis.org) carries out the result of blast search in the database, collect GT transcription factor family member sequence, manually remove tumor-necrosis factor glycoproteins.Adopt 3.0 editions softwares of MrBayes (public use software) that the GT factor protein sequence of having reported in GT family member in paddy rice and the Arabidopis thaliana and the part other plant species is carried out phylogenetic analysis (the results are shown in Figure 1), found that the member among the subfamily GT γ all was not in the news as yet, select member OsGT γ-1 in this subfamily as research object.This gene pairs is answered the cDNA clone J013022H04 (accession number is AK065397) in the Japanese paddy rice total length database (http://cdna01.dna.affrc.go.jp).According to this cloned sequence, design primer OE07F (5 '-GTT GGTACCAGGTTTTTAGGCGTGG-3 ', sequence specific primer add joint KpnI site) and OE07R (5 '-GAA CTGCAGTGACCGATTGCAGTTAG-3 ', the sequence specific primer adds joint PstI), amplification in this clone's the cDNA clone J013022H04 plasmid of sequence from Japanese paddy rice total length database (http://cdna01.dna.affrc.go.jp) is come out.The 391-1850bp of SEQ ID NO:1 display sequence among the corresponding the present invention of amplified production.The PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 40sec, 55 ℃ of 40sec, 72 ℃ of 2min, 32 circulations; 72 ℃ are extended 10min.The PCR product that amplification is obtained is connected into

Carrier (available from Promega company), screening positive clone and order-checking obtain required full-length gene, this clone's called after pGEM-OsGT γ-1.

Sequence involved in the present invention derives from NCBI (http://www.ncbi.nlm.nih.gov/), TIGR (http://www.tigr.org), KOME (http://cdna01.dna.affrc.go.jp/cDNA/), the positional information of localized gene is as the criterion with " TIGR Rice Annotation Release 5 ".

Embodiment 2: tissue and the adverse circumstance express spectra of analyzing rice native gene OSGT γ-1

With rice varieties " precious Shan 97 " (rice varieties that Chinese big area is openly applied) is material, carries out arid respectively in 3 leaf phases, damages to plants caused by sudden drop in temperature with high-salt stress and injury and handle.Doing early, processing is that seedling is cut off the water supply, high-salt stress is that the seedling root is immersed in the 200mM/L NaCl solution, subzero treatment is that seedling is placed 4 ℃ of growth casees, and at 0,1,3 and 6 hour four point in time sampling, it was 0,1,10,30 minute that sampling time point is coerced in injury.Spraying 7 kinds of used hormones of HORMONE TREATMENT is successively: A, dormin (ABA); I, indolylacetic acid (IAA); G, Plant hormones regulators,gibberellins (GA3); K, phytokinin (KT); J, jasmonic (JA); E, ethrel (Eth); S, Whitfield's ointment (SA).Concentration is respectively: 26.43mg/L, 10mg/L, 40mg/L, 100mg/L, 21.03mg/L, 43.35mg/L, 138.12mg/L, handle the back by the different time points sampling, ABA, IAA, GA, KT HORMONE TREATMENT sampling time point are 0,0.5,1,3 hour, and JA, Eth, SA HORMONE TREATMENT sampling time point are 0,1,2,4 hour.Total RNA adopts TRIZOL reagent (available from Invitrogen company) to extract (extracting method is according to above-mentioned TRIZOL reagent specification sheets).Because it is lower that this gene background is expressed, expression pattern analysis utilizes Real-time PCR to finish, utilize ThermoScript II SSII (available from Invitrogen company) with synthetic cDNA first chain (method is according to ThermoScript II reagent specification sheets) of RNA sample reverse transcription, reaction conditions is: 65 ℃ of 5min, 42 ℃ of 50min, 70 ℃ of 10min.Utilize ThermoScript II (available from Invitrogen company).With above-mentioned reverse transcription synthetic cDNA is template, utilize primer H04F:5 '-ATCTGGTGGTCCAACTGCTGA-3 ' and H04R:5 '-CGGCGTTT-TTCAAGTCGAAT-3 ' amplification OsGT γ-1 gene specific fragment, carry out the real-time relative quantitative assay of fluorescence as internal reference (utilizing primer Actin1F:5 '-TGGCATCTCTAGCACATTCC-3 ' and Actin1R:5 '-TGCACAATGGATGGGTCAGA-3 ' amplification OsActin1 gene specific fragment) with the OsActin1 gene.Reaction conditions is: 95 ℃ of 5min; 95 ℃ of 10sec, 60 ℃ of 5sec, 72 ℃ of 34sec, 45 circulations.The result shows that cloned genes OsGT γ-1 of the present invention can be induced (shown in Fig. 2 a and Fig. 2 c) but preserve to coerce with other HORMONE TREATMENT by arid, high salt, low temperature and ABA abduction delivering, is one and is subjected to adverse circumstance inductive transcription factor.With rice varieties " bright extensive 63 " (rice varieties of the open promotion and application of Chinese big area) is material, and the RNA that extracts the tissue of different growing stage detects OsGT γ-1 expression of gene level with Reai-time PCR.13 kinds of histoorgans choosing are respectively: 1, and the root in tillering phase; 2, the stem of jointing stage; The blade of 3,4 leaf phases; The leaf sheath of 4 leaf phases; 5, fringe (0.5-1cm); 6, fringe (3-5cm); 7, fringe (greater than 10cm); 8, the fringe apical meristem before blooming; 9, stamen; 10, gynoecium; 11, the seedling that germinateed 1 day; 12, the seedling in 3 weeks of germinateing; 13; The chrysanthemum seedling in 3 weeks.The result shows: OsGT γ-1 gene all has a certain amount of expression in selected tissue, but with the expression amount in blade, leaf sheath and the young fringe higher (shown in Fig. 2 b).

The proteic Subcellular Localization of embodiment 3:OsGT γ-1

Utilize paddy rice stable conversion system, we will comprise the cDNA fragment that is predicted as OsGT γ-1 nuclear localization sequence and merge with reporter gene GFP, driving by a strong promoter Ubiquitin in the corn is expressed in rice plant, and presentation of results OsGT γ-1 albumen that the root of positive transfer-gen plant is observed under laser confocal microscope is positioned (as shown in Figure 3) in the nucleus.The fusion vector construction process is as follows: OsGT γ-1 Partial cDNA Sequence (correspondence removes 3 '-coding region sequence of terminal 5 amino acid and terminator codon) by primer 5 '-CTA GGTACCGAGCTTCTCTTTGGAATTTGTG-3 ' (the sequence specific primer adds joint KpnI site) and 5 '-TAA CCCGGGCTTTGGTTTGATACCCATCTC-3 ' (the sequence specific primer adds joint SmnaI site) carries out pcr amplification, the 391-1850bp of SEQ ID NO:1 display sequence among the corresponding the present invention of product.(accession number: AK065397) plasmid is that template amplification obtains the purpose fragment to the cDNA clone J013022H04 that answers with this gene pairs among the Japanese paddy rice total length database Knowledge-based OryzaMolecular biological Encyclopedia (http://cdna01.dna.affrc.go.jp).The PCR reaction system is: plasmid DNA template 0.3 μ l (about 100ng), each 1 μ l of 10mM primer, 1 * rTaq polymeric enzyme reaction damping fluid (available from TaKaRa company), 25mM MgCl ₂5 μ l, 10mM dNTP 1 μ l, rTaq polysaccharase (5U/ μ l) (available from TaKaRa company) 0.25 μ l adds ddH2O to 50 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 40sec, 50 ℃ of 40sec, 72 ℃ of 2min, 32 circulations; 72 ℃ are extended 10min.By KpnI and SmaI the above-mentioned cDNA sequence enzyme of OsGT γ-1 is cut and to be connected to the carrier pU1391cGFP that is usually used in the vegetable-protein Subcellular Localization behind the purifying (doctor Dai Mingqiu who has been graduated by crop genetic improvement National Key Laboratory of Hua Zhong Agriculture University transforms, carrier figure sees Fig. 7) in GFP reporter gene front, transformed into escherichia coli DH10 β (bacterial strain is available from Invitrogen company).Cut screening positive clone by enzyme, obtain conversion carrier.

By agriculture bacillus mediated rice genetic method for transformation (as following concrete steps) above carrier (pU1391cGFP-OsGT γ-1) is imported to and to spend in the rice varieties in 11 (rice varieties of the public use that China Paddy Rice Inst provides), through the callus of cultivating in advance, infecting, cultivating altogether, screening having hygromycin resistance, break up, take root, hardening, transplanting, obtain transfer-gen plant.Agriculture bacillus mediated paddy rice (in spend 11) genetic transforming method (system) is at people's reported method such as Hiei (Hiei etc., Efficient transformation ofrice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J, 6:271-282,1994) improve on the basis and carry out (as following concrete steps).

Concrete genetic transformation step is as follows:

(1) callus induction: will spend 11 (rice varieties of the public use that China Paddy Rice Inst provides) to shell in the sophisticated rice paddy seed, used 70% Ethanol Treatment then successively 1 minute, 0.15% mercury chloride (HgCl ₂) seed-coat sterilization 15 minutes; Wash seed 4-5 time with sterilization; Should be placed on (composition is seen below) on the inducing culture by sterile seed; Place dark place to cultivate 4 weeks, 25 ± 1 ℃ of temperature postvaccinal callus inducing medium.

(2) callus subculture: select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in subculture medium (composition is seen below) and go up dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down.

(3) the pre-cultivation: select the embryo callus subculture of consolidation and relatively dry, be put in pre-culture medium (composition is seen below) and go up dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down.

(4) Agrobacterium is cultivated: (composition is seen below) cultivated Agrobacterium EHA105 (deriving from CAMBIA, commercial bacterial strain) two days, 28 ℃ of culture temperature in advance on the LA substratum that has corresponding resistance selection; Described Agrobacterium is transferred to suspension culture base (composition is seen below) lining, cultivated 2-3 hour on 28 ℃ of shaking tables.

(5) Agrobacterium is infected: pre-incubated callus is transferred in the good bottle of sterilization; The suspension of regulating Agrobacterium is to OD ₆₀₀0.8-1.0; Callus was soaked in agrobacterium suspension 30 minutes; Shifting callus blots to the good filter paper of sterilization; Be placed on common substratum (composition is seen below) then and go up cultivation 3 days, culture temperature 19-20 ℃.

(6) callus washing and selection are cultivated: aqua sterilisa washing callus is to cannot see Agrobacterium; Be immersed in the aqua sterilisa that contains 400ppm Pyocianil (CN) 30 minutes; Shifting callus blots to the good filter paper of sterilization; Shift callus and select 2-3 time, each 2 weeks (screening Pyocianil concentration for the first time is 400ppm, and be 250ppm later the second time, Totomycin concentration 250ppm) to selecting substratum (composition is seen below) to go up.

(7) differentiation: kanamycin-resistant callus tissue is transferred to pre-differentiation substratum (composition is seen below) goes up dark place cultivation 5-7 week; Shift the callus (composition is seen below) to division culture medium that pre-differentiation is cultivated, illumination is cultivated 26 ℃ of temperature down.

(8) take root: cut the root that differentiation phase produces; Then it is transferred to and cultivates 2-3 week, 26 ℃ of temperature in the root media under the illumination.

(9) transplant: wash the residual substratum on the root off, the seedling that will have good root system changes the greenhouse over to, divides moistening at initial several Tian Bao water holding simultaneously.

Nutrient media components and prescription thereof: (1) reagent and solution abbreviation: the abbreviation of the used plant hormone of substratum is expressed as follows among the present invention: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyaceticacid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (Casein Enzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (a large amount of composition solution of N6); N6mix (N6 trace ingredients solution); MSmax (a large amount of composition solution of MS); MSmix (MS trace ingredients solution).

(2) main solution formula:

1) preparation of N6 substratum macroelement mother liquor [10 times of concentrated solutions (10X)]:

Saltpetre (KNO ₃) 28.3g

Potassium primary phosphate (KH ₂PO ₄) 4.0g

Ammonium sulfate ((NH ₄) ₂SO ₄) 4.63g

Sal epsom (MgSO ₄7H ₂O) 1.85g

Calcium chloride (CaCl ₂2H ₂O) 1.66g

Dissolving is settled to 1000ml under the room temperature then one by one.

2) preparation of N6 substratum trace element mother liquor [100 times of concentrated solutions (100X)]

Potassiumiodide (KI) 0.08g

Boric acid (H ₃BO ₃) 0.16g

Manganous sulfate (MnSO ₄4H ₂O) 0.44g

Zinc sulfate (ZnSO ₄7H ₂O) 0.15g

Dissolving and be settled to 1000ml under the room temperature.

3) molysite (Fe ₂EDTA) preparation of stock solution (100X)

Prepare the 800ml distilled water and be heated to 70 ℃, add b diammonium disodium edta (Na ₂EDTA2H ₂O) 3.73 grams, fully the dissolving back kept 2 hours in 70 ℃ of water-baths, was settled to 1000ml, and 4 ℃ of preservations are standby.

4) VITAMIN stock solution (100X) preparation

Nicotinic acid (Nicotinic acid) 0.1g

VITMAIN B1 (Thiamine HCl) 0.1g

Vitamin B6 (Pyridoxine HCl) 0.1g

Glycine (Glycine) 0.2g

Inositol (Inositol) 10g

Add water and be settled to 1000ml, 4 ℃ of preservations are standby.

5) preparation of MS substratum macroelement mother liquor (10X)

Ammonium nitrate (NH ₄NO ₃) 16.5g

Saltpetre 19.0g

Potassium primary phosphate 1.7g

Sal epsom 3.7g

Calcium chloride 4.4g

Dissolving and be settled to 1000ml under the room temperature.

6) preparation of MS substratum trace element mother liquor (100X)

Potassiumiodide 0.083g

Boric acid 0.62g

Manganous sulfate 0.86g

Sodium orthomolybdate (Na ₂MoO ₄2H ₂O) 0.025g

Copper sulfate (CuSO ₄5H ₂O) 0.0025g

Dissolving and be settled to 1000ml under the room temperature.

7) 2,4-D stock solution, 6-BA stock solution, naphthylacetic acid (NAA) stock solution, indolylacetic acid (IAA) stock solution: be 1mg/ml.

8) glucose stock solution: 0.5g/ml.

9) preparation of AS stock solution: weigh AS 0.392g, DMSO 10ml.

(3) be used for the culture medium prescription that rice genetic transforms

1) callus inducing medium

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

2,4-D stock solution 2.5ml

Proline(Pro) (Proline) 0.3g

CH 0.6g

Sucrose (Sucrose) 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.

2) subculture medium

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

2,4-D stock solution 2.0ml

Proline(Pro) 0.5g

CH 0.6g

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.

3) pre-culture medium

N6max mother liquor (10X) 12.5ml

N6mix mother liquor (100X) 1.25ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.15g

Sucrose 5g

Agar powder (Agarose) 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, seals sterilization.Heating is fused substratum and is added 5ml glucose stock solution and 250 μ l AS stock solutions before using, and (25ml/ ware) in the culture dish poured in packing into.

4) be total to substratum

N6max mother liquor (10X) 12.5ml

N6mix mother liquor (100X) 1.25ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.75ml

CH 0.2g

Sucrose 5g

Agar powder 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, seals sterilization.Heating is fused substratum and is added 5ml glucose stock solution and 250 μ l AS stock solutions before using, and (the every ware of 25ml/) in the culture dish poured in packing into.

5) suspension culture base

N6max mother liquor (10X) 5ml

N6mix mother liquor (100X) 0.5ml

Fe ²⁺EDTA stock solution (100X) 0.5ml

VITAMIN stock solution (100X) 1ml

2,4-D stock solution 0.2ml

CH 0.08g

Sucrose 2g

Adding distil water is regulated pH value to 5.4 to 100ml, divides to install in the triangular flask of two 100ml, seals sterilization.Add 1ml glucose stock solution and 100 μ l AS stock solutions before using.

6) select substratum

N6max mother liquor (10X) 25ml

N6mix mother liquor (100X) 2.5ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

2,4-D stock solution 0.625ml

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Adding distil water is regulated pH value to 6.0 to 250ml, seals sterilization.The fusion substratum adds 250 μ l HN and 400ppmCN before using, and (25ml/ ware) in the culture dish poured in packing into.

7) break up substratum in advance

N6max mother liquor (10X) 25ml

N6mix mother liquor (100X) 2.5ml

Fe ²⁺EDTA stock solution (100X) 2.5ml

VITAMIN stock solution (100X) 2.5ml

6-BA stock solution 0.5ml

KT stock solution 0.5ml

NAA stock solution 50 μ l

IAA stock solution 50 μ l

CH 0.15g

Sucrose 7.5g

Agar powder 1.75g

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.9, seals sterilization.The fusion substratum adds 250 μ l HN and 200ppm CN before using, and (25ml/ ware) in the culture dish poured in packing into.

8) division culture medium

N6max mother liquor (10X) 100ml

N6mix mother liquor (100X) 10ml

Fe ²⁺EDTA stock solution (100X) 10ml

VITAMIN stock solution (100X) 10ml

6-BA stock solution 2ml

KT stock solution 2ml

NAA stock solution 0.2ml

IAA stock solution 0.2ml

CH 1g

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 6.0.Boil and be settled to 1000ml, divide to install to 50ml triangular flask (50ml/ bottle), seal sterilization.

9) root media

MSmax mother liquor (10X) 50ml

MSmix mother liquor (100X) 5ml

Fe ²⁺EDTA stock solution (100X) 5ml

VITAMIN stock solution (100X) 5ml

Sucrose 30g

Phytagel 3g

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.8.Boil and be settled to 1000ml, divide to install to (25ml/ pipe) in the pipe of taking root, seal sterilization.

10) LA substratum

LB(pH?7.0)1L

Tryptone 10g

Yeast?extracts 5g

NaCl 10g

Agar powder 15g

Adding distil water is settled to 1000ml, seals sterilization.The fusion substratum adds corresponding microbiotic before using, and (25ml/ ware) in the culture dish poured in packing into.

Embodiment 4: identify that OSGT γ-1 paddy rice T-DNA inserts mutant

Function for research OsGT γ-1, with the genome sequence (comprising its promoter region 1000bp) of this gene rice mutant database (http://rmd.ncpgr.cn/ in chamber crop genetic improvement National Key Laboratory of Hua Zhong Agriculture University, one to the public database of opening both at home and abroad, the related rice mutant material public of database can be to crop genetic improvement National Key Laboratory of the Hua Zhong Agriculture University obtainable post-free of possessor China Wuhan City, Hubei Province) in carry out the BLAST retrieval, found that a paddy rice T-DNA inserts mutant (03Z11BT07).Sequence involved in the present invention derives from NCBI (http://www.ncbi.nlm.nih.gov/), TIGR (http://www.tigr.org), KOME (http://cdna01.dna.affrc.go.jp/cDNA/), the positional information of localized gene is as the criterion with " TIGR Rice Annotation Release 5 ".

Match condition according to flanking sequence and rice genome, can determine to insert the site, design a pair of primers F and R on the both sides of inserting the site, and T-DNA goes up primer VP of design, as Fig. 4 a, F is illustrated in the primer that inserts the site upstream design, and R is illustrated in the primer that inserts the design of downstream, site, and VP represents the primer according to the design of T-DNA internal sequence.At T ₁In plant, the T-DNA homozygous plants has only going out that R and VP pairing can increase, because have T-DNA insertion F and R paired product can't obtain amplified fragments too greatly, the plant of wild-type is not owing to there is the insertion of T-DNA, have only F and R pairing can obtain product, heterozygous plant then can be increased when R and VP pairing and F and R pairing and be obtained product.According to the primer sequence that inserts the site design is F (primer): 5 '-GGCACGTCATGGTTGGTAC-3 ', R (primer): 5 '-AACAAAAGCGGCGGTAATG-3 ', VP (primer): 5 '-AATCCAGATCCCCCGAATTA-3 '.The PCR reaction system is: rice mutant sample dna profiling 0.5 μ l (about 50ng), each 0.2 μ l of 10mM primer, 1 * rTaq polymeric enzyme reaction damping fluid, 25mM MgCl ₂2 μ l, 10mM dNTP 0.2 μ l, rTaq polysaccharase (5U/ μ l) 0.1 μ l adds ddH ₂O to 20 μ l.Response procedures is: 94 ℃ of 3min; 94 ℃ of 40sec, 55 ℃ of 40sec, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 10min.PCR result is shown in Fig. 4 b, and wherein swimming lane 3,4, and 13,15,18 isozygoty for T-DNA; 1,2,5,6,7,9,10,11,12,14,16,17 is the T-DNA heterozygosis; 8,9 is the T-DNA feminine gender.The result shows that T-DNA (left margin) is inserted in the promoter region of OsGT γ-1 gene (from the about 500bp of transcription initiation site).

The homozygous mutation body family and the wild-type family of identified gene type have been carried out the high-salt stress experiment to choose 5.Utilize the method for Real-time PCR to detect under the normal condition and OSGT γ-1 expression of gene amount in homozygous mutation body family and the wild-type family under the high-salt stress condition, the result shows that OSGT γ-1 gene all is suppressed (shown in Fig. 4 c) in the expression in the homozygous mutation body.High-salt stress experiment concrete steps are as follows: with homozygous mutation body family and wild-type family seed sterilization (the 75% alcohol processing 3min that shells, 0.15% mercury chloride is handled 10min, sterile water wash 5 times), on the MS substratum, germinate, select after 2-3 days and germinate good and the consistent seed of growing way is transferred to respectively on the MS substratum of the NaCl that contains 0mmol/L and 150mmol/L, observe phenotype after 7-8 days and measure the plant height of plant in the growth of illumination cultivation chamber.Every kind of processing of each family is no less than 15 plant, and experiment is provided with 3 repetitions.The result shows: growth and the contrast under normal operation of homozygous mutation body plant do not have notable difference, but the growth of homozygous mutation body plant under the high-salt stress treatment condition significantly (t tests P＜0.05) is inferior to contrast, illustrates that suppressing OSGT γ-1 expression of gene can make plant that the susceptibility of high-salt stress is strengthened (see figure 4).3 secondary pollutants that this experiment is provided with are learned the reproducible results unanimity, illustrate that this mutant phenotype is that the T-DNA insertion causes really.For genetic stability and the further checking of verifying this mutant is divided into from situation, having bred a generation again is T ₂Generation.With T ₁Withhold the planting seed that obtains and obtain T ₂In generation, isozygotied heterozygosis and wild type seeds.Carry out the above-mentioned experiment of coercing equally, as a result unanimity.

The structure of embodiment 5:OSGT γ-1 gene overexpression carrier, conversion and transfer-gen plant salt tolerant sexual type are identified

In order to illustrate the function of OSGT γ-1 gene better, the applicant is studied the function of this gene with its overexpression in paddy rice by the phenotype of transfer-gen plant.

The overexpression carrier construction method is as follows: at first by searching Japanese paddy rice total length database Knowledge-based Oryza Molecularbiological Encyclopedia (http://cdna01.dna.affrc.go.jp), find its pairing cDNA clone J013022H04 (accession number: AK065397), and buy its full-length cDNA.With this full-length cDNA is template, usefulness primer OE07F (5 '-GTT GGTACCAGGTTTTTAGGCGTGG-3 ', sequence specific primer add joint KpnI site) and OE07R (5 '-GAA CTGCAGTGACCGATTGCAGTTAG-3 ', the sequence specific primer adds joint PstI) amplify the dna fragmentation that comprises OSGT γ-1 total length.The PCR reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of 40sec, 50 ℃ of 40sec, 72 ℃ of 2min, 32 circulations; 72 ℃ are extended 10min.This amplified fragments subclone is arrived

On the carrier (purchasing company) in Promega, screening positive clone also carries out sequence verification, uses primer ALLF (5 '-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AAT ACG ACT CACTAT AGG G) and ALLR (5 '-GGG GAC CAC TIT GTA CAA GAA AGC TGG GTA TTT AGG TGA CAC TATAG) segmental from having this again

Amplifying one on the carrier can be for the fragment on the pCB2004H carrier of recombinating, and the PCR condition is the same.The recombinant clone test kit specification sheets that provides by Invitrogen company carries out transformed into escherichia coli DH10 β (intestinal bacteria DH10 β bacterial strain is available from Invitrogen company) behind the recombining reaction.Cut screening positive clone by enzyme, obtain conversion carrier.PCB2004H is the carrier (Hou etc. that are used for the overexpression vector construction that published, A homolog of human ski-interacting protein in rice positively regulates cellviability and stress tolerance, Proc Natl Acad Sci USA, 2009,106:6410-6415) (carrier figure sees Fig. 6).

By agriculture bacillus mediated rice genetic method for transformation (as following concrete steps) above-mentioned overexpression carrier (pCB2004H-OSGT γ-1) is imported to and to spend in the rice varieties in 11 (rice varieties of the public use that China Paddy Rice Inst provides), through the callus of cultivating in advance, infecting, cultivating altogether, screening having hygromycin resistance, break up, take root, hardening, transplanting, obtain transfer-gen plant.Concrete genetic transformation step is with described in the embodiment 3.

Present embodiment utilizes the method for Real-time PCR to identify T ₀For OSGT γ-1 expression of gene amount in the transfer-gen plant, 5 OSGT γ-1 overexpression T have been chosen ₂Family (shown in Fig. 5 b) is carried out the high-salt stress experiment.Concrete steps are as follows: overexpression transgenosis family and wild-type family seed are shelled, and (75% alcohol is handled 3min in sterilization, 0.15% mercury chloride is handled 10min, sterile water wash 5 times), germinate containing on the MS substratum of 50mg/L Totomycin, wild-type contrast family is sowed on the MS substratum that does not contain Totomycin one day evening, select after 2-3 days and germinate good and the consistent seed of growing way is transferred to respectively on the MS substratum of the NaCl that contains 0mmol/L and 150mmol/L, observe phenotype after 7-8 days and measure the plant height of plant in the growth of illumination cultivation chamber.Every kind of processing of each family is no less than 15 plant, and experiment is provided with 3 repetitions.The result shows: the growth and the contrast of OSGT γ-1 overexpression transfer-gen plant do not have notable difference under normal operation, but growth wants remarkable (t tests P＜0.01) to be better than contrast (Fig. 5) under high-salt stress is handled, illustrate that OsGT γ-1 expression of gene of the present invention can alleviate the plant strain growth that high-salt stress causes and be obstructed, strengthen the tolerance (as Fig. 5) of transgenic plant high-salt stress.

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉application of rice GT transcription factor family gene OsGT γ-1 in control paddy rice salt tolerance

<130>

<141>2009-12-15

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1998

<212>DNA

＜213〉paddy rice (Oryza sativa)

<220>

<221>gene

<222>(1)..(1998)

<223>

<220>

<221>CDS

<222>(475)..(1707)

<223>

<400>1

gattcttcct?ttctctgtgt?ttgtttgttt?ccagttgcca?ctaaccagcc?agagccccgg 60

gggggagaag?agcgagagag?gaggagaaat?cttgcagcaa?tccctcgctt?agattcgcca 120

ttaccgccgc?ttttgttccc?accaagatta?ccaccccctc?ctcctcctgt?gctcgagcgc 180

tcgcatcgca?tttcaggctg?cttcttgatt?ctgcgaggag?gttgaaatct?ctcgtgattg 240

caacagctgc?catccgttcg?ttccggtgga?gtgcacccct?tcatcaccaa?ggaggagaaa 300

aagctgtaat?cttcggccgt?ttctgccttt?ctgcagcaaa?cggttgaaga?aaagccgcgt 360

cttttgggcg?gatttcgcgg?aggccgcttg?aggtttttag?gcgtggttcg?tgtgagcttc 420

tctttggaat?ttgtggggga?aagagtgagg?gagggaggga?gctcagtggg?agag?atg 477

Met

1

gag?ggg?aat?ttg?ccg?ccg?aga?gga?gcc?ctt?gtc?cat?ggc?cat?ggc?ggc 525

Glu?Gly?Asn?Leu?Pro?Pro?Arg?Gly?Ala?Leu?Val?His?Gly?His?Gly?Gly

5 10 15

ggc?gtc?ggc?gcc?ttc?gat?ctg?gag?gcg?acg?atg?cag?ccg?ccg?ccg?ccg 573

Gly?Val?Gly?Ala?Phe?Asp?Leu?Glu?Ala?Thr?Met?Gln?Pro?Pro?Pro?Pro

20 25 30

ttc?cac?ttc?gcc?cag?gat?ccg?cac?ctc?cac?cac?cac?cag?ggc?atg?gtc 621

Phe?His?Phe?Ala?Gln?Asp?Pro?His?Leu?His?His?His?Gln?Gly?Met?Val

35 40 45

ccc?gtc?cgc?ggg?aac?ccg?atg?ctg?gat?ttg?ggc?aat?gtg?gtc?aag?acc 669

Pro?Val?Arg?Gly?Asn?Pro?Met?Leu?Asp?Leu?Gly?Asn?Val?Val?Lys?Thr

50 55 60 65

tcg?ccg?agc?gac?gag?gag?gac?gtg?gat?gac?ggc?cac?cac?cat?ggt?ggc 717

Ser?Pro?Ser?Asp?Glu?Glu?Asp?Val?Asp?Asp?Gly?His?His?His?Gly?Gly

70 75 80

ggc?ggc?ggc?agc?ggt?aag?gag?gcc?tcc?cag?tgg?cac?cgg?gtg?aag?tgg 765

Gly?Gly?Gly?Ser?Gly?Lys?Glu?Ala?Ser?Gln?Trp?His?Arg?Val?Lys?Trp

85 90 95

atc?agt?ggc?atg?gtc?aag?ctg?ctg?gtg?tcc?gcc?gtg?gcc?tac?atc?gac 813

Ile?Ser?Gly?Met?Val?Lys?Leu?Leu?Val?Ser?Ala?Val?Ala?Tyr?Ile?Asp

100 105 110

gag?gac?gtc?gac?atg?gac?tac?ggc?acg?ggc?agc?gcc?gcg?agg?agg?aag 861

Glu?Asp?Val?Asp?Met?Asp?Tyr?Gly?Thr?Gly?Ser?Ala?Ala?Arg?Arg?Lys

115 120 125

cac?gcc?atg?ctg?aag?agg?aag?ggg?aag?tgg?agg?ctc?gtc?tcc?gcg?gcg 909

His?Ala?Met?Leu?Lys?Arg?Lys?Gly?Lys?Trp?Arg?Leu?Val?Ser?Ala?Ala

130 135 140 145

atg?acc?gag?agg?ggc?ttc?ccg?gtg?tcg?ccg?cag?cag?tgc?gag?gac?aag 957

Met?Thr?Glu?Arg?Gly?Phe?Pro?Val?Ser?Pro?Gln?Gln?Cys?Glu?Asp?Lys

150 155 160

ttc?aac?gat?ctc?aac?aag?agg?tac?aag?agg?atg?acc?gag?atc?ctt?ggc 1005

Phe?Asn?Asp?Leu?Asn?Lys?Arg?Tyr?Lys?Arg?Met?Thr?Glu?Ile?Leu?Gly

165 170 175

cgg?gga?acg?gcg?tgc?cag?gtc?gtc?gag?cac?ccc?gag?ctt?ctt?gag?ggg 1053

Arg?Gly?Thr?Ala?Cys?Gln?Val?Val?Glu?His?Pro?Glu?Leu?Leu?Glu?Gly

180 185 190

atg?cgg?ctc?tcg?gga?aag?ctc?aaa?gag?gag?gct?agg?aag?cac?ctg?aac 1101

Met?Arg?Leu?Ser?Gly?Lys?Leu?Lys?Glu?Glu?Ala?Arg?Lys?His?Leu?Asn

195 200 205

tca?aag?cat?ctg?cac?tat?gag?gag?atg?tgc?tcg?tac?cat?aac?agg?aac 1149

Ser?Lys?His?Leu?His?Tyr?Glu?Glu?Met?Cys?Ser?Tyr?His?Asn?Arg?Asn

210 215 220 225

aaa?atg?tgt?ctg?ttt?gat?gat?ccc?gcc?ctt?cag?aag?tca?ttg?cgt?ttg 1197

Lys?Met?Cys?Leu?Phe?Asp?Asp?Pro?Ala?Leu?Gln?Lys?Ser?Leu?Arg?Leu

230 235 240

gcg?ctt?agg?agt?gga?gag?gag?cgt?gca?aag?aag?aac?ccg?ttt?ggg?tat 1245

Ala?Leu?Arg?Ser?Gly?Glu?Glu?Arg?Ala?Lys?Lys?Asn?Pro?Phe?Gly?Tyr

245 250 255

gat?gat?gag?gat?ttt?tct?gac?gat?gac?gac?gaa?gat?gaa?gaa?ttc?gat 1293

Asp?Asp?Glu?Asp?Phe?Ser?Asp?Asp?Asp?Asp?Glu?Asp?Glu?Glu?Phe?Asp

260 265 270

gat?ctg?gag?gtc?agc?gct?gaa?gat?cat?cac?cat?gga?att?cat?ggt?gcc 1341

Asp?Leu?Glu?Val?Ser?Ala?Glu?Asp?His?His?His?Gly?Ile?His?Gly?Ala

275 280 285

aag?agg?ctg?aag?cat?gat?cag?gag?gag?acg?cat?ttt?ggg?tct?aat?ctg 1389

Lys?Arg?Leu?Lys?His?Asp?Gln?Glu?Glu?Thr?His?Phe?Gly?Ser?Asn?Leu

290 295 300 305

tca?gaa?gtt?gct?gtt?att?gac?atg?aac?aaa?atg?ctc?tct?gag?gga?tct 1437

Ser?Glu?Val?Ala?Val?Ile?Asp?Met?Asn?Lys?Met?Leu?Ser?Glu?Gly?Ser

310 315 320

ggt?ggt?cca?act?gct?gaa?aag?agt?ccc?tct?acc?cct?ggt?atg?cgt?gat 1485

Gly?Gly?Pro?Thr?Ala?Glu?Lys?Ser?Pro?Ser?Thr?Pro?Gly?Met?Arg?Asp

325 330 335

att?cga?ctt?gaa?aaa?cgc?cgc?ctg?aag?atc?aaa?gct?cag?atg?ctg?aaa 1533

Ile?Arg?Leu?Glu?Lys?Arg?Arg?Leu?Lys?Ile?Lys?Ala?Gln?Met?Leu?Lys

340 345 350

att?gag?cag?aag?cat?ttc?aag?tgg?ctg?agg?ttc?agc?aag?gag?aag?gac 1581

Ile?Glu?Gln?Lys?His?Phe?Lys?Trp?Leu?Arg?Phe?Ser?Lys?Glu?Lys?Asp

355 360 365

agg?gaa?ttg?gag?aag?atg?aga?ctg?gag?aat?gaa?aag?atg?aaa?ctg?gag 1629

Arg?Glu?Leu?Glu?Lys?Met?Arg?Leu?Glu?Asn?Glu?Lys?Met?Lys?Leu?Glu

370 375 380 385

aat?gag?cgg?ttg?gaa?ttg?gag?ctg?aag?ctt?aaa?gaa?ata?gag?atg?ggt 1677

Asn?Glu?Arg?Leu?Glu?Leu?Glu?Leu?Lys?Leu?Lys?Glu?Ile?Glu?Met?Gly

390 395 400

atc?aaa?cca?aag?aag?att?ttt?agt?gat?tga?agccttggaa?tgatagaact 1727

Ile?Lys?Pro?Lys?Lys?Ile?Phe?Ser?Asp

405 410

agaaatggtt?agagttgctg?gtgatccttt?tgtcttttgc?aggagcttgc?agatttaggg 1787

tcttatatat?acattgatct?gtgcccctgt?ttgaatttta?ccttagctaa?ctgcaatcgg 1847

tcaagtgaat?ggctattcca?gttgttaagt?gtttaaactt?ggtactagtt?ggcctttcaa 1907

atgataaatg?tttaagccgt?ccttgtacat?gcctgctgtt?aactttctgc?agttgtttgt 1967

gtgttgctat?tgacctctga?gtcttttttt?t 1998

<210>2

<211>410

<212>PRT

＜213〉paddy rice (Oryza sativa)

<400>2

Met?Glu?Gly?Asn?Leu?Pro?Pro?Arg?Gly?Ala?Leu?Val?His?Gly?His?Gly

1 5 10 15

Gly?Gly?Val?Gly?Ala?Phe?Asp?Leu?Glu?Ala?Thr?Met?Gln?Pro?Pro?Pro

20 25 30

Pro?Phe?His?Phe?Ala?Gln?Asp?Pro?His?Leu?His?His?His?Gln?Gly?Met

35 40 45

Val?Pro?Val?Arg?Gly?Asn?Pro?Met?Leu?Asp?Leu?Gly?Asn?Val?Val?Lys

50 55 60

Thr?Ser?Pro?Ser?Asp?Glu?Glu?Asp?Val?Asp?Asp?Gly?His?His?His?Gly

65 70 75 80

Gly?Gly?Gly?Gly?Ser?Gly?Lys?Glu?Ala?Ser?Gln?Trp?His?Arg?Val?Lys

85 90 95

Trp?Ile?Ser?Gly?Met?Val?Lys?Leu?Leu?Val?Ser?Ala?Val?Ala?Tyr?Ile

100 105 110

Asp?Glu?Asp?Val?Asp?Met?Asp?Tyr?Gly?Thr?Gly?Ser?Ala?Ala?Arg?Arg

115 120 125

Lys?His?Ala?Met?Leu?Lys?Arg?Lys?Gly?Lys?Trp?Arg?Leu?Val?Ser?Ala

130 135 140

Ala?Met?Thr?Glu?Arg?Gly?Phe?Pro?Val?Ser?Pro?Gln?Gln?Cys?Glu?Asp

145 150 155 160

Lys?Phe?Asn?Asp?Leu?Asn?Lys?Arg?Tyr?Lys?Arg?Met?Thr?Glu?Ile?Leu

165 170 175

Gly?Arg?Gly?Thr?Ala?Cys?Gln?Val?Val?Glu?His?Pro?Glu?Leu?Leu?Glu

180 185 190

Gly?Met?Arg?Leu?Ser?Gly?Lys?Leu?Lys?Glu?Glu?Ala?Arg?Lys?His?Leu

195 200 205

Asn?Ser?Lys?His?Leu?His?Tyr?Glu?Glu?Met?Cys?Ser?Tyr?His?Asn?Arg

210 215 220

Asn?Lys?Met?Cys?Leu?Phe?Asp?Asp?Pro?Ala?Leu?Gln?Lys?Ser?Leu?Arg

225 230 235 240

Leu?Ala?Leu?Arg?Ser?Gly?Glu?Glu?Arg?Ala?Lys?Lys?Asn?Pro?Phe?Gly

245 250 255

Tyr?Asp?Asp?Glu?Asp?Phe?Ser?Asp?Asp?Asp?Asp?Glu?Asp?Glu?Glu?Phe

260 265 270

Asp?Asp?Leu?Glu?Val?Ser?Ala?Glu?Asp?His?His?His?Gly?Ile?His?Gly

275 280 285

Ala?Lys?Arg?Leu?Lys?His?Asp?Gln?Glu?Glu?Thr?His?Phe?Gly?Ser?Asn

290 295 300

Leu?Ser?Glu?Val?Ala?Val?Ile?Asp?Met?Asn?Lys?Met?Leu?Ser?Glu?Gly

305 310 315 320

Ser?Gly?Gly?Pro?Thr?Ala?Glu?Lys?Ser?Pro?Ser?Thr?Pro?Gly?Met?Arg

325 330 335

Asp?Ile?Arg?Leu?Glu?Lys?Arg?Arg?Leu?Lys?Ile?Lys?Ala?Gln?Met?Leu

340 345 350

Lys?Ile?Glu?Gln?Lys?His?Phe?Lys?Trp?Leu?Arg?Phe?Ser?Lys?Glu?Lys

355 360 365

Asp?Arg?Glu?Leu?Glu?Lys?Met?Arg?Leu?Glu?Asn?Glu?Lys?Met?Lys?Leu

370 375 380

Glu?Asn?Glu?Arg?Leu?Glu?Leu?Glu?Leu?Lys?Leu?Lys?Glu?Ile?Glu?Met

385 390 395 400

Gly?Ile?Lys?Pro?Lys?Lys?Ile?Phe?Ser?Asp

405 410

Claims (1)

1. the application of OsGT γ-1 gene in paddy rice salt tolerance genetic improvement that can improve the salt stress tolerance, the nucleotide sequence of this gene is shown in 475-1707 position among the sequence table SEQ NO:1.

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