CN102021182B - Application of rice gene KT506 in improving stress tolerance of plant - Google Patents

Abstract

The invention discloses a gene KT506 which is derived from rice and relates to the stress resistance. Experiments show that through the application of the rice gene KT506, the tolerance of rice (subjected to gene transformation) to high salt can significantly be improved. In the application of the rice gene KT506, proteins and coding genes of the proteins have important theoretical and practical significances to the study on the stress resistance mechanism of the plant and the improvement on the stress resistance and correlated characters of the plant, and play an important role in the stress tolerant genetic engineering improvement of the plants (in particular to cereal crops), thus the prospect of application is broad.

Description

Paddy gene KT506 is in the application that improves on the plant stress tolerance energy

Technical field

The present invention relates to plant stress tolerance relevant gene and its application, particularly derive from paddy rice and the degeneration-resistant relevant application of gene KT506 in the raising plant salt endurance.

Background technology

Paddy rice, corn and wheat are the important food crop of China, and the output of three large crops, quality are all most important for grain-production and the grain security of China.The abiotic stresses such as arid, saline and alkaline, high temperature and freeze injury can directly affect normal growth and the output of food crop.

Along with the application and popularization of Protocols in Molecular Biology develop rapidly and transgenic technology, the cultivation of the genetically modified organism new variety take the farm crop character improvement as major objective more and more comes into one's own.The U.S., Japan, Canada, Korea S and some national governments of Europe have all dropped into the research that considerable fund is carried out plant functional genomics; Simultaneously, some high-end biotech companies drop into the full-length cDNA that a large amount of manpower and materials are separated the types of functionality gene, and trying to be the first obtains useful genetic resources.CERES company such as the U.S. utilizes high-quality cDNA library, a few years in the time at corn, applied for the patent of 15000 genes nearly on the plants such as Arabidopis thaliana, soybean, wheat, cotton, rape; So that powerful MONSANTO company goes out huge fund and its cooperation, the function of joint research gene.But, some are by the gene of the definite functions of the means such as deletion mutant body acquisition, especially anti contravariance related gene, be difficult to really be applied to the character improvement of farm crop, its reason mainly is when having improved anti-adversity, also affect the normal growth of plant, can't keep good economical character.But some anti contravariance related gene does not affect the normal growth of crop basically when being subjected to the regulation and control of adverse circumstance inducible promoter.For this situation, the Mendel Biotechnology company of the U.S. is transferred to the whole transcription factors of Arabidopis thaliana and the combination of different promotor respectively in the tomato, has obtained a collection of gene very large application potential is arranged aspect the farm crop improvement.The method that proof is sought the effective gene of crop character improvement by the mass-producing functional verification of gene is a practicable approach.

Transcription factor is the key gene of a class regulate gene expression, and growing of crop played important regulating effect.Utilize the bioinformation means, the transcription factor gene of inferring from genomic level according to the constructional feature of transcription factor is one group of gene that most possibly has clear and definite function.For many years, the clone of transcription factor and functional study are one of focuses of scientific research always.A large amount of transcription factor that scientists has been utilized different study route isolation identification has been enriched the transcription factor database, has also therefrom found some and economical character related regulatory factors, for the character improvements of farm crop provides the important gene resource.

Summary of the invention

The purpose of this invention is to provide a paddy gene KT506 relevant with resistance of reverse, to be used for improving the resistance of reverse energy of plant.

The KT506 gene relevant with resistance of reverse that invention provides derives from Oryza paddy rice (Oryza sativa L.), and coding has the protein of following aminoacid sequence:

1) the SEQ ID NO:1 in the sequence table;

SEQ ID NO:1 in the sequence table is comprised of 325 amino-acid residues, is albumen KT506.

The encoding gene of KT506 both can be the cDNA sequence of described gene among the present invention, also can be the genomic dna sequence of described gene, or had 90% above homology and the dna sequence dna of the identical function albumen of encoding with described gene.Encoding gene with aminoacid sequence shown in the SEQ ID NO:1 can have the nucleotide sequence of SEQ ID NO:2 in the sequence table.

The expression vector, transgenic cell line and the Host Strains that contain gene of the present invention all belong to protection scope of the present invention.

The primer pair of the arbitrary fragment of amplification KT506 is also within protection scope of the present invention.

Another object of the present invention provides a kind of method that improves plant stress tolerance.

The method of raising plant stress tolerance provided by the present invention is that plant stress tolerance obtains to improve with code book invention KT506 gene transfered plant tissue, cell or the organ relevant with resistance of reverse.

In the method for above-mentioned raising plant stress tolerance, the KT506 gene that paddy rice is relevant with resistance of reverse among the present invention both can be the cDNA sequence of described gene, also can be the genomic gene sequence of described gene; Having the dna sequence dna of 90% above homology and coding identical function albumen with described gene, is the cDNA of described gene or genomic gene sequence to be separated and/or modified and/or design with known method obtain.What it should be appreciated by those skilled in the art is; the minor alteration of Nucleotide identity may cause reduction or the reinforcement of this gene usefulness in the specific gene sequence; and in some application (for example; antisense or co-suppression technology) in, the frequent meeting of partial sequence and full length sequence play a role equally effectively.The method that gene order changes or shortens, and the method for testing the validity of these genes that change all is well known to those skilled in the art.

Paddy rice of the present invention KT506 gene relevant with resistance of reverse or its homologous sequence can import plant tissue, cell or organ by plant expression vector; The carrier that sets out that is used for making up described plant expression vector can be any one carrier etc. that can be used for the binary vector of agrobacterium tumefaciens or Agrobacterium rhizogenes conversion of plant or can be used for the plant micropellet bombardment, such as pBin serial carrier (such as pBin19 etc.), pBI serial carrier (such as pBI 101 etc.), Gateway ^TWSerial carrier (such as pH2GW7 etc.), pCAMBIA serial carrier (such as pCAMBIA 3301 etc.), per8, pX6 or other derivative plant expression vector, the described carrier that sets out also can be the carrier that can copy in prokaryotic organism, such as pENTER-TOPO, pUC serial carrier or pBluescript serial carrier etc.

When using paddy rice is relevant with resistance of reverse among the present invention KT506 gene or its homologous sequence structure plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or induction type (ABA, arid, saline and alkaline or chemical induction etc.) promotor.Described constructive expression's promotor can be cauliflower mosaic virus (CAMV) 35S promoter, corn Ubiquitin promotor or paddy rice actin1 promotor etc.; Described tissue specificity expression promoter can be root-specific expression promotor, blade specific is expressed promotor, dimension pipe specific expressing promoter, seed-specific expression promotor, flower specific expression promotor or pollen specific expression promotor, such as 2S1 promotor (GenBank number: NM_118848.2, GI:30687489) and NapinA (GenBank number: M64633.1, GI:349405) promotor etc.; Described inducible promoter can be and is subjected to low temperature, arid, ABA, ethene, the promotor of inducing such as saline and alkaline or chemical.Above-mentioned promotor can be used separately or be combined with other plant promoter.In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer and/or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene (gus gene of luminophor as adding the coding that in plant, to express, the GFP gene, luciferase genes etc.), antibiotic marker thing (neomycin phosphotransferase (NPTII) gene with resistance, hygromix phosphotransferase (Hygromycin phosphotransferase) gene, gentamicin marker or kantlex marker etc.) or anti-chemical reagent marker gene (such as anti-weedkiller gene) etc.Described host plant cell, tissue or the organ that contains neomycin phosphotransferase (NPTII) gene can be screened by kantlex or its substituted derivatives such as G418 etc., and the host plant cell, tissue or the organ that contain hygromix phosphotransferase (Hygromycin phosphotransferase) gene can be screened by Totomycin.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.After aforesaid method screens, also can adopt Southern, PCR or dot blot equimolecular detection means that transfer-gen plant is detected, whether transform goal gene to determine it.

Wherein, the present invention with pCAMBIA1300 for the carrier that sets out, the plant expression vector called after pCactF-KT506 that contains the paddy rice of the present invention KT506 gene relevant with resistance of reverse of structure.The plant expression vector that carries the paddy rice of the present invention KT506 gene relevant with resistance of reverse or its homologous sequence can be by using protoplastis-chemical mediated method (Ca ²⁺, PEG), Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, pollen tube importing, microinjection, electricity swash, combination transformed plant cells, tissue or the organ of any one or more method in the particle gun, the conventional biological method such as agriculture bacillus mediated, and the vegetable cell, tissue or the organ that transform cultivated into plant; Described tissue and organ can comprise fruit pod, callus, stem apex, blade and the seed etc. of host plant.

In addition, carry out succeeding transfer culture by the transfer-gen plant that conversion is had the paddy rice of the present invention KT506 gene relevant with resistance of reverse or its homologous sequence after, can therefrom further filter out the transfer-gen plant of gene pure.In addition, also can expand this transfer-gen plant numerous, but the resistance of reverse of render transgenic plant is further improved.The expansion of described transgenic plant is numerous to comprise vegetative propagation and/or seminal propagation.

Method of the present invention is all applicable to dicotyledons and monocotyledons, therefore, the described vegetable cell that is converted, tissue or organ both can derive from the dicotyledonss such as tobacco, rape, cotton, soybean, willow, eucalyptus, potato or herbage, also can derive from the monocotyledonss such as paddy rice, corn, wheat, barley, jowar, millet or turfgrass.

The invention provides a gene KT506 relevant with resistance of reverse.Experiment showed, gene transformation paddy rice of the present invention can be improved paddy rice to the tolerance of high-salt stress.Albumen of the present invention and encoding gene thereof are for the research of the anti-contrary mechanism of plant, and improve the resistance of reverse of plant and the improvement of correlated character has important theory and practical significance, to in the anti-contrary genetically engineered improvement of plant (particularly cereal crop), play a significant role, have a extensive future.

Below in conjunction with specific embodiment the present invention is described in further details.

Description of drawings

The T-DNA district collection of illustrative plates of Fig. 1 expression vector pCactF.LB and RB are respectively left margin and the right margin of T-DNA; Hyg represents hygromycin resistance; P35S represents the promotor of 35S gene; T35S represents the terminator of 35S gene; PAct represents the promotor of Actin gene; 3flag represents 3 times flag sequence label; OCS represents the terminator of ocs gene; HindIII, KpnI, SpeI, XbaI, SalI and PstI represent respectively the restriction enzyme site of restriction enzyme.

The Salt Tolerance Analysis of Fig. 2 KT506 transgenic line.A: seedling growth conditions before salt is processed; B: seedling growth conditions after salt is processed; C: normally cultivate rear seedling growth conditions of a week; D: salt stress survival rate statistics.

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " molecular cloning ".The primer and dna sequence dna are synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, order-checking is finished by the Huada Gene Research Center, Beijing, nucleic acid restriction endonuclease in the vector construction process and T4 dna ligase etc. are all available from NEB company, and the method that the equal reference reagent box of method provides is carried out.Carrier framework used in the experiment comes from pCAMBIA1300.

1, the separation of KT506 gene

Infer the encoding sequence of KT506 gene, begin to design 5 ' end primer from the coding initiation site ATG of gene, in termination codon place design 3 ' end primer:

Primer 1:5 ' GCACGCtctagaATGGATCGGCATGAGGAGGAG 3 '

Primer 2: 5 ' GCACGCctgcagTCAGTCTCGGTCATCGATGAAG 3 '

The tctaga sequence is the restriction enzyme site of restriction enzyme XbaI in the primer 1, and the sequence of underscore sign is the encoding sequence of KT506 gene; The ctgcag sequence is the restriction enzyme site of restriction enzyme PstI in the primer 2, and the sequence of underscore sign is the encoding sequence of KT506 gene.

Extract total RNA of the Japanese fine paddy rice of Seedling Stage, obtain cDNA as template by reverse transcription, total length with above primer amplification KT506 gene, its size is 975bp, its nucleotide sequence is shown in SEQ ID NO:2 in the sequence table, and coded protein amino acid sequence is shown in the SEQ ID NO:1 in the sequence table.

Concrete reaction is: get approximately total RNA of 2 μ g, add 1 μ l, 10 * DNase buffer, the DNase of 1 μ l, mend DEPC treated water to 10 μ l system, mixing, behind 37 ℃ of incubation 30min, the RQ DNase stop solution that adds 1 μ l, 65 ℃ of incubation 10min with termination reaction after, add 2 μ l Oligo (dT), 18 primer (0.1 μ g/ μ l), 4 μ l, 5 * First-strand buffer, 1 μ l Ribonuclease inhibitor (40U/ μ l), 2 μ l, 4 * dNTP (each 10mM), 1 μ l MMLV Reverse Transcriptase (200U/ μ l), careful mixing, 37 ℃ are incubated 1 hour.Then processed 5 minutes for 90 ℃, cooled on ice, centrifugal collection namely obtains corresponding reverse transcription product cDNA.The cDNA that obtains is diluted 10 times, get 1 μ l as the template of PCR reaction, each 1 μ l of upstream and downstream primer (10 μ M), LA Taq enzyme (5U/ μ l) 0.5 μ l, 4 * dNTPs (each 10mM), 1 μ l, 2 * GCbuffer (Mg ²⁺) 25 μ l, H ₂O 20.5 μ l.The PCR reaction conditions is 95 ℃ of 5min of preheating, 94 ℃ of 1min of sex change, and the 56 ℃ of 30sec that anneal extend 72 ℃ of 1min50sec, 34 circulations.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, obtained the dna fragmentation that size conforms to expected results respectively.Reclaim respectively and the above-mentioned fragment of purifying, connect among the carrier pEASY-T1 (full formula King Company), transform intestinal bacteria (E.coli) TOP10 bacterial strain by the heat shock method, selecting positive bacterium colony joins in the LB liquid nutrient medium that 5ml contains the 50mg/L kantlex, 37 ℃, cultivated 12-16 hour in the shaking table of 200rpm, extract plasmid, obtain respectively containing the recombinant plasmid of purpose fragment, after sequence verification is correct, downcut purpose fragment KT506 with XbaI and PstI double digestion, be connected into the pCact plant expression vector that carries out identical double digestion, make up and obtain carrier pCactF-KT506.Picking colony PCR is accredited as positive bacterium colony and carries out sequence verification.The T-DNA district collection of illustrative plates of plant expression vector pCactF as shown in Figure 1.

2, the acquisition of KT506 transgenic paddy rice

Will be by the gene KT471 rice transformation relevant with resistance of reverse of embodiment 1 acquisition with agrobacterium-mediated transformation, concrete grammar is as follows:

Utilize the heat shock method to change above-mentioned recombinant vectors pCactF-KT471 over to Agrobacterium AGL0 bacterial strain (Chinese Academy of Sciences's heredity is given), utilize Agrobacterium that paddy rice is carried out cotransformation.

Partial blade with the Transgenic Rice Plants that obtains, extract according to a conventional method total DNA, under the guiding of forward primer 5 '-ACTCACCGCGACGTCTGT-3 ' and reverse primer 5 '-TTCCTTTGCCCTCGGACG-3 ', the pcr amplification hygromycin phosphotransferase gene, obtain the positive transfer-gen plant of 1009bp dna fragmentation through amplification, detected result shows with aforesaid method and has obtained to transform the transgenic paddy rice that pCactF-KT471 is arranged.

3, the Salt Tolerance Analysis of KT506 transfer-gen plant

The T1 of results KT506 is for transgenic paddy rice seed, and the strain of choosing more than 6 is carried out high-salt stress.Its sprouting of 3 angels is cultivated in the water seed soaking, then carry out resistance screening with the 50mg/L Totomycin, simultaneously to spend 11 to compare in the paddy rice that turns empty carrier, screen after 5 days, adjoining tree is all dead, statistics transfer-gen plant resistance seedling and dead seedling number, analyze the resistance of transfer-gen plant and separate ratio, selection resistance seedling separates than the strain that is about 3: 1 with the non-resistant seedling, the result shows the T1 transgenic line that has obtained to have the KT506 that unit point inserts, and treats that seedling is long during to the 8cm left and right sides, transfers to seedling in the triangular flask from culture dish, every bottle of 10 strains, three repetitions.Be cultured to tri-leaf period (the 3rd leaf unfolded fully), its growth conditions such as Fig. 2 A.Use the Hoagland solution that contains 200mM NaCl instead and carry out salt sieve, approximately about 3 days, blade tip jaundice whiting appears in contrast, sagging or half volume of blade even when entirely rolling up (Fig. 2 B), flush away salts solution, the normal cultivation.Changed a salts solution in per 1.5 days during this time.The rehydration second day is changed one time of nutrition liquid again, to remove remaining salinity.Nutritive medium in the triangular flask was changed once in 2 days, to keep the fresh state of nutritive medium.Take a picture (Fig. 2 C) after one week, statistics survives the seedling number, calculates salt tolerant seedling ratio, result such as Fig. 2 D (X-coordinate is different transgenic line numbering, and ordinate zou is salt tolerant seedling per-cent).Experimental result shows, T ₁For transgenic rice plant to the tolerance of salt apparently higher than transgenic rice plant not, after salt is processed, KT506 transgenic paddy rice T ₁Can continued growth behind the generation plant renewal cultivation of positive strain, turn empty carrier in spend the yellow leafs, curling, withered of 11 contrast (CK) plant, cane can not be upright, very fast death (Fig. 2 C) behind the renewal cultivation.

After obtaining the KT506 transgenic paddy rice T2 seed in generation, we have carried out the salt tolerance experiment again, and the result is consistent with above-mentioned experimental result.This description of test KT506 gene has the effect that strengthens Salt Resistance of Rice.

Claims (7)

1. method that strengthens salt resistance of plants, it is characterized in that the encoding gene of plant anti-adversity associated protein is inserted expression vector, acquisition contains the recombinant expression vector of plant anti-adversity associated protein encoding gene, this recombinant expression vector is imported the purpose plant, and screening obtains the plant that salt resistance strengthens from the plant that the plant of expressing described plant anti-adversity associated protein or described plant anti-adversity associated protein expression amount increase; Wherein, the aminoacid sequence of described plant anti-adversity associated protein is shown in SEQ ID NO:1.

2. the method for enhancing salt resistance of plants claimed in claim 1, it is characterized in that: the encoding gene of described plant anti-adversity associated protein is the nucleotide sequence shown in the SEQ ID NO:2 in the sequence table.

3. method claimed in claim 1 is characterized in that: described resistance relevant protein encoding gene is imported plant tissue, cell or organ, again the vegetable cell, tissue or the organ that are converted are cultivated into plant, obtain the transgenic plant that resistance of reverse improves.

4. method claimed in claim 1, the feature of wherein said expression vector is: the carrier that sets out that is used for making up described plant expression vector is a kind of carrier that can be used for the binary vector of agrobacterium tumefaciens or Agrobacterium rhizogenes conversion of plant or can be used for the plant micropellet bombardment, or the carrier that can copy in prokaryotic organism.

5. method claimed in claim 4, the feature of wherein said expression vector is: when making up plant expression vector with described resistance relevant protein encoding gene, drive its expression with a kind of enhancement type, composing type, organizing specific type or inducible promoter.

6. method claimed in claim 4, the wherein said carrier that sets out is the pCAMBIA serial carrier.

7. method claimed in claim 1, wherein said resistance relevant protein can be used for strengthening the salt tolerance of transgenic paddy rice.

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