CN102161994B - Suaeda salsa salt-tolerant gene SsPSI as well as coding protein and application thereof - Google Patents

Abstract

The invention discloses a suaeda salsa salt-tolerant gene SsPSI, a coding gene of the suaeda salsa salt-tolerant gene SsPSI and application of the suaeda salsa salt-tolerant gene SsPSI to culturing genetically modified salt-tolerant plants, wherein the salt-tolerant gene SsPSI of a plant is defined according to a nucleotide sequence table shown in SEQ ID NO:1, and the coding gene of the suaeda salsa salt-tolerant gene SsPSI is defined according to a nucleotide sequence table shown in SEQ ID NO:2.

Description

A kind of Suaeda salsa resistant gene of salt SsPSI and proteins encoded and application

Technical field

The present invention relates to plant genetic engineering field, bright specifically is to utilize the DNA stripping technique to obtain the plant gene SsPSI that function significantly improves, and the invention still further relates to and utilizes this gene to cultivate the transgenic salt-tolerant wheat plant.

Background technology

Salt stress is the critical limitation factor of growth and development of plants, and it can make the especially crop failure of Irrigation farming of crop.Saline soil is considered to a kind of serious disastrous land resources for a long time because its abominable physico-chemical property has limited the growth of most plants.Because most of crops are the right and wrong halophytes all, it grows and receives the influence of soil salt more easily.Yet research in recent years shows, the halophytes resource that is distributing abundant on the saline soil.Halophytes be unique on the saline soil can the growing plants fauna; According to incompletely statistics; There is kind surplus the halophytes 2600 in the whole world; Wherein on non-saline soil, be difficult to growth greatly, it has represented proprietary, irreplaceable genetic resources in one type of salinification habitat, is the important component part of species diversity.At present, along with to the researching and analysing of plant salt tolerance mechanism and relational approach, successfully the clone obtains many salt-resistant related genes, and finds that these genes carry out netted regulation and control and effectively realize the tolerance of plant to salinity through interacting.From now on, tend to a plurality of salt tolerant major genes the plant cotransformation, thereby improve the tolerance of plant as much as possible high salt.Therefore; On the basis of existing relevant resistant gene of salt and functional study thereof, select suitable material, like growing plants under high salt habitat; Clone its resistant gene of salt, analyze its salt tolerant mechanism; The Mechanism of Salt-tolerant of the understanding halophytes of system improving the especially salt tolerance of cash crop of plant better, and is improved the soil extremely important Research Significance is all arranged.

Can be in its body when plant receives salt stress through multiple variation.Plant embodies self ability to the salt tolerance through multiple biochemical route.As under salt stress, plant ability water conservation and suction are protected chloroplast(id) and are kept ionic equilibrium etc.Necessary path comprises the generation that causes the osmotically active metabolite under those salt stresses, special albumen and some antioxidant enzyme and Chaperones Moleculares of removing radicals with the intravital ionic equilibrium of controlling plant and moisture flow to.Salt stress is coerced by osmotic stress and ion and is formed.Under salt stress; A series of variation has all taken place in formalness of plant and internal physiology biochemical characteristic, and some variation is the result of salt stress injury, is the negative response of plant to adverse environmental factor; Some then is the active responding of plant to adverse circumstance, is of value to the adaptation to adverse environment.Plant is when receiving salt stress; Its Physiological and Biochemical Metabolism aspect shows complicated mechanism; Especially be that adjusting on the gene transcription level is an extremely important ring in the plant salt stress response process in molecular mechanism; Many critical function expression of gene receive Salt Stress-induced or inhibition in the plant, and the research of gene expression regulation aspect enjoys investigator's concern, have become the focus of plant stress-resistance research in recent years.

The molecular mechanism that main involved in plant salt is replied is divided into two types: one type is effector molecule, participates in biochemical responses processes such as metabotic change directly, produces the salt tolerant effect; Another kind of is regulatory molecule, is positioned at the effector molecule upper reaches, and the transmission of mediation signal comprises transcription factor and all kinds of kinases that are arranged in the signal cascade system.These protein molecules and transcription factor all receive the control of corresponding gene, and the intravital response gene of plant under salt stress that is demonstrates relevant complicacy.Effector molecule mainly comprises the biosynthetic key gene of osmotic protection material, like carbohydrate, trimethyl-glycine and verivate thereof, alcohols and each seed amino acid.P5CR and P5CS are two important enzymes in the proline(Pro) biosynthetic process, and the latter is a rate-limiting enzyme, thus the overexpression proline(Pro) with the genetically engineered that improves plant salt endurance in the P5CS gene as first-selected object.People such as nineteen ninety-five Kishor import tobacco with the P5CS gene and find that the content of proline(Pro) obviously improves in transgene tobacco, and the osmotic protection effect strengthens under dried early coercing.Liu Jingpin etc. finds also that in research work Arabidopis thaliana (Arabidopsisthaliana) SOS1 two mutants salt tolerance is higher than wild-type, and proline content also is higher than wild-type simultaneously.Because the synthetic glycinebetaine of choline dehydrogenase (CDH) and E.C. 1.1.99.1 (CodA), route of synthesis is simple, and genetic manipulation is convenient, thereby these two kinds of trimethyl-glycine synthase genes are considered to one of stress resistance gene most important and the most likely.Liu Fenghua etc. and Hidenori have carried out BADH gene and CodA transgenic research to plant respectively, and transgenic plant have all obtained higher salt tolerance.

Participate in rebuilding the membranin of the inside and outside ionic equilibrium of film, like Ca ⁺ATP enzyme, Pyrophosphate phosphohydrolase, H ⁺-ATP enzyme and secondary translocator and various channel protein are activated and participate in rebuilding the inside and outside ionic equilibrium of film.Its simple process is: Na ⁺A large amount of " pouring in " kytoplasms of the passage such as albumen that cotransports through Na-K, H ⁺-ATPase, Na ⁺/ H ⁺Antiport albumen etc. is activated, and co-ordination is with driving N a ⁺Efflux and be transported into vacuole, form finally that born of the same parents are outer, kytoplasm, the triangular ionic equilibrium of vacuole.Utilize methods such as proteinic biochemical function analysis and two mutants have complementary functions, cloned and identified at present the membranin of the inside and outside ionic equilibrium of many participations reconstruction films.

The regulatory gene of the encoding transcription factor can be through regulating and control the expression of a series of degeneration-resistant functional genes in downstream and then improving the resistance of plant from many aspects to the gene regulating element.Although abiotic stress by controlled by multiple genes, through the conversion of individual gene, like overexpression SOS1 in transgenic plant, NHX1 gene, can make the plant salt tolerance improve significantly.These plants can grow under 200mmol/L NaCl salt concn, bloom, and the 200mmol/LNacl salt concn is lethal for wild plant.In addition, these plants do not show significantly unusual on living weight yet and change, and the transgenic Fructus Lycopersici esculenti of NHX1 gene overexpression also is the same with the transgene rape result.Therefore regulate the genetic engineering that ionic equilibrium is carried out through tissue specificity overexpression SOS1, NHX1 gene and their positive controlling element SOS2 gene, will significantly improve the salt tolerance of plant.

Anti-saline and alkaline plant is inexhaustible anti-saline and alkaline gene pool; Plant is a variety of to the response pathway and the accommodation mode of salt alkaline stress; Relative gene kind is more, and these genes have difference in different plants, determines that their anti-saline and alkaline oligogenes are also variant.Confirm to resist saline and alkaline genes involved as much as possible, find the corresponding decisive salt resistant gene of different plants, utilize, improve the expression efficiency of plant transgene, carry out plant transgene, and then more effectively improve the saline-alkali tolerance of plant than the proven technique system.

Suaeda salsa is grown in desert, the half-desert saltings; It is unusual a kind of halophytes of salt tolerant; Being under the jurisdiction of Li Ke (Chenopodiaceae) Suaeda (Suaeda Forsk ex Scop) is the Main Constructive Species of haloeremion, Xinjiang, and its leaf morphology, size are with the variation of soil salt and moisture very significantly.Generally under the soil regime of soil salt content＜80g/kg, all can see its growth.Because the fluffy extreme salt tolerant of alkali, to add the fluffy most seedling browses of planting of alkali and can do vegetables, seed oil-containing and edible thereby very big DEVELOPMENT PROSPECT is arranged have been considered to study at present the pattern halophytes of plant salt tolerance mechanism.

Summary of the invention

The present invention is based on the present state of the art, a kind of Suaeda salsa resistant gene of salt SsPSI is provided, this Suaeda salsa resistant gene of salt SsPSI is by the nucleotide sequence table definition of SEQ ID NO:1.This plant salt tolerance gene SsPSI also is provided proteins encoded, and it is by the aminoacid sequence table definition of SEQ ID NO:2.This salt plant salt tolerance gene SsPSI also is provided the application in cultivating the transgenic salt-tolerant wheat plant.

The clone of SsPSI gene: can adopt several different methods such as pcr amplification method, recombination method or synthetic method to obtain PSI gene nucleotide full length sequence or its fragment.Such as, come from cDNA library or genomic library, to screen goal gene based on PSI gene order design polynucleotide probes, also can directly from cDNA or genome, amplify relevant sequence with the pcr amplification method.

Salt tolerant SsPSI can be structured in the Ti-plasmid binary vector of any plant expression vector as goal gene.On pCAMBIA2301, the T-DNA25bp Tumor-necrosis factor glycoproteins of LB and RB is wherein arranged, cauliflower mosaic virus (CAMV) 35S promoter, polyA no terminator also has the kantlex that in eukaryote, uses as selective marker.Promotor in the plant expression vector can be any constitutive promoter, tissue-specific promoter or environmental induction type promotor, like Ubiqutin promotor etc.Enhanser in the carrier both can be a transcriptional enhancer, also can be translational enhancer.For the ease of transgenic plant cells or plant being identified and screening, the affinity tag (like kantlex, weedicide etc.) that the alternative mark (like gus gene, luciferase plain gene etc.) of plant also should be arranged in the carrier or have resistance such as microbiotic.

The recombinant vectors that contains the SsPSI gene fragment can be transferred in a kind of cell or the organism, screens like bacterium, yeast or vegetable cell.For example; Can the gene that the present invention obtains be connected on any Yeast expression carrier such as the pREP5N; Utilize transformed yeasts such as method well known in the art such as lithium acetate technology, electrotransformation; Yeast transformant is coated in the selective medium flat board that contains high density NaCl, is that selective pressure is screened with high salt, confirms the functional verification of its salt tolerance.

Through research method well known in the art such as freeze-thaw method, electric shocking method etc. above-mentioned recombinant vectors is transformed in the Agrobacterium, carries out the Agrobacterium-mediated Transformation plant.Can the new gene SsPSI of salt tolerant be changed in the plant through conventional biotechnological meanss such as microinjection, particle gun, pollen tube channel methods, cultivate the best in quality and improved new variety of plant of biological character of anti-salt.By the plant transformed host both can be that monocotyledons also can be a dicotyledons, as: Arabidopis thaliana, tobacco, cotton, wheat, paddy rice, herbage etc.Gene of the present invention improves crop yield for cultivating the salt-resistant plant kind, and water saving irrigation, improve the ecological environment etc. has very important meaning.

Transgenic recombinant vectors of the present invention can be used as the kind, strain of commercial use or on producing, directly uses or carry out agro-ecology breeding and transgenic plant to improve the salt resistance of crop as genetic resources.

Description of drawings

Fig. 1 is the salt tolerant screening behind the salt tolerant SsPSI gene transformation yeast, yeast after promptly transforming and the unconverted yeast controls growing state 0, on the culture medium flat plate of 600mM NaCl: A:MM substratum; B:600mM NaCl+MM substratum; CK: the yeast that changes empty carrier over to; 1-3: change the pREP5N-PSI yeast, cultivated 3-6 days for 28 ℃.

Fig. 2 is the amplification of part transfer-gen plant

M:DL2000 plus Maker, CK-: negative control, CK+: positive control, 1-15: be transgene tobacco strain system

The salt tolerance of Fig. 3 transgene tobacco is identified

Embodiment

Below in conjunction with specific embodiment, the present invention is elaborated.

1, the clone of resistant gene of salt SsPSI

With TRizol reagent extracted total RNA from halophytes alkali is fluffy, be that template is carried out reverse transcription with total RNA, synthetic cDNA first chain.With synthetic cDNA is template, with the total length primer sequence of SsPSI gene, through the method acquisition goal gene of pcr amplification.The PCR reaction system is 50 μ L:cDNA, 1 μ L, each 1 μ L of primer, and ExTaq enzyme 0.5 μ L, 10 * PCR Buffer (Mg2+plus), 5 μ L, dNTP Mixture 4 μ L add ddH ₂O to 50 μ L.PCR program: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.Agarose gel electrophoresis detects the back and reclaims test kit recovery purpose fragment with DNA.SsPSI purpose fragment is connected in the PGEM-Teasy carrier, and transformed into escherichia coli DH5 α, the SsPSI sequence of being cloned into is definite through checking order.

The total length primer sequence:

PSI-ORF-S：5’-ATGGCTTCTCTAGCAACCTTTGCC-3’

PSI-ORF-As：5’-TTAGATCTTGCCACGAGGTCCG-3’

2, make up the carrier that transformed yeast is expressed

SsPSI gene with total length is a template, and the primer with containing SalI and NotI restriction enzyme site goes out the PSI gene with restriction enzyme site through pcr amplification; With SalI and NotI double digestion PCR product and pREP5N carrier, reclaim product and connect transformed into escherichia coli DH5 α through the T4DNA ligase enzyme; After PCR detection, enzyme are cut evaluation, select positive colony, definite through checking order; With SsPSI gene subclone to Yeast expression carrier pREP5N, called after pREP5N-PSI.Bacterium colony PCR reaction system is 20 μ L: with the bacterium colony template, and each 0.4 μ L of primer, Taq enzyme 0.2 μ L, 10 * PCR Buffer (Mg ²⁺Plus) 2.0 μ L, dNTP Mixture 0.4 μ L.PCR program: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 60 ℃ of renaturation 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.Agarose gel electrophoresis detects the PCR product, have the about 441bp band of size to confirm as positive colony.

Primer sequence

PSI-S：5’-ACGC GTCGACATGGCTTCTCTAGCAACCTTTGCC-3’

PSI-As：5’-ATTT GCGGCCGCTTAGATCTTGCCACGAGGTCCG-3’

3, the screening of transformed yeast and transformant

Change empty plasmid carrier pREP5N and pREP5N-PSI in the fission yeast Sp-Q01 competence over to electric shocking method respectively, again transformant is coated onto the MM substratum respectively and (Leu), cultivates after 3 days, can see growing yeast colony for 28 ℃.Adopt the method for bacterium colony PCR that transformant is identified.Reaction system is 20 μ L: with the bacterium colony template, and each 0.4 μ L of primer, Taq enzyme 0.2 μ L, 10 * PCR Buffer (Mg2+plus), 2.0 μ L, dNTP Mixture 0.4 μ L.PCR program: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.After agarose gel electrophoresis detects, have the about 441bp band of size to confirm as positive yeast transformant.

4, the yeast transformant salt tolerance is identified

For the salt tolerance of getting rid of transformant is by yeast self the caused possibility of suddenling change, the transformant upgrading grain after the checking and empty carrier pREP5N transformed yeast again.The mono-clonal that obtains is supported with the MM liquid nutrient medium and to OD:1.0, is diluted 10 times, gets 5 μ L and drops in the MM substratum and contain on the substratum of 600mMNaCl, cultivates 3-6 days for 28 ℃, observes growing state.The result is as shown in Figure 1, and the yeast that on the MM substratum, transforms pREP5N empty carrier and pREP5N-PSI all can normal growth, and has only the yeast of conversion pREP5N-PSI can normal growth on the MM substratum of 600mM NaCl containing.

5, make up plant expression vector

SsPSI gene with total length is a template; Primer with containing BamHI and SacI restriction enzyme site goes out the SsPSI gene with corresponding restriction enzyme site through pcr amplification; With BamHI and SacI double digestion PCR product and pCAMBIA2300 carrier, reclaim product through gel, goal gene is connected with the T4DNA ligase enzyme with carrier segments; Transformed into escherichia coli DH5 α; After PCR detection, BamHI and SacI double digestion are identified, select positive colony through checking order to confirm with SsPSI gene subclone to plant expression vector pCAMBIA2300 called after pCAMBIA2300-PSI.

Primer sequence:

PSI-CAM-S：5’＞CGGGATCCCGATGGCTTCTCTAGCAACCTTTGCC-3’

PSI-CAM-As：5’＞GAGCTCTTAGATCTTGCCACGAGGTCCG-3’

6, the screening of transgene tobacco

Extract the genomic dna of transgenic tobacco plant blade, get 1 μ L and detect its integrity and concentration through agarose gel electrophoresis.Being template with the genomic dna of transfer-gen plant carries out PCR with the primer of amplification SsPSI full length gene, gets 10 μ L products and is used for agarose gel electrophoresis and detects positive transfer-gen plant.The amplification of part transfer-gen plant is as shown in Figure 2.

7, the salt tolerance of transgene tobacco is identified

With transgenic tobacco plant and wild-type plant be placed on respectively cultivate 12 days on MS and the solid MS culture medium flat plate that contains 250mM NaCl after; The phenotype of transfer-gen plant and wild-type plant under the observation salt stress, thus filter out the transfer-gen plant that salt stress is had obvious resistance.The result is as shown in Figure 3, the equal ability of transgenic tobacco plant and wild-type tobacco normal growth on the MS substratum, and containing on the MS substratum of 250mM NaCl, transgenic tobacco plant ability normal growth, wild-type tobacco receives obvious suppression, and the amount of taking root is few and short.Above-mentioned experimental result shows, the salt tolerance that overexpression can enhancement of plant in the SsPSI transgene tobacco.

Should be understood that, concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.

Claims (3)

1. a Suaeda salsa resistant gene of salt SsPSI is characterized in that, by the nucleotide sequence table definition of SEQ ID NO:1.

2. Suaeda salsa resistant gene of salt SsPSI proteins encoded is characterized in that, by the aminoacid sequence table definition of SEQ ID NO:2.

3. the application of Suaeda salsa resistant gene of salt SsPSI in cultivating the transgenic salt-tolerant wheat plant is characterized in that said Suaeda salsa resistant gene of salt SsPSI is by the nucleotide sequence table definition of SEQ ID NO:1.

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