CN106146637A

CN106146637A - Improve the GmSLT albumen of plant salt tolerance ability and nucleic acid and application

Info

Publication number: CN106146637A
Application number: CN201610756269.1A
Authority: CN
Inventors: 马伟; 刘晓丽; 向东; 厉建蕾; 李泉; 李晴; 熊新彩; 白林泉; 邓子新
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2016-08-29
Filing date: 2016-08-29
Publication date: 2016-11-23
Anticipated expiration: 2036-08-29
Also published as: CN106146637B

Abstract

The present invention relates to a kind of botany technical field improves the GmSLT albumen of plant salt tolerance ability and nucleic acid and application；Described GmSLT albumen is containing one or more in following aminoacid sequence: the domain ABS sequence as shown in SEQ ID NO.3, the TM sequence as shown in SEQ ID NO.4, and the ZBS sequence as shown in SEQ ID NO.5；The invention still further relates to the application encoding the nucleotide sequence of described GmSLT albumen and described nucleotide sequence in strengthening plant salt tolerance ability.The present invention is analyzed by bioinformatics and gene spatial and temporal expression, it is thus achieved that resistant gene of salt GmSLT；By this gene transformation yeast, the salt resistance ability of yeast can be significantly improved；Arabidopsis thaliana transformation and Oryza sativa L., it is also possible to strengthen the salt tolerance of corresponding plant；The gene of the present invention can be used in improveing plant, improves its salt resistance ability.

Description

Improve the GmSLT albumen of plant salt tolerance ability and nucleic acid and application

Technical field

The invention belongs to botany technical field, be specifically related to a kind of GmSLT albumen improving plant salt tolerance ability and core Acid and application.

Background technology

Salting is the one of the main reasons causing crop production reduction, farming area to reduce.Therefore, the salt tolerant energy of crop is improved Power, will make positive contribution to China's agricultural economy sustainable development.And solve this problem important method and be through raw Thing technology cultivates excellent New salt-tolerant cultivar, and the most indispensable is the resistant gene of salt resource utilizing plant.

Plant, when tackling environment stress, from the time, spatially constitutes tight structure, gene expression transcribe water The control measures such as flat, post-transcriptional level and protein level constitute coordinates complicated network.These genes are the most in isolation Salt stress response is worked, but by the integration of signal network, in concert with plays a role, make plant at salt Stain is coerced down, keeps the ionic equilibrium in cell and whole plant；Keep cell and environment and intercellular moisture and infiltration Balance；Safeguard protein, the structure of biological nucleic acid macromole and biological function；Keep physiological metabolism, energy metabolism balance.

Research shows, the salt tolerance of plant is a quantitative trait by controlled by multiple genes, sufficiently complex.Plant is by planting Disadvantageous salting environment is resisted in the regulation and control of strain entirety physiology, the regulation of metabolism, gene expression etc..It has been reported that, when some sodium/hydrogen After the gene of cation transporter, transcription factor, infiltration composition of matter etc. is transferred into plant, and overexpression, these turn base Because the salt resistance ability of plant has had raising in various degree.Illustrate that related gene plays the work of key in terms of plant salt endurance With.For this reason, it is necessary to research and the closely-related gene transferred plant of plant salt tolerance, improve the salt resistance ability of plant with expectation.

Summary of the invention

It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of GmSLT egg improving plant salt tolerance ability Pseudobulbus Bletillae (Rhizoma Bletillae) nucleic acid and application.The gene of the present invention can be used in improveing plant, improves its salt resistance ability.

First aspect, the present invention relates to a kind of GmSLT albumen improving plant salt tolerance ability, and described albumen comprises following ammonia One or more in base acid sequence: the domain ABS sequence as shown in SEQ ID NO.3, such as SEQ ID NO.4 institute

The TM sequence shown, and the ZBS sequence as shown in SEQ ID NO.5.

Preferably, described albumen comprises aminoacid sequence as shown in SEQ ID NO:2.

Preferably, the sequence of described albumen is as shown in SEQ ID NO:2.

Second aspect, the present invention relates to a kind of nucleotide sequence encoding aforementioned GmSLT albumen.

Preferably, described nucleotide sequence is as shown in SEQ ID NO:1.

The third aspect, the invention still further relates to the application in strengthening plant salt tolerance ability of a kind of aforementioned nucleic acid sequence.

Fourth aspect, the present invention relates to a kind of recombinant expression carrier containing aforementioned nucleic acid sequence.

5th aspect, the present invention relates to a kind of transgenic cell line containing aforementioned nucleic acid sequence.

5th aspect, the present invention relates to a kind of recombinant bacterial strain containing aforementioned nucleic acid sequence.

7th aspect, the invention still further relates to a kind of method improving plant salt tolerance ability, comprises the steps: power aforementioned Nucleotide sequence import in plant, cultivate, it is thus achieved that the transgenic plant of salt resistance ability.

Preferably, described plant is monocotyledon, dicotyledon or gymnosperm.

Preferably, described plant is crops, flower plant or forestry plant.

Preferably, described plant is Semen sojae atricolor, Oryza sativa L., arabidopsis, Semen Tritici aestivi, Semen Maydis, Cotton Gossypii, Brassica campestris L, Sorghum vulgare Pers. or Rhizoma Solani tuber osi.

Eighth aspect, the invention still further relates to a kind of method cultivating salt-tolerant plant, it is characterised in that comprise the steps: The transgenic plant obtained by preceding method hybridizes with target plant, and then obtains salt-tolerant plant and filial generation.

9th aspect, the invention still further relates to a kind of raising plant salt tolerance ability structure territory sequence, described domain sequence is Domain ABS sequence as shown in SEQ ID NO.3, the TM sequence as shown in SEQ ID NO.4, or such as SEQ ID NO.5 Shown ZBS sequence.

The present invention has following beneficial effect: the present invention is analyzed by bioinformatics and gene spatial and temporal expression, it is thus achieved that A kind of resistant gene of salt, named GmSLT；By this gene transformation yeast, the salt resistance ability of yeast can be significantly improved；Convert and intend South mustard and Oryza sativa L., it is also possible to strengthen the salt tolerance of corresponding plant；The gene of the present invention can be used in improveing plant, improves its salt tolerant Ability.

Accompanying drawing explanation

By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention, Purpose and advantage will become more apparent upon:

Fig. 1: GmSLT functional domain schematic diagram；

Fig. 2: GmSLT expresses yeast W303-1a salt tolerance droplet test；

Fig. 3: process LAN GmSLT has yeast genes and wild-type yeast contrast under condition of salt stress；

The effect in salt tolerant of Fig. 4: the GmSLT gene key structure territory；

Fig. 5: turn GmSLT Oryza sativa L. To for seedling GmSLT gene test；

Wherein WT is parent, and NTC1, NTC2 are blank, and P1, P2 are positive control；

Fig. 6: part turns the detection of expression of GmSLT trans-genetic hybrid rice plant GmSLT gene；

Fig. 7: turn 1# strain T1 of GmSLT trans-genetic hybrid rice for seedling Salt tolerance result figure.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is expanded on further.These embodiments are merely to illustrate the present invention and need not In limiting the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.

In the embodiment of the present invention, the experimental technique used if no special instructions, is conventional method.

Material used in the embodiment of the present invention, reagent, consumptive material etc., if no special instructions, the most commercially obtain.

The present invention has no particular limits for the plant being applicable to the present invention, as long as it is appropriate to the conversion behaviour of gene Make, such as various crops, flower plant or forestry plant etc..Described plant can be such as (being not limited to): dicotyledonous plants Thing, monocotyledon or gymnosperm.More specifically, described plant includes, but is not limited to: Oryza sativa L., arabidopsis, Semen Tritici aestivi, Corn and soybean, Cotton Gossypii, Rhizoma Solani tuber osi, Brassica campestris L etc..

Embodiment 1, the clone of Semen sojae atricolor candidate gene

Pass through bioinformatic analysis, it was predicted that obtain some soybean salt-tolerance candidate genes.Semen sojae atricolor closes rich 39 seedling (with reference to literary composition Offer--taking charge of the peak that shakes, the summer is light, Wang Baofeng, Bai Heyin, Shi Zhuanzhe, and 2001.Soybean varieties-close rich No. 39." China's agricultural technology spread ", 2001 (4): 33-33) after 150mM sodium chloride solution processes, when utilizing real-time fluorescence quantitative PCR that these candidate genes are carried out Null representation pattern analysis, has the change of multiple candidate gene substantially after finding salt stress.Wherein, GmSLT is that said method prediction obtains Obtain a soybean salt-tolerance gene, and carry out Soybean Seedling Roots phyllopodium because of spatial and temporal expression pattern analysis, table after 150mM salt stress Reach rise substantially, thus it is speculated that the gene being correlated with for soybean salt-tolerance, and this gene is cloned；

According to the sequence of predicted gene GmSLT, utilize software Primer Premier 5 to design pair of primers, utilize height to protect True enzyme KOD expands.Amplification template is that the cDNA obtaining Semen sojae atricolor (utilizes the Soybean Seedling Roots leaf after salt stress to extract RNA to invert again Record is cDNA).

1, design of primers is as follows:

Gm SLT-F 5’-TCTAGAATGGGCGATACTT-3(SEQ ID NO.6)；

Gm SLT-R 5’-GAGCTCTCAAGTCAACATAAGAT-3(SEQ ID NO.7)；

Reaction system:

Reaction condition:

2, PCR primer purification reclaims, and uses PCR primer purification kit (Shanghai JaRa).

3, purified product adds A reaction

50 μ l reaction systems:

72 DEG C connect 30min；

4, the preparation of competent escherichia coli cell

1) inoculation bacillus coli DH 5 alpha, picking list bacterium colony is 37 DEG C of shaking table overnight incubation (about 16 hours) in LB culture medium；

2) take 1ml overnight culture and transfer in 100ml LB culture medium, violent shaken cultivation about 2-3 on 37 DEG C of shaking tables Hour (250-300rpm), is 0.4-0.6 to OD600；

3) 0.1M CaCl2 solution is placed in pre-cooling on ice；Following steps need to operate at superclean bench with on ice；

4) the cultured bacterium solution of 1ml is drawn in 1.5ml centrifuge tube, cooled on ice 10 minutes；

5) 3000g frozen centrifugation 5 minutes at 4 DEG C；

6) supernatant discarded, adds 100 μ l pre-cooling 0.1M CaCl2 solution, inhales dynamic beating the most up and down with liquid-transfering gun, makes thin Born of the same parents' Eddy diffusion, places 20 minutes at ice；

7) 3000g frozen centrifugation 5 minutes at 4 DEG C；

8) supernatant discarded, adds 100 μ l pre-cooling 0.1M CaCl2 solution, inhales dynamic beating the most up and down with liquid-transfering gun, makes thin Born of the same parents' Eddy diffusion；

9) cell suspending liquid can be immediately available for transformation experiment or add cryoprotective agent (15%-40% glycerol) ultralow temperature afterwards Refrigerated storage standby (-70 DEG C).

5, coupled reaction

Use TaKaRa pMD18-T simple vector, operate with reference to description.

Purpose fragment and carrier control mol ratio and are about 6:1, sample-adding mixing.

Adding isopyknic solution I 5 μ l, gently mix, the most centrifugal, 16 DEG C connect a few hours.

6, convert

Take coupled reaction liquid 5 μ l thermal shock method and convert bacillus coli DH 5 alpha competent cell.

1) by competent cell from-70 DEG C of taking-ups, ice melts, is subsequently adding 5 μ l coupled reaction liquid, gently mixes, be put in 30min in ice；

2) thermal shock 45 seconds in 42 DEG C of water-baths, put into rapidly 2min in ice；

3) add 890 μ l LB fluid mediums, 180rpm, shaking table is cultivated 1 hour；

4) take off layer 100 μ l bacterium solution, coat on LB (Amp/X-gal/IPTG) flat board, be inverted overnight incubation for 37 DEG C.

7, bacterium colony PCR identifies

With the toothpick picking single bacterium colony of some white, put in 100 μ l sterilized water and stir evenly, then by 1 μ l bacterium solution as template Carry out bacterium colony PCR.25 μ l reaction systems are as follows, and reaction condition is with this example (1):

8, escherichia coli plasmid extracts

1) picking list colony inoculation is in 3-5ml contains the LB fluid medium of antibiotic, and 37 DEG C are shaken training overnight.

2) taking 1-3ml bacterium solution in clean centrifuge tube, 12000r/min is centrifuged 5min, abandons most supernatant, collects thalline.

3) the Solution I solution of 200 μ l pre-coolings, vibration mixing, suspension thalline is added to precipitation.

4) add 200 μ l Solution II, under rapid and soft reverse centrifuge tube number, mix (less than 5min).

5) add the Solution III solution of 200 μ l pre-coolings, gently mix under reverse centrifuge tube number, place 5min on ice.

6) 12000r/min, centrifugal 10min.

7) careful Aspirate supernatant proceeds in another clean centrifuge tube, adds 400 μ l (or equal-volume) Tris saturated Phenol: chloroform (1: 1) solution, the gentliest vibrates, and mixing, 12000r/min is centrifuged 5min.

8) Aspirate supernatant is to (avoiding adhesion protein) in another centrifuge tube, adds 400 μ l chloroforms, and vibration is mixed repeatedly Even, 12000r/min is centrifuged 5min.

9) careful Aspirate supernatant is in another centrifuge tube, adds 2 times of volume dehydrated alcohol and 1/10 volume of pre-cooling 3M sodium acetate solution, after mixing ,-20 DEG C place 10min.

10) then, 12000r/min is centrifuged 5-10min, abandons most supernatant.Add 70% ethanol 0.5ml to wash 2 times, vacuum It is dried or air-dries, precipitate and dissolve with 50 μ lTE+RNase (20 μ g/ml), then-20 DEG C of preservations.

9, plasmid enzyme restriction is identified

By NEB restricted enzyme BamHI, Sal I, 20 μ l systems

37 DEG C of enzyme action a few hours.

10, check order and analyze

Bacterium colony PCR and enzyme action are accredited as the bacterium colony of the positive, support overnight with single bacterium colony LB+Amp is little, take a small amount of bacterium solution and send public affairs Department's order-checking, uses universal primer order-checking, obtains sequence as shown in SEQ ID NO:1；Sequencing result is predicted with applicant's early stage GmSLT sequence compare, and utilize the bioinformatics tools such as DNAMAN, NCBI, softberry and Phytozome to enter Row is analyzed.

Embodiment 2, yeast Salt tolerance

The total length of design primer amplification resistant gene of salt ORF so that it is can be with yeast vector pRUL129 with double enzyme site It is connected, construction recombination plasmid.After KOD amplification, being connected to pMD-18T, convert DH5 α, take out plasmid double digestion overnight, glue reclaims pure After change, it is connected to the pRUL129 carrier through same enzyme enzyme action.Utilize the transgenic that chemical transformation or electrotransformation will build Vector saccharomyces cerevisiae W303-1A, cultivates 3 days for 28 DEG C, can see the yeast colony of white.Picking list bacterium colony, little support after Carry out sequence verification correct.

1, the preparation of electrotransformation saccharomyces cerevisiae W303-1A competence and conversion

The enforcement of this step refers to document--and the quiet gold of Qin Yu builds tinkling of pieces of jade Bao Xiaominggaodong, and 1999.Affect saccharomyces cerevisiae electric shock The condition of conversion ratio.Journal of Shandong university (natural science edition) Vol.34No.2.

1) will convert with single colony inoculation of yeast strain in 5ml YPD culture medium, 30 DEG C of incubated overnight be to saturated；

2) convert evening before that day, the 2L sterile flask equipped with 500ml YPD culture medium is inoculated appropriate overnight training Nutrient solution, in 30 DEG C of violent shakings, until cell density reaches 1 × 108, (OD600 is about 1.3-1.5,1:10 dilution and is about 0.3- 0.35)；

3) in 4 DEG C, 4000g is centrifuged 5min results and cultivates cell, and cell 80ml sterilized water is resuspended.In order to increase cell pair The susceptibility of electric shock, continues step 4.If need not to connect step 6；

4) adding 10ml, pH7.5,10 × TE buffer, rock uniformly, add 10ml1M Quilonorm (SKB), rotation shakes up, in 30 DEG C, 85rpm shakes 45min；

5) add 2.5ml 1mol/L DTT, and rotate shake simultaneously, shake 15min in 30 DEG C of 85rpm；

6) yeast suspension is washed 3 times, every time with 4000-6000g in 4 DEG C of centrifugation cells, successively re-suspended cells Solution used is as follows:

Precipitation for the first time: 250ml icy water

Second time precipitation: 1mol/L sorbitol ice-cold for 20-30ml

Third time precipitation: 1mol/L sorbitol ice-cold for 0.5ml

Final bacterium solution volume should be 1.3-1.5ml, and now strain density is 1 × 1010.

7), before electricity converts, aseptic ice-cold microcentrifugal tube adds 40 μ l yeast cells and less than or equal to 100ng DNA (volume is less than 5 μ l) to be transformed, mixing.It is transferred in ice-cold electric turn trough, next according to Bio-Rad electroporation apparatus Explanation operation.

8) toward the 1mol/L sorbitol of addition 1ml pre-cooling, gently pressure-vaccum mixing in electric shock tank after pulse；

9) gradient is coated on sorbitol Selective agar medium flat board, cultivates 3-6 days in 30 DEG C, until there is bacterium on flat board Fall.

2, saccharomyces cerevisiae W303-1A STb gene and the extraction of plasmid

1) taking yeast liquid 1-2ml of cultivation 30h, 12000rpm, 1min are centrifugal；

2) wash 2 times with STE；

3) resuspended with 100 μ l TE buffer, add 50 μ l beades (sigama company), add 100 μ l phenol chloroforms, acute Violent shock swings 1h,；

4) 4 DEG C, 12000rpm is centrifuged 10min；

5) take supernatant, add equal-volume chloroform, extract proteins and phenol；

6) step 5 is repeated until liquid level boundary does not has protein residue；

7) taking supernatant, add two volumes ice dehydrated alcohol ,-20 DEG C stand 20min, 12000rpm and are centrifuged 10min；

8) abandon supernatant, 70% washing with alcohol once, natural drying；

9) it is dissolved in 50-100 μ lTE, adds RNase A (final concentration of 20 μ g/ml), preserve after 37 DEG C of reaction 30min In-20 DEG C；

10) PCR detection, because lysis efficiency is low, plasmid content is the highest, it is proposed that template amount is 1～2 μ l/25 μ l reactants System.

3, energy for growth impact experiment under process LAN GmSLT gene pairs yeast salt stress

Droplet test (Yeast drop test assay) is used to detect GmSLT gene pairs yeast salt with growth curve method Coerce lower energy for growth impact (refer to document--Chen Hongyun, Ye Yanrui, Zhu Yi, Zheng Suiping, Lin Ying, 2008.Yeast patience The comparison of evaluation methodology.Food and fermentation industries, 2008,34 (12): 51-57).

Drop method is by the wild type of same concentrations and to convert the yeast dibbling of GmSLT in interpolation 0,300mM, 500mM On the yeast minimal medium of NaCl, cultivate 2-3 days for 28 degree, observe yeast growth situation, Taking Pictures recording.

Growth curve method is: by yeast-inoculated to minimal medium, 28 DEG C of incubated overnight, adjusts OD600 to 0.5, with 1ml Culture medium suspends and accesses the new rich medium liquid YPD (containing 300mM NaCl) of 100ml, and making initial OD 600 is 0.005,28 DEG C, 150rpm, cultivates 3d, is spaced OD value of detection in 6 hours.

By in accompanying drawing 2 it will be seen that when NaCl in medium concentration reaches at 300mM, W303-1A and conversion The W303-1A growth of pRUL129 empty plasmid is all slowed down, but the W303-1A yeast NaCl concentration in the medium of process LAN GmSLT Reach when 500mM, remain to growth.

From accompanying drawing 3 it will be seen that when NaCl in medium concentration reaches at 300mM, from the beginning of 30 hours, process LAN The W303-1A yeast growth speed of GmSLT is significantly higher than W303-1A and converts the W303-1A of pRUL129 empty plasmid.To During 72h, reach 8.85 with the yeast OD600 of GmSLT transgenic, and only reached 4.05 with the comparison of empty plasmid, wild Type W303-1A is 3.02.

Above-mentioned it is demonstrated experimentally that process LAN GmSLT gene, it is remarkably improved yeast and salt stress is adapted to ability.Prove GmSLT Gene function is closely related with salt tolerant.

4, each domain of GmSLT and salt resistant function dependency

1) Over-Lapping PCR:

For checking GmSLT each domain function, use Over-Lapping PCR method, carried out respectively ABS, TM, The disappearance of TM303, TH178 and ZBS domain, refers to Fig. 1.According to the method separately design primer P2, P3 and share draw Thing P1, P4, carry out 2 PCR taking turns each 20-cycle respectively.

Designed primer sequence is:

GmSLTΔABS-P25‘-CTCATGCAA-CCAAGCACCCCAAATG-3‘(SEQ ID NO.8)

GmSLTΔABS-P35‘-GGGGTGCTTGG-TTGCATGAGAAATCTAAG-3‘(SEQ ID NO.9)

GmSLTΔTM-P25‘-GATTTCTCATG-TTGGTGCACTTCCTTCCAG-3‘(SEQ ID NO.10)

GmSLTΔTM-P35‘-GTGCACCAA-CATGAGAAATCTAAGACC-3‘(SEQ ID NO.11)

GmSLTM303-F 5‘-GGATCCATTGATCTATCTCCTGT-3‘(SEQ ID NO.12)

GmSLTM303-R 5‘-GTCGAC-TCAAGTCAACATAAGATCA-3‘(SEQ ID NO.13)

GmSLTH178F-F 5‘-ACATGAACGGGCTCTCTCGCCAAGG-3‘(SEQ ID NO.14)

GmSLTH178F-R 5‘-CCTTGGCGAGAGAGCCCGTTCATGT-3‘(SEQ ID NO.15)

GmSLTΔZBS-P25‘-TCTCGGCACT-CCCGTTCATGTAACTCCTC-3‘(SEQ ID NO.16)

GmSLTΔZBS-P35‘-CATGAACGGG-AGTGCCGAGAAGGGTTTTG-3‘(SEQ ID NO.17)

P15‘-GGATCC-ATGGGCGATACTTCTC-3‘(SEQ ID NO.18)

P45‘-GTCGAC-TCAAGTCAACATAAGATCA-3‘(SEQ ID NO.19)

2) yeast transformation vector restructuring pRUL129 is built

Through sequence verification with BamH I and Sal I restriction enzyme site mutant gene with as the yeast of restriction enzyme site Carrier pRUL129 is connected, and uses electrotransformation transformed saccharomyces cerevisiae, cultivates 3 days for 28 DEG C on the selection flat board containing 1M sorbitol.Choose Take single bacterium colony and do PCR checking, it was demonstrated that four kinds of mutant genes have proceeded to yeast the most.

3) GmSLT domain is tested with salt resistant function

By the method for drop experiment (Yeast drop test assay), multiple transformed yeast is carried out Salt tolerance.Knot Fruit sees Fig. 4.It is observed that when the NaCl concentration in culture medium reaches 300mM, 500mM, process LAN is complete from Fig. 4 The yeast growth of GmSLT gene or disappearance ZBS is substantially than other yeast.This test result indicate that, in each domain, and ABS, TM Once lacking Deng domain, GmSLT gene function almost completely loses, and the two domain is for the salt resistant function of GmSLT It is requisite.Comparatively speaking, after ZBS structure disappearance, less to GmSLT gene function function effect.

Embodiment 3, turn GmSLT Rice Salt test

1, plant conversion carrier builds

With engineered binary vector pCAMBIA1301 (list of references Chen Hong transports, Ye Yanrui, Zhu Yi, Zheng Suiping, Lin Ying, 2008.The comparison of yeast patience evaluation methodology.Food and fermentation industries, 2008,34 (12): 51-57) as plant expression vector (increase before multiple clone site and have 35S promoter), sees embodiment 2, gene GmSLT utilizes I and Sal I two enzyme action of BamH Site double digestion from carrier pMD-18T Simple Vector gets off, and reclaims purification, is then cloned into pCAMBIA1301's Between multiple clone site BamH I and Sal I, referred to as 35S::GmSLT.

2, Agrobacterium prepares

1) 35S::GmSLT is converted Agrobacterium LBR4404 (list of references--what winter jasmine, Gao Bida, 2002.Several containing Nicotiana tabacum L. The structure of the plasmid pBG1121 of fourth matter enzyme gene and rice conversion " Agricultural University Of Hunan's journal: natural science edition ", 2002,28 (2): 93-96), then carry out bacterium colony PCR and identify that checking is correct, and positive bacterium colony is frozen in-70 DEG C with protective agent.

2) from-70 DEG C of refrigerators, take out the Agrobacterium of conservation before converting, be inoculated in the YEP solid culture containing corresponding antibiotic On base, 28 DEG C of incubators cultivate 2-3d.

3) (containing 25mg/L rifampicin+25mg/L chain during picking Agrobacterium list bacterium colony accesses 3mL aseptic YEP fluid medium Mycin+50mg/L kanamycin), 28 DEG C, 200rpm cultivate about 20h.

4) more obtained bacterium solution is accessed 200mL containing in same antibiotic YEP fluid medium, 28 DEG C, 200rpm again Cultivate about 20h, treat that the concentration of Agrobacterium reaches OD₆₀₀About=0.5.

5) 4 DEG C, 3000rpm be centrifuged 15min, abandon supernatant, use conversional solution Eddy diffusion, stand-by.

3, turn GmSLT trans-genetic hybrid rice acquisition (list of references: Zhang Xuemei, Yuan Tao, Xu Xiuzhen, Wang Zhijie, Chunyang LI etc., 2000.The structure of plant expression plasmid pBin438-IFN-γ and to the Efficient Conversion of Agrobacterium LBA4404." Sichuan University is learned Report (natural science edition) ", 2000 (s1)；Easily rely on oneself, Cao Shouyun, Wang Li, storage becomes a useful person, Li Xiang etc., and 2001.Improve Agrobacterium-mediated Transformation The research of Oryza sativa L. frequency, Acta Genetica Sinica, 2001,28 (4): 352-358).

Having multiple method to obtain transgenic paddy rice, this example is fine as material, by mature embryo callus induction using japonica rice Japan Tissue, then infects callus with the Agrobacterium EHA105 containing GmSLT gene, and finally differentiation obtains transfer-gen plant.Tool Body step is as follows:

1) callus induction and subculture

Inducing culture MS mixed-powder 4.4g/L；2,4-D:4mg/L；Sucrose: 30g/L；

Proline: 2.8g/L；Caseinhydrolysate: 0.3g/L；Plant gel: 3g/L (PH5.8)

Subculture medium MS mixed-powder 4.4g/L；2,4-D:2mg/L；Fe-EDTA:5ml/L；Sucrose: 30g/L；Water Solve casein: 0.3g/L；Plant gel: 3g/L (PH5.8)

By Mature seed of rice, sterilize with liquor natrii hypochloritis after shelling, then with sterilizing deionized water wash repeatedly, afterwards It is put on aseptic filter paper and blots, seed is lain against on inducing culture, cultivate about one month under 28 DEG C of dark；

After embryo callus grows out, select ball-type callus, be placed on subculture medium, under 28 DEG C of dark Successive transfer culture about 1 week.

2) Agrobacterium-mediated Transformation and co-culturing, co-cultures base N6 a large amount of: 50ml/L；B5 trace: 10ml/L；B5 is organic: 1ml/L；Inositol: 0.1g/L；2,4-D:2mg/L；Fe-EDTA:5ml/L；Sucrose: 30g/L；Glucose: 10g/L；Plant gel: 3g/L；AS:150 μM (PH5.2) is added after sterilizing；See embodiment 3 and carrier 35S::GmSLT is converted Agrobacterium EHA105, and examine Test and obtain positive bacterium colony.Again by the positive Agrobacterium YEP flat board activation culture 28 DEG C containing antibiotic, 2d, then on flat board Scrape bacterium, be resuspended in containing in appropriate AAM culture fluid, at 28 DEG C, violent shaken cultivation 2 hours on shaking table, adjust bacterium Liquid OD₆₀₀Value about 0.15.And then, the callus after subculture is immersed in Agrobacterium bacterium solution, about 25min.Afterwards, by nothing Bacterium filter paper blots bacterium solution, is transferred to callus co-culture on base containing an aseptic filter paper, and dark 20 DEG C co-culture 3d.

3) selection and differentiation culture

Selective agar medium N6 is a large amount of: 50ml/L；B5 trace: 10ml/L；B5 is organic: 1ml/L；2,4-D:2mg/L；Fe- EDTA:5ml/L；Sucrose: 30g/L；Proline: 0.5g/L；Caseinhydrolysate: 0.3g/L；Inositol: 0.1g/L；Phytagel: 3g/L, adds cephamycin: 500mg/L after sterilizing；Add hygromycin 50mg/L (PH5.8) pre-division culture medium N6 a large amount of: 50ml/ L；B5 trace: 10ml/L；B5 is organic: 1ml/L；NAA:1mg/L；Fe-EDTA:5ml/L；Sucrose: 30g/L；Proline: 0.5g/ L；Caseinhydrolysate: 0.3g/L；Inositol: 0.1g/L；Plant gel: 3g/L, adds cephamycin: 250mg/L after sterilizing；ABA: 5mg/L；6-BA:2.5mg/L；Add hygromycin 50mg/L；(PH5.8)

Division culture medium N6 is a large amount of: 50ml/L；B5 trace: 10ml/L；B5 is organic: 1ml/L；NAA:0.5mg/L；Fe- EDTA:5ml/L；6-BA:4mg/L；Sucrose: 30g/L；Caseinhydrolysate: 0.3g/L；Inositol: 0.1g/L；Plant gel: 4.6g/ L.(PH=5.8)

After co-culturing, wound healing is moved on in triangular flask, add sterilized water washing callus 3 times, then with the addition of The sterilized water washing of 0.5g/L cephamycin, repeatedly to clarification, adds (containing 0.5g/L cephamycin) sterilized water again, is placed on after outwelling Shaking on shaking table 2 hours, 28 DEG C, 150 turns, (if finding, liquid is muddy, should be the most clear with the washing of 0.5g/L cephamycin sterilized water again Clearly) afterwards wound healing is moved on aseptic filter paper, blot excessive moisture, super-clean bench blows about 1 hour, then is shifted by callus (bigger callus can be selected be dispersed in culture medium) on Selective agar medium, cultivate two weeks for dark 28 DEG C.

The resistant calli that picking grows out from former callus again, transfers on pre-division culture medium, and dark 28 DEG C pre-differentiation culture 1 week.Then, callus is transferred on division culture medium, 28 DEG C of differentiation culture: first cultivate under dark 3d, then under continuous light, at night in daytime photoperiod 16h/8h, cultivates more than two weeks, and such callus can differentiate seedling.Will The seedling differentiated is transferred to the triangular flask containing MS culture medium and cultivates long greatly plant (well developed root system), is finally transplanted to Nutrition Soil Cultivate.

4) qualification of Oryza sativa L. positive plant

A, turn GmSLT Oryza sativa L. To for seedling regular-PCR detect

Utilizing the simple extraction method of SDS, cut off a small amount of rice leaf, liquid nitrogen grinds, and extracts DNA.Then as template, use GmSLT detection primer expands, and carries out PCR amplification, and compares with original parent Oryza sativa L. (WT), and result refers to accompanying drawing 5.Primer As follows:

GmSTL-d-F:5 '-CTTCACATGAGGAACTGG-3 ' (SEQ ID NO.20)

GmSTL-d-R:5 '-CTCAGTCTCATAGCCTTG-3 ' (SEQ ID NO.21)

It will be seen that no matter with the rice seedlings genomic DNA converting GmSTL or cDNA as mould from electrophoretogram (Fig. 5) Plate, the most amplifiable go out with using the plasmid containing GmSTL gene as positive control carry out PCR expand identical fragment, it was demonstrated that turn Gene vaccine has successfully imported GmSTL gene.

5) GmSLT Oryza sativa L. To is turned for part strain gene expression component analysis (Real time PCR)

A) the plant RNA extraction test kit that seedling Total RNAs extraction uses Beijing health to be ShiJi Co., Ltd, operation is with reference to explanation Book.The process of related supplies: overnight, then 121 DEG C of sterilizings 30 minutes, go the DEPC solution soaking blueness frotton with 0.1% DEPC solution, 80 DEG C of dry for standby.And add dehydrated alcohol with DEPC water and prepare 75% ethanol.

B) reverse transcription is cDNA.Extraction total serum IgE in blade, RNA elution volume 50 μ l, after checking R NA purity and integrity degree, Carry out reverse transcription.Recycling Reverse Transcription box, synthesizes cDNA with reference to description.

Then, utilizing the cDNA of preparation to carry out RT-PCR detection as template, ibid the primer used by common detection is carried out Positive plant detects, and compares with original parent Oryza sativa L. (WT).

The rice plant selecting test positive prepares cDNA template, further according to relevant principle, has separately designed rice The real-time fluorescence quantitative PCR primer of tub and GmSLT, as follows:

Rice tub-F:5 '-GGCAAGATGAGCACCAAGGA-3 ' (SEQ ID NO.22)

Rice tub-R:5 '-AAGCCACCGCAATACACCAC-3 ' (SEQ ID NO.23)

GmSLT-F 5’-AGAATCACCACCCATCCA-3’(SEQ ID NO.24)

GmSLT-R 5’-GTGCCAAGACCAACATCC-3’；(SEQ ID NO.25)

Real time fluorescent quantitative pcr reaction system (SYBR Green staining), 20 μ l systems, concrete composition is as follows:

Of short duration centrifugal after mixing gently

Reaction condition:

Program finally uses melting curve (melting curve) to react, and analyzes the specificity of PCR primer.

The each sample of real-time fluorescence quantitative PCR repeats, using Oryza sativa L. house-keeping gene tub as internal reference, with 2 for parallel three times^-ΔΔCTSide Method calculates target gene relative expression quantity.

From accompanying drawing 6 it can be seen that, wild-type parent Seedling is not detected by the expression of GmSLT, and in transgenic seedling 1,3,4 All detecting the expression of GmSLT, wherein the expression of No. 1 Seedling is the highest.

4, transgenic paddy rice Salt tolerance

Material: Oryza sativa L. Japan fine wild type WT seedling and turn GmSLT Oryza sativa L. To for 1# strain seedling；

Processing method: watering the MS+NaCl solution of NaCl concentration 1M and 1.50M respectively, every basin waters 30ml once, unites after 9d Meter Rice Salt situation.

By in following table and Fig. 7 it is observed that use 1M Nacl process respectively parent Japan 1# that is fine and that turn GmSLT little After Seedling 9 days, wild type Japan is fine has occurred obvious wilt phenomenon, and obvious salt does not occur in the seedling turning GmSLT gene Evil symptom.After the NaCl using 1.5M processes 9 days, wild type Japan is fine the most withered, though and turning the seedling of GmSLT gene There is blade wilt phenomenon, but seedling is still survived.From this it can be concluded that process LAN GmSLT gene can significantly improve Oryza sativa L. Salt-tolerant trait.

Turn the rice seedlings salt stress result of GmSLT gene

Salinity	Plant	Plant performance after process
			1M	WT Oryza sativa L.	Blade is withered heavier
1M	Turn GmSLT Oryza sativa L.	Blade is withered slight
			1.5M	WT Oryza sativa L.	Plant part is withered, and plant part is withered serious
1.5M	Turn GmSLT Oryza sativa L.	Plant survives, and blade is withered

In sum, the present invention is analyzed by bioinformatics and gene spatial and temporal expression, it is thus achieved that a kind of resistant gene of salt, life Entitled GmSLT；By multiple biologies such as this gene transformation yeast, Oryza sativa L., all it is remarkably improved the salt resistant character of associated biomolecule.This Bright gene can be used in improveing industrial strain, such as: yeast, and crop, such as: Oryza sativa L. etc., is remarkably improved its salt resistance ability. There is the industrial producing strain fostering requirements such as reduction yeast, reduce production cost, improve target compound yield and extension is made Application prospect and the value such as thing planting area, raising yield.

Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, this not shadow Ring the flesh and blood of the present invention.

Claims

1. the GmSLT albumen improving plant salt tolerance ability, it is characterised in that described albumen comprises in following aminoacid sequence One or more: the domain ABS sequence as shown in SEQ ID NO.3, the TM sequence as shown in SEQ ID NO.4, and ZBS sequence as shown in SEQ ID NO.5.

2. the as claimed in claim 1 GmSLT albumen improving plant salt tolerance ability, it is characterised in that described albumen comprise as Aminoacid sequence shown in SEQ ID NO:2.

3. the GmSLT albumen improving plant salt tolerance ability as claimed in claim 1, it is characterised in that the sequence of described albumen As shown in SEQ ID NO:2.

4. one kind encodes the nucleotide sequence of GmSLT albumen described in claim 1.

5. nucleotide sequence as claimed in claim 4, it is characterised in that described nucleotide sequence is as shown in SEQ ID NO:1.

6. a nucleotide sequence as claimed in claim 4 application in strengthening plant salt tolerance ability.

7. the recombinant expression carrier containing nucleotide sequence as claimed in claim 4.

8. the transgenic cell line containing nucleotide sequence as claimed in claim 4.

9. the recombinant bacterial strain containing nucleotide sequence as claimed in claim 4.

10. the method improving plant salt tolerance ability, it is characterised in that comprise the steps: the core described in claim 4 Acid sequence imports in plant, cultivates, it is thus achieved that the transgenic plant of salt resistance ability.

11. methods as claimed in claim 10, it is characterised in that described plant is monocotyledon, dicotyledon or naked Sub-plant.

12. methods as claimed in claim 11, it is characterised in that described plant is that crops, flower plant or forestry are planted Thing.

13. methods as claimed in claim 11, it is characterised in that described plant is Semen sojae atricolor, Oryza sativa L., arabidopsis, Semen Tritici aestivi, jade Rice, Cotton Gossypii, Brassica campestris L, Sorghum vulgare Pers. or Rhizoma Solani tuber osi.

14. 1 kinds of methods cultivating salt-tolerant plant, it is characterised in that comprise the steps: to obtain method described in claim 10 The transgenic plant obtained hybridizes with target plant, and then obtains salt-tolerant plant and filial generation.

15. 1 kinds are improved plant salt tolerance ability structure territory sequence, it is characterised in that described domain sequence is such as SEQ ID Domain ABS sequence shown in NO.3, the TM sequence as shown in SEQ ID NO.4, or the ZBS as shown in SEQ ID NO.5 Sequence.