CN104894090A - AtSRT2 protein and its coding sequence and application - Google Patents

Abstract

The invention discloses an AtSRT2 protein. The protein is characterized in that it is a protein having an amino acid sequence represented as SEQ ID NO. 1 in a sequence table, or a protein, with the same activity as an amino acid residue sequence of SEQ ID No. 2, derived from the SEQ ID No.2, and having an amino acid sequence obtained by subjecting an amino acid residue sequence of the SEQ ID NO. 1 to substitution, deletion or addition by more than one amino acid residue. By genetic engineering, a coding sequence of the AtSRT2 protein is introduced into a target plant to obtain a transgenic plant having higher salt tolerance than that of the target plant, so that the salt tolerance of the target plant is improved. Under salt treatment, compared with the wild-type plants, the plant introduced the coding sequence of the AtSRT2 protein has the advantages that germination rate is significantly improved, the salt tolerance of the plant is enhanced, the size is greater, the areas of the root and leaf are significantly greater, and the plant survival rate is improved significantly. The AtSRT2 protein and its coding sequence play an important role in cultivating salt-tolerant plants and plants with enhanced salt tolerance.

Description

AtSRT2 albumen and encoding sequence thereof and application

Technical field

The present invention relates to molecular genetics field, especially a kind of AtSRT2 albumen and encoding sequence thereof and application.

Background technology

The soil salinization has become the worldwide great environmental and resource problem that jeopardize human survival.According to Food and Argriculture OrganizationFAO's incomplete statistics, saline soil ground area is up to 9.54 hundred million hm in the world ², the Sustainable development of serious restriction husbandry.China's all kinds of salinization land area is equivalent to 1/4 of existing arable land, and in recent years, along with soil salinification area constantly expands, national soil salt matter area reaches 0.12 hundred million hm ², be salinate fields big country of the world (Li Bin etc., 2005).Salt stress seriously suppresses the g and D of plant, is to cause crop production reduction important environmental stress factor.Therefore, by the salt tolerance of animal nutrition Crop Improvement, thus expand arable land usable floor area, improve saltings crop yield, to Global Agriculture development and Improvement of Ecological Environment important in inhibiting.

In plant evolution process, plant is in order to existence procreation in the environment of constantly change, and developing corresponding molecular regulation network and deal with salt stress.When being subject to salt stress, many plant hormones such as dormin, Plant hormones regulators,gibberellins, brassinolide and Whitfield's ointment etc. all can participate in this regulation process.In addition, harmful ion transported to extracellular or isolation in vacuole control to cross polyionic accumulation (Zhang and Blumwald, 2001 in tenuigenin by activating SOS signal pathway and vacuole skin reverse transport passage NHX1; Yamaguchi et al.; 2013); and increasing permeation protective agent as the synthesis of proline(Pro), polyvalent alcohol, carbohydrate and methylamine etc., the growth being carried out Plants under Salt Stress by osmoregulation is all the important regulating and controlling mode (Deinlein et al., 2014) that plant tackles salt stress.

In recent years along with the development of epigenetics, it is found that genome is protein modified and act on the adaptability of various environment stress plays plant.Research shows, when salt stress occurs, has karyomit(e) to be modified at and participate in the response of anti-salt in contemporary plant.After seedling stage tempers process with slight salt stress, be incubated at the Arabidopis thaliana under normal condition, there is not the growth phase of salt stress in the later stage, between tempering rear plant and contrasting, there is no visible phenotypic difference; Again with after salt stress process, in the plant body that discovery was taken exercise, the abduction delivering of HKT1 is higher than control group, Na ⁺accumulate lower, show more anti-salt.Carry out analysis with ChIP-seq to full-length genome 4 kinds of chromosome modification to show, after salt stress is taken exercise, the modification of H3K27me3 has been distributed with change (Sani et al., 2014).Song etc. (2012) report, salt stress process can affect the promotor of soybean 4 transcription factors and the methylation state of DNA of coding region, shows that the epigenetic modification of these genes can strengthen the salt tolerance of plant.Zhaoliang etc. (2011) research shows, histone arginine methyltransferase SKB1, by changing histone H 4 R3sme2 level, affects the expression of ABA signal pathway key gene, thus the salt tolerance of mediated plant.

In the epigenetic regulation process of plant salt endurance, mainly concentrate on DNA methylation and histone methylated research, other modifies as less in researchs such as acetylation of histone.AtSRT2 is a kind of histon deacetylase (HDAC), at present, does not also have the report of AtSRT2 in mediated plant salt tolerance.This research finds that AtSRT2 has mediated plant salt endurance regulation process, process LAN atSRT2gene can improve arabidopsis thaliana salt-tolerance, especially the salt tolerance of Their Seed Germinating Period.And seed germination is a period crucial in plant growth and development process, directly affect the plant whole subsequent development stage, and this period, very responsive again to environment, be more vulnerable to the harm of salt stress.The present invention is significant for molecular improvement salt tolerance of crop.

Summary of the invention

Technical requirements

Technical problem to be solved by this invention is to provide a kind of AtSRT2 albumen.

The technical problem that the present invention also will solve is to provide the encoding sequence of above-mentioned protein fragments.

The technical problem that the present invention also will solve is to provide said gene fragment and is improving the application in plant salt endurance.

The present invention is achieved in that AtSRT2 albumen, it is the albumen with the aminoacid sequence shown in SEQ ID NO:1 in sequence table, or by the amino acid residue sequence of SEQ ID NO:1 through the replacement of more than one amino-acid residue, disappearance or interpolation, and there is the albumen derivative by SEQ ID NO:2 with the identical activity of amino acid residue sequence of SEQ ID NO:2.

The encoding sequence of AtSRT2 albumen, it is one of following Nucleotide:

(1) nucleotide sequence shown in SEQ ID NO:2 in sequence table;

(2) polynucleotide of the aminoacid sequence shown in SEQ ID NO:2 in polynucleotide;

(3) nucleotide sequence of the nucleotide sequence hybridization that can limit with SEQ ID NO:2 in sequence table under high high stringency conditions.

The encoding sequence of AtSRT2 albumen is improving the application in plant salt endurance.

AtSRT2 albumen of the present invention can pass through synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.

The encoding sequence of AtSRT2 albumen of the present invention is by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in SEQ ID NO:2 in sequence table, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in SEQ ID NO:1 is held to obtain at its 5 ' end and/or 3 '.

SEQ ID NO:2 is made up of 1131 Nucleotide, and whole SEQ ID NO:2 is the encoding sequence of AtSRT2 albumen, and SEQ ID NO:1 is made up of 376 amino acid altogether.

Described high high stringency conditions is in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS, hybridizes and wash film under 65 DEG C of conditions.

Recombinant expression vector containing described gene, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.

Available existing expression vector establishment contains the recombinant expression vector of described gene.Described expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of micropellet bombardment.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any one enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can be used alone or are combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of qualification and screening, can processing expression carrier used thereof, enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the chemical resistance reagent marker gene etc. of colour-change can be produced as added coding.Also any selected marker can not be added, directly according to phenotypic screen.

Described recombinant expression vector specifically can be the recombinant plasmid multiple clone site that described gene inserts pBI121 plasmid obtained.

Beneficial effect

By engineered method, the encoding sequence of AtSRT2 albumen provided by the invention is imported in object plant, obtain the transgenic plant of salt tolerance higher than described object plant, the salt tolerance of this plant is obtained improve, its major embodiment is: under Ficus caricaL condition, compared with wild-type, proceed to the plant of the encoding sequence of AtSRT2 albumen, seed germination rate significantly improves; Salt tolerance strengthens, and plant is more tall and big compared with wild-type, and root, leaf area are significantly higher than wild-type; Survival rate of plant significantly improves.The present invention plays an important role in the plant breeding of cultivation salt tolerance and salt tolerance enhancing.

Accompanying drawing explanation

Fig. 1 is Their Seed Germinating Period atSRT2genetic expression is by Salt Stress-induced;

Fig. 2 is that AtSRT2 subcellular localization signal affects (arrow represents cell membrane localization signal) by salt stress;

Fig. 3 is the structural representation of recombinant plasmid PBI121-AtSRT2;

Fig. 4 is for turning atSRT2the screening of gene masculine plant

(WT represents wildtype Arabidopsis thaliana, and Col/pBI121 is the Arabidopis thaliana life proceeding to pBI121 empty carrier,

OE-1, OE-2 and OE-3 represent that 3 turn respectively atSRT2gene strain);

Fig. 5 is 3 and turns atSRT2gene strain, wildtype Arabidopsis thaliana seed germination rate under Ficus caricaL compares, and (WT represents wildtype Arabidopsis thaliana, and OE-1, OE-2 and OE-3 represent that 3 turn respectively atSRT2gene strain);

Fig. 6 is 3 and turns atSRT2gene strain, wildtype Arabidopsis thaliana under Ficus caricaL plant strain growth phenotype (WT represents wildtype Arabidopsis thaliana, OE-1, OE-2 and OE-3 represent respectively 3 turn atSRT2gene strain);

Fig. 7 is 3 and turns atSRT2gene strain, wildtype Arabidopsis thaliana plant forms and surviving rate under Ficus caricaL compare, and (WT represents wildtype Arabidopsis thaliana, and OE-1, OE-2 and OE-3 represent that 3 turn respectively atSRT2gene strain).

Embodiment

According to following embodiment, the present invention may be better understood.But, those skilled in the art will readily understand, described by embodiment only for illustration of the present invention, and should can not limit in claims the present invention described in detail yet.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Arabidopis thaliana ( arabidopsis thalianal.) environmental Columbia0: order from Arabidopis thaliana database, http://www.arabidopsis .org/;

Arabidopis thaliana is planted in Institute of Crop Science, Chinese Academy of Agricultural Science's phytotron, and growth room's temperature is set to 21 DEG C, and the long day is that 16h illumination/8h is dark, and short day is that 10h illumination/14h is dark.

PBI121 carrier: purchased from Clontech company, catalog number 6081-1.

embodiment 1, Their Seed Germinating Period atSRT2express by Salt Stress-induced

1, the extraction of Arabidopis thaliana total serum IgE

By the seed of wildtype Arabidopsis thaliana Columbia0 70%(volume fraction in Bechtop) ethanol postincubation 3min, then with aseptic washing 2 times, wash 1min at every turn, after carrying out sterilising treatment 10min with the clorox of 5 ‰ (mass volume ratios), again with aseptic washing twice, wash one minute at every turn.Aseptic seed is sowed in MS0 liquid nutrient medium by control group liquid-transfering gun, and 4 DEG C of vernalization transferred to tissue culture room after 3 days, sampled respectively at 0h, 6h, 12h, 24h, 36h, 48h.Meanwhile, aseptic seed is sowed in the MS0 liquid nutrient medium containing 60mM NaCl by Ficus caricaL group liquid-transfering gun, and 4 DEG C of vernalization transferred to tissue culture room after 3 days, sampled respectively at 0h, 6h, 12h, 24h, 36h, 48h, 60h, 72h.

The wildtype Arabidopsis thaliana TRIzol method processed is extracted total serum IgE, and the RNA extracted is after 2% agarose gel electrophoresis detection is qualified, and carrying out reverse transcription by ThermoScript II (TaKaRa company) to total serum IgE is cDNA, and-20 DEG C save backup.

2, RT-PCR detects atSRT2the expression of gene

According to atSRT2gene (AT5G09230, arabidopsis gene group seat is numbered; Http:// www.arabidopsis.org/) sequences Design Auele Specific Primer atSRT2-F1 and atSRT2-R1, object fragment is 273bp.With Arabidopis thaliana actingene designs primer for internal reference atActin-F and atActin-R, object fragment is 227bp.

The cDNA of the different time sections process obtained in step 1 is diluted to suitable concentration and carries out real-time quantitative PCR mensuration as template.According to Real Master Mix (SYBR Green) PCR kit, (TIANGEN company specification sheets carries out real-time quantitative PCR.Reaction system comprises 2.5 × Real Master Mix 4 μ l, each 0.5 μ l, ddH2O4.5 μ l, the template 1 μ l of upstream and downstream primer.PCR amplification program is: 95 DEG C of denaturation 3min, 95 DEG C of sex change 20s, 53 DEG C of annealing 20s, and 72 DEG C extend 20s, 40 circulations, and each circulation the 3rd step carries out fluorescent collecting, and each sample arranges 3 repetitions.

atSRT2-F: 5 '-TTCCGTCGCGAAGTATGTACC-3 ' (At5G09230:53-73bp, i.e. the 53-73 position of SEQ ID NO:2 in sequence table)

atSRT2-R: 5 '-CTGTGCTAACCCCAGCTCCAGTC-3 ' (At5G09230:303-325bp, the i.e. reverse complementary sequence of the 303-325 position of SEQ ID NO:2 in sequence table)

AtActin-F ：5’-CCCCTGCTATGTATGTGGCTAT-3’

AtActin-R ：5’-TGCTGTGGTGGTGAAAGAGTAA-3’

RT-PCR analytical results shows, and solubility curve is unimodal, and amplified production specificity is good, and fluorescence curve can react amplification well.Right atSRT2gene under Ficus caricaL in Seed Germination the expression of different time sections carry out statistical study, as shown in Figure 1, under non process conditions, normal seed germination needs 48 hours to result, in whole process atSRT2express stable, not obvious modulation up and down; Under 60mM NaCl treatment condition, seed germination postpones, and needs 72 hours, atSRT2express with process time lengthening and progressively raise, reached maximum at 48 hours, be 9.21 times of initial transcriptional level, expression amount declines again gradually subsequently.Above result of study shows atSRT2in Seed Germination, it is expressed by Salt Stress-induced.

embodiment 2, protoplastis Subcellular Localization

1, Subcellular Localization vector construction

According to CDS sequences Design Auele Specific Primer AtSRT2-F2 and AtSRT2-R2 of the AtSRT2 gene that TAIR website provides, amplification AtSRT2 full length gene (going terminator codon).Object fragment is connected on the Subcellular Localization carrier 16318GFP that BamHI enzyme cuts with Infusion enzyme, ligation system is: 2 μ l Infusion HD Enzyme Premix, 5 μ l 16318GFP digestion products, 3 μ l pcr amplification products, 50 DEG C are reacted 15 minutes.Heat shock transformation of E. coli Top10 after ligation.PCR detection is carried out to recombinant plasmid.Positive colony is checked order.Extract plasmid, obtain recombinant plasmid 116318-AtSRT2-GFP.

Primer sequence is:

16318-AtSRT2-GFP-F:5 '-TATCTCTAGA gGATCCaTGCTTTCTATGAACATGAGAAG-3 ' (underscore part is the recognition site of BamHI, and sequence is thereafter the 1-23 position of SEQ ID NO:2)

16318-AtSRT2-GFP-R:5 '-TGCTCACCAT gGATCCgAGAGCTGGGACACTGAGCG-3 ' (underscore part is the recognition site of BamHI, and sequence is thereafter the reverse complementary sequence of the 1109-1128 position of SEQ ID NO:2)

2, protoplastis preparation

1) getting the Arabidopis thaliana grown to about 2-3 week is test materials (Arabidopis thaliana is less than normal, prepares protoplastis integrity good).

2), under low light condition, the bar shaped (need not cut off completely, can each leaf bar be kept to be connected at same limb edge, such protoplastis cracking be more abundant) that blade blade is cut into 0.5-1mm is got.

3) add 10-20 ml enzymolysis solution, cover tin platinum paper, room temperature lucifuge 45rpm, 3h.

4) with 100-150 order steel filter screen careful filtration.

5) centrifugal 1 minute of room temperature 100g (Brake, lifting speed is all transferred to 2, prevents rotating speed to be elevated rapidly), removes supernatant (retaining a small amount of liquid).

6) first softly mix, then it is resuspended to add 1ml W5, softly mixes, rear ice bath 30 minutes, centrifugal 1 minute of 100g (the same), removes supernatant (the same).

7) first softly mix, then it is resuspended to add appropriate MMG.

3, room temperature conversion

1) 10 μ l 16318-AtSRT2-GFP(10-20 μ g plasmid DNA are added), be generally 15 μ g.

2) 100 μ l protoplastiss are added, mixing.

3) 110 μ l PEG/Ca solution (PEG notes constant volume) mixings are added.

4) 23 DEG C of incubations 30 minutes.

5) add 440 μ l W5 solution to mix gently.

6) centrifugal 1 minute of 100g (the same), removes supernatant PEG.

7) add 100 μ l W5 resuspended, then add 0.9 ml W5(Ficus caricaL experiment, in W5, adding NaCl to final concentration is 60mM).

8) 23 DEG C of lucifuge overnight incubation.

9) under Laser Scanning Confocal Microscope, GFP signal is observed.

As shown in Figure 2, under non process conditions, AtSRT2 is positioned at cytolemma and nucleus; Under 60Mm NaCl treatment condition, AtSRT2 film signal for locating disappears, and only has nuclear localization signal.With DAPI, nucleus is dyeed, find that AtSRT2 location GFP signal can be completely overlapping with DAPI dyeing part, show that AtSRT2 is positioned at nucleus under 60Mm NaCl treatment condition.Above result shows, AtSRT2 Subcellular Localization can change with NaCl process, and AtSRT2 has response to NaCl process.

embodiment 3, atSRT2gene clone and acquisition process LAN strain

1, the clone of AtSRT2 gene and the structure of recombinant expression vector pBI121-AtSRT2

With the wildtype Arabidopsis thaliana Columbia0 of Adult plant for experiment material, extract total serum IgE by TRIzol method, the Reverse Transcription box reverse transcription detecting qualified RNA TaKaRa company is cDNA, and-20 DEG C save backup.According to what TAIR website provided atSRT2the CDS sequences Design Auele Specific Primer of gene atSRT2-F2 and atSRT2-R2, amplification atSRT2full length gene.Reaction system comprises 2 × GC Buffer 25 μ l, dNTP Mix 4 μ l, upstream and downstream primer each 1 μ l, Primerstar 0.3 μ l, ddH ₂the above PCR reaction reagent of O 16.7 μ l, template cDNA 2 μ l(all comes from TaKaRa company pcr amplification test kit).PCR amplification program is 94 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 49 DEG C of annealing 45s, and 72 DEG C extend 1min, 40 circulations, last 72 DEG C of annealing 10min, 4 DEG C of preservations.

PBI121-AtSRT2-F: 5 '-CC cCCGGGaTGCTTTCTATGAACATGAGAAG-3 ' (underscore part is the recognition site of SmaI, and sequence is thereafter the 1-23 position of SEQ ID NO:2)

PBI121-AtSRT2-R: 5 '-GG aCTAGTcTAGAGAGCTGGGACACTGAGCG-3 ' (underscore part is the recognition site of Spe I, and sequence is thereafter the reverse complementary sequence of the 1109-1131 position of SEQ ID NO:2)

PCR primer is carried out agarose gel electrophoresis, reclaims test kit with the DNA gel of TaKaRa company and reclaim purifying target DNA.Utilize the P Easy-Blunt cloning Vector system of TIANGEN company, the PCR primer of acquisition is inserted on cloning vector.Reaction system 10 μ l: 8 μ l PCR product, 2 μ l vector, after room temperature places 15min, be added to connection product in 100 μ l TOP10 competent cells, and mixing ice bath 30s, puts 2min on ice after 42 DEG C of heat shock 90s immediately.Add the LB nutrient solution of 500 μ l balances to room temperature afterwards, in 37 DEG C of shaking tables, 200rpm cultivates 1h.After being smoothened on the LB substratum containing Amp resistance by the bacterium liquid glass stick of muddiness, put overnight incubation in 37 DEG C of constant incubators.Picking white mono-clonal, utilizes primer atSRT2-F2 and atSRT2-R2 carries out bacterium liquid PCR and detects (object clip size is about 1131bp).PCR is identified correct bacterial strain shakes bacterium upgrading grain, with restriction endonuclease sma I and Spe I double digestion plasmid, size is about the object band of 1131bp, be connected with the pBI121 carrier large fragment through same double digestion, obtain recombinant plasmid.Sequence verification in the restriction enzyme site Sma I and Spe I insertion sequence table of pBI121 DNA fragment shown in SEQ ID NO:2 ( atSRT2gene) recombinant plasmid called after pBI121-AtSRT2(Fig. 3).In recombinant expression vector pBI121-AtSRT2, the promotor starting AtSRT2 genetic expression is 35s promotor.Wherein, SEQ ID NO:2 is the encoding sequence of the albumen (AtSRT2 albumen) shown in SEQ ID NO:1.

2, acquisition and the qualification of AtSRT2 gene Arabidopis thaliana is turned

The recombinant expression vector pBI121-AtSRT2(that step one is obtained or pBI121 carrier) use freeze-thaw method transformation Agrobacterium C58C1(purchased from Biovector CO., LTD company, article No. article No. Biovector009) (the method for freeze-thaw method transformation Agrobacterium, with reference to Holsters M, de Waele D, Depicker A.Transfection and transformation of Agrobacterium tumefaciens.Mol Gen Genet, 1978,183:181-187).Use primer atSRT2-F2 and atSRT2-R2 carries out bacterium liquid PCR to be identified, will show the recombinational agrobacterium called after C58C1/pBI121-AtSRT2 containing DNA fragment (about 1131bp) shown in SEQ ID NO:2 in ordered list through qualification; The recombinational agrobacterium name C58C1/pBI121 of pBI121 empty carrier will be proceeded to.Adopt method (the Bechtold N etc. that Agrobacterium inflorescence infects, (1993) In plant a Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.C.R.Acad.Sci.316:1194 – 1199) by recombinational agrobacterium C58C1/pBI121-AtSRT2(or C58C1/pBI121 of above-mentioned gained) the environmental Columbia0 of arabidopsis thaliana transformation, screen through the MS0 solid medium containing 50mg/mL Kan, filtered out for 9 strain T0 generations turn altogether atSRT2gene plant, gets wherein three strains and is denoted as OE-1, OE-2 and OE-3(wherein three T0 generations turn atSRT2gene plant is as the seedling of arrow indication in Fig. 4).By the positive plant filtered out transplant cultivate in greenhouse to Nutrition Soil, breed often for seed all at the enterprising row filter of MS0 solid medium containing 50mg/mL Kan, breeding to T3 for time start to carry out Function Identification.Carry out PCR detection to the transgenic arabidopsis seedling in T3 generation further, with the genomic dna of transgenic arabidopsis seedling for template, the primer that PCR detects is atSRT2-F2 and atSRT2-R2.PCR detected result shows, 3 different turning atSRT2gene strain all increases and obtains the fragment that length is about 1131bp.And carry out PCR detection with same primer pair wildtype Arabidopsis thaliana Columbia0 plant and the plant that proceeds to pBI121 empty carrier, do not obtain above-mentioned amplified fragments (Fig. 4).

embodiment 4, turn atSRT2gene arabidopsis thaliana salt-tolerance is identified

1, germination period salt tolerance detects

With following three kinds of Arabidopsis plant for experiment material: wildtype Arabidopsis thaliana Columbia0(WT), in 3 T3 generations, turn AtSRT2 gene strain (OE-1, OE-2 and OE-3), proceed to the strain of pBI121 empty carrier.After seed carries out aseptically process, be sowed on MS0 solid medium and the MS0 solid medium containing 120mM NaCl, 150mM NaCl respectively, 4 DEG C of vernalization put tissue culture room growth, when process added up seed germination rate after 2 days after 3 days.Under non-process and treatment condition, proceed to strain no significant difference compared with wild-type of pBI121 empty carrier.But as shown in Figure 5, under non process conditions, wild type seeds germination physiology slightly lower than atSRT2process LAN strain, after illumination cultivation 4d, wild-type with atSRT2process LAN strain germination rate reaches unanimity, and all reaches more than 96%.Under NaCl process, wild mutant and atSRT2process LAN strain all shows as seed germination and postpones, and seed germination rate declines.Under 120mM, 150mM NaCl process, atSRT2postpone about 1 day under the relative normal growing conditions of process LAN strain seed germination speed, but final seed germination rate is suitable with non-process; And wild-type except germination physiology postpone except, under 120mM, 150mM NaCl process seed germination rate comparatively non-process have dropped 39.37% and 58.1% respectively.Above result of study shows to turn atSRT2gene strain significantly improves seed germination speed under Ficus caricaL condition and final seed germination rate, improves the salt tolerance of Their Seed Germinating Period.

2, Seedling Salt-tolerance detects

With following three kinds of Arabidopsis plant for experiment material: wildtype Arabidopsis thaliana Columbia0(WT), in 3 T3 generations, turn AtSRT2 gene strain (OE-1, OE-2 and OE-3), proceed to the strain of pBI121 empty carrier.Seed, after MS0 solid medium is sprouted and cultivated 5 days, is transferred to the MS0 solid medium containing 90mM NaCl, and the phenotype of each material is observed in process for 10 days afterwards, and carries out root, leaf area scanning and data statistic analysis.Under non-process and treatment condition, proceed to strain no significant difference compared with wild-type of pBI121 empty carrier.But as shown in Figure 6, under non process conditions, turn atSRT2gene strain size is suitable with wild-type, and root, leaf area scanning result show that storeroom is without significant difference; Under 90mM NaCl process, all test materialss grow and are all had a strong impact on, and plant becomes short and small, and comparatively speaking, wild-type comparatively turns atSRT2gene strain is affected even more serious.Root, leaf area scanning result show, turn atSRT2gene strain blade face is actively significantly higher than wild-type, turns atSRT2gene strain OE-1, OE-3 leaf area are significantly higher than wild-type.

Meanwhile, by above-mentioned experiment material after MS0 solid medium is sprouted and cultivated 8 days, be transferred to soil and cultivate in greenhouse, cultivate after 10 days, with 150mM NaCl process, process and add up survival rate of plant after 21 days.As shown in Figure 7, before 150mM NaCl process, each experiment material phenotype no significant difference, after 150mM NaCl process, each experiment material blade starts jaundice, and plant part is downright bad, and comparatively speaking, wild-type comparatively turns atSRT2gene strain affects even more serious by salt stress, plant relative survival statistics shows, under 150mM NaCl treatment condition, turns atSRT2gene strain OE-1, OE-2 and OE-3 plant relative survival are respectively 78.1%, 81.8% and 68%, and pole is significantly higher than 26.8% of wild-type.

The result of above embodiment shows, compared with wild-type, turns atSRT2gene strain significantly improves the ability that plant maintains normal plants growth under salt stress, improves plant relative survival.

SEQUENCE LISTING

sequence table

<110> Guizhou Province Grass Industry Research Institute

<120> AtSRT2 albumen and encoding sequence thereof and application

<130> nm:

<160> 4

<170> PatentIn version

<210> SEQ ID NO：1

<211> 376

<212> amino acid

<213> wildtype Arabidopsis thaliana Columbia0

<220>

<223>

<400>

MLSMNMRRVF GGVSTDLFPS RSMYRPLQSG GNLVMLFKGC RRFVRTTCRV 50

SIPGGSLGNE SKAPPRFLRD RKIVPDADPP NMEDIHKLYR LFEQSSRLTI 100

LTGAGVSTEC GIPDYRSPNG AYSSGFKPIT HQEFTRSSRA RRRYWARSYA 150

GWRRFTAAQP GPAHTALASL EKAGRINFMI TQNVDRLHHR AGSDPLELHG 200

TVYTVMCLEC GFSFPRDLFQ DQLKAINPKW AEAIESIDHG DPGSEKSFGM 250

KQRPDGDIEI DEKFWEEGFH IPVCEKCKGV LKPDVIFFGD NIPKERATQA 300

MEVAKQSDAF LVLGSSLMTM SAFRLCRAAH EAGAMTAIVN IGETRADDIV 350

PLKINARVGE ILHRVLDVGS LSVPAL 376

 

 

<210> SEQ ID NO：2

<211> 1131

<212> DNA

<213> wildtype Arabidopsis thaliana Columbia0

<220>

<223>

<400> 2

ATGCT TTCTA TGAAC ATGAG AAGAG TGTTT GGAGG TGTAT CTACA GATTT 50 GTTTC CGTCG CGAAG TATGT ACCGG CCGCT GCAGT CTGGG GGAAA CTTGG 100 TTATG TTGTT TAAAG GATGC AGACG GTTTG TGAGA ACAAC GTGTC GCGTT 150 TCGAT CCCTG GTGGT TCTTT GGGGA ATGAA TCAAA AGCAC CTCCA AGGTT 200 TCTGA GGGAT AGAAA GATTG TTCCA GATGC TGATC CCCCC AATAT GGAAG 250 ATATC CACAA GTTGT ATCGA CTTTT TGAGC AAAGC TCGAG ACTCA CCATC 300 TTGAC TGGAG CTGGG GTTAG CACAG AATGT GGAAT TCCGG ATTAC AGAAG 350 TCCCA ATGGA GCATA CAGTT CTGGT TTCAA ACCAA TTACC CATCA GGAGT 400 TTACT CGATC AAGCC GAGCT CGTAG GCGGT ATTGG GCGAG GAGTT ACGCT 450 GGATG GAGGA GGTTC ACTGC AGCAC AACCA GGACC AGCTC ATACT GCTTT 500 AGCAT CACTA GAAAA AGCTG GACGA ATAAA TTTTA TGATC ACACA AAATG 550 TTGAC AGGTT GCATC ATCGT GCTGG GAGCG ATCCA CTTGA ATTAC ATGGC 600 ACAGT ATATA CTGTT ATGTG CTTAG AGTGC GGCTT CTCTT TTCCC CGAGA 650 CTTGT TTCAG GATCA GCTAA AAGCA ATCAA TCCTA AGTGG GCTGA AGCTA 700 TAGAA AGTAT TGATC ATGGA GATCC AGGAT CAGAG AAGAG CTTTG GCATG 750 AAACA AAGGC CTGAT GGTGA TATCG AGATT GACGA AAAGT TTTGG GAAGA 800 GGGTT TTCAT ATACC AGTAT GTGAG AAGTG CAAAG GAGTC CTAAA GCCTG 850 ATGTA ATTTT CTTTG GAGAC AACAT CCCGA AGGAA AGAGC TACTC AAGCA 900

ATGGA AGTTG CAAAA CAGAG CGATG CATTC CTCGT GTTGG GTTCA TCCTT 950 AATGA CAATG TCTGC TTTTC GCCTT TGCAG AGCGG CTCAT GAGGC TGGTG 1000 CTATG ACCGC AATTG TAAAT ATAGG CGAAA CAAGA GCTGA TGACA TTGTT 1050 CCATT GAAAA TCAAT GCTAG GGTTG GTGAG ATATT GCACA GAGTT CTTGA 1100 CGTGG GATCG CTCAG TGTCC CAGCT CTCTA G 1131

 

<210> SEQ ID NO： 3

<211> 21

<212> DNA

<213> artificial sequence

 

<220>

<223>

According to the CDS sequence of the AtSRT2 gene that TAIR website provides, then use DNAMAN software design, for the amplification of AtSRT2 gene C DS sequence.

 

<400> 3

TTCCG TCGCG AAGTA TGTAC C 21

 

<210> SEQ ID NO：4

<211> 23

<212> DNA

<213> artificial sequence

 

<220>

<223>

According to the CDS sequence of the AtSRT2 gene that TAIR website provides, then use DNAMAN software design, for the amplification of AtSRT2 gene C DS sequence.

 

 

<400> 4

CTGTG CTAAC CCCAG CTCCA GTC 23

 

<210> SEQ ID NO：5

<211> 22

<212> DNA

<213> artificial sequence

 

<220>

<223> is with Arabidopis thaliana actingene is internal reference, then uses DNAMAN software design, for qRT-PCR.

 

<400> 5

CCCCT GCTAT GTATG TGGCT AT 22

 

<210> SEQ ID NO：6

<211> 22

<212> DNA

<213> artificial sequence

 

<220>

<223> is with Arabidopis thaliana actingene is internal reference, then uses DNAMAN software design, for qRT-PCR.

<400> 6

TGCTG TGGTG GTGAA AGAGT AA 22

 

<210> SEQ ID NO：7

<211> 39

<212> DNA

<213> artificial sequence

 

<220>

<223>, according to the CDS sequence of the AtSRT2 gene that TAIR website provides, then uses DNAMAN software design, for 16318:AtSRT2-GFP vector construction.

 

<400> 7

TATCT CTAGA GGATC CATGC TTTCT ATGAA CATGA GAAG 39

 

<210> SEQ ID NO：8

<211> 36

<212> DNA

<213> artificial sequence

 

<220>

<223>, according to the CDS sequence of the AtSRT2 gene that TAIR website provides, then uses DNAMAN software design, for 16318:AtSRT2-GFP vector construction.

 

<400> 8

TGCTC ACCAT GGATC CGAGA GCTGG GACAC TGAGC G 36

 

<210> SEQ ID NO：9

<211> 31

<212> DNA

<213> artificial sequence

 

<220>

<223>, according to the CDS sequence of the AtSRT2 gene that TAIR website provides, then uses DNAMAN software design, for pBI121:AtSRT2-GFP vector construction.

<400> 9

CC CCC GGGAT GCTTT CTATG AACAT GAGAA G 31

 

<210> SEQ ID NO：10

<211> 31

<212> DNA

<213> artificial sequence

 

<220>

<223>, according to the CDS sequence of the AtSRT2 gene that TAIR website provides, then uses DNAMAN software design, for pBI121:AtSRT2-GFP vector construction.

 

<400> 10

GG ACT AGTCT AGAGA GCTGG GACAC TGAGC G 31

Claims (3)

1. an AtSRT2 albumen, it is characterized in that: it is the albumen with the aminoacid sequence shown in SEQ ID NO:1 in sequence table, or by the amino acid residue sequence of SEQ ID NO:1 through the replacement of more than one amino-acid residue, disappearance or interpolation, and there is the albumen derivative by SEQ ID NO:2 with the identical activity of amino acid residue sequence of SEQ ID NO:2.

2. an encoding sequence for AtSRT2 albumen as claimed in claim 1, is characterized in that: it is one of following Nucleotide:

(1) nucleotide sequence shown in SEQ ID NO:2 in sequence table;

(2) polynucleotide of the aminoacid sequence shown in SEQ ID NO:2 in polynucleotide;

(3) nucleotide sequence of the nucleotide sequence hybridization that can limit with SEQ ID NO:2 in sequence table under high high stringency conditions.

3. the encoding sequence of an AtSRT2 albumen as claimed in claim 2 is improving the application in plant salt endurance.