CN106047886B - Na+/H+Reverse transporter gene and application thereof - Google Patents

Abstract

The invention discloses Na on a wheat plasma membrane⁺/H⁺An inverse transporter gene and applications thereof. The Na is⁺/H⁺The total length of the inverse transport protein gene is 3460bp, the gene is positioned on a cytoplasmic membrane after being expressed, and Na is exerted⁺/H⁺Exchange function of Na⁺Is discharged to the outside of cells, and thus the present invention also discloses the use of the Na⁺/H⁺A method for constructing a plant genetic transformation vector by using the reverse transporter gene and improving the salt tolerance of a plant.

Description

Na+/H+Reverse transporter gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a gene capable of converting Na⁺Na excreted to the outside of the cell⁺/H⁺An inverse transporter gene and applications thereof.

Background

The first time of cloning from arabidopsis thaliana in 2000SOS1Gene and is thought to be Na located on the plasma membrane⁺/H⁺The reverse transporter, at its N-terminus, is a 12-transmembrane domain with a long hydrophilic tail at the C-terminus. The result of subcellular localization shows that the protein is localized on the cell membrane and is the only responsible Na which is identified to be localized on the plasma membrane in the current plants⁺The protein of the outflow. Na (Na)⁺/H⁺Reverse transporter to H⁺Transporting Na into cells⁺Transport to the extracellular space. The mutation of this gene in Arabidopsis makes the plant body very sensitive to salt and causes Na⁺Efflux of Na and⁺from the rootThe long-distance transportation of the stems and leaves. In general, SOS1 makes SOS1 in dormant state through interaction of C-terminal self-inhibitory domain and adjacent domain, SOS2-SOS3 kinase complex phosphorylates SOS1 to activate SOS1, and SOS1 protein C-terminal [ V, I]VR[V,I]Two serines in the DSPS motif are sites where phosphorylation occurs. SOS1 activity is inhibited if the phosphorylation site or the site recognized by the SOS2-SOS3 complex is mutated.

Na has been cloned in rice⁺/H⁺Inverse transporters, and in Arabidopsis thalianaSOS1Gene homology, Na located on plasma membrane⁺/H⁺Transport function, reducing Na in yeast after yeast transformation⁺In this context, the SOS2-SOS3 complex in Arabidopsis activates OsSOS1 in rice.OsSOS1Transformation of Arabidopsis thalianasos1The sensitivity of the mutant to salt can be reduced after the mutant. The above results indicate that the SOS signaling pathway is present and functional in cereal crops, which suggests that the SOS pathway is conserved in both monocots and dicots. Research results in woody plant poplar show that the related protein of SOS signal channel can correspond to the protein in Arabidopsis one by one and complement the phenotype of the corresponding mutant in Arabidopsis. Experiments in arabidopsis, rice and poplar have fully demonstrated that the SOS pathway is present in both herbaceous, woody plants, monocotyledonous and dicotyledonous plants, and that the function is conserved. In wheatSOS1Some achievements are also obtained in the gene research, and recently, the non-damage micrometering technology is used for detecting the wheat root Na⁺When the ion current is carried out, stronger Na is detected in the salt-tolerant variety Kharchia 65⁺Ion efflux phenomenon, which is demonstrated by plasma membrane H⁺-ATPase supplied with energy fromSOS1Homologous gene-mediated production.

The invention is concerned with wheatTaSOS1Cloning and functional research are carried out on the gene, and the complementary salt sensitive yeast mutant is found; transformation of Arabidopsis thalianasos1After the mutant is subjected to the over-expression, the sensitivity of the mutant to salt can be reduced, and the salt tolerance of the transgenic arabidopsis is improved; in wheatSOS1Silencing of the gene also results in Na in wheat leaves⁺Increase of ion content.

Disclosure of Invention

The inventors of the present invention compared Arabidopsis thaliana with riceSOS1Gene sequence, designing primer, cloning wheat in wheat variety Keyi 26SOS1The gene has Genebank accession number FN356229, and is used in combination with Arabidopsis thaliana and riceSOS1The genes are aligned, and the sequence is also found to show high similarity with the SOS1 protein in rice and Arabidopsis thaliana. Through the result analysis of genetically transformed arabidopsis thaliana and yeast, the wheat is foundSOS1The gene can improve the salt tolerance of arabidopsis thaliana and yeast in wheatSOS1Silencing of the gene also results in Na in wheat leaves⁺Increase of ion content.

The invention aims to provide a plasma membrane Na⁺/H⁺Reverse transporter gene capable of transporting Na in plants⁺The ions are expelled to the outside of the cell.

The gene provided by the invention is derived from salt-tolerant wheat variety Keyi 26 and has a nucleotide sequence shown as SEQ ID No.1 or a degenerate sequence thereof. The DNA shown in SEQ ID No.1 has a length of 3460bp (wherein the ORF region is 3429), is a nuclear gene, is located on the chromosome of the third homologous group A of wheat, and is named asTaSOS1-A1Encodes a protein of 1143 amino acid residues.

The invention provides a primer pair for amplifying the full length of the gene, wherein in the primer pair, the sequence of a forward primer is shown as SEQ ID No.2, and the sequence of a reverse primer is shown as SEQ ID No. 3.

The present invention provides an expression vector comprising said gene operably linked to a 35S promoter. Transforming the expression vector into plants, and screening out plants with salt tolerance.

The wheat Na provided by the invention⁺/H⁺The reverse transporter gene is genetically transformed into a dicotyledonous plant Arabidopsis thaliana by using a genetic engineering technology, and the overexpression can improve the salt tolerance of the Arabidopsis thaliana and can complement the Arabidopsis thalianasos1And (3) mutants.

The present invention provides a yeast expression vector comprising the gene operably linked downstream of a promoter of the expression vector.

The wheat Na provided by the invention⁺/H⁺Genetic transformation of reverse transporter gene to lack of endogenous Na by using genetic engineering technology⁺In yeasts of the transport system and screening for the complementary absence of endogenous Na⁺Yeast strains of the transport system.

The present invention provides a viral vector comprising said gene operably linked downstream of the T7 promoter and origin of replication of the viral vector.

The wheat Na provided by the invention⁺/H⁺The reverse transport protein gene is genetically transformed into monocotyledonous gramineous plant wheat by utilizing a genetic engineering technology to reduce endogenous genes of the wheatTaSOS1-A1Increase the expression of Na in wheat leaves⁺Accumulation of (2). Thus, utilizeTaSOS1-A1Can create salt-tolerant materials, provides valuable materials for the research of the salt tolerance of wheat, and has important significance for the genetic improvement of wheat.

Drawings

FIG. 1 is a drawing ofTaSOS1-A1Chromosomal localization of genes.

FIG. 2 is the construction process of dicotyledonous plant binary expression vector.

FIG. 3 shows the results of PCR and RT-PCR identification of transgenic Arabidopsis plants, wherein A is the result of PCR identification, and the sequence from left to right is: wild type negative control, wild type transgenic lines T1-6, T1-9 and T1-10, mutant negative control, mutant transgenic lines T1-1, T1-2 and T1-7; b is the wild RT-PCR identification result, and the sample loading sequence from left to right is as follows: negative control, wild type transgenic lines T1-6, T1-9 and T1-10; c is the RT-PCR identification result of the mutant, and the sample sequence from left to right is as follows: mutant negative controls, mutant transgenic lines T1-1, T1-2, and T1-7.

FIG. 4 shows the experimental results of the growth of transgenic Arabidopsis thaliana in MS and MS plus NaCl culture medium,sos1for the mutant, T1-1, T1-2 and T1-7 are transgenic lines of the transmutant respectively, col is a wild type, and T1-6, T1-9 and T1-10 are transgenic lines of the transwild type respectively.

FIG. 5 shows PCR identification of positive clones of transgenic yeast strains, AXT3-A and ANT3-A respectivelyTaSOS-A1Transformation of AXT3 and ANT3 mutants individuallyCK is the transformation empty vector control.

FIG. 6 shows the results of growth experiments of transgenic yeast in AP and AP-NaCl medium, W303 is wild type, AXT3 and ANT3 represent mutants of transgenic empty vector, and AXT3-A and ANT3-A represent mutants of transgenic empty vector, respectivelyTaSOS-A1AXT3 and ANT3 mutants were transformed separately. Growth of wild type, mutant and transgenic yeasts on AP medium, 10-fold gradient dilution on AP or AP +30 mM NaCl medium, 28 ℃ after 3 days of culture were photographed.

FIG. 7 shows transgenic yeast cell Na⁺、K⁺Concentration and K⁺/Na⁺And (4) the ratio.

FIG. 8 is viral inductionPDSGene silencing phenotype, wherein A is an inoculation control for infecting wheat leaves with buffer solution without BSMV virus; b isPDSBSMV virus of the gene segment infects wheat leaf phenotype.

FIG. 9 is viral inductionTaSOS1-A1Quantitative PCR expression analysis of gene silencing.

FIG. 10 is viral inductionTaSOS1-A1Na from Gene silenced plant leaves⁺、K⁺And (4) concentration.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 wheat plasma Membrane Na⁺/H⁺Reverse transporter geneTaSOS1-A1Obtained by

1. Wheat plasma membrane Na⁺/H⁺Reverse transporter geneTaSOS1-A1The separation specifically comprises the following steps:

(1) mixing semen Tritici aestivi 26Tritium aestivumL.) the seeds were surface-sterilized with 10% queen flower solution for 10 minutes, washed 3 times with sterilized distilled water, placed in a petri dish with two layers of sterilized filter paper, after 2 days at room temperature (90% seeds germinated), the petri dish was placed in a refrigerator at 4 ℃ for 24 hours to homogenize the germinated seeds, and then the petri dish was grown in a culture room (23 ℃, 16 hours of light/8 hours of dark) for 2 hoursOn day, the seedlings with consistent growth were hydroponically cultured in a container containing 1/2Hogland nutrient solution, and after about 7 days, the leaves of the seedlings were washed, blotted dry, quick-frozen with liquid nitrogen, and stored in a refrigerator at-80 ℃ for total RNA extraction.

(2) Total RNA of the wheat leaves preserved in the step (1) was extracted by Trizol method (available from Tiangen Co., Ltd.) and treated with DNase I for future use.

(3) The total RNA from step (2) was reverse transcribed into cDNA using random primers (TAKARA; product of Promega), and MM L V reverse transcriptase (product of Promega). The reaction system and parameters were 2. mu.g of total RNA, 1.25. mu.l of random primers (10. mu.M), 5 × MM L V RT Buffer 5. mu.l, 1.25. mu.l of dNTPs (10 mM each), 1. mu.l of RNase Inhibitor (40U/. mu.l), 1. mu.l of MM L V reverse transcriptase (200U/. mu.l), 25. mu.l of DEPC water, flash centrifugation, 37 ℃ for 60 min, 70 ℃ for 15 min to inactivate the reverse transcriptase.

(4) According toAtSOS1The amino acid sequence of (A) is used for carrying out TB L ASTN search on the EST database of wheat on an NCBI website, and the method of electronic cloning is used for predicting the wheatTaSOS1Designing gene specific primers with the primer sequences shown as SEQ ID No.2 (forward primer) and SEQ ID No.3 (reverse primer), and amplifying by PCR (polymerase chain reaction) with the cDNA obtained in the step (3) as a template to obtain the geneTaSOS1The PCR reaction system is reverse transcription product 2 mul, DNA polymerase buffer (10 ×, MgCl-containing)₂) 2 mul, 2 mul dNTPs (2.5 mM), 1 mul forward primer (10 muM), 1 mul reverse primer (10 muM), 0.2 mul ExTaq polymerase (Takara, 5U/mul), and sterilizing ultrapure water to 20 mul. And (3) amplification procedure: pre-denaturation at 94 ℃ for 5 min, [ 30 s at 94 ℃, 40s at 58.4 ℃ and 3 min at 72 ℃ for 30 s]× 35 cycles, filling in 10 min at 72 ℃, cloning into T vector (product of Promega company), selecting positive clone, sending to Beijing Nosai gene company for sequencing, obtaining nucleotide sequence shown as SEQ ID No.1, wherein the sequence length is 3429 bp, and encodes protein of 1143 amino acid residues.

2. Wheat plasma membrane Na⁺/H⁺Reverse transporter geneTaSOS1-A1Chromosome mapping of

(1) The method for extracting genome DNA of an edentulous tetrasoma plant in Chinese spring comprises the steps of preparing three storage solutions, namely, 0.35M Sorbitol (31.9 g Sorbitol), 0.1M Tris HCl pH 8.0 (50 ml 1M Tris HCl pH 8.0), 5 mM EDTA pH 8.0 (5 ml 0.5M EDTA pH 8.0), sterilized ultrapure water to 500ml, 0.2M Tris HCl pH 8.0 (100 ml 1M Tris HCl pH 8.0), 0.05M EDTA pH 8.0 (50 ml 0.5M EDTA pH 8.0), 2M NaCl (200 ml 5M NaCl), 2% CTAB (10 g CTAB), sterilized ultrapure water to 500ml, III, sarcosstock 5% (w/v), working solution, preparing the formula (120 ml) by adding 0.6 g Sorbiumdimethyl phosphate buffer (10 g sodium sulfate) to 500ml, adding a fresh water phase, adding 10 ml ethanol, adding 10 ml, adding a gel, adding 10 ml, adding 10 g sodium chloride, precipitating, adding 10 g agarose gel, adding 10 ml, adding a gel, a.

(2) Performing PCR amplification by taking genomic DNA of the material of the four bodies of the Chinese spring scarce body as a template, wherein the PCR reaction system is 2 mul of genomic DNA (50 ng/mul) and 2 mul of DNA polymerase buffer (10 ×, containing MgCl)₂) 2 μ l, dNTPs (2.5 mM) 2 μ l, forward primer 5 'AGTGCATCATTGCTGTCTTCAACC 3' (10 μ M) 1 μ l, reverse primer 5 'AGTCGTCATCTTCTCCTACCGCC 3' (10 μ M) 1 μ l, ExTaq polymerase (Takara, 5U/μ l) 0.2 μ l, and sterilizing ultrapure water to 20 μ l. And (3) amplification procedure: pre-denaturation at 94 ℃ for 5 min, [ 30 s at 94 ℃, 40s at 66.4 ℃ and 30 s at 72 ℃]× 30 cycles, filling in at 72 ℃ for 10 min, and detecting by agarose electrophoresis, wherein the electrophoresis result is shown in figure 1,TaSOS1-A1mapped to group A chromosomes of the third homologous group of wheat.

Example 2 wheat plasma Membrane Na⁺/H⁺Reverse transporter geneTaSOS1-A1Function of (2)

1. Construction of dicotyledon binary expression vector

Will be provided withTaSOS1-A1The upstream primer of the gene was added with XbaI cleavage site 5 '-GATCTAGAATGGAGACGGAGGAGGCCG-3' and the downstream primer was 5 '-TCAGCTGCCTCGCGGTGG-3'), to generateTaSOS1- A1The full-length T vector plasmid is used as a template for PCR amplification. The PCR product was recovered, ligated to pGEM-T Vector and sequenced. Digesting the plasmid TaSOS1-A1-T with correct sequencing and the binary expression vector pCAMBIA 1300-35S-GUS (p 1300-GUS for short) plasmid by Xba I and Sac I, recovering corresponding fragments, and connecting and transforming; and (3) identifying colony PCR as an extracted plasmid of a positive clone, further performing enzyme digestion identification, and storing the correctly identified plasmid p 1300-TaSOS 1-A1 for later use. The construction process of dicotyledonous plant binary expression vector is shown in figure 2.

2. Double expression vector for transforming agrobacterium of dicotyledon

(1) Preparation of Agrobacterium-infected competent cells

Selecting Agrobacterium tumefaciens GV3101 single colony, inoculating into 3 ml fresh YEB liquid culture medium, shake culturing at 28 deg.C overnight, inoculating into 50 ml YEB (rifampicin Rif 60 mg/L) culture medium at a ratio of 1: 10, expanding culturing at 28 deg.C until OD₆₀₀=0.4-0.6, placing the bacterial liquid on ice, centrifuging at 5,000 rpm and 4 ℃ for 5 min, discarding the supernatant, suspending the thallus in 10 ml of 0.15 mol/L NaCl at 5,000 rpm and centrifuging at 4 ℃ for 5 min, discarding the supernatant, and using 1 ml of precooled 20 mmol/L CaCl₂Gently suspending cells at (4 ℃) and 200 mu l per tube, adding sterile glycerol with the final concentration of 20%, quickly freezing for 1 minute in liquid nitrogen, and storing at-70 ℃ for later use.

(2) Transformation of Agrobacterium

Mu.l of the constructed plasmid DNA (p 1300-TaSOS 1-A1) was added to 200. mu.l of Agrobacterium competent cells, mixed well, ice-cooled for 30 min, rapidly frozen with liquid nitrogen for 3-5 min, water-washed at 37 ℃ for 5 min, added to 1 ml of YEB medium at 28 ℃ and 150 rpm for 4 h, 10,000 rpm for 30 s, the supernatant was discarded, the cells were resuspended, plated on YEB (Kan 50 mg/L and Rif 60 mg/L) medium and cultured at 28 ℃ for 48 h.

(3) Identification of recombinant Agrobacterium

The single colonies grown on the transformation plates were picked, inoculated into YEB (Kan 50 mg/L and Rif 60 mg/L) liquid medium, and subjected to shaking culture at 28 ℃ overnight, 2 μ l of overnight culture solution was taken as a template for PCR, and the upstream and downstream primers were 5 '-GATCTAGAATGGAGACGGAGGAGGCCG-3', 5 '-TCAGCTGCCTCGCGGTGG-3', respectively.

The PCR reaction system (20. mu.l) is 2. mu.l of bacterial liquid, DNA polymerase buffer (10 ×, MgCl-containing)₂) 2 mul, 2 mul dNTPs (2.5 mM), 1 mul forward primer (10 muM), 1 mul reverse primer (10 muM), 0.2 mul ExTaq polymerase (Takara, 5U/mul), and sterilizing ultrapure water to 20 mul. And (3) amplification procedure: pre-denaturation at 94 ℃ for 5 min, [ 30 s at 94 ℃, 40s at 58.4 ℃ and 3 min at 72 ℃ for 30 s]× 35 cycles, and fill-in at 72 ℃ for 10 min.

3. Transformation and selection of Arabidopsis thaliana

(1) Cultivation of Arabidopsis plants

Firstly, 100 mul of arabidopsis wild type seeds and mutant seeds are respectively put into a 2 ml sterile centrifuge tube, 1 ml of sterile water is added, the surface is soaked for 2 min, and the supernatant is sucked and discarded. Adding 1 ml of 70% ethanol, resuspending the seeds, sterilizing the surface for 1min, carefully removing the ethanol, adding 1 ml of sterile water, resuspending the seeds, and removing the supernatant (washing with water was repeated 3 times). Adding 1 ml of 20% queen bee bleaching water, resuspending the seeds, deeply sterilizing for 10 min, centrifuging at 10000 rpm for 1min, carefully removing the queen bee bleaching water, resuspending the seeds with sterile water, and washing for more than 3 times. Resuspending the seeds with sterile water, transferring to MS culture medium with a pipettor, uniformly spreading, and vernalizing at 4 deg.C for 3 d; the cells were transferred to a culture chamber and cultured under 16 h/8 h (light/dark) conditions at 23 ℃. After 10 days, when 2-4 true leaves grow out from the seedlings, transplanting the seedlings into pots (vermiculite: nutrient soil: 2: 1) for continuous culture under the culture conditions that: culturing at 22-23 deg.C for 16 h/8 h (light/dark). Arabidopsis thaliana grows for about 2 weeks and then enters the flowering phase, and when 1-2 siliques are formed after the Arabidopsis thaliana is bolting to 10-15 cm, the Arabidopsis thaliana can be used for transformation, and the bud state is optimal. Before transformation, the siliques and the completely opened flower buds are cut off, and only the newly exposed and tender flower buds are left.

(2) Agrobacterium culture

Inoculating single colony in 5 ml YEB (Kan 50 mg/L and Rif 60 mg/L) culture medium, culturing overnight, inoculating in 200ml YEB conical flask at a ratio of 1: 1000, culturing for 10-12 hr, and allowing the bacterial liquid to grow to OD₆₀₀At a value of about 0.8, the cells were collected by centrifugation at 5000 rpm for 15 min, and the cells were resuspended in OD (OD = 5.7) using a transformation medium (1/2 × MS, 5% sucrose, 0.01 mg/ml 6-BA, 0.03% Silrwet-77, KOH pH = 5.7)₆₀₀Reaching about 0.8.

(3) Agrobacterium-mediated transformation of Arabidopsis thaliana

Arabidopsis thaliana was transformed by the floral dip method (Clough SJ, 1998): pouring a transformation medium containing agrobacterium into a 200ml beaker, inverting the arabidopsis thaliana with flower buds thereon, enabling the whole inflorescence to enter the transformation medium, soaking for 30 s, taking out the arabidopsis thaliana, shaking off excessive water beads on leaves, stems and flowers, laying on the side, putting in a clean plastic basin, and covering with a film, keeping the humidity and culturing for 24 h in a dark place. The film was uncovered, and the Arabidopsis thaliana was placed under light, watered thoroughly, and cultured normally. After the transformed plants grow normally, blossom and bear fruits, and after 2-3 weeks, seeds can be harvested when the siliques are completely withered and yellow and are about to crack. The seeds are stored in 2 ml centrifuge tubes and stored for a short time at normal temperature or for a long time at 4 ℃ and-20 ℃.

(4) Arabidopsis transgenic plant screening

The transformed seeds were sterilized, sown on MS medium containing Hyg 20. mu.g/ml, vernalized at 4 ℃ for 3 days, and then cultured under normal light. Arabidopsis seedlings transformed with the foreign gene were identified about 10 days later (T1 generation). Because the binary vector has Hyg resistant locus, the resistant seedling cotyledon is green, the hypocotyl is longer, and 4 true leaves grow; whereas the non-transformants were not resistant, only 2 true leaves. Transplanting resistant plants, continuously culturing and collecting seeds of T2 generations. Seeds of T2 generation were sown in a culture medium containing Hyg 20. mu.g/ml and screening was continued, and it was possible to determine whether the progeny were homozygote and the copy number of the foreign gene insertion based on the segregation ratio of the progeny (T2 generation). Selecting transgenic plants with single gene insertion for transplanting, collecting seeds (T3 generation) from the single plants, and continuously screening on a culture medium containing Hyg20 mug/ml, wherein the progeny which is not separated (all are green seedlings) is homozygote.

(5) Identification of transgenic plants of Arabidopsis

And (3) carrying out small-quantity extraction on the screened T1 generation arabidopsis thaliana positive plants according to a conventional method, taking 1 mu l of genomic DNA as a template, carrying out conventional PCR amplification by using a primer pair 5 '-GATCTAGAATGGAGACGGAGGAGGCCG-3' and 5 '-TCAGCTGCCTCGCGGTGG-3', and respectively taking wild type and mutant arabidopsis thaliana genomic DNAs as controls to further detect the positive plants. Meanwhile, RNA is extracted from T3 transgenic plant in small quantity, RT-PCR is carried out to detect the expression condition of the transgene, and wild type and mutant arabidopsis RNA are respectively used as controls. The results are shown in FIG. 3, and most hygromycin-resistant seedlings are transgenic positive plants and can normally express hygromycinTaSOS1-A1A gene.

(6) Phenotypic Observation of transgenic Arabidopsis

Respectively sowing wild type, mutant and transgenic homozygous strains of arabidopsis thaliana in 1/2 MS culture medium, culturing for 4 days, selecting a part of arabidopsis thaliana with consistent growth, transferring the part of arabidopsis thaliana into 1/2 MS culture medium containing salt (mutant 50 mM NaCl, wild type 150 mM NaCl), transferring the part of arabidopsis thaliana into 1/2 MS culture medium without salt as a control, vertically placing a plate, observing the growth of arabidopsis thaliana seedling roots, and observing after about 5 days. As shown in FIG. 4, the transgenic Arabidopsis thaliana showed no difference from the wild type and the mutant in the 1/2 MS medium without salt, and the root of the transgenic Arabidopsis thaliana was longer than that of the wild type in the 1/2 MS medium with 150 mM NaCl and that of the mutant in the 1/2 MS medium with 50 mM NaCl. Shows that the transgenic arabidopsis plants have strong salt tolerance and come from wheatTaSOS1-A1The gene can improve the salt tolerance of Arabidopsis plants.

4.TaSOS1-A1Complementary yeast mutant phenotypes

(1) Yeast strains

Yeast (A)Saccharomyces cerevisiae) Mutant strains required for complementation experiments include AXT3 (MATα, ade2-1,his3-11,leu2-112,trp1-1,ura3-1,Δena1∷HIS3∷ena4,nha1∷LEU2,nhx∷ TRP1,can1-100) Absence of endogenous NHX1 and Na on the plasma membrane⁺Transporters ENA1-4 and NHA 1; ANT3 (MATα, ade2-1,his3-11,leu2-112,trp1-1,ura3-1,Δena1∷HIS3∷ena4, nha1∷LEU2) Absence of Na on the plasma membrane⁺Transporters ENA1-4 and NHA 1. Control line W303: (MATα,ade2-1,his3-11,leu2-112, trp1-1,ura3-1）。

(2) Vector construction

By containingTaSOS1-A1Taking plasmid of exogenous fragment as template, using primer containing enzyme cutting site to make PCR amplification, synthesizing primer: 5 '-GATCTAGAATGGAGACGGAGGAGGCCG-3' (blackbody)XbaICleavage site), 5 '-GAGGTACCTCAGCTGCCTCGCGGTGG-3' (blackbody-type)KpnIEnzyme cutting site), extracting plasmid after the T-linked vector is sequenced correctly, and respectively carrying out double enzyme cutting on the plasmid containing exogenous fragment and the vector pYPGE15 (enzyme cutting site) (XbaI/KpnI) Performing enzyme digestion product electrophoresis, respectively cutting and recovering target bands into gel, connecting pYPGE15 vector, transforming the connecting product into Escherichia coli DH5 α, screening resistance, and extracting plasmidXbaIAndKpnIand (5) carrying out enzyme digestion identification.

(3) Transformed yeast

Taking 1.5 ml of yeast overnight bacterial liquid, centrifuging at 12,000rpm for 10 s, discarding supernatant, washing with 1 ml of water, centrifuging at 10 s, discarding supernatant, preparing 360 mul of transformation reaction liquid (PEG 3350 (50%), 1M L iAc 36 mul, 11 mul of salmon sperm DNA) in advance, and constructing 30 mul and ddH of plasmid vector₂O to 360 mul) is added into the yeast sediment, evenly mixed, washed in water at 42 ℃ for 1 h without shaking, centrifuged for 10 s, the supernatant is discarded, 100 mul of bacterial liquid is taken to be coated on a plate (YNB does not contain uracil), remark is made, the formula of the YNB culture medium, 0.67% yeast nitrogen base (without amino acid, Difco), 2% glucose, 3% agar and pH6.2, adenine (0.05 g/L), tryptophan (0.1 g/L), histidine (0.05 g/L), leucine (0.1 g/L) and pYPGE15 vector contain genes for synthesizing uracil according to the characteristics of the culture strain and the pYPGE15 vector, and no additional bacteria are neededColony PCR identified positive clones as shown in figure 5.

(4) Identification of salt tolerance of yeast

The yeast salt tolerance is identified by using AP (arginine Phosphate Medium) culture medium, and the formula is as follows (1L): PO₄H₃（85%）0.55 ml， MgSO₄（1M）2 ml，CaCl₂(0.1M) 2 ml, Oligoelents (100 ×) 10 ml, KCl to a final concentration of 1 mM, NaCl to the appropriate concentration according to the requirements of the experiment, pH6.5 adjusted with L-argine, sterile water to 900 ml, and 20 g/L agar if solid media are used, autoclaving, cooling to 50 ℃ and adding 100 ml of 20% glucose (0.5 atm, 110 ℃ Sterilization) and 10 ml of 100 × vitamin stock (filter Sterilization), where formulation 1L 100 × Oligoelents requires Boric acid (50 mg), CuSO₄（4 mg），IK（10 mg），FeCl₃（20 mg），MnSO₄·H₂O（40 mg），Na₂MoO₄·2H₂O（20 mg），ZnSO₄·7H₂O (4 mg); 100 ml 100 × Vitamins required Biotin (1 ml, 0.2 mg/ml ethanol), Nicotinic acid (4 mg), Pyridoxine (4 mg), Thia mine (4 mg), Panthoteic acid (4 mg).

Culturing the yeast strain with correct identification in an AP liquid culture medium overnight, adjusting the concentration of the bacterial liquid by measuring OD value, then carrying out a series of gradient dilution according to 10 × times, respectively sucking 10 mul into a solid AP (30 mM NaCl) culture medium by using a pipettor, observing after culturing for 3 days at 28 ℃, photographing, wherein in the AP culture medium without salt, the transgenic yeast has no difference compared with the wild type yeast w303 and the mutant AXT-3, ANT-3, and in the AP culture medium with 30 mM NaCl, the mutant AXT-3, ANT-3 yeast is inhibited from growing, and the transgenic yeast is changed into the wild type yeast w303, the mutant AXT-3, ANT-3TaSOS1-A1The yeast with the gene can well restore mutant phenotype, and the phenotype on the salt plate is close to the wild type.

(5) Determination of ion content of yeast

The correctly identified yeast strains were cultured overnight in AP broth, then grown to 50m L (28 ℃, 200 rpm) in AP broth with 10mM NaCl, and after OD ≈ 0.20, the cells were culturedCold buffer (10 mM NaCl)₂、10 mM CaCl₂And 1 mM HEPES), drying for 48 hours at 65 ℃, weighing, putting into a digestion tank, adding nitric acid and hydrogen peroxide (7: 1, super pure), and digesting in a digestion furnace, wherein the total volume of the digestion tank is not less than 1/4 of the volume of the digestion tank. Transferring the digestion solution into volumetric flask after digestion, adding double distilled water to constant volume of 25 ml, and detecting Na by ICP/MS (Thermo, San Jose, CA, USA)⁺、K⁺Ion content. The results of the detection are shown in FIG. 7TaSOS1-A1Na in the yeast strain of (1)⁺The content is significantly lower than that of the mutant control, about 50% of the control; in contrast K⁺The content is increased by about 40% compared with the controlTaSOS1-A1Of yeast strain K⁺/Na⁺The ratio is about 3 times that of the control, which showsTaSOS1-A1The gene has efflux Na⁺The function of (c).

Viral mediationTaSOS1-A1Gene silencing

(1) Construction of viral vectors

The genome of barley streak mosaic virus contains three different single-stranded RNA molecules, three-stranded cDNA is respectively cloned on pBSMV vector (with Amp resistance), namely pBSMV- α, pBSMV- β and pBSMV-gamma, constructed gamma chain (pBSMV-gamma-GFP) with exogenous GFP fragment is used as control of virus infection and used for constructing vector, gamma chain (pBSMV-gamma-PDS) with exogenous PDS (phytoene dehydrogenase) fragment is used for infecting wheat for several days, the leaf blade can produce photobleaching phenomenon, and can provide evidence for successful virus infection, said vector possesses T7 promoter and transcription origin, and can utilize T7 RNA polymerase to make in vitro transcription of linearized vector to obtain virus RNA, and make RNA transcript inoculated into wheat leaf blade, and said virus RNA can be packaged into active genome in plant body and propagated in plant bodyNheⅠA site for receiving an exogenous target fragment (a fragment of a gene to be silenced), the exogenous fragment being replicable with replication of the virus's own genome, the target gene fragment silencing expression of plant endogenous RNA by an RNAi mechanism.

Design beltNheIPrimer 5' -GCTAGCTCCCTCGGCGACGGCG for cleavage siteAC-3 ', 5' -UTR), 5 '-GCTAGCTCTCGAAGAGGAGGGCGGGG-3' PCR amplifies a fragment of the corresponding gene to be silenced, which is recovered and ligated to PromegaT-vector, transformed, and the positive clones identified, followed by sequencing. After the sequence is determined to be completely correct, useNheIThe target fragment is cut off from the T-vector by the enzyme and simultaneouslyNheIThe enzyme cuts off GFP in the virus vector pBSMV-gamma-GFP, and the target gene fragment and the vector pBSMV-gamma are respectively recovered, connected and transformed. The positive clone inserted in the reverse direction was identified with primer 5 'CAACTGCCAATCGTGAGTAGG 3' for in vitro transcription of the virus and the vector ligated correctly was named pBSMV-gamma-TaSOS 1-A1.

(2) Viral in vitro transcription

Firstly, linearizing the circular vector, comprising the steps of respectively extracting plasmids of pBSMV- α, pBSMV- β, pBSMV-gamma-TaSOS 1-A1, pBSMV-gamma-PDS and pBSMV-gamma-GFP, dividing each plasmid into three tubes with the total amount of 6 mu g, linearizing the circular vector under the conditions of pBSMV- α 2 mu g and 5 mu l of 10 × buffer solution,MluⅠendonuclease 2 μ l (20 units), supplement ddH₂Detecting after 6 hours at 37 ℃ when the temperature is between O and 50 mu l to ensure complete enzyme digestion, and using a pBSMV-gamma-TaSOS 1-A1, a pBSMV-gamma-PDS and a pBSMV-gamma-GFP linearization system as a pBSMV- α and pBSMV- β linearization systemSpeⅠEndonuclease substitutionMluⅠAnd the other steps are the same as the pBSMV- α linearization system, 350 mul of DEPC water, 500 mul of phenol, chloroform (1: 1) are added into 150 mul of enzyme digestion product, the mixture is uniformly mixed, the mixture is centrifuged at 12,000rpm for 10 min, 450 mul of supernatant is carefully sucked into a new EP tube, 45 mul of 3M NaAc (pH5.2) is added, 1 ml of absolute ethyl alcohol is added, the mixture is precipitated at 4 ℃ overnight at 12,000rpm, the mixture is centrifuged for 10 min, 70% ethyl alcohol (prepared by DEPC water) is washed once, and DNA is dissolved in 20 mul of DEPC water.

(3) Viral RNA Synthesis

Using AmpliCAP-Max from EPICENTRE^TMViral RNA was synthesized by T7 and T3 High Yield Message MakerKits, following the instructions, 1.5. mu.g linearized fragment, 2. mu.l 10 × Transcriptionbuffer, 8. mu.l Cap/NTP PreMix, 2. mu.l 100 mM DTT, 2. mu.l AmpliCap-Max T7 enzymeSolution, and supplemented with water to 20. mu.l, reacted at 42 ℃ for 2.5 h, and stored at-80 ℃ for further use.

(4) Inoculation of viruses

The synthesized α, β and gamma RNA containing the target fragment were mixed by 20 μ l each, diluted with 3-fold volume (180 μ l) of DEPC water, and then added with 2 × GKP buffer (50 mM Glycine, 30 mM K) of the same volume (240 μ l)₂HPO₄(pH 9.2), 1% Bentonite, 1% Celite), which can be mixed for inoculation. In the two-leaf period of wheat, when the second leaf is flat and the third leaf is not grown, 8-10 mu l of transcription mixed liquor is smeared on the second leaf by using sterile gloves. And (3) the inoculation temperature is 18-20 ℃, after inoculation, DEPC water is used for spraying the wheat leaves, a preservative film is covered, the wheat is thoroughly watered, the moisture is kept for 24 hours, and then normal culture is carried out. The same number of wheat seedlings were inoculated with diluted GKP buffer as mock inoculation controls (mockccontrols) in the same manner as described above.

(4) Realtime QT-PCR analysis of Gene expression

When the control infected with the virus showed symptoms (about 20 days), i.e., the 3 rd and 4 th leaves of wheat infected with BSMV: TaPDS recombinant virus showed a photo-bleaching phenomenon, while the inoculated control did not show this phenomenon, indicating that the virus infection was successful as shown in FIG. 8. Respectively extracting control and virus infected wheat leaf RNA, reverse transcribing into cDNA, and detecting by fluorescent quantitative PCR methodTaSOS1-A1Whether or not gene silencing has occurred. As shown in FIG. 9, the results were successful in the BSMV TaSOS1-A1 virus-infected wheatTaSOS1-A1Is down-regulated by 60%.

(5) Determination of ion concentration

When symptoms appeared in the virus-infected controls (about 20 days), 200 mM NaCl was added for 24 hours, the leaves were removed, oven-dried, digested and then assayed for Na by ICP/MS (Thermo, San Jose, Calif., USA)⁺、K⁺Ion content. The results are shown in FIG. 10 for BSMV Na accumulated in TaSOS1-A1 infected wheat leaves⁺More than 2 times of BSMV, GFP inoculation control, K⁺The content did not change significantly.

<110> Shijiazhuang city farm and forestry science research institute

<120>Na⁺/H⁺Reverse transporter gene and application thereof

<160>3

<210>1

<211>3460

<212>DNA

<213>Triticum, triticum aestivumTriticum aestivumL.）

<400>1

1 CREATEDFEA TURESLCATI NQUALIFIER SATGGAGACG GAGGAGGCCG GCTCCCCCAG

61 CCCCGACGAC GCGGTGCTCT TCTTCGGGGT GGCCCTCGTG CTGGGCATCG CCTCCCGCCA

121 CCTCCTCCGC GGCACCCGCG TCCCCTACAC CGTCGCCCTC CTTGTCCTCG GCGTCGCCCT

181 CGGCGGCCTA GAGTACGGGA CCAAGCATGG CCTGGGCAAG CTCGGAGCCG GCATCCGTAT

241 CTGGTCTGCC ATAAATCCTG ATCTCCTTCT GGCCGTCTTC CTCCCCGCCC TCCTCTTCGA

301 GAGCTCCTTC TCCATGGAAG TGCACCAGAT CAAGAAATGC ATGGCGCAGA TGGTGTTGCT

361 TGCTGTCCCA GGCGTGGTGA TCTCAACAGT TTTGCTTGGC GCCGCTGTAA AGCTCACTTT

421 TCCCTATGAC TGGAACTGGA AAACATCATT CTTGTTCAGT GGACTGCTTA GTGCAACCGA

481 CCCTGTTGCT GTGGTTGCTC TTCTCAAAGA CCTAGGAGCA AGCAAAAAGC TCAGTACAAT

541 AATTGAAGGA GAGTCCTTGA TGAATGACGG GACTGCTATT GTTGTCTATC AGCTATTCTA

601 TCGAATGGTG CTTGGAAAAA CTTTCGATGC AGGGTCCATC ATAAAGTTCT TGTCACAAGT

661 TTCACTTGGA GCTGTTGCTC TGGGCCTTGC GTTTGGAATT GCATCAGTAC TATGGCTGGG

721 ATTTATTTTC AATGATACAA TCATAGAGAT TTCACTTACC CTTGCTGTCA GCTATATTGC

781 TTTCTTCACT GCGCAAGATG CATTGGAGGT CTCTGGTGTT TTAGCCGTCA TGACCTTGGG

841 GATGTTCTAT GCTGCTTTTG CAAAAACTGC TTTTAAGGGT GACAGCCAGC AAAGTTTACA

901 TCATTTCTGG GAAATGGTTG CTTACATTGC AAACACACTC ATTTTCATAC TGAGTGGGGT

961 TGTTATTGCA GATGGTGTAC TACAAGATAA TATTCATTTT GAGAGGCATG GCACATCATG

1021 GGGGTTCCTT GTTCTGCTCT ATGTTTTTGT GCAAATATCG CGTGCTGTAG TTGTCGGTGT

1081 TTTGTATCCA CTGTTGCGTC ACTTTGGGTA TGGTATGGAC ATCAAAGAAG CCACAGTTCT

1141 TGTTTGGTCA GGACTGCGAG GAGCTGTTGC TCTATCACTC TCTCTGTCCG TTAAACGTGC

1201 TAGTGATTCA GTTCAAACTT ATCTGAAACC AGAAGTTGGA ACAATGTTTG TGTTCTTCAC

1261 AGGTGGCATT GTGTTTCTGA CATTGATTTT GAATGGTTCT ACCACACAAT TTTTGTTGCA

1321 CCTACTTGGT CTGGGAAAAT TGTCAGCAAC GAAGCTTCGT GTATTGAAGT ATACACAATA

1381 TGAAATGCTA AACAAGGCAT TGGAGGCTTT TGGTGATCTC AGGGATGATG AGGAACTTGG

1441 GCCTGTTGAT TGGGTTAATG TGAAGAAATA TATCACATGT TTGAATAACT TGGAAGATGA

1501 ACAAGCACAT CCCCACGATG TTCCTGACAA GGATGATCAC ATACATACCA TGAATTTGAA

1561 GGATACTCGA GTGCGGCTTT TGAATGGTGT GCAAGCTGCT TACTGGGGAA TGCTTGAAGA

1621 GGGACGAATA ACTCAATCTA CAGCAAATAT TTTAATGAGA TCAGTTGATG AAGCTATGGA

1681 TCTTGTCTCT AGTCAATCAT TATGTGATTG GAAGGGTTTG CGGTCCAATG TCCATTTCCC

1741 AAATTACTAT AGGTTCCTTC AGATGAGCAG GTTGCCAAGA AGGCTTGTCA CATACTTCAC

1801 AGTAGAAAGA TTGGAGTTAG GATGTTACAT CTGTGCGGCA TTTCTTCGCG CTCATAGAAT

1861 TGCGAGGAGA CAACTACATG ATTTTCTTGG TGATAGTGAG ATTGCAAGAA TTGTCATCGA

1921 TGAAAGCACT GCTGTGGGGG AGGAAGCTAA AAAGTTTCTG GAAGATGTTC GTGTTACATT

1981 CCCTCAGGTG CTTCGTGCAT TAAAGACTCG ACAAGTAACA TATGCAGTAT TGACACACTT

2041 GAGTGAGTAT ATTCAAGACC TCGGGAAGAC TGGGTTGCTC GAGGAAAAAG AAATAGTCCA

2101 TCTCGATGAT GCTTTGCAGA CAGACTTGAA GAAGCTTCAG AGGAATCCAC CGCTGGTGAA

2161 AATGCCAAGA GTTCGTGAAC TTCTAAACAC TCATCCTTTA GTCGGTGCAC TGTCTGCTGA

2221 TGTTCGTGAT CCATCGTTAA GTAATACAAA GGAAACAATA AAAGTTCATG GAACAATTCT

2281 ATACAGAGAA GGCTCAAGGC GGACTGGTAT ATGGCTTGTT TCGACTGGAA TAGTAAAGTG

2341 GACAAGTCGG AGACTATGCA CCAGGCATTC GTTGGATCCA ATTTTGTCAC ATGGAAGCAC

2401 TTTGGGTCTA TATGAGGCAT TAACTGGAAA GCCTTATATT TGTGACATTA TTACAGAATC

2461 GGTGGTGCAT TGTTTCTTCG TTGAAGCTGA AAAAATAGAG CAATTGCGCC AGTCTGATCC

2521 TTCTATTGAG GATTTTATGT GGCAGGAAAG TGCTCTAGTC ATTGCAAGGA TTTTGCTCCC

2581 TCAGATATTT GAGAAAATGG CAATGCGTGA GATGAGGGTT CTCATTTCAG AAAGGTCTAG

2641 TATGAATGTC TACATTAAGG GGGAAGCCAT TGAGCTTGGG CATAATAACG TAGGCATCTT

2701 ATTGGAAGGA TTTCTGAAGA CAGAGAACCG AACTTTGATC ACAGCTCCAG CTGTGCTGCT

2761 GCCGTCAAAC ACTGATTTGA ACTTATTTGG CCTGCAGTCT TCAGCCATGA ATCAGATAGA

2821 CTACTGCTAT ACTGCTCCCA GTTATCAGGT GGAGGCTAGA GCAAGGGCCA CCATATTTGA

2881 AATAGGTAGC CTAGATATAG AAGCCGATCT GCAAAGAAGT GCATCATTGC TGTCTTCAAC

2941 CCTCGGACCA TCACGAACAC AGAGCAAAGA GCATGTCGGT TTGCTCAGGT GGCCGGAGAG

3001 TTTCCGGAGA TCCAGCGGGC CTGGGAATGC AAGCCTAGCT GAAATCAGAA GTCAGCCTGG

3061 TAACTTCTCT GCTAGAGCCT TGCAAGTCAG CATGTATGGC AGCATGACGG ATGGCATGCA

3121 CCGTGCTCGG CGGCAACCGA GGCTTGCTCA TGTGGAAGGA AACCAGAAGC ACAGCGTGTC

3181 GTATCCAAAG GTGCCTTCAA GGGCAGCCGA CACGCGGCCT CTGTTGTCGG TGAGGTCGGA

3241 GGGCTCCAAT GCGATGAAGA GAAAATCTGC TCCTGCCCCA GCTATAGCTC CTGCACTCGC

3301 TCCCTTTCCG CCGCCCCTAG CAGAAGGCCG GCAACGCAGG GCGGTAGGAG AAGATGACGA

3361 CTCGAGCGAT GAGTCCGTGG GGGAAGAAGT CATCGTCAGA GTGGACTCTC CCAGCATGCT

3421 CTCGTTCAAT CCACCCTCCG GCCCACCGCG AGGCAGCTGA

<210>2

<211>21

<212>DNA

<400>2

GCAGTACCGT CCCTTCTCTT C

<210>3

<211>19

<212>DNA

<400>3

TACTGCAACC GTCAGCTGC

Claims (8)

1. Plant Na⁺/H⁺An inverse transporter gene characterized by: the nucleotide sequence of the gene is shown in SEQ ID No. 1.

2. An expression vector comprising the gene of claim 1.

3. The expression vector of claim 2, which is a yeast expression vector.

4. The expression vector of claim 2, which is a viral expression vector.

5. The expression vector of claim 3 or 4, wherein: the gene of claim 1 operably linked to a promoter or transcription origin.

6. The expression vector of claim 5, wherein: the gene of claim 1 operably linked to a 35S promoter.

7. The gene of claim 1 exerts Na in plants⁺/H⁺Exchange function of Na⁺Use for discharge to the extracellular space, characterized in that: transforming the gene of claim 1 into wheat and allowing expression; or transforming the gene of claim 1 into a yeast mutant to obtain a yeast strain complementing the yeast mutant.

8. Use according to claim 7, characterized in that: in improving the Na content of the leaf⁺The application in the breeding of plants with high content or the application in the breeding of salt-tolerant plants.

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