WO2010060270A1 - 一种新的植物强耐盐基因nhxfs1及其编码蛋白和应用 - Google Patents

一种新的植物强耐盐基因nhxfs1及其编码蛋白和应用 Download PDF

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WO2010060270A1
WO2010060270A1 PCT/CN2009/001278 CN2009001278W WO2010060270A1 WO 2010060270 A1 WO2010060270 A1 WO 2010060270A1 CN 2009001278 W CN2009001278 W CN 2009001278W WO 2010060270 A1 WO2010060270 A1 WO 2010060270A1
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gene
salt
nhxfs1
plant
seq
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夏涛
张辉
徐凯
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华东师范大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to the field of plant genetic engineering, in particular to the use of a DNA family shuffling technique to obtain a gene of a plant Na+/H + antiporter protein with significantly improved function.
  • the present invention also relates to the use of the gene to grow a transgenic salt-tolerant plant.
  • Soil salinization is an important abiotic stress factor affecting crop production and ecological environment.
  • the permeability of the plasma membrane of plant cells subjected to osmotic stress and ion toxicity increases, causing secondary damage such as oxidative stress, nutritional deficiency and a series of physiological metabolic reactions, thus causing serious damage to the normal growth and development of plants. .
  • Plant Cell. 1995, 7 : 1099-1111 mainly includes two strategies for osmotic adjustment and ion balance of reconstructed cells.
  • the former refers to plant cells using some inorganic ions or synthesizing some small molecular organic compounds as osmotic regulators to reduce the cell water potential and resist osmotic stress.
  • the latter mainly refers to the purpose of plant cells to achieve ion balance and eliminate Na+ toxicity by transporting excess Na+ in the cytoplasm to cells or compartmentalizing into vacuoles.
  • a striking feature of plant cells is that they have a large membrane-isolated vacuole.
  • the plant Na7H + antiporter is a Na+ antiporter, which is distributed on both the plant plasma membrane and the tonoplast.
  • the tonoplast Na7H+ antiporter is the main protein responsible for the compartmentalization of Na + into vacuoles.
  • the plant Na7H+ transporter was first found on the plasma membrane of barley, and the activity of Na7H + transport was generally detected on various plant plasma membranes and tonoplasts. Since the world's first plant Na7H+ antiporter gene i/Vm has been cloned (Gaxiola RA, Rao R, Sherman A, Grisaf i P, Alper S, and Fink G R.
  • the Arabidopsis thai i ana proton transporters can function in cation detoxify cation in yeast. Proc Natl Acad Sci USA. 1999 , 96 : 1480 ⁇ 1485), the tonoplast Na + /H+ antiporter genes of higher plants such as rice, Beibin, and Suaeda were successively cloned. , respectively (3 ⁇ 4 ⁇ 3 ⁇ 4 ⁇ (Fukuda A, Nakamura A, Tanaka Y. Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. Biochim Biophys Acta.
  • the Na+/H+ antiporter located in the tonoplast membrane separates Na+ into Into the vacuole, thereby reducing the concentration of Na+ in the cytoplasm, causing excess Na + to leave the metabolic site, reducing its damage to the enzyme and membrane system, and also reducing the cell water potential and resisting the osmotic stress caused by salt. Therefore, the Na7H+ antiporter plays an important role in the salt tolerance of plants.
  • NaVH+antiporter NaVH+antiporter
  • Overexpression of the NaVH+antiporter (NaVH+antiporter) gene into a target plant for overexpression is an effective way to develop transgenic salt tolerant plants.
  • Apse et al. analyzed Arabidopsis thaliana plants transfected with A-myelin XI gene.
  • Transgenic Arabidopsis plants overexpressing Na+/H+ antiporter could grow normally in 200 mmol/L NaCl.
  • the Na7H+ antiporter gene that has been cloned in plants still has low activity and low salt tolerance, which greatly limits the production and promotion of the gene.
  • DNA shuffling technology is a fast and efficient method for in vitro directed evolution of proteins, enzymes and monoclonal antibodies. It has broad application prospects in improving enzyme activity, protein yield and improving protein (enzyme) performance.
  • the principle is to randomly fragment a set of closely related nucleic acid sequences, and then reassemble these random fragments by primerless PCR to obtain a full-length nucleic acid sequence, in which mutations are introduced and extensive recombination of different mutations is performed. , thereby completing the rapid evolution of the nucleic acid sequence of interest to enhance the function of the nucleic acid sequence or the protein encoded thereby (Stemmer W P. DNA shuffling by random fragmentation and reassembly : in vitro recombination for molecular Evolution.
  • DNA shuffling can be divided into single gene reorganization and gene family reorganization. The former only performs fragmentation of individual genes and reassembles by primerless PCR. Family reorganization can reorganize gene families with similar or closely related genes. .
  • the present invention aims to obtain a novel plant salt-tolerant gene NHXFS1 which encodes a Na + /H+ antiporter having ion transport activity.
  • Another object of the present invention is to provide a Na+/H + antiporter NHXFS1 encoded by the above gene, which has a stronger ion transport activity than a wild type NaVH+reporter.
  • the present invention also provides a recombinant vector containing the above gene.
  • a host cell comprising the recombinant plasmid described above is also provided.
  • a method of constructing a recombinant vector, a transgenic plant, or the like is further provided to apply the above-mentioned gene and the encoded protein to cultivate a new transgenic plant variety having higher salt tolerance.
  • the Na+/H+ antiporter (Na7H + antiporter) plays an important role in plant salt tolerance, and its activity affects the salt tolerance of plants.
  • the invention adopts DNA family shuffling technology to carry out in vitro molecular evolution of wild type salt-tolerant genes Na1 ⁇ 23 ⁇ 4 ⁇ and OSNHX ⁇ 3 ⁇ 4 ⁇ encoding Na+/H + antiporter to obtain a novel Na + /H+ antiporter gene encoding higher ion transport activity.
  • the Na7H + antiporter NHXFS1 encoded by this new gene can partition more Na + from the cytoplasm into the vacuole, and transform the obtained new gene into the target plant, which can produce more salt-tolerant ability.
  • the wild-type Na7H + antiporter gene was mutated and recombined by in vitro DNA shuffling technology, and the Na7H + with significantly enhanced ion transport activity was screened by a functional complementation method in a yeast mutant strain in which the Na7H + antiporter gene was deleted.
  • a new gene for reverse transporter mutations was screened by a functional complementation method in a yeast mutant strain in which the Na7H + antiporter gene was deleted.
  • the present invention relates to a novel plant salt-tolerant gene, ⁇ 3 ⁇ 47, which encodes the Na + /H+ antiporter NHXFS1, which is capable of improving the salt tolerance of plants, and is one of the following nucleotide sequences:
  • a preferred plant strong salt tolerance gene, NHXFS1 has a nucleotide sequence of SEQ ID NO: 1, and a mutation of 4 nucleotides compared to the wild type Na7H + antiporter gene (3 ⁇ 4 ⁇ 3 ⁇ 4 ⁇ , wherein the 185th position And 1059 are adenine, and 1221 and 1517 are guanine.
  • the encoded protein of the above gene is an ion transporting NaVH + antiporter NHXFS1, which is characterized by one of the following amino acid sequences:
  • Preferred amino acid sequence is SEQ ID NO: 2, and rice wild type Na7H + antiporter There are two amino acid changes between OsNHXl, of which 62 is aspartic acid, 506 is glycine, and the amino acid sequence homology with SEQ ID NO: 2 is between 70-100%.
  • the protein also includes a protein which increases, decreases or replaces one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2.
  • Cloning of NaVH + antiporter 3 ⁇ 4 factor The full length sequence of the Na + /H + antiporter nucleotide or a fragment thereof can be obtained by various methods such as PCR amplification, recombinant method or artificial synthesis.
  • a polynucleotide probe based on a Na + /H+ antiporter gene sequence is designed to screen a cDNA library or a genomic library for a gene of interest, and a PCR amplification method can also be used to directly amplify a sequence from a cDNA or a genome.
  • DNA shuffling of the NaVH + antiporter gene The substrate used for DNA shuffling may be the Na7H+ antiporter gene of different individuals, strains or species of the organism, or may be a conventional method such as error-prone PCR or site-directed mutagenesis. Different mutant forms of the NaVH+ antiporter gene were prepared.
  • the main steps for DNA shuffling of the NaVH + antiporter gene are: First, the NaVH + antiporter gene is randomly fragmented with DNase I or a restriction enzyme, and the small fragment, ie, the overlapping fragment, is recovered. Primerless PCR was then performed with DNA polymerase without primers to allow small fragments to be amplified by primers and templates, resulting in extensive recombination of the different mutations. Finally, a recombinant gene library containing the recombinant Na+/H+ antiporter gene fragment was obtained by Primer PCR amplification.
  • a recombinant gene bank containing a recombinant NaVlT antiporter gene fragment can be transformed into a cell or organism such as a bacterial, yeast or plant cell for screening.
  • the recombinant gene library obtained by the present invention can be ligated into any yeast expression vector such as PYPGE15, and transformed into a yeast Na7H+reporter mutant such as W303Aenal-4Anhxl by a method known in the art such as lithium acetate technique, electroporation or the like.
  • Yeast transformants were plated in APG-selective plates containing high NaCl concentrations. High-throughput screening was performed with high salt as the selection pressure to obtain mutants with significantly improved salt tolerance.
  • the plasmid was extracted from the yeast mutant strain with significantly improved salt tolerance, and the nucleotide sequence of the shuffled high salt-resistant Na + /H+ antiporter gene was obtained by sequencing, using conventional biological software such as DNA Star. , Clustalx and TMpred for bioinformatics analysis of nucleotide sequence alignment, amino acid sequence alignment and hydrophilic hydrophobicity analysis of high salt-tolerant Na+/H+ antiporter gene and wild-type Na7H + antiporter gene. The results showed that the Na7H + reverse transporter NHXFS1 was not reorganized. The Na7H + antiporter has stronger ion transport activity, and can partition more Na + from the cytoplasm into the vacuole, so that the host cells introduced into the gene have better salt tolerance.
  • the Na7H + antiporter gene/1 ⁇ 2 3 ⁇ 473 ⁇ 47 can be constructed as a gene of interest in any plant-expressed Ti-plasmid binary vector such as pBISN1, which has a T_DNA 25 bp repeat of LB and RB, and a mannopine synthase promoter ( Pmas), polyA nos terminator, and neomycin phosphotransferase II (nptll) used as a selection marker in eukaryotes.
  • the antibiotic used for selection is kanamycin.
  • the promoter in the plant expression vector can be any constitutive promoter, tissue-specific promoter or environmentally inducible promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter, the Ubiqutin promoter.
  • the enhancer in the vector can be either a transcriptional enhancer or a translational enhancer.
  • the vector should also have a plant selectable marker (GUS gene, luciferase gene, etc.) or a marker with antibiotic resistance (such as kanamycin, weeding). Agent, etc.).
  • the recombinant vector is introduced into Agrobacterium by a research method known in the art such as a freeze-thaw method, an electric shock method or the like to perform Agrobacterium transformation.
  • the new strong salt-tolerant Na+/H + antiporter gene NHXFS1 can be transferred into plants by conventional biotechnological methods such as microinjection, gene gun, Agrobacterium-mediated or pollen tube pathway, to produce salt-tolerant quality and organisms.
  • the transformed plant host can be either a monocot or a dicot, such as: Arabidopsis thaliana, tobacco, tomato, canola, rice, wheat, corn, soybeans, vegetables, trees, flowers, pasture, and turfgrass.
  • the gene of the present invention is of great significance for cultivating salt-tolerant plant varieties, increasing crop yield, and improving ecological environment.
  • the transgenic recombinant vector of the present invention can be used as a commercial product variety, strain or as a genetic resource for direct production or for agricultural biological breeding and transgenic plants to enhance the salt tolerance of the crop.
  • Figure 1 is a comparison of the functional complementation of the shuffled NHXFS1 gene with the unshuffled AtNHX1 and OsNHX DmNHX1 genes in the yeast double mutant strain W303-IB A enal-4 A nhxl, ie, the following cells at pH 5.2, containing 0, 100 mM, 200 mM Growth on APG plates at 250 mM, 300 mM, and 350 mM NaCl:
  • I is wild type W303-1B containing PYPGE15 plasmid
  • II is W303-1B Aenal-4: HIS3 Anhxl: :TRP1 containing PYPGE15 plasmid
  • III is W303-1B ⁇ enal-2::HIS3 containing pYPGE15 plasmid
  • IV is W303-1B Aenal-4:: HIS3 containing AtNHXl-pYPGE15 recombinant plasmid ⁇ nhxl : :TRP1
  • V is W303-IB Aenal-4 containing: OsNHX pYPGE15 recombinant plasmid: :HIS3 ⁇ nhxl : :TRP1
  • VI is W303- IB Aenal-4 containing DraNHXl-pYPGE15 recombinant plasmid : : HIS3 ⁇ nhxl : : TRP1, VD, VD1
  • the following primers were designed with reference to the cDNA sequences of AtNHX1, OsNHX1, and DmNHX1 (the 5' ends contain Smal and Sal I restriction sites, respectively):
  • OsR (5, -ACGCGTCGACTCATCTTCCTCCATGGC-3, )
  • OsF (5, -TCCCCCGGGATGGGGATGGAGGTGGCG-3 ')
  • Arabidopsis thaliana Na+/H + antiporter gene i/WZi, rice Na+/H+ antiporter gene OsNHX1 and chrysanthemum Na7H+ antiporter gene DmNHX1 were obtained by PCR amplification and ligated into pGM-T vector, respectively. Enter E. coli DH5a, clone iA, OsNHXl, DmNHXl, and determine by sequencing.
  • Example 2 DNA shuffling of AtNHX OsNHX1, ⁇ 1 ⁇ 2 ⁇ 3 ⁇ 43 ⁇ 4 genes
  • the purified DNA concentration was determined by DNase I random digestion ultraviolet absorption method, and the content of 9 ug of AtNHX1 and OsNHXl DmNHX1 was mixed, so that the ratio of the three genetic substances was 1: 1:1.
  • 40 ⁇ l of the mixed AtNHX1, OsNHX1, and DmNHX1 were added to a 50 ⁇ l digestion reaction system (10 mM Tris-HCl, pH 7.5, 50 mM MnCl 2 ), and reacted at 15 ° C for 10 min.
  • the product was electrophoresed on a 2.5% agarose gel, and the gel containing the 100-200 bp fragment was excised and recovered.
  • the 5 ⁇ 1 primer-free PCR product was used as a template to amplify the full-length sequence of the recombinant gene with AtR and AtF, OsR and OsF, DmR and DmF primers.
  • the PCR reaction system was (lO X Taq buffer, dNTP each 0.2 ⁇ m, each primer 30 ⁇ , 0.6 U Taq polymerase), the PCR reaction procedure was: 94 ⁇ pre-denaturation for 3 min, 94 ° C denaturation for 1 min, 58 ° C Annealed for 30 s, extended at 72 ° C for 1. 5 min, total 30 cycles, and finally extended at 72 ° C for 5 min.
  • the recombinant product obtained in Example 2 was digested with Sal I and Sma I, purified, and ligated into the yeast expression vector pYPGE15 which was similarly digested, and the constructed expression shuffling library was introduced into the yeast mutant strain W303-l BAenal by the lithium acetate method.
  • the plasmid in the high-salt-resistant yeast mutant was extracted using a yeast plasmid extraction kit for DNA sequencing. Nucleotide sequence alignment, amino acid sequence alignment and hydrophilicity of high salt-tolerant Na+/H + antiporter gene and wild-type NaVH+ antiporter gene using software such as DNA Star, Clustalx and TMpred Sexual hydrophobicity analysis.
  • the recombinant plasmid pGM-T-NHXFS1 obtained in Example 3 was digested with Sal I and Sma I, recovered and ligated into the yeast expression vector PYPGE15 which was similarly digested, and the constructed NHXFS1-pYPGE 15 was treated with lithium acetate.
  • the recombinant plasmid was introduced into the yeast double mutant W303-1B ⁇ enal-4: :HIS3 ⁇ nhxl : : TRP1 and screened in a uracil-free APG selective medium to obtain a positive transformant.
  • Example 6 Comparison of yeast functional complementation experiments
  • the wild-type yeast W303-1B is a salt-tolerant yeast whose mutant strain W303-1B ⁇ enal-4 :: HIS3 Anhxl : :TRP1 has low salt tolerance, and a recombinant vector containing the NHXFS1 gene is introduced into the yeast mutation. Strain, a functional complementation comparison experiment can be performed.
  • the recombinant plasmids pGM-T_AtNHX1, pGM-T- 0sNHX1, pGM-T-DmNHX1 were digested with Sal I and Sma I, recovered and ligated into the yeast expression vector PYPGE15 which was similarly digested, and constructed by the lithium acetate method.
  • the recombinant plasmid was introduced into the yeast double mutant W303-1B ⁇ enal-4 : :HIS3 ⁇ nhxl : :TRP1, and the empty yeast expression vector PYPGE15 was introduced into wild type yeast W303-1B and yeast mutant W303- IB Aena.
  • Aenal-4 HIS3 ⁇ nhxl : : TRP1, screened in APM-free medium without uracil.
  • the selected positive transformants and the selected high-yield yeast strains were inoculated separately into APG liquid medium and cultured at 0D 6 . . Adjusted to 1.0, respectively, 10, 100 and 1000 dilutions, each taking 5 ⁇ 1 points to ⁇ 5. 5 different salt concentrations (0mM, 100 mM, 200 mM, 250 mM, 300 mM, 350 mM NaCl)
  • APG plate 30 ⁇ , cultured for 2 days to a week, observe growth. The results are shown in Figure 1.
  • the yeast mutant W303-1B ⁇ enal-4::HIS3 Anhxl::TRP1 (II) lacking the Na7H+reporter gene is salt-sensitive to the wild-type yeast strain W303-IB (III). More, the yeast mutant strain W303 IB Aenal-4: :HIS3 ⁇ nhxl::TRPl (IV, V, VI) expressing AtNHX1, 0s ⁇ 1, DmNHXl can partially complement the salt tolerance of yeast, while the NHXFS1 gene can confer yeast mutation.
  • the stronger salt tolerance (W, ⁇ ) of the strain indicates that the NaVH+antiporter NHXFS1 obtained by this shunt has stronger ion transport activity than the unmodified AtNHX1 and OsNHXK DmNHXl Na+/H+ antiporter, and more Na can be added. + is separated from the cytoplasm into the vacuole.

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Description

一种新的植物强耐盐基因皿 ¾7及其编码蛋白和应用 技术领域
本发明涉及到植物基因工程领域，具体的说是利用 DNA家族改组技术获得功 能显著提高的植物 Na+/H+逆向转运蛋白的基因， 本发明还涉及到利用此基因培育 转基因耐盐植物。
背景技术
土壤盐渍化是影响农作物生产和生态环境的一个重要的非生物胁迫因素。目 前， 世界上大约有 20%的耕地和接近 50%的灌溉用地受到盐渍的严重危害 (Flowler T J， Yeo A R. Breeding for sal inity resistance in crop plants. Where next? Aust. J. Plant Physiol. 1995, 22 : 875〜884)。 我国大约有 5亿亩 盐碱地， 并且其面积有不断增加的趋势。 培育抗盐植物是促进盐碱地开发利用、 改良土壤和生态治理的有效途径。 因此， 利用现代生物学技术， 通过对植物耐盐 生理和分子生物学的深入研究， 克隆和改造植物重要的耐盐基因，进而将这些基 因转化到植物中以培育转基因耐盐植物新品种，对于农业生产、环境保护和生态 治理等都具有重要的意义。
高浓度的盐分对植物造成严重的伤害，主要表现在渗透胁迫和离子毒害两方 面 ( Blumwald E, Aharon G S, Apse M P. Sodium transport in plant cells. Biochew Biophy Acta. 2000， 1465 : 140〜151 )。 一方面， 土壤中高浓度的盐 分使土壤水势低于植物细胞的水势从而引起植物细胞水分亏缺即渗透胁迫；另一 方面， 过量渗入植物细胞的各种盐离子又会对细胞造成离子毒害， 主要是 Na+毒 害。遭受渗透胁迫和离子毒害的植物细胞的质膜的通透性增加，导致过氧化胁迫、 营养亏缺及一系列生理代谢反应的紊乱等次生伤害作用，从而对植物正常的生长 发育造成严重危害。
自然界中植物为了适应高浓度的盐，形成了一系列的防御机制 (Bohnert H J,
确认本 Nelson D E , Jensen R G. Adaptation to environmental stress. Plant Cell. 1995, 7 : 1099〜1111)， 主要包括渗透调节和重建细胞的离子均衡两种策 略。前者指植物细胞利用一些无机离子或者合成一些小分子有机化合物作为渗透 调节剂， 以降低细胞水势， 抵御渗透胁迫。后者主要指植物细胞通过将细胞质中 过量的 Na+转运出细胞或者区隔化到液泡中， 实现离子均衡、消除 Na+毒害的目的。 植物细胞的一个显著特征就是拥有体积很大的膜隔离的液泡， 盐胁迫条件下，植 物细胞保持细胞质低浓度 Na+的最直接方式就是将 Na+隔离到液泡中。 这样既可以 使胞质酶免受离子毒害， 又能提高细胞整体的渗透压起到促进细胞吸水的作用， 是自然界中耐盐植物主要的耐盐机制（Flower T J , Troke P F , Yeo A R. The mechani sms of salt tolerance in halophytes. Annu Rev Plant
Physiol . 1977 , 28 : 89〜121. )。
植物 Na7H+逆向转运蛋白是一种 Na+的逆向转运体， 在植物质膜和液泡膜上都 有分布， 其中液泡膜 Na7H+逆向转运蛋白是负责将 Na+区隔化到液泡的主要蛋白。 植物 Na7H+转运蛋白最先在大麦质膜上发现， 之后在多种植物质膜和液泡膜上普 遍检测到 Na7H+转运的活性。 自世界上首个植物 Na7H+逆向转运蛋白基因 i/Vm 被克隆后 (Gaxiola R A, Rao R, Sherman A, Grisaf i P, Alper S, and Fink G R. The Arabidopsis thai i ana proton transporters , AtNhxl and Avpl , can function in cation detoxify cation in yeast . Proc Natl Acad Sci USA. 1999 , 96 : 1480〜1485)， 水稻、 北滨黎、 碱蓬等高等植物的液泡膜 Na+/H+逆向转 运蛋白基因相继得到克隆， 分别为 (¾Λ¾η ( Fukuda A , Nakamura A , Tanaka Y. Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa. Biochim Biophys Acta. 1999 , 1446 : 149〜155) 、 AgNHXY (Hamada A, Shono M, Xia T, Ohta M, Hayashi Y, Tanaka A, Hayakawa T. Isolation and characterization of a Na+/H+ antiporter gene from the halophyte A triplex gmelini. Plant Molecular Biology. 2Q<^, A6 >5〜 2 ^> 、 SsNHXi (Ma X L, Zhang Q, Shi H Z, Zhu J K, Zhao Y X, Ma C L, Zhang H. Molecular cloning and different expression of a vacuolar Na+/H+ antiporter gene in Suaeda salsa under salt stress . Biological 2004， 48 (2) : 219〜225 ) 等 。 在 液泡膜质子泵提供的能量下， 位于液泡膜的 Na+/H+逆向转运蛋白将 Na+区隔化进 入液泡中从而降低细胞质中 Na+的浓度，使过多的 Na+离开代谢位点，减轻其对酶和 膜系统的伤害， 还可降低细胞水势， 抵抗盐分造成的渗透胁迫。 所以， Na7H+逆 向转运蛋白对植物的抗盐性起着重要的作用。
将 NaVH+逆向转运蛋白 （NaVH+ antiporter) 基因转化到目标植物中进行过 量表达是培育转基因耐盐植物的有效方式。 Apse等对转 A髓 XI基因的拟南芥植 株进行了分析， 过量表达 Na+/H+逆向转运蛋白的转基因拟南芥植株在 200 mmol/ L NaCl 中能正常生长发育。 免疫印迹表明， 在转化植株叶片提纯液泡中含有比 从野生型叶片提纯液泡中更多的^ ίΛ ¾ 产物， 液泡上 Na7H+逆向转运蛋白活力 显著增强（Apse M P , Aharon G S , Snedden W A， Bluraward E. Salt tolerance conferred by overexpression of a vacuolar Na+/H+ ant iporter in Arabidopsis . Science. 1999 , 285 : 1256〜1258)。 同样， 将拟南芥 AtNHXl基因转入西红柿 中， 转基因西红柿在 200 ramol/L NaCl胁迫下能够正常生长、 开花和结实。 尽管 叶片中 Na+浓度高， 但西红柿果实中 Na+浓度很低（Zhang H X, Blumward E. Transgenic salt-tolerant tomato plants accumulate salt in fol iage but not in fruit . Nature Biotechnology . 2001, 19 ： 765〜 768)。 这些研究都表明 了 Na7H+逆向转运蛋白基因及其蛋白在植物对于高盐反应中的重要性， 被认为 是目前最有希望利用其特性培养转基因耐盐品种的基因。
然而， 在实际生产应用上， 目前在植物中已克隆的 Na7H+逆向转运蛋白基 因仍然存在着活性不高、耐盐性不强的问题， 很大程度上限制了该基因的生产应 用和推广。如何利用特定的现代生物学技术， 克隆新的强耐盐基因或对该基因进 行分子进化和改造，进而将这些新基因转化到植物中以培育转基因耐盐植物新品 种， 对于农业生产、 环境保护和生态治理等都具有重要的意义。
DNA 改组（DNA Shuffl ing)技术是目前蛋白质、 酶和单克隆抗体等体外定向 进化的快速高效方法,在提高酶活性、 蛋白质产量和改善蛋白质 (酶）的性能等方 面具有广泛的应用前景。其原理是将一组紧密相关的核酸序列随机片段化，然后 利用无引物 PCR将这些随机片段进行重新组装而得到全长的核酸序列，在这个过 程中引入突变并对不同的突变进行广泛的重组，从而完成对目的核酸序列的迅速 进化， 以提高核酸序列或其编码的蛋白的功能 (Stemmer W P. DNA shuffling by random fragmentation and reassembly : in vitro recombination for molecular evolution. Proc Natl Acad Sci USA. 1994, 91 : 10747-10751)。 DNA改组可分 为单基因的改组和基因家族的改组，前者仅仅对单个基因进行随即片段化，利用 无引物 PCR进行重新组装，而家族改组可对亲缘关系相近或者差距很大的基因家 族进行改组。
DNA改组技术自诞生以来， 已成功地对多种基因如工业用酶、 抗体及一些重 要的蛋白等进行了定向进化， 使酶的活性、底物特异性及抗体的特异性、蛋白功 能、热稳定性等得到了显著的提高（Crameri A， Rai l lard S, Bermudez E, Stemmer W P. DNA shuffling of a family of genes from diverse species accelerates directed evolution . Nature. 1998, 391 : 288 )。 如 Stemmer等应用该技术取得 了一系列研究成果， 获得了酶活提高了 32000 倍的 β _内酰胺酶（Stemmer WP. Ra id evolution of a protein in vitro by DNA shuffl ing. Nature. 1994， 370 : 389〜391)、 荧光强度超过野生型 45倍的绿色荧光蛋白（Crameri A, Whitehorn E A, Tate E, Stemmer W P. Improved green fluorescent protein by molecular evolution using DNA shuffl ing. Nat Biotechnol. 1996, 14 : 315〜319)。 通 过 DNA改组方法， Crameri等成功得到了砷酸盐抗性提高 40倍的菌株 (Crameri A, Dawes G, Rodriguez E J, Silver S, Stemmer W P. Molecular evolution of an arsenate detoxification pathway by DNA shuffl ing. Nat Biotechnol. 1997, 15 : 436〜438)。 但是， 应用该技术在改进 Na+/H+逆向转运蛋白基因及其蛋白 质的性能方面， 国内外均未见报导。
发明内容
本发明目的在于获得一种新的植物强耐盐基因 NHXFS1, 此基因能编码具有 离子转运活性的 Na+/H+逆向转运蛋白。
本发明的另一个目的在于提供上述基因编码的 Na+/H+逆向转运蛋白 NHXFS1 , 其离子转运活性比野生型的 NaVH+逆向转运蛋白更强。
本发明还提供了含有上述基因的重组载体。
本发明的另一方面， 还提供了含有上述重组质粒的宿主细胞。
本发明的另一方面， 还提供了重组载体的构建、转基因植物等方法， 以应用 上述基因和编码蛋白培育耐盐性更强的转基因植物新品种。 Na+/H+逆向转运蛋白 （Na7H+antiporter) 在植物耐盐中起重要作用， 其活 性大小影响着植物的耐盐性。 本发明通过 DNA家族改组技术， 对编码 Na+/H+逆向 转运蛋白的野生型耐盐基因 ί½¾ί 、 OSNHX ΖΜ¾ί 进行体外分子进化， 以获 得编码离子转运活性更高的 Na+/H+逆向转运蛋白新基因 Λ¾ί ¾ 。 这种新基因所 编码的 Na7H+逆向转运蛋白 NHXFS1能将更多的 Na+从细胞质中区隔化到液泡中， 将得到的新基因转化到目标植物中，能够培育出耐盐能力更强的转基因植物新品 种。
具体来说， 通过体外 DNA改组技术对野生型的 Na7H+逆向转运蛋白基因进行 突变重组， 通过功能互补法， 在 Na7H+逆向转运蛋白基因缺失的酵母突变株中定 向筛选离子转运活性显著增强的 Na7H+逆向转运蛋白突变新基因。
本发明涉及到一种新的植物强耐盐基因 ^Τ¾7， 是编码 Na+/H+逆向转运蛋 白 NHXFS1, 能够提高植物抗盐能力， 是下列核苷酸序列之一：
1) 序列表中的 SEQ ID N0:1;
2) 与序列表中 SEQ ID N0:1限定的核苷酸序列同源性在 70-100%、 且编码 相同功能蛋白质的 DNA序列；
3) 编码序列表中 SEQ ID NO :2所示蛋白质序列的 DNA序列。 优选的植物强耐盐基因 NHXFS1, 其核苷酸序列如 SEQ ID N0:1, 与野生型 Na7H+逆向转运蛋白基因（¾Λ¾Ϊ 之间相比， 有 4个核苷酸的突变， 其中第 185 位和 1059位为腺嘌呤， 1221位和 1517位为鸟嘌呤。 上述基因的编码蛋白， 为具有离子转运活性的 NaVH+逆向转运蛋白 NHXFS1, 其特征在于， 是下列氨基酸序列之一：
1) 序列表中 SEQ ID N0:2;
2) 与序列表中 SEQ ID N0:2限定的氨基酸序列同源性在 70-100%之间的蛋 白质；
3) 在 SEQ ID N0:2限定的氨基酸序列中增加、 减少或替换一个或几个氨基 酸且具有相同活性的蛋白质。
优选的氨基酸序列如 SEQ ID N0:2, 与水稻野生型 Na7H+ 逆向转运蛋白 OsNHXl之间相比有 2个氨基酸的改变， 其中第 62位为天冬氨酸， 506位为甘氨 酸， 也包括与 SEQ ID N0:2所示氨基酸序列同源性在 70-100%之间的蛋白质， 也 包括在 SEQ ID N0: 2所示氨基酸序列中增加、 减少或替换一个或几个氨基酸的蛋 白质。
本发明的技术方案如下：
1、 NaVH+逆向转运蛋白 ¾因的克隆： 可以采用 PCR扩增法、 重组法或人工 合成法等多种方法来获得 Na+/H+逆向转运蛋白核苷酸全长序列或其片段。 比如， 基于 Na+/H+逆向转运蛋白基因序列设计多核苷酸探针来从 cDNA文库或基因组文 库中筛选目的基因， 也可以用 PCR扩增方法直接从 cDNA或基因组中扩增出有关 序列。
2、 NaVH+逆向转运蛋白基因的 DNA 改组： 用于 DNA改组的底物可以是生 物体不同个体、株系或种类的 Na7H+逆向转运蛋白基因，也可以是通过易错 PCR、 定点突变等常规方法制取的不同突变形式的 NaVH+逆向转运蛋白基因。
对 NaVH+逆向转运蛋白基因进行 DNA 改组的主要步骤为： 首先用 DNase I 或限制酶对 NaVH+逆向转运蛋白基因进行随机片段化， 回收其中的小片段即重 叠片段。 然后在不加引物的情况下， 用 DNA 聚合酶进行 Primerless PCR使小片 段互为引物和模板进行扩增，从而使不同的突变得到广泛的重组。最后经 Primer PCR扩增得到含有重组 Na+/H+逆向转运蛋白基因片段的重组基因库。
含有重组 NaVlT逆向转运蛋白基因片段的重组基因库可以转入到一种细胞 或生物体内如细菌、酵母或植物细胞进行筛选。例如， 可以将本发明得到的重组 基因库连接到任何酵母表达载体如 PYPGE15中，利用本领域公知的方法如乙酸锂 技术、电转法等转化酵母 Na7H+逆向转运蛋白突变体如 W303Aenal- 4Anhxl中， 构建表达重组基因库。将酵母转化子涂布在含高 NaCl浓度的 APG选择性平板中 以高盐为选择压力进行高通量筛选， 得到盐耐受性显著提高的突变株。
从筛选到的盐耐受性显著提高的酵母突变株中提取质粒，通过测序来获取改 组后的耐高盐 Na+/H+ 逆向转运蛋白基因的核苷酸序列， 运用常规的生物软件如 DNA Star, Clustalx和 TMpred 等对耐高盐 Na+/H+逆向转运蛋白基因和野生型 Na7H+逆向转运蛋白基因进行核苷酸序列比对、氨基酸序列比对以及亲水性疏水 性分析等生物信息学分析。 结果表明， Na7H+ 逆向转运蛋白 NHXFS1 比未改组 Na7H+逆向转运蛋白具有更强的离子转运活性， 可以将更多 Na+的从胞质中区隔 化到液泡中， 使导入该基因的宿主细胞具有更好的耐盐性。
Na7H+逆向转运蛋白基因/½ ¾7¾7作为目的基因， 可以构建在任何植物表达 的 Ti-质粒双元载体中比如 pBISNl上，其中有 LB和 RB的 T_DNA 25bp重复序列， 甘露氨酸合成酶启动子 （Pmas ), polyA nos 终止子， 还有在真核生物中作为选 择标记使用的新霉素磷酸转移酶 II (nptll) , 用于选择的抗生素是卡那霉素。 植 物表达载体中的启动子可以是任何一种组成型启动子、组织特异性启动子或环境 诱导型启动子， 如花椰菜花叶病毒 (CAMV) 35S启动子， Ubiqutin启动子。 载体中 的增强子既可以是转录增强子，也可以是翻译增强子。为了便于对转基因植物细 胞或植物进行鉴定及筛选， 载体中还应有植物可选择性标记（GUS基因、 荧光素 酶基因等） 或具有抗生素等抗性的标记物 （如卡那霉素、 除草剂等）。
通过本领域公知的研究方法如冻融法、电击法等将上述重组载体导入农杆菌 中， 进行农杆菌转化。 可通过微注射、 基因枪、 农杆菌介导或花粉管通道方法等 常规生物技术方法将新的强耐盐 Na+/H+ 逆向转运蛋白基因 NHXFS1 转入植物 中，培育出抗盐品质优良及生物学性状得到改善的植物新的品种。被转化的植物 宿主既可以是单子叶植物也可以是双子叶植物， 如： 拟南芥、烟草、番茄、油菜、 水稻、 小麦、 玉米、 大豆、 蔬菜、 树木、 花卉、 牧草和草坪草等。 本发明的基因 对于培育抗盐植物品种， 提高农作物产量、 改善生态环境等具有重要的意义。
本发明的转基因重组载体可作为商业用途的品种、品系或作为基因资源在生 产上直接使用或进行农业生物育种和转基因植物以提高作物的抗盐性。
附图说明
图 1为改组获得的 NHXFS1基因与未改组 AtNHXl、 OsNHX DmNHXl基因在酵 母双突变菌株 W303- IB A enal- 4 A nhxl 中功能互补实验比较， 即以下细胞在 pH5. 5、 含有 0、 100mM、 200mM、 250mM、 300mM和 350mM NaCl的 APG平板上生长 情况：
其中 I为含有 PYPGE15质粒的野生型 W303- 1B， II为含有 PYPGE15质粒的 W303-1B Aenal-4 :： HIS3 Anhxl： :TRP1， III为含有 pYPGE15质粒的 W303-1B Δ enal-2 :： HIS3， IV为含有 AtNHXl-pYPGE15重组质粒的 W303—1B Aenal-4:： HIS3 △nhxl : :TRPl， V为含有 OsNHX卜 pYPGE15重组质粒的 W303- IB Aenal-4 : :HIS3 △nhxl : :TRPl , VI为含有 DraNHXl- pYPGE15重组质粒的 W303- IB Aenal-4 : : HIS3 △ nhxl : : TRPl， VD， VD1为含有 NHXFS1- pYPGE15 重组质粒的 W303- IB △ enal-4 : : HIS3 Anhxl : : TRP10
具体实施方式
实施例 1. 拟南芥,水稻， 菊花 Na7H+逆向转运蛋白基因的克隆
用 TRizol 试剂从拟南芥， 水稻和菊花植物幼叶中抽提总 RNA, 以总 RNA为 模版进行反转录，合成 cDNA第一链。以合成的 cDNA为模版，参照 AtNHXl、 OsNHXl、 DmNHXl的 cDNA序列， 设计以下引物（5' 端分别包含 Smal 和 Sal I酶切位点）：
AtR (5， -GCGTCGACTCAAGCCTTACTAAGATCAGGAGG-3 ' )
AtF ( 5， -TCCCCCGGGATGTTGGA TTCTCTAGTG- 3， )
OsR (5， -ACGCGTCGACTCATCTTCCTCCATGGC-3， )
OsF (5， -TCCCCCGGGATGGGGATGGAGGTGGCG-3 ' )
DmR ( 5， -GCGTCGACTTAGTTTCTTTCTTCATCTTC-3 ' )
DmF (5， -TCCCCCGGGATGGTGTTCGATTC-3 ' )
通过 PCR扩增获得拟南芥 Na+/H+逆向转运蛋白基因 i/WZi 、水稻 Na+/H+逆 向转运蛋白基因 OsNHXl和菊花 Na7H+逆向转运蛋白基因 DmNHXl,分别将其连接 到 pGM- T 载体中， 转入大肠杆菌 DH5a,克隆得到 iA 、 OsNHXl、 DmNHXl膽 列， 并通过测序来确定。 实施例 2. AtNHX OsNHXl、 Ζ½Λ¾¾ 基因的 DNA改组
1 ) 起始材料的制备 以 pGM- T- AtNHXl、 pGM- T_0sNHXl、 pGM-T- DmNHXl 为模版， 用 PfuDNA聚合酶对三个基因进行 PCR扩增， 纯化后作为 DNA改组的起 始材料。
2 ) DNase I随机酶切 紫外吸收法测定纯化后的 DNA浓度， 取含量为 9ug 的 AtNHXl、 OsNHXl DmNHXl混合， 使三个基因物质的量比值为 1： 1： 1。取 40ul 混合后的 AtNHXl、 OsNHXl、 DmNHXl加入到 50μ1酶切反应体系中（ 10mM Tris-HCl , pH7. 5， 50mM MnCl2), 15°C下反应 10min。 加入 DNase I 0. 15U， 混匀， 15°C， lminl6s, 90°C, lOmin. 产物通过 2. 5%琼脂糖凝胶电泳， 切下含 100- 200bp的片 段的凝胶并回收。
3 )无引物 PCR 取 I4g回收的小片段，加入到 50μ1反应体系中（lO X Taq buffer, dNTP 各 0. 2mM， 0. 6υ/μ1 Taq聚合酶）。 PCR程序为反应条件： 94°C预 变性 3min， 94°C变性 30s， 45 °C 退火 lmin, 72°C延伸 lmin， 共 50cycles， 最后 72°C 延伸 5min。
4)有引物 PCR 取 5μ1 无引物 PCR的产物作为模板，分别用 AtR和 AtF、 OsR和 OsF、 DmR和 DmF三对引物对重组的基因全长序列进行 PCR扩增。 PCR反应 体系为（lO X Taq缓冲液， dNTP各 0. 2mM， 每种引物各 30μΜ， 0. 6U Taq聚合酶）， PCR反应程序为： 94Ό预变性 3min， 94°C变性 lmin, 58 °C 退火 30s， 72°C延 伸 1. 5min， 共 30cycles， 最后 72°C 延伸 5min。
通过 DNA改组， 以 AtF和 AtR、 DmF和 DmR为引物的两组没有扩增出条带， 而以 OsR和 OsF为引物获得了含有重组 Na7H+逆向转运蛋白基因片段的重组基 因库。 实施例 3. 表达改组文库的构建及耐高盐 Na7H+逆向转运蛋白基因 NHXFS1的筛 选
用 Sal I 和 Sma I对实施例 2得到的重组产物进行酶切， 纯化后连接到同 样酶切的酵母表达载体 pYPGE15中， 用乙酸锂方法将构建的表达改组文库导入 酵母突变株 W303-lBAenal- 4 : : HIS3 Anhxl : :TRPl中，涂布到不含尿嘧啶的 APG 选择培养基上， 30°C， 培养 2d， 筛选到含有 Na7H+逆向转运蛋白基因的酵母株， 通过增加 NaCl浓度对所筛选到的酵母株进一步进行筛选，最终在 200mM NaCl浓 度的选择培养基上得到了一个正常生长的酵母突变子，该突变子含有新的植物强 耐盐基因 NHXFS1。 实施例 4. 耐高盐基因醒 FS1的序列分析
用酵母质粒提取试剂盒提取耐高盐的酵母突变子中的质粒进行 DNA测序。用 DNA Star、 Clustalx和 TMpred 等软件对耐高盐 Na+/H+逆向转运蛋白基因和野 生型 NaVH+逆向转运蛋白基因进行核苷酸序列比对、 氨基酸序列比对以及亲水 性疏水性分析。
结果显示， NHXFSl基因与野生型 Na+/H+逆向转运蛋白基因 Λ¾ί 之间有 4 个核苷酸的突变， 其中第 185位鸟嘌呤转换为腺嘌呤， 1059位胸腺嘧啶颠换为 腺嘌呤， 1221位胸腺嘧啶颠换为鸟嘌吟， 1517位腺嘌呤转换为鸟嘌呤。 NHXFSl 编码蛋白与水稻野生型 Na7H+逆向转运蛋白之间有 2个氨基酸的改变， 第 62位 由甘氨酸变为天冬氨酸，第 506位由天冬氨酸变为甘氨酸，如序列表 SEQ ID N0: 1 和 SEQ ID N0: 2。 实施例 5. 重组载体和宿主细胞的构建
用 Sal I 和 Sma I对实施例 3中得到的重组质粒 pGM-T- NHXFSl进行酶切， 回收之后连接到经同样酶切的酵母表达载体 PYPGE15中，用乙酸锂方法将构建的 NHXFSl- pYPGE 15 重组质粒导入到酵母双突变株 W303-1B Δ enal-4： :HIS3 Δ nhxl : :TRPl 中， 在不含尿嘧啶的 APG选择性培养基中进行筛选， 得到阳性转化 子。 实施例 6. 酵母功能互补比较实验
野生型酵母 W303- 1B 是一种具有耐盐性的酵母， 其突变株 W303-1B Δ enal-4 :： HIS3 Anhxl : :TRPl耐盐性很低， 将含有 NHXFSl基因的重组载体导入 该酵母突变株， 可进行功能互补比较实验。
用 Sal I 和 Sma I对重组质粒 pGM- T_AtNHXl、 pGM- T- 0sNHXl、 pGM-T-DmNHXl 进行酶切， 回收之后连接到经同样酶切的酵母表达载体 PYPGE15中，用乙酸锂方 法将构建的重组质粒导入到酵母双突变株 W303-1B Δ enal-4 : :HIS3 Δ nhxl : :TRPl 中， 同时将空的酵母表达载体 PYPGE15 分别导入到野生型酵母 W303- 1B、酵母突变株 W303- IB Aena卜 2 : :HIS3和 W303-1B Aenal-4:： HIS3 Δ nhxl : : TRPl 中， 在不含尿嘧啶的 APG选择性培养基中进行筛选。 将筛选到的阳 性转化子和筛选到的耐高盐酵母菌株分别接种到 APG液体培养基中培养，将 0D6。。 都调整到 1. 0， 分别进行 10倍、 100倍和 1000倍稀释， 各取 5μ1点种到 ρΗ5. 5 不同盐浓度 （0mM、 100 mM、 200 mM、 250 mM、 300 mM、 350 mM NaCl ) 的 APG 平板上， 30Ό， 培养 2d到一周， 观察生长情况。 结果如图 1所示， 缺失 Na7H+逆向转运蛋白基因的酵母突变株 W303-1B Δ enal-4::HIS3 Anhxl::TRPl (II) 比野生型的酵母菌株 W303- IB (III) 对盐敏 感的多， 表达 AtNHXl、 0s匪 1、 DmNHXl的酵母突变株 W303 IB Aenal-4: :HIS3 △nhxl::TRPl (IV, V、 VI) 能够部分互补酵母的耐盐性， 而 NHXFS1基因能够 赋予酵母突变株更强的耐盐性 （W、 \ )， 这表明此改组获得的 NaVH+逆向转运 蛋白 NHXFS1 比未改组 AtNHXl、 OsNHXK DmNHXl Na+/H+逆向转运蛋白具有更强 的离子转运活性， 可以将更多 Na+的从胞质中区隔化到液泡中。

Claims

权 利 要 求 书
1、 一种新的植物强耐盐基因 Λ%¾7¾7， 其特征在于， 是下列核苷酸序列之一：
1) 序列表中的 SEQ ID N0:1;
2) 与序列表中 SEQ ID N0:1限定的核苷酸序列同源性在 70-100%、 且编码 相同功能蛋白质的 DNA序列；
3) 编码序列表中 SEQ ID NO :2所示蛋白质序列的 DNA序列。
2、 一种新的植物强耐盐基因 NHXFS1的编码蛋白，为具有离子转运活性的 NaVH+ 逆向转运蛋白 NHXFS1, 其特征在于， 是下列氨基酸序列之一：
1) 序列表中 SEQ ID NO: 2;
2) 与序列表中 SEQ ID N0:2限定的氨基酸序列同源性在 70_100%之间的蛋 白质；
3) 在 SEQ ID NO: 2限定的氨基酸序列中增加、 减少或替换一个或几个氨基 酸且具有相同活性的蛋白质。
3、 含有权利要求 1所述基因的重组载体。
4、 含有权利要求 1所述基因的宿主细胞。
5、 权利要求 1所述一种新的植物强耐盐基因 NHXFS1在培育抗盐植物方面的应 用， 其特征在于， 所述植物强耐盐基因 NHXFS1通过微注射、 基因枪、 农杆菌介 导或花粉管通道的方法转入植物中，培育出耐盐性及生物学性状得到改善的植物 新品种。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955522A (zh) * 2010-09-30 2011-01-26 中国农业大学 一种花生二价铁转运蛋白及其编码基因与应用
CN111034406A (zh) * 2019-12-11 2020-04-21 青岛九天智慧农业集团有限公司 一种针对新疆盐碱地种植水稻的土壤调理方法
CN113444734A (zh) * 2021-08-02 2021-09-28 内蒙古大学 耐盐型转基因杨树的制备方法及应用
CN114214335A (zh) * 2022-01-14 2022-03-22 浙江省农业科学院 一种碱蓬耐盐性相关编码基因及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101413004B (zh) * 2008-11-25 2011-02-02 华东师范大学 一种植物强耐盐基因nhxfs1及其编码蛋白
CN102559702A (zh) * 2012-03-12 2012-07-11 南京农业大学 耐盐基因CcSOS1及其应用
CN103993020B (zh) * 2014-06-09 2016-10-05 天津大学 一种植物强耐盐基因SseNHX1及其编码蛋白和应用
CN107630023B (zh) * 2017-08-23 2020-03-27 东北农业大学 肇东盐单胞菌耐盐碱基因duf2062及其鉴定方法
CN111088260A (zh) * 2020-01-16 2020-05-01 南京农业大学 萝卜耐盐基因RsNHX1及应用
CN114107361A (zh) * 2021-11-15 2022-03-01 南京瑞源生物技术有限公司 一种筛选物种文库酵母菌株耐盐基因的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050028235A1 (en) * 2002-07-12 2005-02-03 Hong-Xia Zhang Plant fruit with elevated potassium levels
US20070107082A1 (en) * 2003-06-03 2007-05-10 Cropdesign N.V. Transgenic monocotyledonous plants overexpressing a nhx protein and having improved growth characteristics and a method for making the same
CN101260403A (zh) * 2008-02-02 2008-09-10 华东师范大学 一种新的植物强耐盐基因AtNHXS1及其编码蛋白和应用
CN101413004A (zh) * 2008-11-25 2009-04-22 华东师范大学 一种新的植物强耐盐基因nhxfs1及其编码蛋白和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050028235A1 (en) * 2002-07-12 2005-02-03 Hong-Xia Zhang Plant fruit with elevated potassium levels
US20070107082A1 (en) * 2003-06-03 2007-05-10 Cropdesign N.V. Transgenic monocotyledonous plants overexpressing a nhx protein and having improved growth characteristics and a method for making the same
CN101260403A (zh) * 2008-02-02 2008-09-10 华东师范大学 一种新的植物强耐盐基因AtNHXS1及其编码蛋白和应用
CN101413004A (zh) * 2008-11-25 2009-04-22 华东师范大学 一种新的植物强耐盐基因nhxfs1及其编码蛋白和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ATSUNORI FUKUDA ET AL.: "Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa", BIOCHIMICA ET BIOPHYSICA ACTA, 1999, pages 1446 *
DATABASE EMBL/GENBANK 31 December 1999 (1999-12-31), Database accession no. AB021878 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955522A (zh) * 2010-09-30 2011-01-26 中国农业大学 一种花生二价铁转运蛋白及其编码基因与应用
CN101955522B (zh) * 2010-09-30 2012-07-18 中国农业大学 一种花生二价铁转运蛋白及其编码基因与应用
CN111034406A (zh) * 2019-12-11 2020-04-21 青岛九天智慧农业集团有限公司 一种针对新疆盐碱地种植水稻的土壤调理方法
CN113444734A (zh) * 2021-08-02 2021-09-28 内蒙古大学 耐盐型转基因杨树的制备方法及应用
CN114214335A (zh) * 2022-01-14 2022-03-22 浙江省农业科学院 一种碱蓬耐盐性相关编码基因及其应用
CN114214335B (zh) * 2022-01-14 2023-07-04 浙江省农业科学院 一种碱蓬耐盐性相关编码基因及其应用

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