CN101413004A - Novel plant strong-salt resistance gene NHXFS1, encoding protein and use thereof - Google Patents
Novel plant strong-salt resistance gene NHXFS1, encoding protein and use thereof Download PDFInfo
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Abstract
The invention relates to the field of plant genetic engineering and provides a novel plant strong salt-tolerant gene NHXS1 obtained by a restructuring technology. A nucleotide sequence of the plant strong salt-tolerant gene NHXS1 is SEQ ID NO: 1, or a DNA sequence which has 70 to 100 percent homology with the SEQ ID NO: 1 nucleotide sequence or a DNA sequence for coding a protein sequence of SEQ ID NO: 2. Na + / H + antiporter NHXS1 colded by the gene has stronger ion transport activity than the wild-type Na + / H + antiporter AtNHX1. The invention also provides methods for construction of recombinant vectors and transgenic plants to apply the genes and proteins, and can culture a novel breed of transgenic plant with stronger salt-tolerant property or other improved biological characteristics.
Description
Technical field
The present invention relates to plant genetic engineering field, utilize DNA family shuffling technology to obtain the plant Na that function significantly improves specifically
+/ H
+The gene of reverse transport protein the invention still further relates to and utilizes this gene to cultivate the transgenic salt-tolerant wheat plant.
Background technology
The soil salinization is an important abiotic stress factor that influences farm crop production and ecotope.At present, nearly in the world 20% arable land and be subjected to serious harm (the Flowler T J of salt marsh near 50% irrigation land used, Yeo A R.Breeding for salinity resistance in crop plants.Where next? Aust.J.Plant Physiol.1995,22:875～884).The nearly 500,000,000 mu of saltingss of China, and its area has ever-increasing trend.Cultivate salt-resistant plant and be and promote the saltings development and use, improve the soil and the effective way of ecological management.Therefore; utilize the modern biology technology; by to plant salt tolerance physiology and molecular biological further investigation; clone and the important resistant gene of salt of plant modification; and then with these gene transformation in the plant to cultivate the transgenic salt-tolerant wheat new variety of plant, all have great importance for agriculture production, environment protection and ecological management etc.
The salinity of high density causes serious injury plant, show that mainly osmotic stress and ion poison two aspects (Blumwald E, Aharon G S, Apse M P.Sodium transport in plant cells.Biochem Biophy Acta.2000,1465:140～151).On the one hand, thus it is osmotic stress that the flow of water that the salinity of soil middle and high concentration makes soil water potential be lower than vegetable cell causes the vegetable cell water deficit; On the other hand, the various salt ions of excessive infiltration vegetable cell can cause ion to poison by pair cell again, mainly are Na
+Poison.Increased by the permeability of the plasma membrane of the vegetable cell that osmotic stress and ion poison, cause that peroxidation is coerced, the secondary injury effects such as disorder of nutritional deficiency and a series of physiological metabolism reaction, cause serious harm thereby plant grown normally.
The occurring in nature plant is in order to adapt to the salt of high density, formed a series of defense mechanism (Bohnert H J, Nelson D E, Jensen R G.Adaptation to environmental stress.PlantCell.1995,7:1099～1111), the balanced two kinds of strategies of ion that mainly comprise osmoregulation and reconstituted cell.The former refers to that vegetable cell utilizes some mineral ions or more synthetic small molecules organic compound as Osmolyte regulator, to reduce the cell flow of water, resists osmotic stress.The latter refers to that mainly vegetable cell passes through excessive N a in the tenuigenin
+Transport out cell or separating in vacuole, realize the ion equilibrium, eliminate Na
+The purpose of poisoning.A notable feature of vegetable cell is exactly to have the isolated vacuole of the very big film of volume, and under the condition of salt stress, vegetable cell keeps tenuigenin lower concentration Na
+Direct mode be exactly with Na
+Isolate in the vacuole.So both can make the kytoplasm enzyme avoid ion poisons, the osmotic pressure that can improve cell integral body again plays the effect that promotes the cell suction, be the main salt tolerant mechanism of occurring in nature salt-tolerant plant (Flower T J, Troke P F, Yeo A R.Themechanisms of salt tolerance in halophytes.Annu Rev PlantPhysiol.1977,28:89～121.).
Plant Na
+/ H
+Reverse transport protein is a kind of Na
+The antiport body, distribution is all arranged, wherein vacuole skin Na on plasmalemma of plant and vacuole skin
+/ H
+Reverse transport protein is to be responsible for Na
+Separating is to the major protein of vacuole.Plant Na
+/ H
+Translocator is found on the barley plasma membrane at first, generally detects Na afterwards on various plants plasma membrane and vacuole skin
+/ H
+The activity of transhipment.From first plant Na in the world
+/ H
+Reverse transport protein gene A tNHX1 is cloned back (Gaxiola R A, Rao R, Sherman A, Grisafi P, Alper S, and Fink GR.The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, the vacuole skin Na of high plant such as canfunction in cation detoxify cation in yeast.Proc Natl Acad Sci USA.1999,96:1480～1485), paddy rice, Bei Binli, alkali be fluffy
+/ H
+The reverse transport protein gene obtains the clone in succession, be respectively OsNHX1 (Fukuda A, Nakamura A, TanakaY.Molecular cloning and expression of the Na+/H+ exchanger gene in Oryzasativa.Biochim Biophys Acta.1999,1446:149～155), AgNHX1 (HamadaA, Shono M, Xia T, Ohta M, Hayashi Y, Tanaka A, Hayakawa T.Isolation andcharacterization of a Na+/H+ antiporter gene from the halophyte Atriplexgmelini.Plant Molecular Biology.2001,46:35～42), SsNHX1 (Ma X L, ZhangQ, Shi H Z, Zhu J K, Zhao Y X, Ma C L, Zhang H.Molecular cloning anddifferent expression of a vacuolar Na+/H+ antiporter gene in Suaeda salsaunder salt stress.Biological Plantarum.2004,48 (2): 219～225) etc.Under the energy that the vacuole skin proton pump provides, be positioned at the Na of vacuole skin
+/ H
+Reverse transport protein is with Na
+Thereby separating enters and reduces Na in the tenuigenin in the vacuole
+Concentration, make too much Na
+Leave the metabolism site, alleviate its injury, also can reduce the cell flow of water, the osmotic stress that the opposing salinity causes enzyme and film system.So, Na
+/ H
+Reverse transport protein plays an important role to the salt resistance of plant.
With Na
+/ H
+Reverse transport protein (Na
+/ H
+Antiporter) gene transformation is carried out the effective means that overexpression is a cultivation transgenic salt-tolerant wheat plant in target plant.Apse etc. analyze overexpression Na to the Arabidopis thaliana plant that changes the AtNHX1 gene
+/ H
+The transgenic arabidopsis plant of reverse transport protein can grow by normal growth in 200mmol/L NaCl.Immunoblotting shows, contains in transformed plant blade purification vacuole than more AtNHX1 product from wild-type blade purification vacuole, Na on the vacuole
+/ H
+The reverse transport protein vigor significantly strengthens (Apse M P, Aharon G S, Snedden W A, Blumward E.Salt toleranceconferred by overexpression of a vacuolar Na
+/ H
+Antiporter in Arabidopsis.Science.1999,285:1256～1258).Equally, Arabidopis thaliana AtNHX1 gene is changed in the tomato, the transgenosis tomato can normal growth under 200mmol/L NaCl coerces, bloom and solid.Although Na in the blade
+Concentration height, but Na in the tomato fruit
+Concentration very low (Zhang H X, Blumward E.Transgenic salt-tolerant tomato plants accumulate salt in foliage but notin fruit.Nature Biotechnology.2001,19:765～768).These researchs have all shown Na
+/ H
+Reverse transport protein gene and albumen thereof, are considered to be hopeful to utilize its characteristic to cultivate the gene of transgenic salt-tolerant wheat kind at present most for the importance in the high reactant salt plant.
Yet, on production application, the Na that in plant, has cloned at present
+/ H
+The reverse transport protein gene still exists the problem that activity is not high, salt tolerance is not strong, has limited the production application and the popularization of this gene to a great extent.How to utilize specific modern biology technology; clone new strong resistant gene of salt or this gene is carried out molecular evolution and transformation; and then with these new gene transformation in the plant to cultivate the transgenic salt-tolerant wheat new variety of plant, all have great importance for agriculture production, environment protection and ecological management etc.
DNA reorganization (DNA Shuffling) technology is the method rapidly and efficiently of lactam enzyme by directional anagenesis in vitro such as present protein, enzyme and monoclonal antibody, is with a wide range of applications at the aspects such as performance that improve enzymic activity, protein output and improve protein (enzyme).Its principle is the nucleotide sequence random fragmentation that a group is closely related, utilize no primer PCR that these random fragments are re-assemblied then and obtain the nucleotide sequence of total length, introducing suddenlys change and different sudden changes is recombinated widely in this process, thereby finish rapid evolution to the purpose nucleotide sequence, with the function that improves nucleotide sequence or its encoded protein (Stemmer W P.DNA shuffling byrandom fragmentation and reassembly:in vitro recombination for molecularevolution.Proc Natl Acad Sci USA.1994,91:10747-10751).DNA reorganization can be divided into the reorganization of monogenic reorganization and gene family, and the former only carries out fragmentation immediately to individual gene, utilizes no primer PCR to re-assembly, and family's reorganization can be reorganized by gene family close to sibship or that have a long way to go.
The DNA shuffling technology is since being born, successfully several genes such as industrial enzymes, antibody and some important protein etc. have been carried out orthogenesis, specificity, protein function, thermostability that makes activity, substrate specificity and the antibody of enzyme etc. obtained significant raising (Crameri A, Raillard S, Bermudez E, StemmerW P.DNA shuffling of a family of genes from diverse species acceleratesdirected evolution.Nature.1998,391:288).A series of achievements in research have been obtained as this technology of application such as Stemmer, obtain enzyme work and improved 32000 times β-Nei Xiananmei (Stemmer WP.Rapid evolution of a protein in vitro by DNA shuffling.Nature.1994,370:389～391), fluorescence intensity surpasses green fluorescent protein (the Crameri A of 45 times of wild-types, Whitehorn EA, Tate E, Stemmer W P.Improved green fluorescent protein by molecularevolution using DNA shuffling.Nat Biotechnol.1996,14:315～319).By DNA reorganization method, Crameri etc. have successfully obtained the arsenate resistance and have improved 40 times bacterial strain (Crameri A, Dawes G, Rodriguez E J, Silver S, Stemmer W P.Molecular evolution of anarsenate detoxification pathway by DNA shuffling.Nat Biotechnol.1997,15:436～438).But, use this technology and improving Na
+/ H
+Reverse transport protein gene and proteinic aspect of performance thereof there is no report both at home and abroad.
Summary of the invention
The object of the invention is to obtain a kind of new plant strong-salt resistance gene NHXFS 1, and this gene can be encoded and be had the active Na of ion transport
+/ H
+Reverse transport protein.
Another object of the present invention is to provide the Na of said gene coding
+/ H
+Reverse transport protein NHXFS1, the Na of its ion transport specific activity wild-type
+/ H
+Reverse transport protein is stronger.
The present invention also provides the recombinant vectors that contains said gene.
Another aspect of the present invention also provides the host cell that contains above-mentioned recombinant plasmid.
Another aspect of the present invention also provides the methods such as structure, transgenic plant of recombinant vectors, cultivates the stronger transgenic plant new variety of salt tolerance to use said gene and proteins encoded.
Na
+/ H
+Reverse transport protein (Na
+/ H
+Antiporter) in plant salt tolerance, play an important role the salt tolerance of its active affects plant.The present invention is by DNA family shuffling technology, to coding Na
+/ H
+The wild-type resistant gene of salt AtNHX1 of reverse transport protein, OSNHX1, DmNHX1 carry out external molecular evolution, to obtain the higher Na of coding ion transport activity
+/ H
+The new gene NHXFS1 of reverse transport protein.The Na of this new coded by said gene
+/ H
+Reverse transport protein NHXFS1 can be with more Na
+Separating be in vacuole from tenuigenin, and the new gene transformation that obtains in target plant, can be cultivated the stronger transgenic plant new variety of salt resistance ability.
Specifically, by the Na of external DNA shuffling technology to wild-type
+/ H
+The reorganization that suddenlys change of reverse transport protein gene is by the method for having complementary functions, at Na
+/ H
+The active significantly enhanced Na of directed screening ion transport in the yeast mutant of reverse transport protein genetically deficient
+/ H
+The reverse transport protein new gene that suddenlys change.
The present invention relates to a kind of new plant strong-salt resistance gene NHXFS 1, is coding Na
+/ H
+Reverse transport protein NHXFS1 can improve the plant anti-salt ability, is one of following nucleotide sequences:
1) the SEQ ID NO:1 in the sequence table;
2) with sequence table in the nucleotide sequence homology that limits of SEQ ID NO:1 in 70-100% and coding identical function protein DNA sequence;
3) dna sequence dna of protein sequence shown in the SEQ ID NO:2 in the code sequence tabulation.
Preferred plant strong-salt resistance gene NHXFS 1, its nucleotide sequence such as SEQ ID NO:1 are with wild-type Na
+/ H
+Compare between the reverse transport protein gene OsNHX1, the sudden change of 4 Nucleotide is arranged, wherein the 185th and 1059 is VITAMIN B4, and 1221 and 1517 is guanine.
The proteins encoded of said gene is for having the active Na of ion transport
+/ H
+Reverse transport protein NHXFS1 is characterized in that, is one of following amino acid sequences:
1) SEQ ID NO:2 in the sequence table;
2) protein of amino acid sequence homology between 70-100% that SEQ ID NO:2 limits and in the sequence table;
3) in the aminoacid sequence that SEQ ID NO:2 limits, increase, reduce or replace one or several amino acid and have identical active protein.
Preferred amino acids sequence such as SEQ ID NO:2 are with paddy rice wild-type Na
+/ H
+2 amino acid whose changes have been compared between the reverse transport protein OsNHX1, wherein the 62nd is aspartic acid, 506 is glycine, also comprise and SEQ ID NO:2 shown in the protein of amino acid sequence homology between 70-100%, being also included within shown in the SEQ ID NO:2 in the aminoacid sequence increases, reduces or replace one or several amino acid whose protein.
Technical scheme of the present invention is as follows:
1, Na
+/ H
+The clone of reverse transport protein gene: can adopt several different methods such as pcr amplification method, recombination method or synthetic method to obtain Na
+/ H
+Reverse transport protein Nucleotide full length sequence or its fragment.Such as, based on Na
+/ H
+Reverse transport protein gene order design polynucleotide probes screens goal gene from cDNA library or genomic library, also can directly amplify relevant sequence from cDNA or genome with the pcr amplification method.
2, Na
+/ H
+The DNA reorganization of reverse transport protein gene: the substrate that is used for DNA reorganization can be the Na of organism Different Individual, strain system or kind
+/ H
+The reverse transport protein gene also can be the Na of the different mutant forms produced by ordinary methods such as fallibility PCR, rite-directed mutagenesises
+/ H
+The reverse transport protein gene.
To Na
+/ H
+The key step that the reverse transport protein gene carries out DNA reorganization is: at first use DNase I or restriction enzyme to Na
+/ H
+The reverse transport protein gene carries out random fragmentation, and the small segment that reclaims wherein is an overlapping fragments.Then under the situation that does not add primer, with archaeal dna polymerase carry out Primerless PCR make small segment each other primer and template increase, thereby different sudden changes is recombinated widely., PrimerPCR obtains containing reorganization Na after increasing
+/ H
+The recombination storehouse of reverse transport protein gene fragment.
Contain reorganization Na
+/ H
+The recombination storehouse of reverse transport protein gene fragment can be transferred in a kind of cell or the organism and screen as bacterium, yeast or vegetable cell.For example, the recombination storehouse that the present invention can be obtained is connected among any Yeast expression carrier such as the pYPGE15, utilizes method well known in the art such as transformed yeast Na such as lithium acetate technology, electric commentaries on classics method
+/ H
+Among reverse transport protein mutant such as the W303 △ ena1-4 △ nhx1, construction expression recombination storehouse.Yeast transformant being coated in the APG selectivity flat board that contains high NaCl concentration with high salt is that selective pressure is carried out high flux screening, obtains the mutant strain that the salt tolerance significantly improves.
From the yeast mutant that the salt tolerance that screens significantly improves, extract plasmid, obtain anti-high salt Na after the reorganization by order-checking
+/ H
+The nucleotide sequence of reverse transport protein gene, the biosoftware that utilization is conventional such as DNA Star, Clustalx and TMpred etc. are to anti-high salt Na
+/ H
+Reverse transport protein gene and wild-type Na
+/ H
+The reverse transport protein gene carries out bioinformatic analysis such as nucleotide sequence comparison, aminoacid sequence comparison and wetting ability hydrophobicity analysis.The result shows, Na
+/ H
+Reverse transport protein NHXFS1 is than not reorganizing Na
+/ H
+Reverse transport protein has stronger ion transport activity, can be with more Na
+Separating from kytoplasm in vacuole, make the host cell that imports this gene have better salt tolerance.
Na
+/ H
+Reverse transport protein gene NHXFS1 is as goal gene, can be structured in the Ti-plasmid binary vector of any expression of plants such as on the pBISN1, the T-DNA25bp tumor-necrosis factor glycoproteins that LB and RB are wherein arranged, mannosaminic acid synthase promoter (Pmas), polyA no terminator, also have the neomycin phosphotransferase II (nptII) that uses as selective marker in eukaryote, the microbiotic that is used to select is a kantlex.Promotor in the plant expression vector can be any constitutive promoter, tissue-specific promoter or environmental induction type promotor, as cauliflower mosaic virus (CAMV) 35S promoter, Ubiqutin promotor.Enhanser in the carrier both can be a transcriptional enhancer, also can be translational enhancer.For the ease of transgenic plant cells or plant being identified and screening, the marker (as kantlex, weedicide etc.) that the alternative mark (gus gene, luciferase gene etc.) of plant also should be arranged in the carrier or have resistance such as microbiotic.
By research method well known in the art such as freeze-thaw method, electric shocking method etc. above-mentioned recombinant vectors is imported in the Agrobacterium, carry out Agrobacterium-mediated Transformation.Can pass through conventional biotechnological meanss such as microinjection, particle gun, agriculture bacillus mediated or pollen tube channel method with new strong salt tolerant Na
+/ H
+Reverse transport protein gene NHXFS1 changes in the plant, cultivates the kind that anti-salt is best in quality and the improved plant of biological character is new.By the plant transformed host both can be that monocotyledons also can be a dicotyledons, as: Arabidopis thaliana, tobacco, tomato, rape, paddy rice, wheat, corn, soybean, vegetables, trees, flowers, herbage and turfgrass etc.Gene of the present invention is for cultivating the salt-resistant plant kind, and improve crop yield, improve the ecological environment etc. has great importance.
Transgenosis recombinant vectors of the present invention can be used as the kind, strain of commercial use or directly uses on producing or carry out agro-ecology breeding and transgenic plant to improve the salt resistance of crop as genetic resources.
Description of drawings
The NHXFS1 gene that Fig. 1 obtains for reorganization with do not reorganize AtNHX1, OsNHX1, DmNHX1 gene in the two mutant strain W303-1B Δ ena1-4 Δ nhx1 of yeast function complementation experiment relatively, promptly following cell at pH5.5, contain 0, growing state on the APG flat board of 100mM, 200mM, 250mM, 300mM and 350mM NaCl:
Wherein I is the wild-type W303-1B that contains the PYPGE15 plasmid, II is the W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 that contains the PYPGE15 plasmid, III is the W303-1B △ ena1-2::HIS3 that contains the pYPGE15 plasmid, IV is the W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 that contains the AtNHX1-pYPGE15 recombinant plasmid, V is the W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 that contains the OsNHX1-pYPGE15 recombinant plasmid, VI is the W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 that contains the DmNHX1-pYPGE15 recombinant plasmid, VII, VIII are the W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 that contains the NHXFS1-pYPGE15 recombinant plasmid.
Embodiment
Embodiment 1. Arabidopis thalianas, paddy rice, chrysanthemum Na
+/ H
+The clone of reverse transport protein gene
From Arabidopis thaliana, extracted total RNA in paddy rice and the chrythanthemum spire is that masterplate carries out reverse transcription with total RNA with TRizol reagent, synthetic cDNA first chain.With synthetic cDNA is masterplate, with reference to the cDNA sequence of AtNHX1, OsNHX1, DmNHX1, designs following primer (5 ' end comprises SmaI and SalI restriction enzyme site respectively):
AtR(5’-GC
GTCGACTCAAGCCTTACTAAGATCAGGAGG-3’)
AtF(5’-TCC
CCCGGGATGTTGGA?TTCTCTAGTG-3’)
OsR(5’-ACGC
GTCGACTCATCTTCCTCCATGGC-3’)
OsF(5’-TCC
CCCGGGATGGGGATGGAGGTGGCG-3’)
DmR(5’-GC
GTCGACTTAGTTTCTTTCTTCATCTTC-3’)
DmF(5’-TC
CCCCGGGATGGTGTTCGATTC-3’)
Obtain Arabidopis thaliana Na by pcr amplification
+/ H
+Reverse transport protein gene A tNHX1, paddy rice Na
+/ H
+Reverse transport protein gene OsNHX1 and chrysanthemum Na
+/ H
+Reverse transport protein gene DmNHX1 is connected to respectively in the pGM-T carrier, changes escherichia coli DH5a over to, and the clone obtains the sequence of AtNHX1, OsNHX1, DmNHX1, and definite by checking order.
The DNA reorganization of embodiment 2.AtNHX1, OsNHX1, DmNHX1 gene
1) preparation of parent material is a masterplate with pGM-T-AtNHX1, pGM-T-OsNHX1, pGM-T-DmNHX1, with the PfuDNA polysaccharase three genes is carried out pcr amplification, the parent material of reorganizing as DNA behind the purifying.
2) DNase I at random enzyme cut DNA concentration after ultraviolet absorption method is measured purifying, get AtNHX1, OsNHX1, the DmNHX1 that content is 9ug and mix, the amount ratio that makes three genetic stews is 1:1:1.Get the mixed AtNHX1 of 40ul, OsNHX1, DmNHX1 join (10mM Tris-HCl, pH7.5,50mM MnCl in the 50 μ l endonuclease reaction systems
2), 15 ℃ are reacted 10min down.Add DNase I 0.15U, mixing, 15 ℃, 1min16s, 90 ℃, the 10min. product downcuts the segmental gel and the recovery that contain 100-200bp by 2.5% agarose gel electrophoresis.
3) no primer PCR is got the small segment that 1 μ g reclaims, and joins (10 * Taqbuffer, each 0.2mM of dNTP, 0.6U/ μ lTaq polysaccharase) in the 50 μ l reaction systems.The PCR program is a reaction conditions: 94 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30s, 45 ℃ of annealing 1min, 72 ℃ are extended 1min, are total to 50cycles, and last 72 ℃ are extended 5min.
4) there is primer PCR to get product that 5 μ l do not have primer PCR, with AtR and AtF, OsR and OsF, DmR and three pairs of primers of DmF the full length gene sequence of reorganization carried out pcr amplification respectively as template.The PCR reaction system is (10 * Taq damping fluid, each 0.2mM of dNTP, every kind of each 30 μ M of primer, a 0.6U Taq polysaccharase), the PCR response procedures is: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 1min, 58 ℃ of annealing 30s, 72 ℃ are extended 1.5min, are total to 30cycles, and last 72 ℃ are extended 5min.
By DNA reorganization, be that two groups of primer do not amplify band with AtF and AtR, DmF and DmR, and be that primer has obtained to contain reorganization Na with OsR and OsF
+/ H
+The recombination storehouse of reverse transport protein gene fragment.
Embodiment 3. expresses the structure and the anti-high salt Na in reorganization library
+/ H
+The screening of reverse transport protein gene NHXFS1
The recombinant products that embodiment 2 is obtained with Sal I and Sma I carries out enzyme and cuts, be connected to behind the purifying among the Yeast expression carrier pYPGE15 that same enzyme cuts, the expression reorganization library that will make up with the lithium acetate method imports among the yeast mutant W303-1B △ ena1-4::HIS3 △ nhx1::TRP1, being applied to the APG that does not contain uridylic selects on the substratum, 30 ℃, cultivate 2d, screen and contain Na
+/ H
+The yeast strains of reverse transport protein gene, by increasing NaCl concentration the yeast strains that is screened is further screened, final yeast mutation that has obtained a normal growth on the selection substratum of 200mM NaCl concentration, this muton contains new plant strong-salt resistance gene NHXFS 1.
The sequential analysis of embodiment 4. anti-high salt gene NHXFS1
The plasmid that extracts in halophilic yeast mutation with yeast plasmid extraction test kit carries out dna sequencing.With softwares such as DNA Star, Clustalx and TMpred to anti-high salt Na
+/ H
+Reverse transport protein gene and wild-type Na
+/ H
+The reverse transport protein gene carries out nucleotide sequence comparison, aminoacid sequence comparison and wetting ability hydrophobicity analysis.
The result shows, NHXFS1 gene and wild-type Na
+/ H
+The sudden change of 4 Nucleotide is arranged between the reverse transport protein gene OsNHX1, and wherein the 185th guanine is converted to VITAMIN B4, and 1059 thymus pyrimidine transversion are VITAMIN B4, and 1221 thymus pyrimidine transversion are guanine, and 1517 VITAMIN B4 are converted to guanine.NHXFS1 proteins encoded and paddy rice wild-type Na
+/ H
+2 amino acid whose changes are arranged between the reverse transport protein, and the 62nd becomes aspartic acid by glycine, and the 506th becomes glycine by aspartic acid, as sequence table SEQ ID NO:1 and SEQ ID NO:2.
The structure of embodiment 5. recombinant vectorss and host cell
With Sal I and Sma I the recombinant plasmid pGM-T-NHXFS1 that obtains among the embodiment 3 being carried out enzyme cuts, be connected in the Yeast expression carrier pYPGE15 that same enzyme is cut after reclaiming, with the lithium acetate method NHXFS1-pYPGE15 recombinant plasmid that makes up is imported among the yeast double-mutant strain W303-1B △ ena1-4::HIS3 △ nhx1::TRP1, in not containing the APG selective medium of uridylic, screen, obtain positive transformant.
The comparative experiments that has complementary functions of embodiment 6. yeast
Wild-type yeast W303-1B is a kind of yeast with salt tolerance, its mutant strain W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 salt tolerance is very low, the recombinant vectors that will contain the NHXFS1 gene imports this yeast mutant, and comparative experiments can have complementary functions.
With Sal I and Sma I to recombinant plasmid pGM-T-AtNHX1, pGM-T-OsNHX1, pGM-T-DmNHX1 carries out enzyme and cuts, be connected in the Yeast expression carrier pYPGE15 that same enzyme is cut after reclaiming, with the lithium acetate method recombinant plasmid that makes up is imported among the yeast double-mutant strain W303-1B △ ena1-4::HIS3 △ nhx1::TRP1, Yeast expression carrier pYPGE15 with sky imports to wild-type yeast W303-1B respectively simultaneously, among yeast mutant W303-1B △ ena1-2::HIS3 and the W303-1B △ ena1-4::HIS3 △ nhx1::TRP1, in not containing the APG selective medium of uridylic, screen.The positive transformant that screens and the anti-high salt yeast strain that screens be inoculated into respectively in the APG liquid nutrient medium cultivate, with OD
600All adjust to 1.0, carry out 10 times, 100 times and 1000 times of dilutions respectively, respectively get 5 μ l dibblings to the APG flat board of the different salt concn of pH5.5 (0mM, 100mM, 200mM, 250mM, 300mM, 350mM NaCl), 30 ℃, cultivate 2d to a week, observe growing state.
The result lacks Na as shown in Figure 1
+/ H
+The yeast mutant W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 (II) of reverse transport protein gene than the yeast strain W303-1B (III) of wild-type to salt-sensitive many, the yeast mutant W303-1B △ ena1-4::HIS3 △ nhx1::TRP1 (IV, V, VI) that expresses AtNHX1, OsNHX1, DmNHX1 can the complementary zymic salt tolerance of part, and the NHXFS1 gene can be given the stronger salt tolerance of yeast mutant (VII, VIII), and this shows the Na that this reorganization obtains
+/ H
+Reverse transport protein NHXFS1 is than not reorganizing AtNHX1, OsNHX1, DmNHX1 Na
+/ H
+Reverse transport protein has stronger ion transport activity, can be with more Na
+Separating from kytoplasm in vacuole.
Sequence table
＜110〉East China Normal University
＜120〉a kind of new plant strong-salt resistance gene NHXFS 1 and proteins encoded and application
<130>none
<160>2
<170>PatentIn?version?3.3
<210>1
<211>1608
<212>DNA
＜213〉artificial sequence
<400>1
<210>2
<211>536
<212>PRT
＜213〉artificial sequence
<400>2
Claims (5)
1, a kind of new plant strong-salt resistance gene NHXFS 1 is characterized in that, is one of following nucleotide sequences:
1) the SEQ ID NO:1 in the sequence table;
2) with sequence table in the nucleotide sequence homology that limits of SEQ ID NO:1 in 70-100% and coding identical function protein DNA sequence;
3) dna sequence dna of protein sequence shown in the SEQID NO:2 in the code sequence tabulation.
2, a kind of proteins encoded of new plant strong-salt resistance gene NHXFS 1 is for having the active Na of ion transport
+/ H
+Reverse transport protein NHXFS1 is characterized in that, is one of following amino acid sequences:
1) SEQ ID NO:2 in the sequence table;
2) protein of amino acid sequence homology between 70-100% that SEQ ID NO:2 limits and in the sequence table;
3) in the aminoacid sequence that SEQ ID NO:2 limits, increase, reduce or replace one or several amino acid and have identical active protein.
3, the recombinant vectors that contains the described gene of claim 1.
4, the host cell that contains the described gene of claim 1.
5, the described a kind of new application of plant strong-salt resistance gene NHXFS 1 aspect the cultivation salt-resistant plant of claim 1, it is characterized in that, described plant strong-salt resistance gene NHXFS 1 changes in the plant by microinjection, particle gun, method agriculture bacillus mediated or pollen tube channel, cultivates the improved new variety of plant of salt tolerance and biological character.
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| PCT/CN2009/001278 WO2010060270A1 (en) | 2008-11-25 | 2009-11-18 | Novel strongly salt-tolerant gene nhxfs1 of plant, encoding protein and uses thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010060270A1 (en) * | 2008-11-25 | 2010-06-03 | 华东师范大学 | Novel strongly salt-tolerant gene nhxfs1 of plant, encoding protein and uses thereof |
| CN102559702A (en) * | 2012-03-12 | 2012-07-11 | 南京农业大学 | Salt-tolerant gene CcSOS1 and application thereof |
| CN103993020A (en) * | 2014-06-09 | 2014-08-20 | 天津大学 | Strong-salt-tolerant plant gene SseNHX1 as well as encoding protein and application of strong-salt-tolerant plant gene SseNHX1 |
| CN107630023A (en) * | 2017-08-23 | 2018-01-26 | 东北农业大学 | Zhaodong Halomonas saline-resisting and alkaline-resisting gene duf2062 and its authentication method |
| CN111088260A (en) * | 2020-01-16 | 2020-05-01 | 南京农业大学 | Radish salt-tolerant gene RsNHX1 and application thereof |
| CN114107361A (en) * | 2021-11-15 | 2022-03-01 | 南京瑞源生物技术有限公司 | Method for screening salt-tolerant genes of species library yeast strains |
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| CN113444734B (en) * | 2021-08-02 | 2023-03-21 | 内蒙古大学 | Preparation method and application of salt-tolerant transgenic poplar |
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| US20050028235A1 (en) * | 2002-07-12 | 2005-02-03 | Hong-Xia Zhang | Plant fruit with elevated potassium levels |
| WO2004106528A1 (en) * | 2003-06-03 | 2004-12-09 | Cropdesign N.V. | Transgenic monocotyledonous plants overexpressing a nhx protein and having improved growth charcteristics and a method for making the same |
| CN101260403B (en) * | 2008-02-02 | 2010-12-01 | 华东师范大学 | Plant strong salt-resistant gene AtNHXS1 and its coding protein and application |
| CN101413004B (en) * | 2008-11-25 | 2011-02-02 | 华东师范大学 | Novel plant strong-salt resistance gene NHXFS1 and encoding protein |
-
2008
- 2008-11-25 CN CN2008102033112A patent/CN101413004B/en not_active Expired - Fee Related
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2009
- 2009-11-18 WO PCT/CN2009/001278 patent/WO2010060270A1/en active Application Filing
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010060270A1 (en) * | 2008-11-25 | 2010-06-03 | 华东师范大学 | Novel strongly salt-tolerant gene nhxfs1 of plant, encoding protein and uses thereof |
| CN102559702A (en) * | 2012-03-12 | 2012-07-11 | 南京农业大学 | Salt-tolerant gene CcSOS1 and application thereof |
| CN103993020A (en) * | 2014-06-09 | 2014-08-20 | 天津大学 | Strong-salt-tolerant plant gene SseNHX1 as well as encoding protein and application of strong-salt-tolerant plant gene SseNHX1 |
| CN103993020B (en) * | 2014-06-09 | 2016-10-05 | 天津大学 | A kind of plant strong resistant gene of salt SseNHX1 and encoding proteins thereof and application |
| CN107630023A (en) * | 2017-08-23 | 2018-01-26 | 东北农业大学 | Zhaodong Halomonas saline-resisting and alkaline-resisting gene duf2062 and its authentication method |
| CN107630023B (en) * | 2017-08-23 | 2020-03-27 | 东北农业大学 | Salt and alkali resistant gene duf2062 of Zhaodong halomonas and its identification method |
| CN111088260A (en) * | 2020-01-16 | 2020-05-01 | 南京农业大学 | Radish salt-tolerant gene RsNHX1 and application thereof |
| CN114107361A (en) * | 2021-11-15 | 2022-03-01 | 南京瑞源生物技术有限公司 | Method for screening salt-tolerant genes of species library yeast strains |
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| CN101413004B (en) | 2011-02-02 |
| WO2010060270A1 (en) | 2010-06-03 |
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