CN103619872A - Cotton AVP1 protein and coding gene and application thereof - Google Patents

Abstract

The present invention provides an AVP1 protein derived from cotton and an application thereof in cultivating transgenic plants with improved drought resistance and salt tolerance.

Description

Cotton AVP1 protein and coding gene and application thereof

Cotton AVP1 albumen and its encoding gene and applied technical field

The present invention relates to vegetable protein and its encoding gene and application, the more particularly to one AVP1 albumen from cotton, and its application in the genetically modified plants that drought resistance, salt tolerance are improved are cultivated.

Background technology

Arid causes the earth land area Planting Crops more than 40% to be restricted, and also constitutes serious threat to Global Agriculture production and staple food supply, arid is to one of main environment restrictive condition of crop yield.

The area of saline-alkali soil is very big in the world, there are about 400,000,000 hectares, accounts for the 1/3 of irrigated farmland.In dry semiarid ＆ arid zones because rainfall is few, evaporation violent, salinity is constantly accumulated；Riviera causes soil salt content to increase due to inwelling.China's saline-alkali soil is distributed mainly on northwest, North China, northeast and coastal region, and with the passage increased year by year with grower's year generation of greenhouse area, the soil salinization is on the rise.For most crops, excessive Na+ can produce toxic action to the normal growth metabolism of plant in soil.Therefore how the problem of crop yield just turns into particularly significant in China's agricultural production is improved under salt marsh environment.

Use conventional methods seed selection salt tolerant, drought resisting plant variety it is no doubt simple and feasible, but make slow progress, limitation is big.With the development of Protocols in Molecular Biology, large quantities of genes relevant with plant salt tolerance, drought resisting are cloned in succession, plant transgenic technology has important breakthrough, this administers salination and Desertification Soil provides new idea and method effectively to improve crop yield using nonirrigated farmland and salt-soda soil.

The resistance of plant is a sufficiently complex quantitative character, and its Mechanisms of Salt Resistance is related to from plant to organ, tissue, Physiology and biochemistry are until each level of molecule.Plant can produce corresponding responsing reaction when being forced, to reduce or eliminate the harm brought to plant.This responsing reaction of plant is one and is related to polygenes, multi signal approach, the complex process of polygenes product.These genes and its expression product can be divided into 3 classes：（1) gene and product of signal cascade amplification system and transcription control are participated in；(2) gene and its expression product directly worked to protection biomembrane and protein；（3) gene and protein related with transhipment to the intake of water and ion.The scientist of various countries has also done substantial amounts of work, and the progress of making a breakthrough property for this（Park S. 2005. Up- regulation of a tf-pyrophosphatase (tf-PPase) as a strategy to engineer drought-resistant crop plants. Proc. Natl. Acad. Sci. USA. 102 : 18830-18835 ; ABE H. 2003. A rabid op sis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcrip tional activato rs in abscisic acid signaling. Plant Cell, 15 : 63-78； Zhang ZL. 2011. Arabidopsis Floral Initiator SKB1 Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing through Altering Hi stone H4R3 and Small Nuclear

l Ribonucleoprotein LSM4 Methylation. Plant Cell, 23 : 396 - 411).By animal nutrition, significant achievement is all achieved to the research for coercing crops, xerophyte and halophytes with tolerance, stress-related genes and signal transduction system there has also been and further understand.

But for current research situation, because its mechanism is sufficiently complex, many plants still need to be studied to the biochemistry under adverse circumstance and physiological response mechanism.Research in terms of the function and expression regulation of degeneration-resistant response gene occupies the majority, but the mechanism of contact between degeneration-resistant related signaling pathways and whole signal transmission network system need further research.Although many research institutions are by modern biotechnology, all kinds of genetically modified plants with anti-adversity abilities such as certain salt tolerant, drought resistings are obtained, the standard of industrialization is also not up to.Therefore in terms of stress resistance of plant is improved, also many needs of work are done.The content of the invention

Inventor clones AVP1 genes from cotton, is transformed into tobacco, obtains the transgene tobacco that salt tolerance, drought resistance are improved, and has obvious application prospect improving stress resistance of plant research field.

The encoding gene that the present inventor has cloned an AVP1 albuminoid for cotton using SSH and the RACE method being combined (is named as GhAVPl-1 genes herein）DNA sequence dna.And find to be conducted into after transfer-gen plant, the salt tolerance and drought resistance of transfer-gen plant are can obviously improve, and these characters can stablize heredity.

The first aspect of the present invention provides an AVP1 albuminoid for cotton, and its sequence is SEQ ID No: 1.

The second aspect of the present invention provides the nucleotide sequence of the albumen described in coding first aspect present invention.Preferably, the nucleotide sequence of encoding said proteins has SEQ ID NO:Nucleotide sequence shown in 2.

The third aspect of the present invention provides a kind of recombinant expression carrier, and it contains the nucleotide sequence described in second aspect of the present invention, and the nucleotide sequence is operably connected with the expression control sequence of the expression vector.In another embodiment, the expression vector is rd29A-GhAVPl-l-2300, as shown in Figure 2.

The fourth aspect of the present invention provides a kind of recombinant cell, contains the recombinant expression carrier described in the nucleotide sequence or third aspect present invention described in second aspect of the present invention.In one embodiment, the recombinant cell is restructuring agrobatcerium cell.

A kind of method that the fifth aspect of the present invention provides improvement plant salt endurance and/or drought resistance, including：Recombinant expression carrier described in nucleotide sequence or third aspect present invention described in second aspect of the present invention is imported into plant or plant tissue and makes the gene expression.In one embodiment, the plant is tobacco.

The sixth aspect of the present invention provides a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the nucleotide sequence or third aspect present invention described in second aspect of the present invention or plant tissue are cultivated under conditions of plant is effectively produced.In one embodiment, the plant is tobacco.

Albumen, the nucleotide sequence described in second aspect of the present invention, the recombinant expression carrier described in third aspect present invention or the recombinant cell described in fourth aspect present invention that the seventh aspect of the present invention is provided described in first aspect present invention are used to improve plant salt endurance and/or drought resistance and the purposes for plant breeding.In one embodiment, the plant is tobacco. Brief description of the drawings

Fig. 1 plant expression vector rd29A- GhAVPl-l-2300 build flow（Scheme la-c).

Fig. 2 plant expression vector rd29A- GhAVPl-1-2300 plasmid map.

The drought resistance growing state of Fig. 3 transgene tobaccos and check plant；It is left：Transfer-gen plant（ M3-3 );It is right：Compare tobacco 3.

The salt tolerance growing state of Fig. 4 transgene tobaccos and check plant；It is left：Transfer-gen plant（ M3-13 );It is right：Compare tobacco 13.Embodiment

AVP1 genes are tonoplast pyrophosphatase genes.

Cotton SSH library constructions under embodiment 1, drought stress：

Specific method is：

Subtractive library is built by suppressed subtractive hybridization method using the method shown in the PCR-select cDNA Subtraction Kit of Clontech companies.MRNA using the leaf of the cotton seedling of Osmotic treatment in experimentation is used as sample（), tester the mRNA using the leaf of untreated cotton seedling is used as control（driver).Specific steps are summarized as follows：

(1) material to be tested：

(National Cotton mid-term storehouse obtains unit Cotton research institute, Unified number to Ji cotton 14：ZM-30270) it is seeded on sterilized vermiculite, is cultivated under the conditions of 25 °C, photoperiod 16h/8h, 1/2MS culture mediums are poured weekly（9.39 mM KN0₃, 0.625 mM KH2P0₄, 10.3 mM MliNOs, 0.75 mM MgS0₄, 1.5 mM CaCl₂, 50 μ Μ ΚΙ, 100 μ M H₃B0₃, 100 μM of MnS0₄, 30 μM of ZnS0₄, 1 μM of Na₂Mo0₄, 0.1 μM of CoCl₂, 100 μM of Na₂EDTA, 100 M FeSO₄) once.It is used to test when seedling plant height reaches 25-30cm.

(2) material process：

2 groups, every group of 4 basins, per 1 plant of basin will be divided into for examination seedling.First group is control group, in 25 °C, illumination cultivation, normal to pour.Second group is Osmotic treatment group, and 25 °C, illumination cultivation stop burning filling, handled 10 days, the blade of two groups of seedling apicals 1/3 of timely clip after being disposed, after liquid nitrogen quick freeze, preserved in -70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and the cotton leaf 0.5g of Osmotic treatment group are taken respectively, use plant RNA extraction kit（Invitrogen the total serum IgE of cotton) is extracted according to specification.Absorbance of the total serum IgE in 260nm and 280nm is determined with the ultraviolet specrophotometer U-2001 of fflTACffl companies, OD260/OD280 ratios are 1.8-2.0, show that total serum IgE purity is higher, the integrality of total serum IgE is detected with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.Use Oligotex mRNA purification kits (the purification of polyA+ RNA of Qiagen companies From total RNA) separation mRNA.

(4) suppressed subtractive hybridization：

In order to have increased access to EST (imig_ene) validity, it is to avoid gene, in non-translational region, has carried out following suppressed subtractive hybridization without restriction enzyme site and obtained sequence.The mRNA obtained from previous step obtains cDNA according to the PCR-selectTM cDNA Subtraction Kit of Clontech companies explanation.Double-strand cDNA is digested respectively with Rsal (being purchased from New England Biolabs) and Haein (being purchased from New England Biolabs), two groups of suppressed subtractive hybridizations are done.First group：The double-strand cDNA Rsal digestions that control group, arid group are obtained, then carry out suppressed subtractive hybridization；Second group：The double-strand cDNA Haelll digestions that control group, arid group are obtained, then carry out suppressed subtractive hybridization.Method shown in the PCR-selectTM cDNA Subtraction Kit product descriptions of other steps and method of suppressed subtractive hybridization strictly by Clontech companies is carried out, and finally merges second of PCR primer of two groups of positive subtractive hybridization cDNA fragments.

(5) structure of cDNA subtractive libraries and preliminary screening, clone, identification

Second of PCR primer of the positive subtractive hybridization cDNA fragments of merging（QIAquick PCR Purification Kit are purified, purchased from Qiagen) it is connected with pGEM-T Easy (being purchased from Promega kits) carrier, according to the product description of pGEM-T Easy kits, comprises the following steps that：Following ingredients are sequentially added with 200ul PCR pipes：Second of PCR primer 3ul, T4 ligase buffer solution 5 ul, pGEM-T Easy carrier l ul, T4 DNA ligase lul of cDNA fragments is purified, is stayed overnight in 4 °C of connections.Take Ι Ο μ Ι ^ coupled reaction products, it is added in Ι Ο Ο μ competence e. coli jm109 (being purchased from TAKARA), ice bath 30min, heat shock 60s, ice bath 2min, separately adding 250 μ Ι Β fluid nutrient mediums, (1% Tryptone is purchased from OXOID, 0.5% Yeast Extract are purchased from OXOID, and 1% NaCl is purchased from traditional Chinese medicines）Put in 37 °C of shaking tables, 225 r/min shake bacterium 30min, take 200 ^ bacterium solutions plant in containing 50 ug/mL ampicillins, the ug/mL IPTG of 40 ug/mL X-gak 24 (X-gal/IPTG be purchased from TAKARA) LB solid mediums (added in LB fluid nutrient mediums 1.5% agar）On culture plate, 37 °C of 18 h of cultivation.Count diameter in culture plate>1 mm clear white and blue colonies number, random 360 white colonies of picking (numbering：Gh-D001 to Gh-D360).360 white colonies are chosen in 96 porocyte culture plates (CORNING) of the LB fluid nutrient mediums containing 50 ug/mL ampicillins, glycerol adding is saved backup to final concentration 20% in -80 °C after 37 °C of overnight incubations.With nest-type PRC bow I things Primer 1, (the PCR-select cDNA Subtraction Kit kits of Clontech companies are carried）(the PCR-select cDNA Subtraction Kit kits of Clontech companies are carried with Primer 2R）Bacterium solution PCR amplifications are carried out, 292 positive colonies is obtained, Invitrogen is being sent to all positive colonies（Shanghai）Trade Co., Ltd is sequenced.

(6) the cDNA sequencing analysis of differential cloning：

DNA sequencing result is removed after carrier and indefinite sequence and unnecessary cDNA, 180 EST (unig are obtained_ene).Find that wherein 102 unigene have homologous sequence in GenBank through BlastN（Homology more than 50%）, 33 EST Unknown Functions or for assume albumen, separately there are 45 not obtain homologous matching, thus it is speculated that to be probably in 3 ', 5' ends untranslated The shorter sequence in area.

The clone of embodiment 2, GhAVPl-1 genes

There is a unigene of homologous sequence above-mentioned, clone Gh-D087 sequences： SEQ ID No: 3：

1 ACCAGTATGT TGGCATTTTC ATGGTTGCTT TTGCAATCTT GATTTTCCTT TTCCTTGGCT

61 CGGTAGAGGG TTTCAGCACA AAGAGCCAGC CTTGCACTTA TGACAAATCT AAGATGTGCA

121 AACCTGCTCT TGCCACTGCT ATATTCAGCA CAGTATCTTT CTTGCTCGGT GCTGTCACTT

181 CAGTAGTTTC TGGCTTTCTT GGGATGAAAA TTGCTACCTA TGCTAATGCT CGAACCACCC

241 TGGAGGCAAG AAAGGGAGTT GGGAAGGCAT TTATTACTGC ATTCAGATCT GGTGCTGTCA

301 TGGGCTTTCT TCTTGCTGCA AATGGTCTTT TGGTGCTTTA CATTGCCATC AACTTATTCA

361 AGCTCTACTA TGGTGATGAC TGGGGTGGTC TTTTTGAGGC AATCACTGGT TATGGACTTG

421 GAGGTTCATC CATGGCGCTT TTTGGAAGAG TTGGTGGAGG CATCTATACA AAAGCTGCTG

481 ATGTGGGTGC TGATCTTGTA GGCAAGGTGG AAAGGAACAT TCCTGAGGAT GACCCTAGAA

541 ACCCAGCTGT GATTGCTGAC AATGTTGGGG ATAATGTTGG TGATATCGCT GGGATGGGAT

601 CTGATCTTTT TGGGTCCTAT GCTGAATCAT CTTGTGCTGC ACTTGTTGTC GCTTCCATCT

661 CTTCTTTTGG CATCAATCAT GAATTGACTC CGATGTTATA TCCTCTCATT ATAAGTTCCG

721 TTGGTATCAT TGTTTGTTTA ATCACCACCT TATTTGCTAC TGATTTCTTT GAGATCAAGG

781 CTGTTAAGGA AATTGAGCCA TCATTAAAGA GGCAGCTTAT CATCTCCACT GTTCTCATGA

841 CTATCGGAAT TGGGATTGTT AGTTGGGTAG CTCTTCCATC TTCCTTTACC ATTTTCAATT

901 TTGGAGATCA GAAAGCTGTG AAGAATTGGC AGCTATTCTT ATGTGTTGCT GTTGGTCTTT

961 GGGCTGGCCT AATTATCGGT TTTGTAACCG AGT

Sequence analysis（Nucleotide sequence is in NCBI Blast) show that the amino acid sequence of coding of the sequence belongs to AVP1 albuminoids, the corresponding encoding genes of clone Gh-D087 are named as GhAVPl-1 genes herein.

According to the GhAVPl-1 genetic fragments obtained, two specific primers are designed, 3 ' RACE 5 ' end primers are used as：

GhAVPl-1 GSP1 : SEQ ID No： 4：

CATCTTGTGC TGCACTTGTTGT

GhAVPl-1 GSP2: SEQ ID No： 5：

GA CTCCGATGTT ATATCCTCTC

Experimental procedure is operated by kit specification（3'RACE System for Rapid Amplification ofcDNAEnds kits are purchased from invitrogen companies）.

With SEQ ID NO:4 bend with 3' ends | and (kit is carried thing AUAP）, the amplification of first round PCR is carried out by template of the cDNA of mRNA reverse transcriptions.Comprise the following steps that:Ex Taq are purchased from TAKARA, 50 μ PCR reaction systems：5 μ Ι Ο χ Ε χ Buffer, 3 μ 2.5mM dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 μ Ex Taq, 10 μ Μ bow | thing SEQ ID NO:Each 2.0 μ 1 of 4 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension.

The PCR primer of gained takes 2.0 μ as template after diluting 100 times with double leavened water, with SEQ ID NO:5 bend with 3' ends | Thing AUAP carries out second and takes turns PCR amplifications, comprises the following steps that：The Ρ Ο reaction systems of 50 μ 1：5 μ Ι Ο χ Ε χ Buffer, 3 μ 2.5mM dNTP, the PCR primer that the 2.0 μ first round diluted, 1.0 l Ex Taq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 5 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension.Second of PCR primer（QIAquick PCR Purification Kit are purified, purchased from Qiagen) 3ul is connected to pGEM-T Easy Vector, it is transformed into e. coli jm109 (specific method is ibid), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50 ug/mL ampicillins, glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO:5 bend with 3' ends | and thing AUAP carries out bacterium solution PCR amplifications（The Ρ Ο reaction systems of 50 μ 1：5 μ Ι Ο χ Ε χ Buffer, 3 μ 2.5mM dNTP, 2.0 μ bacterium solutions, 1.0 μ Ex Taq, 10 μ Μ bow | thing SEQ ID NO:Each 2.0 μ 1 of 5 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension), 4 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of gene 3' ends.

According to the GhAVPl-1 genetic fragments obtained, three specific primers are designed, 5 ' RACE 3 ' end primers are used as：

GhAVPl-1 GSP4: SEQ ID No： 6：

AGCAACACAT AAGAATAGCT G

GhAVPl-1 GSP5: SEQ ID No： 7：

GATGGAAGAG CTACCCAACT AAC

GhAVPl-1 GSP6: SEQ ID No： 8：

TCATGAGAAC AGTGGAGATG AT

Experimental procedure is operated by kit specification（5 ' RACE System for Rapid Amplification of cDNA Ends kits, purchased from invitrogen companies）. SEQ ID No:6 as mRNA reverse transcriptions into cDNA primer.

ffl SEQ ID NO:7 (kit is carried with 5' universal primers AAP）, with cDNA (the reverse transcription primer SEQ ID NO of mRNA reverse transcriptions:6) amplification of first round PCR is carried out for template, comprised the following steps that：Ex Taq are purchased from TAKARA, 50 μ PCR reaction systems：The cDNA of the 5 μ Ι Ο χ Ε χ μ total serum IgE reverse transcriptions of Buffer, 3 μ 2.5mM dNTP, 2.0,1.0 μ Ex Taq, 10 μ Μ bow | thing SEQ ID NO:Each 2.0 μ 1 of 7 and AAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 55 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension.

The PCR primer of gained takes 2.0 μ as template after diluting 100 times with distilled water, with SEQ ID NO:8 bend with 3' ends | and thing AUAP carries out second and takes turns PCR amplifications, comprises the following steps that：50 μ PCR reaction systems：5 μ Ι Ο χ Ε χ Buffer, 3 μ 2.5mM dNTP, the PCR primer that the 2.0 μ first round diluted, 1.0 μ Ex Taq, 10 μ Μ bow | thing SEQ ID NO:Each 2.0 μ 1 of 8 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94V is denatured 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension.Second of PCR primer（QIAquick PCR Purification Kit are purified, purchased from Qiagen) 3ul is connected to pGEM-T Easy Vector, it is transformed into JM109 (specific method is ibid), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50 ug/mL ampicillins, glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO:8 bend with 3' ends | and thing AUAP carries out bacterium solution PCR amplifications（50 μ PCR reaction systems：5 μ Ι Ο χ Ε χ Buffer, 3 μ 2.5mM dNTP, 2.0 μ bacterium solutions, 1.0 μ Ex Taq, 10 μ Μ bow | thing SEQ ID NO:Each 2.0 μ 1 of 8 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of extension lO min), 4 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of gene 5' ends.

After 5 ' the RACE product clonings sequencing of gained, splice with 3 ' RACE products sequencing results.Obtain GhAVPl-1 full length cDNA sequences.A pair of bows are designed according to GhAVPl-1 full length cDNA sequences | thing is as follows：

GhAVPl-lF: SEQ ID No: 9：

ATGGGGGCCTCCTCGATTTTGCCAGATCTC

GhAVPl-lR: SEQ ID No: 10：

CTAGAATATC TTAAACAGCA GTCCACCATG TGT

Pass through SEQ ID No:9 and SEQ ID No:10 clone GhAVPl-1 full length genes.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of cotton.The Ρ Ο reaction systems of 50 μ 1：Ι Ο μ Ι 5 xPS Buffer, 3 μ 1 2.5mM dNTP, 2.0 μ 1 cDNA, Ι Ο μ Ι PrimeSTAR, 10 μ Μ bow I thing SEQ ID No:9 and SEQ ID No:10 each μ 1 of 2.0 μ 1,30 distilled water.PCR reaction conditions：94 °C of pre-degeneration 5min;94 °C of denaturation 30s, 58 °C of annealing 30s, 72 °C of extension 2min, after 33 circulations；72 °C of extension 10min.

Pcr amplification product adds A.Above-mentioned PCR primer is added to 2.5 times of absolute ethyl alcohol, -20 °C are placed 10 minutes, centrifugation is removed supernatant, dried, and is dissolved with the distilled waters of 21 μ 1.Add the mM of 2.5ul Ι Ο χ Ε χ Buffer, 0.5ul 5 dATP, 2.5ul Ι Ο χ Ε χ Taq.Reaction condition：70 °C are reacted 30 minutes.The DNA fragmentation for obtaining about 2300bp is reclaimed（Omega QIAquick Gel Extraction Kits）, it is connected to pGEM T-easy carriers（Purchased from Promega kits）, conversion JM109C methods are ibid), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50 ug/mL ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO :9 and SEQ ID NO:10 carry out bacterium solution PCR amplifications（Using TaKaRa PrimeSTAR HS archaeal dna polymerases, bacterium solution enters performing PCR reaction.The PCR reaction systems of 50 μ 1： ΙΟμΙ 5><PS Buffer, 3 μ 1 2.5mM dNTP, the bacterium solutions of 2.0 μ 1, Ι Ο μ Ι PrimeSTAR, 10 μ Μ bow | thing SEQ ID No:9 and SEQ ID No:10 each μ 1 of 2.0 μ 1,30 distilled water.PCR reaction conditions：94 °C of pre-degeneration 5min;94 °C of denaturation 30s, 58 °C of annealing 30s, 72V extension 2min, after 33 circulations；72 °C of extension 10min.）5 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO: 2.Its protein expression sequence is SEQ ID NO: 1. GhAVPl -1 amino acid sequence： SEQ ID No: 1

1 MGASSILPDL GAEILIPVCA

21 VIGIAFSLVQ WLLVSKVKLS

41 PGRDSGSPGN NGAGGK GYS

61 DYLIEEEEGL DHNWIKCA

81 EIQTAISEGA TSFLFTEYQY

101 VGIFMVAFAI LIFLFLGSVE

121 GFSTKSQPCT YDKSKMCKPA

141 LATAIFSTVS FLLGAVTSW

161 SGFLGMKIAT YANARTTLEA

181 RKGVGKAFIT AFRSGAVMGF

201 LLAANGLLVL YIAINLFKLY

221 YGDDWGGLFE AITGYGLGGS

241 SMALFGRVGG GIYTKAADVG

261 ADLVGKVERN IPEDDPRNPA

281 VIADNVGDNV GDIAGMGSDL

301 FGSYAESSCA ALWASISSF

321 GINHELTPML YPLI ISSVGI

341 IVCLITTLFA TDFFEIKAVK

361 EIEPSLKRQL I ISTVLMTIG

381 IGIVSWVALP SSFTIFNFGD

401 QKAVK WQLF LCVAVGLWAG

421 LI IGFVTEYY TSNAYSPVQD

441 VADSCRTGAA TNVIFGLALG

461 YKSCI IPIFA lAISIFVSFS

481 FAAMYGIAVA ALGMLSTIAT

501 GLAIDAYGPI SDNAGGIAEM

521 AGMSHRIRER TDALDAAGNT

541 TAAIGKGFAI GSAALVSLAL

561 FGAFVSRAAI STVDVLTPKV

581 FIGLLVGAML PYWFSAMTMK

601 SVGSAALKMV EEVRRQFNTI

621 PGLMEGTAKP DYATCVKIST

641 DASIKEMIPP GALVMLTPLI

661 VGIFFGVETL SGVLAGSLVS

681 GVQIAISASN TGGAWDNAKK

701 YIEAGASEHA RTLGPKGSEP

721 HKAAVIGDTI GDPLKDTSGP

741 SLNILIKLMA VESLVFAPFF

761 ATHGGLLFKI p*

The nucleotide sequence of GhAVPl-1 encoding genes： SEQ ID No: 2

1 ATGGGGGCCT CCTCGATTTT GCCAGATCTC GGAGCTGAGA TCTTGATCCC CGTTTGCGCC

61 GTTATTGGAA TTGCCTTTTC TCTTGTACAA TGGCTTCTTG TTTCCAAGGT GAAGCTCTCC 121 CCGGGTCGAG ACTCGGGTTC CCCCGGTAAC AACGGTGCGG GTGGGAAAAA CGGCTATTCC

181 GATTACCTCA TTGAAGAAGA AGAAGGTCTT AATGACCATA ATGTTGTTAT TAAATGTGCT

241 GAAATTCAGA CCGCCATATC TGAAGGAGCA ACCTCATTTC TTTTCACCGA GTACCAGTAT

301 GTTGGCATTT TCATGGTTGC TTTTGCAATC TTGATTTTCC TTTTCCTTGG CTCGGTAGAG

361 GGTTTCAGCA CAAAGAGCCA GCCTTGCACT TATGACAAAT CTAAGATGTG CAAACCTGCT

421 CTTGCCACTG CTATATTCAG CACAGTATCT TTCTTGCTCG GTGCTGTCAC TTCAGTAGTT

481 TCTGGCTTTC TTGGGATGAA AATTGCTACC TATGCTAATG CTCGAACCAC CCTGGAGGCA

541 AGAAAGGGAG TTGGGAAGGC ATTTATTACT GCATTCAGAT CTGGTGCTGT CATGGGCTTT

601 CTTCTTGCTG CAAATGGTCT TTTGGTGCTT TACATTGCCA TCAACTTATT CAAGCTCTAC

661 TATGGTGATG ACTGGGGTGG TCTTTTTGAG GCAATCACTG GTTATGGACT TGGAGGTTCA

721 TCCATGGCGC TTTTTGGAAG AGTTGGTGGA GGCATCTATA CAAAAGCTGC TGATGTGGGT

781 GCTGATCTTG TAGGCAAGGT GGAAAGGAAC ATTCCTGAGG ATGACCCTAG AAACCCAGCT

841 GTGATTGCTG ACAATGTTGG GGATAATGTT GGTGATATCG CTGGGATGGG ATCTGATCTT

901 TTTGGGTCCT ATGCTGAATC ATCTTGTGCT GCACTTGTTG TCGCTTCCAT CTCTTCTTTT

961 GGCATCAATC ATGAATTGAC TCCGATGTTA TATCCTCTCA TTATAAGTTC CGTTGGTATC

1021 ATTGTTTGTT TAATCACCAC CTTATTTGCT ACTGATTTCT TTGAGATCAA GGCTGTTAAG

1081 GAAATTGAGC CATCATTAAA GAGGCAGCTT ATCATCTCCA CTGTTCTCAT GACTATCGGA

1141 ATTGGGATTG TTAGTTGGGT AGCTCTTCCA TCTTCCTTTA CCATTTTCAA TTTTGGAGAT

1201 CAGAAAGCTG TGAAGAATTG GCAGCTATTC TTATGTGTTG CTGTTGGTCT TTGGGCTGGC

1261 CTAATTATCG GTTTTGTAAC CGAGTACTAC ACTAGCAATG CATACAGCCC TGTACAAGAC

1321 GTTGCTGATT CCTGCAGGAC TGGAGCAGCG ACTAATGTTA TTTTCGGCCT TGCTTTGGGT

1381 TACAAGTCTT GCATTATTCC TATTTTTGCC ATTGCAATCA GTATTTTTGT TAGTTTCAGC

1441 TTTGCAGCTA TGTATGGCAT TGCTGTTGCT GCCCTTGGAA TGCTGAGCAC CATAGCTACT

1501 GGATTGGCTA TTGATGCTTA TGGTCCCATC AGTGATAATG CTGGAGGCAT TGCTGAGATG

1561 GCTGGCATGA GCCACAGAAT TCGAGAGAGA ACTGATGCTC TTGATGCTGC AGGAAACACC

1621 ACTGCTGCTA TTGGAAAGGG TTTCGCCATT GGTTCAGCAG CCCTTGTGTC CCTTGCCCTC

1681 TTTGGTGCCT TTGTGAGCCG TGCTGCTATT TCAACAGTTG ATGTATTGAC CCCTAAAGTT

1741 TTTATTGGGT TGCTTGTCGG GGCAATGCTT CCTTACTGGT TCTCTGCTAT GACCATGAAG

1801 AGTGTGGGAA GTGCTGCTTT GAAGATGGTT GAGGAAGTGC GTCGGCAATT TAACACGATC

1861 CCAGGTCTCA TGGAGGGCAC TGCTAAGCCA GACTATGCTA CCTGTGTTAA GATCTCTACT

1921 GATGCTTCCA TCAAAGAAAT GATCCCACCT GGTGCGCTAG TTATGCTCAC ACCCCTTATT

1981 GTTGGGATCT TTTTTGGTGT TGAAACTCTC TCTGGTGTCC TCGCTGGATC CCTCGTCTCT

2041 GGCGTCCAGA TTGCTATCTC CGCATCGAAC ACAGGGGGTG CTTGGGATAA TGCTAAGAAG

2101 TATATCGAGG CCGGTGCTTC AGAACATGCA AGAACTCTTG GTCCCAAAGG ATCAGAACCA

2161 CATAAGGCAG CTGTTATCGG TGACACCATT GGGGACCCTT TAAAGGATAC ATCCGGACCA

2221 TCACTGAACA TCCTCATCAA GCTAATGGCT GTTGAATCAC TTGTCTTTGC ACCCTTCTTC

The GhAVPl-1 gene plant expression vector establishments of 2281 GCCACACATG GTGGACTGCT GTTTAAGATA TTCTAG embodiments 3

It is as shown in Figure 1 that plant expression vector rd29A- GhAVPl-1-2300 build flow.

Plant binary expression vector PCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoter that NPTII genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.Inducible promoter rd29A and Tnos is selected as the promoter and terminator of GhAVPl-1 genes. Specific steps are summarized as follows：

SEQ ID NO:11 and SEQ ID NO:12 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector PBI 121）For template amplification Pnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1：10 μ 15 X PS Buffer, 3 μ 12 5mM dNTP, the PrimeSTAR of 1. 0 μ, 1 121,1. 0 μ of PBI 1,10 μ Μ primer SEQ ID NO:11 and SEQ ID NO:12 each 2. 0 μ 1, and 31 μ 1 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 56 °C of annealing 30 s, 72 °C of 30 s of extension, 33 circulations；72 °C of 10 min of extension.PCR primer is connected to pCAMBIA2300 by EcoRI, Bglll (being purchased from New England Biolabs) digestion and obtains pCAMBIA2300-l, and coupled reaction condition is shown in step shown in pr omega T4 ligase boxes.

SEQ ID NO: 11 ：

GCAC GAATTC ATACAAATGGACGAACGGAT

SEQ ID NO: 12：

ATCC AGATCT AGATCCGGTGCAGATTATTTG

SEQ ID No:13 and SEQ ID No_:14 using PBI121 as template amplification Tnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：The 10 μ 5xPS μ of Buffer, 3 μ 2.5mM dNTP, 1.0 PBI121,1.0 ^1^1^8 fourths, 10 (the ^ ^^0 of 8 ￡ of primer 0 10:13 and 8 ￡ 0,11) ^^0:It is 14 each 2. (^1, and 31 μ 1 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 30 s of extension, 33 circulations；72 °C of 10 min of extension.PCR primer is connected to PCAMBIA2300-1 by Sacl, EcoRI (being purchased from New England Biolabs) digestion and obtains pCAMBIA2300-2, and coupled reaction condition is step shown in pr omega T4 connection enzyme reagent kits.

SEQ ID No: 13：

AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID No: 14：

TCAGA¾TTCCCAGTGAATT CCCGATCTAG TA

SEQ ID NO:15 and SEQ ID NO:16 with arabidopsis (Colombia's type, purchased from www.arabidopsis.org) DNA is template (with reference to Zeng I, et L. 2002, Preparation of total DNA from " recalcit rant plant taxa ", Acta Bot. Sin., 44 (6):Method in 694-697 extracts arabidopsis DNA) amplification arabidopsis rd29 A promoters.Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems： 10 μΐ 5><The μ Μ of the PS μ arabidopsis of Buffer, 3 μ 2.5mM dNTP, 1.0 DNA, 1.0 ^ PrimeSTAR 10 primer SEQ ID NO:15 and SEQ ID NO:16 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 30 s of extension, 33 circulations；72 °C of 10 min of extension.PCR primer is connected to pCAMBIA2300-2 by HindIII, Sail (being purchased from New England Biolabs) digestion and obtains pCAMBIA2300-3, and coupled reaction condition is step shown in promega T4 connections enzyme reagent kit Suddenly.

SEQ ID No: 15：

ACTAAGCTTCCTTCTTGACATCATTCAATTTTA

SEQ ID No: 16：

TGAgt cga cTCCAAAGATT TTTTTCTTTC CAATAG

SEQ ID No:17 and SEQ ID No:18 amplification AVPl-1 genes,（Template is that embodiment 2 obtains the pGEM T-easy recombinant vectors containing AVP 1-1 genes）, using TaKaRa PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1：10 μ 1 5XPS Buffer, 3 μ 1 2. 5mM dNTP, 1. 0 μ 1 AVPl-l-pGEM, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:17 and SEQ ID NO:18 each 2. 0 μ 1, and 31 μ 1 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension.PCR primer is connected to PCAMBIA2300-3 by Sall, Sac I (being purchased from New England Biolabs) digestion, plant expression vector rd29A-GhAVPl-l_2300 is obtained, coupled reaction condition is step shown in pr omega T4 connection enzyme reagent kits.

SEQ ID No: 17：

TGAGTCGACATGGGGGCCTCCTCGATTTTGCCAGATCTC SEQ ID No: 18：

AAGGAGCTC CTAGAATATC TTAAACAGCA GTCCACCATG TGT

The rd29A-GhAVPl-l-2300 expression vectors of embodiment 4 convert Agrobacterium

It is prepared by Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：In advance l_2d by Agrobacterium LBA4404 in the LB solid medium flat boards containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins（The agar of addition 1.5% in LB fluid nutrient mediums）It is upper to draw single spot inoculation, 28 °C of cultures 1 to 2d.Picking single bacterium colony is inoculated in LB fluid nutrient mediums of the 5ml containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins, and it is 0. 4 that overnight incubation (about 12_16h) to 0D600 values are shaken under 28 °C, forms seed bacterium solution.Take the bacterium solution after 5ml activation（ 1：20 ratio）In the LB fluid nutrient mediums for being inoculated in the same concentration antibiotic of 100ml, 28 °C are shaken culture 2-2. 5h to 0D600=0. 8.Ice bath bacterium solution 10min, shakes up once every 3min, makes bacterium even into resting state.In 4 °C of lower 4000g centrifugations 10min, supernatant is abandoned；Certain glycerine resuspension thalline of head for precooling 10% is added, 4 °C of lower 4000g centrifugations 10min collect precipitation；Repeated to wash 3-4 times with 10% glycerine；Adding 10% glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt above-mentioned Agrobacterium LBA4404 competent cell on ice, expression vector establishment rd29A-GhAVPl-l-2300, the min of ice bath about 10 after mixing prepared by 1 μ 1 embodiment 3 are added into 40 μ 1 competent cell.Will Said mixture is transferred in the electric shock of precooling cup with rifle, and rapping makes mixture reach bottom, has been careful not to bubble.To be shocked by electricity cup（Purchased from bio-rad, model Micropulser) it is put on the slideway of electroporation chamber, promote slideway to put electric shock cup to electroporation chamber base electrode.Using 0. lcm electric shock cup, MicroPulser (being purchased from bio-rad) program is set to " Agr ", and electric shock is once.Take out immediately in electric shock cup, the LB fluid nutrient mediums for adding 28 °C of preheatings.It is quickly soft to be beaten cell with rifle.Above-mentioned suspension is transferred to 1.5ml centrifuge tube, 28 °C, 225rpm cultures lh.100-200 μ 1 bacterium solution is taken to be coated with and corresponding resistance screening culture medium (LB solid mediums, containing 50_μ§The rifampins of/ι η 1,50_μ§The streptomysins of/ι η 1,50_μ§The kanamycins of/ι η 1）On flat board, 28 °C of cultures.Embodiment 5 obtains transgene tobacco using Agrobacterium-medialed transformation method

With 75% alcohol-pickled tobacco（Countries tobacco mid-term storehouse, obtains tobacco institute of unit the Chinese Academy of Agricultural Sciences, storehouse numbering I5A00660) seed 30s, then 8min is soaked with 0.1% mercuric chloride, carry out surface sterilization.Sterilized tobacco seed is placed in MS solid cultures (18.78 mMKN0₃, 1.25 mMKH₂P0₄, 20.6 mMMliNOs, 1.5mMMgS0₄, 3.0mMCaCl₂, 50μΜΚΙ, 100μΜΗ₃ΒΟ₃, 100 MMnSO₄, 30 MZnSO₄, 1 MNa₂Mo0₄, 0.1 MCoCl₂, 100 MNa₂EDTA, 100 MFeSO₄, 7.4 g/L agar, sucrose 30g/L) on aseptic germination, prepare aseptic seedling.Take tests for sterility to be cut into the leaf dish of 5mm X 5mm sizes, with the During Agrobacterium leaf dish lOmin containing expression vector that exponential phase is in embodiment 4, blot bacterium solution, co-cultured 2 days under dark condition（MS solid mediums)；And not to be used as control blade by the leaf dish of During Agrobacterium.The blade of During Agrobacterium is gone into differential medium（MS solid medium+lmg/L BA+0. lmg/L NAA+50mg/L kanamycins+500mg/L cephalosporins）On, cultivated 45 days or so under illumination condition, cut after bud is grown up and be transferred to root media（MS solid medium+50mg/L kanamycins+500mg/L cephalosporins）Middle culture 30 days or so, after being transferred to seedling after well developed root system on the MS solid mediums only added with 500mg/L cephalosporins.Meanwhile, control blade is gone into differential medium（MS solid medium+lmg/L BA+0.1mg/L NAA+50mg/L kanamycins+500mg/L cephalosporins）On, cultivated 45 days or so under illumination condition, cut after bud is grown up and be transferred to root media (MS solid medium+50mg/L kanamycins+500mg/L cephalosporins）Middle culture 30 days or so, after being transferred to seedling after well developed root system on the MS solid mediums only added with 500mg/L cephalosporins.

The small seedling leaf of tobacco of During Agrobacterium is taken, extracts after genomic DNA (arabidopsis DNA extraction method in be the same as Example 3), uses primer SEQIDNo:17 and SEQIDNo:18 enter performing PCR identification（Ex Taq are purchased from TAKARA, 50 μ PCR reaction systems：The 5 μ Ι Ο χ Ε χ μ of Buffer, 3 μ 2.5mM dNTP, 2.0 DNA, 1.0 μ Ex Taq, 10 μ Μ primer SEQIDNO:17 and SEQIDNo:18 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of extension lOmin), the transgene tobacco for choosing 20 plants of successful conversions numbers into T_QM1-T_QM20。

Sterilized vermiculite is impregnated with 1/2MS culture mediums, by above-mentioned culture T_QM1-T_QM20 transgene tobaccos seedling and control seedling are transplanted on the vermiculite, 25 °C, optical culture/14 hour light culture circulation in 10 hours, burn a 1/2MS within every 5 days.Greatly Seed is obtained after about 60 days.Embodiment 6 is overexpressed drought resisting simulated experiment and the Function Identification in GhAVPl-1 transgene tobacco T1 generations

Sterilized vermiculite is impregnated with 1/2MS culture mediums.By T₀For transgene tobacco T₀M1、 T₀M2、 T₀M3、 T₀M4 standing grain mouthful T₀M5 seed and control tobacco seed is sowed on vermiculite respectively, 25 °C, optical culture/14 hour light culture circulation in 10 hours, pour a 1/2MS within every 5 days, after culture 25 days, win bottom leaf, extract genomic DNA (arabidopsis DNA extraction method in be the same as Example 3）, utilize primer SEQ ID NO:17 and SEQ ID NO:18 do PCR identifications, and (Ex Taq are purchased from TAKARA, the Ρ Ο reaction systems of 50 μ 1：5 μ 10xEx μ DNA, Ι Ο the μ Ι Ε χ of Buffer, 3 μ 2.5mM dNTP, 2.0 Taq, 10 μ Μ primer SEQ ID NO:17 and SEQ ID No:18 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension), reject negative plant（Control tobacco similarly wins leaf）.(each 10 plants of 2 Τ ι Μ 314 and T^MS of Τ ι Μ Κ Τ ι Μ, number is respectively picking transgene tobacco of the same size！^ ^ are extremely！^ ^), TiM2-l Τ ι Μ 2-10 1^^13-1 to 1^^13-10, Μ Φ Ι to TiM^lO and T^MS-l to 1 5-10), control tobacco（10 plants, numbering is that 10) control 1 to control does drought-enduring experiment.Transgene tobacco, arid 14 days (not the watering) of control tobacco, 25 °C, optical culture/14 hour light culture circulation in 10 hours.Plant pair plant is then taken out to be weighed.The Identification of Drought of above-mentioned 1 generation transfer-gen plant shows that adjoining tree is all wilted seriously, and transfer-gen plant can normal growth, show obvious drought resistance（Referring to Fig. 3 and table 1).In figure 3, transfer-gen plant and the control exemplary display of tobacco 3 are chosen, the result of other tobaccos is similar with them.Embodiment 7 is overexpressed salt resistance simulated experiment and the Function Identification in GhAVPl-1 transgene tobacco T1 generations

Sterilized vermiculite is impregnated with 1/2MS culture mediums.By T₀For transgene tobacco T₀M1、 T₀M2、 T₀M3、 T₀M4 standing grain mouthful T₀M5 seed and control tobacco seed is sowed on vermiculite respectively, 25 °C, optical culture/14 hour light culture circulation in 10 hours, pour a 1/2MS within every 5 days, after culture 25 days, win bottom leaf, extract genomic DNA (arabidopsis DNA extraction method in be the same as Example 3）, utilize primer SEQ ID NO:17 and SEQ ID NO:18 do PCR identifications, and (Ex Taq are purchased from TAKARA, 50 μ PCR reaction systems：The 5 μ Ι Ο χ Ε χ μ of Buffer, 3 μ 2.5mM dNTP, 2.0 DNA, 1.0 μ Ex Taq, 10 μ Μ primer SEQ ID NO:17 and SEQ ID No:18 each 2.0 μ 1, and 35 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5;94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, 33 circulations；72 °C of 10 min of extension), reject negative plant（Control tobacco similarly wins leaf）.Picking transgene tobacco of the same size（TiMl, T M2, Τ ι Μ 314 and each 10 plants of T^MS, numbering is respectively！^ is extremely！^ ^., 1 12-11 to 1 12-20, Τ ι Μ 3-11 to 1 3-20, TiM^ll to 1 4-20 and TiMS-ll to 1 5-20), control tobacco（10 plants, numbering is that 20) control 11 to control does salt tolerant experiment, pours lOO mMNaCl, 25 °C, optical culture/14 hour light culture circulation in 10 hours observe result after 14 days：Show for the Salt-Tolerance Identification of transfer-gen plant, adjoining tree is all unable to normal growth, growth is substantially suppressed, and Transfer-gen plant is grown all apparently higher than adjoining tree, shows obvious salt tolerance（Referring to Fig. 4 and table 1).In Fig. 4, transfer-gen plant M3-13 and the control exemplary display of tobacco 13 are chosen, the result of other tobaccos is similar with them.

Claims (10)

1. Claims

   1. a kind of albumen, the albumen is cotton AVP1 albuminoids, its sequence is SEQ ID No: 1.

   2. encode the nucleotide sequence of the albumen described in claim 1.

   3. the nucleotide sequence described in claim 2, it is characterised in that with SEQ ID NO:Nucleotide sequence shown in 2.

   4. a kind of recombinant expression carrier, it contains the nucleotide sequence described in Claims 2 or 3 and the nucleotide sequence is operably connected with the expression control sequence of the expression vector；Preferably, the carrier is the rd29A-GhAVPl-l-2300 carriers shown in Fig. 2.

   5. a kind of recombinant cell, it contains the gene order described in Claims 2 or 3, the recombinant expression carrier described in claim 4, it is preferable that the recombinant cell is restructuring agrobatcerium cell.

   6. a kind of method of improvement plant salt endurance and/or drought resistance, including：Recombinant expression carrier described in nucleotide sequence or claim 4 described in Claims 2 or 3 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is tobacco.

   7. a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the nucleotide sequence or claim 4 described in claim 2 or 3 or plant tissue are cultivated under conditions of plant is effectively produced.

   8. the method described in claim 7, wherein the plant is tobacco.

   9. the recombinant expression carrier described in nucleotide sequence, claim 4 described in the albumen of claim 1, Claims 2 or 3 or the recombinant cell described in claim 5 are used to improve plant salt endurance and drought resistance and the purposes for plant breeding, it is preferred that, the plant is tobacco.

   10. the purposes described in claim 9, wherein the plant is tobacco.