CN103619872B - A kind of AVP1 albumen of cotton and its encoding gene and application - Google Patents
A kind of AVP1 albumen of cotton and its encoding gene and application Download PDFInfo
- Publication number
- CN103619872B CN103619872B CN201280001630.XA CN201280001630A CN103619872B CN 103619872 B CN103619872 B CN 103619872B CN 201280001630 A CN201280001630 A CN 201280001630A CN 103619872 B CN103619872 B CN 103619872B
- Authority
- CN
- China
- Prior art keywords
- seq
- plant
- nucleotide sequence
- tobacco
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
Abstract
The present invention relates to vegetable protein and its encoding gene and application, more particularly to the AVP1 albumen for deriving from cotton, and its application in drought resistance, the genetically modified plants that salt tolerance improves are cultivated.
Description
Technical field
The present invention relates to vegetable protein and its encoding gene and application, more particularly to an AVP1 egg for deriving from cotton
In vain, and its cultivate drought resistance, salt tolerance improve genetically modified plants in application.
Background technology
Arid is so that the earth land area Planting Crops more than 40% are restricted, to Global Agriculture production and grain
Supply also constitutes serious threat, and 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.Dry half-dried
Since rainfall is few, evaporation is violent, salinity constantly accumulates for drought, arid area；Riviera causes soil to contain due to inwelling
The increase of salt amount.China's saline-alkali soil is distributed mainly on northwest, North China, northeast and coastal region, with the growth year by year of greenhouse area
With the passage in grower's year generation, the soil salinization is on the rise.For most crops, excessive Na+ can be right in soil
The normal growth metabolism of plant produces toxic action.Therefore how to improve crop yield under salt marsh environment just becomes China
The problem of particularly significant in agricultural production.
It is no doubt simple and feasible to use conventional methods selection and breeding salt tolerant, the plant variety of drought resisting, but makes slow progress, limitation
Greatly.With the development of molecular biology technology, large quantities of genes related with plant salt tolerance, drought resisting are cloned in succession, plant
Transgenic technology has important breakthrough, this improves crop yield to efficiently use nonirrigated farmland and salt-soda soil, administers salination and desert
Change soil and provide new idea and method.
The resistance of plant is a sufficiently complex quantitative character, its Mechanisms of Salt Resistance is related to from plant to organ, tissue,
Physiology and biochemistry is 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, polygenes product and answers
Miscellaneous process.These genes and its expression product can be divided into 3 classes:(1) base of signal cascade amplification system and transcription control is participated in
Cause and product；(2) gene and its expression product directly to work to protection biomembrane and protein；（3）With water and ion
Take in and transport relevant gene and protein.The scientist of various countries has also done substantial amounts of work for this, and making a breakthrough property
Progress（Park S.2005.Up-regulation of a H+-pyrophosphatase(H+-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 RegulatingTranscription and Pre-mRNA Splicing through Altering Histone
H4R3 and Small Nuclear Ribonucleoprotein LSM4 Methylation.Plant Cell,23:396–
411）.By animal nutrition, the research for coercing crops, xerophyte and halophytes with tolerance is all taken
Significant achievement was obtained, further understanding there has also been to stress-related genes and signal transduction system.
But for current research situation, since its mechanism is sufficiently complex, many plants are to the biochemistry under adverse circumstance
Still needed to be studied with physiological response mechanism.Research in terms of the function of degeneration-resistant response gene and expression regulation accounts for
Majority, but contact between degeneration-resistant relevant signaling pathways and whole signal transmit network system mechanism need into
One step research.Although many research institutions are obtained all kinds of with degeneration-resistant energy such as certain salt tolerant, drought resistings by modern biotechnology
The genetically modified plants of power, but also it is not up to the standard of industrialization.Therefore in terms of stress resistance of plant is improved, many work need
Do.
The content of the invention
Inventor clones AVP1 genes from cotton, is transformed into tobacco, obtains salt tolerance, the transgenosis that drought resistance improves
Tobacco, has obvious application prospect improving stress resistance of plant research field.
The present inventor has cloned the coding base of an AVP1 albuminoid for cotton using SSH and the RACE method being combined
Cause（GhAVP1-1 genes are named as herein）DNA sequence dna.And find after being conducted into transfer-gen plant, it can obviously improve and turn base
Because of the salt tolerance and drought resistance of plant, and these characters can stablize heredity.
The first aspect of the present invention provides an AVP1 albuminoid for cotton, 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, it contains the nucleotide described in second aspect of the present invention
Sequence, and the nucleotide sequence is operably connected with the expression control sequence of the expression vector.In another implementation
In scheme, the expression vector is rd29A-GhAVP1-1-2300, as shown in Figure 2.
The fourth aspect of the present invention provides a kind of recombinant cell, containing the nucleotide sequence described in second aspect of the present invention or
Recombinant expression carrier described in person's third aspect present invention.In one embodiment, the recombinant cell is recombinational agrobacterium
Cell.
The fifth aspect of the present invention provides a kind of method for improving plant salt endurance and/or drought resistance, including：By the present invention
Recombinant expression carrier described in nucleotide sequence or third aspect present invention described in second aspect imports plant or plant group
Knit and make 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：Effectively producing the bar of plant
Recombination expression of the culture containing the nucleotide sequence described in second aspect of the present invention or described in third aspect present invention carries under part
The plant of body or plant tissue.In one embodiment, the plant is tobacco.
The seventh aspect of the present invention provides the albumen described in first aspect present invention, the nucleosides described in second aspect of the present invention
The recombinant cell described in recombinant expression carrier or fourth aspect present invention described in acid sequence, third aspect present invention is 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 vectors rd29A-GhAVP1-1-2300 builds flow（Fig. 1 a-c）.
The plasmid map of Fig. 2 plant expression vectors rd29A-GhAVP1-1-2300.
The drought resistance growing state of Fig. 3 transgene tobaccos and check plant；It is left：Transfer-gen plant（T1M3-3）；It is right：It is right
According to tobacco 3.
The salt tolerance growing state of Fig. 4 transgene tobaccos and check plant；It is left：Transfer-gen plant（T1M3-13）；It is right：It is right
According to tobacco 13.
Embodiment
AVP1 genes are tonoplast H+Pyrophosphatase gene.
Cotton SSH library constructions under embodiment 1, drought stress：
Specific method is：
Utilize the PCR-select of Clontech companiesTMMethod shown in cDNA Subtraction Kit passes through suppression
Subtractive hybridization method builds subtractive library.Sample is used as using the mRNA of the leaf of the cotton seedling of Osmotic treatment in experimentation
（tester）, control is used as using the mRNA of the leaf of untreated cotton seedling（driver）.Specific steps are summarized as follows：
（1）Material to be tested：
Ji cotton 14（National Cotton mid-term storehouse, obtains unit Cotton research institute, Unified number：ZM-30270）It is seeded into
On sterilized vermiculite, cultivated under the conditions of 25 DEG C, photoperiod 16h/8h, pour 1/2MS culture mediums weekly（9.39mM KNO3,
0.625mM KH2PO4, 10.3mM NH4NO3, 0.75mM MgSO4, 1.5mM CaCl2, 50 μM of KI, 100 μM of H3BO3, 100 μM
MnSO4, 30 μM of ZnSO4, 1 μM of Na2MoO4, 0.1 μM of CoCl2, 100 μM of Na2EDTA, 100 μM of FeSO4）Once.When seedling strain
It is used to test during up to 25-30cm.
（2）Material process：
2 groups will be divided into for examination seedling, every group of 4 basins, per 1 plant of basin.First group is control group, in 25 DEG C, illumination cultivation, normally
Pour.Second group is Osmotic treatment group, and 25 DEG C, illumination cultivation, stop pouring, handle 10 days, timely clip two after being disposed
The blade of group seedling apical 1/3, after liquid nitrogen quick freeze, preserves in -70 DEG C of refrigerators.
（3）Total RNAs extraction：
Control group and the cotton leaf 0.5g of Osmotic treatment group are taken respectively, with plant RNA extraction kit
（invitrogen）Total serum IgE according to specification extraction cotton.Measured with the ultraviolet specrophotometer U-2001 of HITACHI companies
For total serum IgE in the absorbance of 260nm and 280nm, OD260/OD280 ratios are 1.8-2.0, show that total serum IgE purity is higher, are used
The integrality of 1.0% agarose gel electrophoresis detection total serum IgE, the brightness of 28S bands is about 2 times of 18S bands, shows RNA's
Integrality is good.Use Oligotex mRNA purification kits (the purification of polyA+ of Qiagen companies
RNAfrom total RNA) separation mRNA.
（4）Suppressed subtractive hybridization：
In order to have increased access to the validity of EST (unigene), gene is avoided to be turned over without restriction enzyme site and obtained sequence non-
Area is translated, has carried out following suppressed subtractive hybridization.PCR- from the mRNA that previous step obtains according to Clontech companies
The explanation of selectTM cDNA Subtraction Kit obtains cDNA.Use RsaI（Purchased from New England Biolabs）With
HaeIII（Purchased from New England Biolabs）Double-strand cDNA is digested respectively, does two groups of suppressed subtractive hybridizations.First
Group：The double-strand cDNA RsaI digestions that control group, arid group obtain, then carry out suppressed subtractive hybridization；Second group：Control group,
The double-strand cDNA HaeIII digestions that arid group obtains, then carry out suppressed subtractive hybridization.Other steps of suppressed subtractive hybridization
And method is strictly pressed shown in the PCR-selectTM cDNA Subtraction Kit product descriptions of Clontech companies
Method carries out, and finally merges second of PCR product of two groups of forward direction subtractive hybridization cDNA fragments.
（5）The structure of cDNA subtractive libraries and preliminary screening, clone, identification
Second of PCR product of the positive subtractive hybridization cDNA fragments of merging（QIAquick PCR Purification
Kit is purified, purchased from Qiagen）It is connected with pGEM-T Easy (being purchased from Promega kits) carrier, is tried according to pGEM-T Easy
The product description of agent box, comprises the following steps that：Following ingredients are sequentially added with 200ul PCR pipes：Purify the of cDNA fragments
Secondary PCR product 3ul, T4 ligase buffer solution 5ul, pGEM-T Easy carriers lul, T4DNA ligase 1ul, in 4 DEG C of connections
Overnight.10 μ L coupled reaction products are taken, are added in 100 μ L competence e. coli jm109s (being purchased from TAKARA), ice bath
30min, heat shock 60s, ice bath 2min, separately add 250 μ L LB fluid nutrient mediums（1%Tryptone is purchased from OXOID, 0.5%Yeast
Extract is purchased from OXOID, and 1%NaCl is purchased from traditional Chinese medicines）Put in 37 DEG C of shaking tables, 225r/min shakes bacterium 30min, takes 200 μ L bacterium solution kinds
Plant in ampicillin containing 50ug/mL, 40ug/mL X-gal, 24ug/mL IPTG（X-gal/IPTG is purchased from TAKARA）LB
Solid medium（1.5% agar is added in LB fluid nutrient mediums）On culture plate, 37 DEG C of cultivation 18h.Count diameter in culture plate
The clear white and blue colonies number of ＞ 1mm, random 360 white colonies of picking (numbering：Gh-D001 to Gh-D360).Will
360 white colonies choose 96 porocyte culture plates (CORNING) in the LB fluid nutrient mediums containing 50ug/mL ampicillins
In, glycerol adding is saved backup to final concentration 20% in -80 DEG C after 37 DEG C of overnight incubations.With nest-type PRC primer Primer 1
（The PCR-select of Clontech companiesTMCDNA Subtraction Kit kits carry）With Primer 2R
（The PCR-select of Clontech companiesTMCDNA Subtraction Kit kits carry）Bacterium solution PCR amplification is carried out, is obtained
To 292 positive colonies, Invitrogen is being sent to all positive colonies（Shanghai）Trade Co., Ltd is sequenced.
（6）The cDNA sequencing analysis of differential cloning：
After DNA sequencing result is removed carrier and indefinite sequence and unnecessary cDNA, 180 EST are obtained
(unigene).Find that wherein 102 unigene have homologous sequence in GenBank through BlastN（Homology more than 50%）, 33
Bar EST Unknown Functions are hypothesis albumen, separately have 45 not obtain homologous matching, supposition is probably non-in 3 ', 5 ' ends
The shorter sequence of translated region.
The clone of embodiment 2, GhAVP1-1 genes
There is the 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 the coding of the sequence belongs to AVP1 classes
Albumen, is named as GhAVP1-1 genes by the corresponding encoding genes of clone Gh-D087 herein.
According to the GhAVP1-1 genetic fragments obtained, two specific primers are designed, the 5 ' ends as 3 ' RACE are special
Specific primer：
GhAVP1-1GSP1：SEQ ID No：4：
CATCTTGTGC TGCACTTGTTGT
GhAVP1-1GSP2：SEQ ID No：5：
GA CTCCGATGTT ATATCCTCTC
Experimental procedure is operated by kit specification（3’RACE System for Rapid Amplification of
CDNA Ends kits are purchased from invitrogen companies）.
With SEQ ID NO:4 and 3 ' end primer AUAP（Kit carries）, carried out by template of the cDNA of mRNA reverse transcriptions
First round PCR amplification.Comprise the following steps that：Ex Taq are purchased from TAKARA, 50 μ l PCR reaction systems：5μl 10×
ExBuffer, the dNTP of 3 μ l 2.5mM, the cDNA of 2.0 μ l mRNA reverse transcriptions, 1.0 μ l Ex Taq, 10 μM of primer SEQ ID
NO:Each 2.0 μ l of 4 and AUAP, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30s, 58
DEG C annealing 30s, 72 DEG C extension 2min, 33 circulation；72 DEG C of extension 10min.
The PCR product of gained takes 2.0 μ l as template after diluting 100 times by the use of double distilled waters, with SEQ ID NO:5 draw with 3 ' ends
Thing AUAP carries out the second wheel PCR amplification, comprises the following steps that：50 μ l PCR reaction systems：5 μ 10 × Ex of l Buffer, 3 μ
The dNTP of l2.5mM, the 2.0 μ l first round diluted PCR products, 1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO:5 Hes
Each 2.0 μ l of AUAP, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing
30s, 72 DEG C of extension 2min, 33 circulations；72 DEG C of extension 10min.Second of PCR product（QIAquick PCR
Purification Kit are purified, purchased from Qiagen）3u1 is connected to pGEM-T Easy Vector, is transformed into Escherichia coli
JM109 (specific method is same as above), random 10 white colonies of picking are in the LB fluid nutrient mediums containing 50ug/mL ampicillins
Middle culture, glycerol adding to final concentration 20%, -80 DEG C saves backup after 37 DEG C of overnight incubations.SEQ ID NO:5 and 3 ' end primers
AUAP carries out bacterium solution PCR amplification（50 μ l PCR reaction systems：5 μ 10 × Ex of l Buffer, the dNTP of 3 μ l 2.5mM, 2.0 μ l
Bacterium solution, 1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO:Each 2.0 μ l of 5 and AUAP, and the distilled water of 35 μ l.PCR reacts
Condition：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, 33 circulate；72 DEG C of extensions
10min）, 4 positive colonies are obtained, send Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and obtains the 3 ' of the cDNA of the gene
End.
According to the GhAVP1-1 genetic fragments obtained, three specific primers are designed, the 3 ' ends as 5 ' RACE are special
Specific primer：
GhAVP1-1GSP4：SEQ ID No：6：
AGCAACACAT AAGAATAGCT G
GhAVP1-1GSP5：SEQ ID No：7：
GATGGAAGAG CTACCCAACT AAC
GhAVP1-1GSP6：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 primer as mRNA reverse transcriptions into cDNA.
With SEQ ID NO：7 and 5 ' universal primer AAP（Kit carries）, with the cDNA of mRNA reverse transcriptions（Reverse transcription is drawn
Thing SEQ ID NO:6）First round PCR amplification is carried out for template, is comprised the following steps that：Ex Taq are purchased from TAKARA, 50 μ l PCR
Reaction system：The cDNA, 1.0 μ l Ex of the 5 μ l μ l total serum IgE reverse transcriptions of 10 × ExBuffer, the dNTP of 3 μ l 2.5mM, 2.0
Taq, 10 μM of primer SEQ ID NO:Each 2.0 μ l of 7 and AAP, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre-degenerations
5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min, 33 circulate；72 DEG C of extension 10min.
The PCR product of gained takes 2.0 μ l as template after diluting 100 times by the use of distilled water, with SEQ ID NO:8 draw with 3 ' ends
Thing AUAP carries out the second wheel PCR amplification, comprises the following steps that：50 μ l PCR reaction systems：5 μ 10 × Ex of l Buffer, 3 μ
The dNTP of l2.5mM, the 2.0 μ l first round diluted PCR products, 1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO:8 Hes
Each 2.0 μ l of AUAP, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing
30s, 72 DEG C of extension 2min, 33 circulations；72 DEG C of extension 10min.Second of PCR product（QIAquick PCR
Purification Kit are purified, purchased from Qiagen）3u1 is connected to pGEM-T Easy Vector, and it is (specific to be transformed into JM109
Method is same as above), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50ug/mL ampicillins, and 37
Glycerol adding to final concentration 20%, -80 DEG C saves backup after DEG C overnight incubation.SEQ ID NO:8 and 3 ' end primer AUAP carry out bacterium solution
PCR amplification（50 μ l PCR reaction systems：5 μ 10 × Ex of l Buffer, the dNTP of 3 μ l 2.5mM, 2.0 μ l bacterium solutions, 1.0 μ l Ex
Taq, 10 μM of primer SEQ ID NO:Each 2.0 μ l of 8 and AUAP, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre- changes
Property 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, 33 circulate；72 DEG C of extension 10min）, obtain 4 sun
Property clone, send Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and obtains the 5 ' ends of the cDNA of the gene.
After 5 ' the RACE product clonings sequencing of gained, splice with 3 ' RACE products sequencing results.Obtain GhAVP1-1 total lengths
CDNA sequence.It is as follows that pair of primers is designed according to GhAVP1-1 full length cDNA sequences：
GhAVP1-1F：SEQ ID No：9：
ATGGGGGCCTCCTCGATTTTGCCAGATCTC
GhAVP1-1R：SEQ ID No：10：
CTAGAATATC TTAAACAGCA GTCCACCATG TGT
Pass through SEQ ID No：9 and SEQ ID No：10 clone GhAVP1-1 full length genes.
Using the PrimeSTAR HS archaeal dna polymerases of TaKaRa, PCR reactions are carried out by template of the cDNA of cotton.50μl
PCR reaction systems：10 μ 5 × PS of l μ l of Buffer, the dNTP of 3 μ l 2.5mM, 2.0 cDNA, 1.0 μ l PrimeSTAR, 10 μM
Primer SEQ ID No：9 and SEQ ID No：10 each 2.0 μ l, the distilled water of 30 μ l.PCR reaction conditions：94 DEG C of pre-degenerations
5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, after 33 circulations；72 DEG C of extension 10min.
Pcr amplification product adds A.Above-mentioned PCR product is added to 2.5 times of absolute ethyl alcohol, -20 DEG C are placed 10 minutes, and centrifugation, goes
Supernatant, dries, and is dissolved with 21 μ l distilled waters.The dATP of addition 10 × Ex of 2.5ul Buffer, 0.5ul 5mM, 2.5ul 10 ×
Ex Taq.Reaction condition：70 DEG C are reacted 30 minutes.The DNA fragmentation for obtaining about 2300bp is recycled（Omega QIAquick Gel Extraction Kits）,
It is connected to pGEM T-easy carriers（Purchased from Promega kits）, conversion JM109 (method is same as above), random 10 whites of picking
Bacterium colony is cultivated in the LB fluid nutrient mediums containing 50ug/mL ampicillins, and glycerol adding is to final concentration after 37 DEG C of overnight incubations
20%, -80 DEG C save backup.SEQ ID NO:9 and SEQ ID NO:10 carry out bacterium solution PCR amplification（Using TaKaRa's
PrimeSTAR HS archaeal dna polymerases, bacterium solution carry out PCR reactions.50 μ l PCR reaction systems：10 μ 5 × PS of l Buffer, 3 μ l
The dNTP of 2.5mM, 2.0 μ l bacterium solutions, 1.0 μ l PrimeSTAR, 10 μM of primer SEQ ID No：9 and SEQ ID No：10 is each
2.0 μ l, the distilled water of 30 μ l.PCR reaction conditions：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C are prolonged
2min is stretched, after 33 circulations；72 DEG C of extension 10min.）5 positive colonies are obtained, send Invitrogen（Shanghai）Trade Co., Ltd
Sequencing, sequence is SEQ ID NO:2.Its protein expression sequence is SEQ ID NO：1.The amino acid sequence of GhAVP1-1：SEQ
ID No：1
1 MGASSILPDL GAEILIPVCA
21 VIGIAFSLVQ WLLVSKVKLS
41 PGRDSGSPGN NGAGGKNGYS
61 DYLIEEEEGL NDHNVVIKCA
81 EIQTAISEGA TSFLFTEYQY
101 VGIFMVAFAI LIFLFLGSVE
121 GFSTKSQPCT YDKSKMCKPA
141 LATAIFSTVS FLLGAVTSVV
161 SGFLGMKIAT YANARTTLEA
181 RKGVGKAFIT AFRSGAVMGF
201 LLAANGLLVL YIAINLFKLY
221 YGDDWGGLFE AITGYGLGGS
241 SMALFGRVGG GIYTKAADVG
261 ADLVGKVERN IPEDDPRNPA
281 VIADNVGDNV GDIAGMGSDL
301 FGSYAESSCA ALVVASISSF
321 GINHELTPML YPLIISSVGI
341 IVCLITTLFA TDFFEIKAVK
361 EIEPSLKRQL IISTVLMTIG
381 IGIVSWVALP SSFTIFNFGD
401 QKAVKNWQLF LCVAVGLWAG
421 LIIGFVTEYY TSNAYSPVQD
441 VADSCRTGAA TNVIFGLALG
461 YKSCIIPIFA IAISIFVSFS
481 FAAMYGIAVA ALGMLSTIAT
501 GLAIDAYGPI SDNAGGIAEM
521 AGMSHRIRER TDALDAAGNT
541 TAAIGKGFAI GSAALVS LAL
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 F*
The nucleotide sequence of GhAVP1-1 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
2281 GCCACACATG GTGGACTGCT GTTTAAGATA TTCTAG
Embodiment 3GhAVP1-1 gene plant expression vector establishments
Plant expression vector rd29A-GhAVP1-1-2300 structure flows are as shown in Figure 1.
Select plant binary expression vector pCAMBIA2300（Purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）
As plant expression vector, 35S promoter of the NPTII genes containing double enhancers is replaced with Pnos promoters, to reduce NPTII eggs
Expression in plant in vain.Select promoters and terminator of the inducible promoter rd29A and Tnos as GhAVP1-1 genes.
Specific steps are summarized as follows：
SEQ ID NO:11 and SEQ ID NO:12 with plant expression vector PBI121（Have purchased from Beijing China ocean science and technology
Limit company）For template amplification Pnos, using the PrimeSTAR HS archaeal dna polymerases of TaKaRa.50 μ l PCR reaction systems：10μ
5 × PS of l μ l of Buffer, the dNTP of 3 μ l 2.5mM, 1.0 PBI121,1.0 μ l PrimeSTAR, 10 μM of primer SEQ ID
NO:11 and SEQ ID NO:12 each 2.0 μ l, and the distilled water of 31 μ l.PCR reaction conditions：94 DEG C of pre-degeneration 5min；94 DEG C of changes
Property 30s, 56 DEG C annealing 30s, 72 DEG C extension 30s, 33 circulation；72 DEG C of extension 10min.PCR product passes through EcoRI, BglII
（Purchased from New England Biolabs）Digestion is connected to pCAMBIA2300 and obtains pCAMBIA2300-1, coupled reaction condition
See step shown in promega 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's
PrimeSTARHS archaeal dna polymerases.50 μ l PCR reaction systems：10 μ 5 × PS of l Buffer, the dNTP, 1.0 μ of 3 μ l 2.5mM
L PBI121,1.0 μ l PrimeSTAR, 10 μM of primer SEQ ID NO:13 and SEQ ID NO:14 each 2.0 μ l, and 31 μ l
Distilled water.PCR reaction conditions：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30s, 58 DEG C annealing 30s, 72 DEG C extension 30s, 33
Circulation；72 DEG C of extension 10min.PCR product passes through SacI, EcoRI（Purchased from New England Biolabs）Digestion is connected to
PCAMBIA2300-1 obtains pCAMBIA2300-2, and coupled reaction condition is step shown in promega T4 connection enzyme reagent kits.
SEQ ID No：13：
AAGGAGCTCGAATTTCCCCGATCGTTCAAA
SEQ ID No：14：
TCAGAATTCCCAGTGAATT CCCGATCTAG TA
SEQ ID NO:15 and SEQ ID NO:16 with arabidopsis, (Colombia's type, is purchased from
Www.arabidopsis.org) DNA is template（With reference to Zeng J., et L.2002, Preparation of total DNA
from“recalcit rant plant taxa”,Acta Bot.Sin.,44(6):Method extraction arabidopsis in 694-697
DNA）Expand arabidopsis rd29A promoters.Using the PrimeSTARHS archaeal dna polymerases of TaKaRa.50 μ l PCR reaction systems：
10 μ 5 × PS of l μ l arabidopsis DNA of Buffer, the dNTP of 3 μ l 2.5mM, 1.0,1.0 μ l PrimeSTAR, 10 μM of primer
SEQ ID NO:15 and SEQ ID NO:16 each 2.0 μ l, and the distilled water of 31 μ l.PCR reaction conditions：94 DEG C of pre-degenerations
5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, 33 circulate；72 DEG C of extension 10min.PCR product passes through
HindIII、SalI（Purchased from New England Biolabs）Digestion is connected to pCAMBIA2300-2 and obtains pCAMBIA2300-
3, coupled reaction condition is step shown in promega T4 connection enzyme reagent kits.
SEQ ID No：15：
ACTAAGCTTCCTTCTTGACATCATTCAATTTTA
SEQ ID No：16：
TGAgtcgacTCCAAAGATT TTTTTCTTTC CAATAG
SEQ ID No：17 and SEQ ID No：18 amplification AVP1-1 genes,（Template is that embodiment 2 is contained
The pGEM T-easy recombinant vectors of AVP1-1 genes）, using the PrimeSTAR HS archaeal dna polymerases of TaKaRa.50μl PCR
Reaction system：10 μ 5 × PS of l μ l of Buffer, the dNTP of 3 μ l 2.5mM, 1.0 AVP1-1-pGEM, 1.0 μ l PrimeSTAR,
10 μM of primer SEQ ID NO:17 and SEQ ID NO:18 each 2.0 μ l, and the distilled water of 31 μ l.PCR reaction conditions：94℃
Pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, 33 circulate；72 DEG C of extension 10min.PCR is produced
Thing passes through SalI, SacI（Purchased from New England Biolabs）Digestion is connected to pCAMBIA2300-3, obtains plant expression
Carrier rd29A-GhAVP1-1-2300, coupled reaction condition are steps shown in promega T4 connection enzyme reagent kits.
SEQ ID No：17：
TGAGTCGACATGGGGGCCTCCTCGATTTTGCCAGATCTC
SEQ ID No：18：
AAGGAGCT CCTAGAATATC TTAAACAGCA GTCCACCATG TGT
Embodiment 4rd29A-GhAVP1-1-2300 expression vectors convert Agrobacterium
Agrobacterium LBA4404（Purchased from Biovector Science Lab, Inc）It is prepared by competent cell：1-2d will in advance
Agrobacterium LBA4404 is in the LB solid medium tablets containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins（LB fluid nutrient mediums
The agar of middle addition 1.5%）It is upper to draw single spot inoculation, 28 DEG C of cultures 1 to 2d.Picking single bacterium colony is inoculated in 5ml and contains 50 μ g/ml profit good fortune
It is 0.4 that overnight incubation (about 12-16h) to OD600 values are shaken in the LB fluid nutrient mediums of gentle 50 μ g/ml streptomysins, at 28 DEG C,
Form seed bacterium solution.Take the bacterium solution after 5ml activation（1:20 ratio）It is inoculated in the LB liquid training of the same concentration antibiotic of 100ml
Support in base, 28 DEG C are shaken culture 2-2.5h to OD600=0.8.Ice bath bacterium solution 10min, shakes up once every 3min, makes bacterium equal
It is even to enter resting state.4000g centrifuges 10min at 4 DEG C, abandons supernatant；Add certain 10% glycerine resuspension bacterium of head for precooling
Body, 4000g centrifugations 10min, collects precipitation at 4 DEG C；Repeat to wash 3-4 times with 10% glycerine；Add appropriate ice bath precooling 10% is sweet
Again suspended bacterial precipitates oil, is dispensed with 40 μ l/ pipes, is saved backup in -70 DEG C.
Convert Agrobacterium：Melt above-mentioned Agrobacterium LBA4404 competent cell on ice, into the competent cell of 40 μ l
Add expression vector establishment rd29A-GhAVP1-1-2300, ice bath about 10min after mixing prepared by the embodiment 3 of 1 μ l.Will be upper
State mixture to be transferred in the electric shock cup of precooling with rifle, tapping 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, promotes slideway to put electric shock cup to electroporation chamber base
At seat electrode.Use the electric shock cup of 0.1cm, MicroPulser（Purchased from bio-rad）Program be arranged to " Agr ", electric shock one
It is secondary.Electric shock cup is taken out immediately, is added in the LB fluid nutrient mediums of 28 DEG C of preheatings.It is quick and soft to be beaten cell with rifle.Will
Above-mentioned suspension is transferred to the centrifuge tube of 1.5ml, and 28 DEG C, 225rpm cultivates 1h.Take the bacterium solution of 100-200 μ l to be coated with corresponding to resist
Property screening and culturing medium（LB solid mediums, containing 50 μ g/ml rifampins, 50 μ g/ml streptomysins, 50 μ g/ml kanamycins）Tablet
On, 28 DEG C of cultures.
Embodiment 5 obtains transgene tobacco using Agrobacterium-medialed transformation method
With 75% alcohol impregnated 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 to consolidate
Body culture medium（18.78mM KNO3, 1.25mM KH2PO4, 20.6mM NH4NO3, 1.5mM MgSO4, 3.0mM CaCl2, 50 μM
KI, 100 μM of H3BO3, 100 μM of MnSO4, 30 μM of ZnSO4, 1 μM of Na2MoO4, 0.1 μM of CoCl2, 100 μM of Na2EDTA, 100 μ
M FeSO4, 7.4g/L agar, sucrose 30g/L）Upper aseptic germination, prepares aseptic seedling.Tests for sterility is taken to be cut into 5mm × 5mm big
Small leaf dish, with the During Agrobacterium leaf dish 10min containing expression vector that exponential phase is in embodiment 4, blots 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.By agriculture
The blade of bacillus dip dyeing goes to differential medium（MS solid medium+1mg/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, turns seedling after well developed root system
Enter on the MS solid mediums only added with 500mg/L cephalosporins.Meanwhile control blade is gone into differential medium（MS solids
Culture medium+1mg/L BA+0.1mg/LNAA+50mg/L kanamycins+500mg/L cephalosporins）On, cultivate 45 under illumination condition
It or so, cuts after bud is grown up and is transferred to root media（MS solid medium+50mg/L kanamycins+500mg/L cephalos
Mycin）Seedling, is transferred to only added with the MS solid mediums of 500mg/L cephalosporins by middle culture 30 days or so after well developed root system
On.
The small seedling leaf of tobacco of During Agrobacterium is taken, extracts genomic DNA（With arabidopsis DNA extraction sides in embodiment 3
Method）Afterwards, with primer SEQ ID No：17 and SEQ ID No：18 carry out PCR identifications（Ex Taq are purchased from TAKARA, and 50 μ lPCR are anti-
Answer system：5 μ 10 × Ex of l μ l of Buffer, the dNTP of 3 μ l 2.5mM, 2.0 DNA, 1.0 μ l Ex Taq, 10 μM of primer SEQ
ID NO:17 and SEQ ID No：18 each 2.0 μ l, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre-degeneration 5min；94
DEG C denaturation 30s, 58 DEG C annealing 30s, 72 DEG C extension 2min, 33 circulation；72 DEG C of extension 10min）, 20 plants of successful conversions of selection
Transgene tobacco is numbered into T0M1-T0M20。
Sterilized vermiculite is impregnated with 1/2MS culture mediums, by above-mentioned culture T0M1-T0M20 transgene tobaccos seedling and control
Seedling is transplanted on the vermiculite, 25 DEG C, 10 it is small when optical culture/14 it is small when light culture circulate, pour a 1/2MS within every 5 days.About
Seed is obtained after 60 days.
Embodiment 6 is overexpressed drought resisting simulated experiment and the Function Identification in GhAVP1-1 transgene tobacco T1 generations
Sterilized vermiculite is impregnated with 1/2MS culture mediums.By T0For transgene tobacco T0M1、T0M2、T0M3、T0M4 and T0M5
Seed and control tobacco seed sow respectively on vermiculite, 25 DEG C, 10 it is small when optical culture/14 it is small when light culture circulate, every 5
It pours a 1/2MS, after cultivating 25 days, wins bottom leaf, extracts genomic DNA（With arabidopsis in embodiment 3
DNA extraction method）, utilize primer SEQ ID NO：17 and SEQ ID NO：18 do PCR identifications, and (Ex Taq are purchased from TAKARA, 50 μ
L PCR reaction systems：5 μ 10 × Ex of l μ l of Buffer, the dNTP of 3 μ l 2.5mM, 2.0 DNA, 1.0 μ l Ex Taq, 10 μM
Primer SEQ ID NO:17 and SEQ ID No：18 each 2.0 μ l, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre-degenerations
5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, 33 circulate；72 DEG C of extension 10min), reject negative plant
Strain（Control tobacco similarly wins leaf）.Picking transgene tobacco of the same size（T1M1、T1M2、T1M3、T1M4 and
T1Each 10 plants of M5, numbering is T respectively1M1-1 to T1M1-10、T1M2-1 to T1M2-10、T1M3-1 to T1M3-10、T1M4-1 is extremely
T1M4-10 and T1M5-1 to T1M5-10）, control tobacco（10 plants, numbering is to compare 1 to control 10）Do drought-enduring experiment.Transgenosis
Tobacco, control tobacco are arid 14 days (not watering), 25 DEG C, 10 it is small when optical culture/14 it is small when light culture circulate.Then take out plant
Weigh to plant.Above-mentioned T1Show for the Identification of Drought of transfer-gen plant, adjoining tree is all wilted seriously, and transgenosis
Plant can normal growth, show obvious drought resistance（Referring to Fig. 3 and table 1）.In figure 3, transfer-gen plant is chosen
T1M3-3 and control 3 exemplary display of tobacco, the result of other tobaccos are similar with them.
Embodiment 7 is overexpressed salt resistance simulated experiment and the Function Identification in GhAVP1-1 transgene tobacco T1 generations
Sterilized vermiculite is impregnated with 1/2MS culture mediums.By T0For transgene tobacco T0M1、T0M2、T0M3、T0M4 and T0M5
Seed and control tobacco seed sow respectively on vermiculite, 25 DEG C, 10 it is small when optical culture/14 it is small when light culture circulate, every 5
It pours a 1/2MS, after cultivating 25 days, wins bottom leaf, extracts genomic DNA（With arabidopsis in embodiment 3
DNA extraction method）, utilize primer SEQ ID NO：17 and SEQ ID NO：18 do PCR identifications, and (Ex Taq are purchased from TAKARA, 50 μ
L PCR reaction systems：5 μ 10 × Ex of l μ l of Buffer, the dNTP of 3 μ l 2.5mM, 2.0 DNA, 1.0 μ l Ex Taq, 10 μM
Primer SEQ ID NO:17 and SEQ ID No：18 each 2.0 μ l, and the distilled water of 35 μ l.PCR reaction conditions：94 DEG C of pre-degenerations
5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, 33 circulate；72 DEG C of extension 10min), reject negative plant
Strain（Control tobacco similarly wins leaf）.Picking transgene tobacco of the same size（T1M1、T1M2、T1M3、T1M4 and
T1Each 10 plants of M5, numbering is T respectively1M1-11 to T1M1-20、T1M2-11 to T1M2-20、T1M3-11 to T1M3-20、T1M4-11
To T1M4-20 and T1M5-11 to T1M5-20）, control tobacco（10 plants, numbering is to compare 11 to control 20）Salt tolerant experiment is done, is poured
Fill 100mM NaCl, 25 DEG C, 10 it is small when optical culture/14 it is small when light culture circulate, observe result after 14 days：T1For transfer-gen plant
Salt-Tolerance Identification show that adjoining tree is all unable to normal growth, growth substantially is suppressed, and transfer-gen plant grow it is all bright
It is aobvious to be higher than adjoining tree, show obvious salt tolerance（Referring to Fig. 4 and table 1）.In Fig. 4, transfer-gen plant T is chosen1M3-13
It is similar with them with control 13 exemplary display of tobacco, the result of other tobaccos.
Claims (11)
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 there is SEQ ID NO:Nucleotide sequence shown in 2.
4. a kind of recombinant expression carrier, its contain the nucleotide sequence described in Claims 2 or 3 and the nucleotide sequence with
The expression control sequence of the expression vector is operably connected.
5. the recombinant expression carrier of claim 4, the carrier is the rd29A-GhAVP1-1-2300 carriers shown in Fig. 2.
6. a kind of recombinant cell, it contains the nucleotide sequence described in Claims 2 or 3, the restructuring described in claim 4 or 5
Expression vector.
7. the recombinant cell of claim 6, the recombinant cell is restructuring agrobatcerium cell.
8. a kind of method for improving Tobacco Salt and/or drought resistance, including：By the nucleotide sequence described in Claims 2 or 3
Or the recombinant expression carrier described in claim 4 or 5 imports tobacco or Tissues of Tobacco and expresses the nucleotide sequence.
9. a kind of method of prepare transgenosis plant, including：Under conditions of plant is effectively produced culture containing claim 2 or
The plant of the recombinant expression carrier described in nucleotide sequence or claim 4 or 5 described in 3 or plant tissue.
10. the method described in claim 9, wherein the plant is tobacco.
11. the restructuring table described in nucleotide sequence, claim 4 or 5 described in the albumen of claim 1, Claims 2 or 3
It is used to improve Tobacco Salt and drought resistance up to the recombinant cell described in carrier or claim 6 or 7 and is educated for tobacco
The purposes of kind.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2012/076512 WO2013181802A1 (en) | 2012-06-06 | 2012-06-06 | Cotton avp1 protein and coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103619872A CN103619872A (en) | 2014-03-05 |
| CN103619872B true CN103619872B (en) | 2018-04-27 |
Family
ID=49711277
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201280001630.XA Expired - Fee Related CN103619872B (en) | 2012-06-06 | 2012-06-06 | A kind of AVP1 albumen of cotton and its encoding gene and application |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN103619872B (en) |
| WO (1) | WO2013181802A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001033945A1 (en) * | 1999-11-10 | 2001-05-17 | University Of Connecticut | Stress-resistant oversized transgenic plants capable of growing in salinized soil |
| WO2002015674A1 (en) * | 2000-08-18 | 2002-02-28 | University Of Connecticut | Enhanced meristematic activity and competence by overexpression of tonoplast pyrophosphatase |
| CN102002485A (en) * | 2010-10-26 | 2011-04-06 | 复旦大学 | Pyrophosphatase of ammopiptanthus mongolicus, and coding gene and application thereof |
-
2012
- 2012-06-06 WO PCT/CN2012/076512 patent/WO2013181802A1/en active Application Filing
- 2012-06-06 CN CN201280001630.XA patent/CN103619872B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001033945A1 (en) * | 1999-11-10 | 2001-05-17 | University Of Connecticut | Stress-resistant oversized transgenic plants capable of growing in salinized soil |
| WO2002015674A1 (en) * | 2000-08-18 | 2002-02-28 | University Of Connecticut | Enhanced meristematic activity and competence by overexpression of tonoplast pyrophosphatase |
| CN102002485A (en) * | 2010-10-26 | 2011-04-06 | 复旦大学 | Pyrophosphatase of ammopiptanthus mongolicus, and coding gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres.;Paterson AH等;《Nature》;20121220;第492卷(第7429期);全文 * |
| 冀棉14;《作物杂志》;19890215(第01期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2013181802A1 (en) | 2013-12-12 |
| CN103619872A (en) | 2014-03-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103620039B (en) | Hkt protein of cotton and encoding gene thereof and application | |
| CN103842376B (en) | Cotton AVP1 albumen and its encoding gene and application | |
| CN105073773B (en) | A kind of small salt mustard tonoplast pyrophosphatase VP1 and its encoding gene and application | |
| CN103619872B (en) | A kind of AVP1 albumen of cotton and its encoding gene and application | |
| CN105026564B (en) | Cotton ion channel albuminoid and its encoding gene and application | |
| CN105026565B (en) | One cotton ion channel albuminoid and its encoding gene and application | |
| CN103842377B (en) | Cotton AVP1 albumen and its encoding gene and application | |
| CN105408349B (en) | A kind of Bruguiera conjugata molybdenum cofactors vulcanization enzyme MCSU and its encoding gene and application | |
| CN105189535A (en) | Transporter HKT2 with high affinity for potassium ions and which is derived from cotton and the coding gene and use thereof | |
| CN105121458B (en) | A kind of Bruguiera conjugata betaine aldehyde dehydrogenase BADH and its encoding gene and application | |
| CN105452453A (en) | Thellungiella halophila calcineurin b-like protein cbl-4, coding gene of same, and application thereof | |
| CN105008386B (en) | A kind of small salt mustard tonoplast sodium hydrogen antiporter protein NHX2 and its encoding gene and application | |
| CN104379746B (en) | One cotton ion channel albuminoid and its encoding gene and application | |
| CN103703020B (en) | A DREB1 classes transcription factor for cotton and its encoding gene and application | |
| CN105008538B (en) | A kind of cotton isopentene group transferase I PT3 and its encoding gene and application | |
| CN105473719B (en) | A kind of cotton zinc finger protein ZPT5-5 and its encoding gene and application | |
| CN105408350B (en) | A kind of Avicennia marina monooxygenase CMO and its encoding gene and application | |
| CN105073771B (en) | A kind of small salt mustard tonoplast sodium hydrogen antiporter protein NHX1 and its encoding gene and application | |
| CN103857693A (en) | Dreb1-like transcription factor of cotton and coding gene and application thereof | |
| CN103842375B (en) | A DREB1 classes transcription factor for cotton and its encoding gene and application | |
| CN105102622B (en) | A kind of cotton zeaxanthin epoxidase ZEP 1 and its encoding gene and application | |
| CN105073993B (en) | A kind of cotton molybdenum cofactors vulcanization enzyme MCSU 1 and its encoding gene and application | |
| CN105102472B (en) | A kind of cotton zinc finger protein ZPT5-1 and its encoding gene and application | |
| CN104968787A (en) | Cotton homeotic-leucine zipper protein hdbzip-1 and coding gene and use thereof | |
| CN105073774B (en) | A kind of cotton leucine zipper protein bZIP-2 and its encoding gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 518048, the 308 tower of Oriental Pearl science and technology building, Sha Mo Industrial Zone, Shenzhen, Guangdong, Futian District, 4 Applicant after: Biocentury Seed Industry Co., Ltd. Address before: 4 floor of Oriental Pearl Tower of science and technology, 308 Industrial Park, Futian District, Guangdong, Shenzhen Province, China Applicant before: Biocentury Transgene Co., Ltd. |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 518117 7th floor, Chuangshi seed industry building, A701, No.22, Puzi Road, Pingdi street, Longgang District, Shenzhen, Guangdong Province Patentee after: Biocentury Seed Industry Co., Ltd. Address before: 518048, the 308 tower of Oriental Pearl science and technology building, Sha Mo Industrial Zone, Shenzhen, Guangdong, Futian District, 4 Patentee before: Biocentury Seed Industry Co., Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180427 Termination date: 20200606 |