CN106754961A - A kind of regulation plant is to arid induced signal calcium ion and the gene of hormone abscisic acid reaction - Google Patents
A kind of regulation plant is to arid induced signal calcium ion and the gene of hormone abscisic acid reaction Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 title abstract description 16
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 title abstract description 8
- 229910001424 calcium ion Inorganic materials 0.000 title abstract description 8
- 239000005556 hormone Substances 0.000 title abstract description 8
- 229940088597 hormone Drugs 0.000 title abstract description 8
- 230000033228 biological regulation Effects 0.000 title abstract description 4
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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
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Abstract
The invention discloses a kind of regulation plant to arid induced signal calcium ion and the gene of hormone abscisic acid reaction, specifically, it is related to the clone of gene related to drought tolerance Sv4317 and functional expression mode, by the preparation and treatment, the extraction of RNA and the reverse transcription of cDNA of material, carry out cloning drought resisting Sv4317 genes using PCR, the gene is imported in arabidopsis wild plant by transgenic approach, by screen resistance seedling it is final to T3For homozygosis seedling, genetically modified plants and wild type control plants are carried out into different element phenotypic analyses, it can be found that genetically modified plants have more sensitive phenotype for mineral element calcium and arid hormone ABA.
Description
Technical field
The invention belongs to plant biotechnology field.Sabina vulgaris belongs to strong drought life and the barren plant of resistance to soil nutrient, extensively
Northern China desertificated area is applied to check winds and fix drifting sand in ecological engineering.The present invention is first with based on Illumina Solexa
Technology platform, the sequencing of high flux transcript profile has been carried out to Sabina vulgaris root RNA.Because calcium is that plant sensing and resistance adverse circumstance are most important
Signaling molecule, we have carried out combing to there is the protein-bonded gene of coded Ca in Sabina vulgaris transcript profile.Further to wherein
Sequence code name is that the gene of Sv4317 is analysed in depth and studied.Bioinformatics finds that Sv4317 may encode a Ca and turn
Fortune albumen.For the function of clear and definite this gene, this gene is intended south by us by the method for genetic engineering in model plant
Expressed in mustard.Phenotypic analysis discovery is carried out to transgenic arabidopsis, the arabidopsis thaliana containing Sv4317 genes, relatively
In the normal plants for not containing this gene, show to low calcium and low concentration arid sensing hormone abscisic acid (ABA) hypersensitization
Phenomenon.This explanation Sv4317 has regulation plant to arid induced signal calcium ion and arid sensing hormone abscisic acid reaction
Function, with the potentiality being applied in regulation crop senses to both arid coherent signals, genetic breeding work.
Background technology
Sabina vulgaris (Sabina vuLgaris.), also known as savin juniper, savin, be distributed mainly on the Inner Mongol, Shaanxi,
The ground such as Xinjiang, Ningxia, Gansu, Qinghai.There are the ground such as Jiangsu, Zhejiang, Anhui, Hunan main cultivation base.Sabina vulgaris can stand wind
Erosion sand is buried, the long-term desert Environment for adapting to arid, is the fine tree species that arid, semiarid zone are checked winds and fixed drifting sand with water and soil conservation.
Light, happiness feel nice and cool dry weather, it is cold-resistant, drought-enduring, barren-resistant, to soil requirement not sternly, intolerant to waterlogging, fertile penetrating soil into
It is long very fast.Strong adaptability, cuttage is preferably lived, and cultivation management is simple, and the strong aithullium of this vitality is in north greening and makes
Have in woods and lift the light effect of lumping weight, so research Sabina vulgaris, for the development and utilization tool of wild plant gene related to drought tolerance
It is significant.Physiology and ecosystem characterization aspect are concentrated mainly on for the research of Sabina vulgaris at present, on its anti-drought gene
Clone and using aspect without report.
China's plant molecular breeding ability is huge with developed country's gap, and the adversity gene for possessing independent intellectual property right is generation
The focus of boundary plant molecular breeding field competition.For the exploitation and utilization of plant stress-resistance gene, current research is mainly concentrated
In the crop such as model plant arabidopsis or paddy rice, wheat, lack the excavation to the adversity gene enriched in wild plant body.This
Invention is exactly using the high throughput sequencing technologies of ILLumina companies, first-selected seeds of being checked winds and fixed drifting sand to northern China desertificated area
Sabina vulgaris carries out transcript profile sequencing, obtains a kind of gene related to drought tolerance Sv4317 filtered out for a long time through natural environment.
The content of the invention
The utilization molecule clone technology that the purpose of the present invention is, clones to anti-drought gene Sv4317, finally gives use
In the expression vector of gene expression, so that the channel genes are expressed in arabidopsis wild plant and in wild plant body,
The function of the gene is further verified by phenotypic analysis again.
Embodiment of the present invention be the Sabina vulgaris from nursery stock base for experiment material, using ILLumina SoLexa
The method of transcript profile high-flux sequence, obtains the nucleotide sequence of Sabina vulgaris gene related to drought tolerance Sv4317, by sequence
Analysis design primer, then the gene is obtained by molecule clone technology, and be conducted into arabidopsis, finally obtain transgenosis
Plant, by carrying out the biological function that phenotypic analysis understands gene to transfer-gen plant.
It is an object of the present invention to provide new Sabina vulgaris gene related to drought tolerance, it is Sv4317 to name, and its sequence is
SEQ No：1 sequence.
The present invention relates to a kind of expression quantity gene higher in Sabina vulgaris root, entitled Sv4317, its nucleotide sequence
Such as SEQ NO in sequence table：1 or SEQ NO：Shown in 2,728 amino acid sequences of its coding, SEQ NO such as in sequence table：3 institutes
Show.
Specifically, the invention provides the polynucleotides of the separation containing one of following sequences：
(1) the SEQ NO in sequence table：1 or SEQ NO：2
(2) DNA sequence dna that SEQ NO.1 or the SEQ NO.2 and in sequence table are limited has more than 90% homology, and compiles
Code-phase congenerous protein DNA sequence；
The above-mentioned polynucleotides being related to also include substitution, missing and insertion mutation body and allelic variant, splice variant, piece
Section, derivative etc..
It will be appreciated by persons skilled in the art that the polynucleotides of above-mentioned separation also include those and SEQ NO：1 or
SEQ NO：Sequence shown in 2 has the sequence compared with high homology, the sequence that for example homology is more than 95%, or 90%, or even 85%
Row；
Can also be with SEQ NO under high stringency conditions including those：1 or SEQ NO：The sequence of sequence hybridization shown in 2；Or can
With SEQ NO：1 or SEQ NO：The complementary sequence of 2 sequence.
Invention also provides the technical side that new gene Sv4317 of the invention is applied to Studies of Gene Engineering on Plant Drought-resistance
Case, specific plant can include Sabina vulgaris and arabidopsis.
The present invention has successfully been isolated from Sabina vulgaris and obtains a kind of decorrelation gene filtered out for a long time through natural environment
Sv4317, this cultivates drought-resistant plant there is provided new direction to using technique for gene engineering, to the molecular breeding under drought stress
Work is significant.Specifically, one of specific embodiments of the present invention are that Sv4317 genes are applied into plant base
Because engineering is improving survival ability of the plant under drought stress.
Outlined above describes the present invention, can by referring to provided herein is some specific embodiments further understand this
Invention, these embodiments are merely to illustrate and not limit the present invention.
Brief description of the drawings
Fig. 1：The total flow chart of experiment
Fig. 2：There is bar in low concentration of calcium ion and arid hormone ABA in the transgenic arabidopsis C6-6 containing Sv4317 genes
Growth phenotype under part.
There is bar in low concentration of calcium ion and arid hormone ABA in the transgenic arabidopsis C6-6 that Fig. 3 contain Sv4317 genes
Leaf fresh weight statistical chart under part.
Fig. 4：Growth tables of the transgenic arabidopsis C6-6 containing Sv4317 genes under the conditions of other mineral ions shortage
Type.
Fig. 5：Transgenic arabidopsis C6-6 containing Sv4317 genes root systems long under the conditions of other mineral ions shortage
Meter analysis.
Fig. 6：The planting patterns of culture dish in phenotypic analysis
Specific embodiment
Embodiment 1, Sabina vulgaris sample collection
The present invention is experiment material from the seeds of Inner Mongol Meng Cao drought resistings limited company nursery stock base.For maximum
The richness for increasing its RNA related to resistance in vivo of limit, selection is entered in weather relatively cold December (2013)
The sampling of row Sabina vulgaris root, sample is stored in rapidly standby in liquid nitrogen after collection.
The structure of embodiment 2, expression vector
A. the extraction of total serum IgE
RNA is extracted using TRIzol methods
1), appropriate liquid nitrogen is contained in mortar, abundant precooling mortar, until liquid nitrogen does not seethe with excitement flash, by what is gathered
Plant (ensures that liquid nitrogen did not had root or powder) in being put into liquid nitrogen
2) after, adding liquid nitrogen, plant is pounded fragment with pestle, when liquid nitrogen is evaporated into and is only capable of covering mortar bottom, rapidly
Grinding, adds liquid nitrogen in time, and process of lapping is repeated 2-3 times.
3), will prepare to hold centrifuge tube precooling in liquid nitrogen of abrasive flour：Order (should not be by for ttom of pipe-tube wall-mouth of pipe
Body is stretched into liquid nitrogen with lid coupling part, in order to avoid fracture).In the middle of centrifuge tube lid a duck eye, Yi Mianjia are bored with tweezers
Enter and pipe explosion is centrifuged after liquid nitrogen.
4), with by the sufficiently cool centrifuge tube of liquid nitrogen powder is taken from mortar, (1.5mL centrifuge tube powder volumes are less than
50 μ L), 1mLTRIzol extract solutions are rapidly added, centrifuge tube bottom powder is impacted when adding extract solution, and rapidly on the oscillator
Mix (extract solution was dissolved RNA in 30 seconds, to protect RNA).Ensure that powder did not melted before TRIzol is incorporated.(powder
End is about 1: 10 to 1 with the volume ratio of extract solution: 15).Fully mix, room temperature is placed and is no less than 5 minutes.
5), 10000x g, 4 DEG C are centrifuged 20 minutes.Centrifugation time is longer, can make RNA with the more preferable of other precipitations point,
Polluted in RNA phases less.
6) 80% or so supernatant, is taken in a new centrifuge tube, adds the chloroform of 1/4 volume, abundant Vortex
7), 10000x g, 4 DEG C are centrifuged 15 minutes.This is whether detection RNA extracts can successful committed step.If preceding
Face is all successful, and that at this time sees is that two-layer is fully transparent but clear separate solution, and thing is seen in centre any
Matter.This is most perfect.If strictly observing preceding step, this situation is reached not so difficult.It is relatively imperfect, it is also possible to
The situation of receiving is that centre has light sub-translucent whiteness；If this when of intermediate layer has clearly lighttight
Milky white precipitate, experiment has substantially failed.Extracted repeatedly with chloroform and also do not account for problem.And extract can all have every time
The loss of solution, even if to finally protein precipitation is eliminated, also cannot get enough RNA.
8) 80% or so supernatant, is taken in a new centrifuge tube.Some supernatants would rather be sacrificed, also to be kept away completely
Exempt to draw any intermediate layer material.Add isometric in -20 DEG C of isopropanols of preservation, abundant Vortex.- 20 DEG C of standings 1 are small
When, if Time Continuous work.If as matter of time, also can overnight, this when, RNA was substantially stabilization.
9), 10000x g, 4 DEG C are centrifuged 20 minutes, abandon supernatant.
10), with 1mL ethanol (preparation of DEPC water).Fully mix, RNA precipitate is crushed completely, so long as can be only achieved
The purpose of ethanol dissolving salt.10000x g, 4 DEG C of centrifugations, 10 minutes (washing secondary) (down interior is carried out) with this ultra-clean
11) most of supernatant, is slowly outwelled, centrifuge tube is inverted on the filter paper of sterilizing, exhausts raffinate.Quick centrifugation at a high speed
1 minute.Residual supernatant fluid is drawn with minimum pipette tips.Drying at room temperature 5-10 minutes
12) 20-30 μ L or so DEPC water back dissolvings, are added.
13) 1 μ L electrophoresis, is taken, banding pattern is observed.High voltage, the short time, controls in 10 minutes.
The reverse transcription of b.cDNA
RNA 7μL
Anchored OLigo(dT)20Primer(0.5μg/μL) 1μL
DEPC 1μL
65 DEG C are incubated 5 minutes, and ice bath 2 minutes is added
2Xts II Reaction Mix 10μL
TransScript II RT/RI Enzyme Mix 1μL
gDNA Remover 1μL
50 DEG C are incubated reverse transcription 30 minutes, 85 DEG C of heating, 5 seconds inactivation TransScript II RT/RI with
gDNARemover。
C. the structure of Sabina vulgaris Sv4317 expression vectors
Expand the primer of Sv4317 gene coded sequences
Forward primer：5’-ATTTGCGGCCGCATG GAG GAT CCG CTG ATC ATG AAT-3’
Reverse primer：5’-CCCTCGAGCTAAGC ACA ATT TTC TTC CAA CTC-3’
From TaKaRa a high-fidelity archaeal dna polymerase Primer Star to C6-6The clone for carrying out genes of interest.
Concrete scheme is as follows
1st, the reaction system of genes of interest：(50μL)
PCR cycle system
98℃ 5min
26 circulations
2nd, the glue reclaim kit from TRAN reclaims genes of interest
1. the target DNA band in Ago-Gel is cut, is put into clean centrifuge tube and is weighed, such as pectin weight is
100mg, can be considered 100 μ L (100mg=100 μ L), by that analogy.
2. 3 times of bulk solution GSB are added, in 55 DEG C of water-bath colloidal sol 6-10 minute, interruption mixing in (2-3 minutes), it is ensured that glue
Block melts completely, after glue melts completely, observes the color of solution, and such as color is purple, adds appropriate 3M sodium acetates
(PH5.2), adjustment color is identical with GSB colors (yellow).(for increase DNA yields, can add 1 times of isopropanol of volume in
In the gel solution for having melted, such as pectin weight is 100mg, add 100 μ L isopropanols).
3. room temperature (centrifugal column combination DNA abilities are weak during high temperature) is down to wait the gel solution for melting, it is quiet in addition centrifugal column
Put 1 minute, 10000x g are centrifuged 1 minute, abandon efflux.
4. add 650 μ L solution Ws B (adding absolute ethyl alcohol in advance), 10000x g to be centrifuged 1 minute, abandon efflux.
5. 10000x g are centrifuged 1-2 minutes, remove the WB of residual.
6. centrifugal column is placed in a clean centrifuge tube, standing of uncapping 1 minute makes residual ethanol volatilize clean.In post
Center adds 30-50 μ L EB or deionized water (PH ＞ 7.0), and (EB or deionized water are preheated in 60-70 DEG C of water-bath, using effect
More preferably), it is stored at room temperature 1 minute.
7. 10000x g are centrifuged 1 minute, eluted dna.The DNA that will be eluted is in -20 DEG C of preservations.
3rd, the purpose fragment and cloning vector pEASY-BLunt SimpLe (Kan) for obtaining glue reclaim are according to a certain percentage
25 DEG C of one hours of connection.
4th, heat shock method conversion E. coli competent：Take E. coli competent to be thawed on ice, add coupled reaction to produce
Thing, mixes, ice bath 30 minutes, and 42 DEG C of heat shocks 90 seconds are immediately placed on 2 minutes on ice；Add 500 μ L non-resistant LB Liquid Cultures
Base, be coated with for bacterium solution and contain corresponding antibiosis by 200rpm or 37 DEG C of constant incubator quiescent culture after 60 minutes on 37 DEG C of shaking tables
On the solid LB flat boards of plain (Kan 50mg/mL)；Its result is observed after 37 DEG C of inversion overnight incubations.
5th, bacterium colony PCR identifications
In order to obtain correct positive colony, select the single bacterium colony grown on culture medium at random first and enter performing PCR identification
(general 12 or so), template changes monoclonal bacterium colony into, with sterilized toothpick picking.
The system (from a archaeal dna polymerase EasyTaqMix of TRAN) of bacterium colony PCR
Mix, centrifugation, amplified reaction, reaction condition are carried out by following circulation：
94℃ 5min
35 circulations
1% agarose gel electrophoresis analyzes PCR amplifications, band is correctly chosen bacterium and is added to and contain kan (50mg/
ML) in the 5mL LB nutrient solutions of antibiotic, 37 DEG C of incubator overnight cultures.
6th, plasmid extraction：Plasmid extraction kit from TRAN extracts plasmid
1) bacterium solution of 2mL incubated overnights, is taken, 10000x g are centrifuged 1 minute, remove supernatant.As bacterium solution amount is big, can several times from
The heart is collected.
2) 250 μ L colourless solutions RB (containing RnaseA), are added, concussion suspended bacterial precipitation should not leave small bacterium block.
3) 250 μ L blue solution LB, are added, mixing 4-6 times is leniently spun upside down, thalline is fully cracked, form blue
The bright solution of color, color from half it is bright be changed into bright blueness, indicate cracking completely (no more than 5 minutes).
4) 350 μ L yellow solution NB, are added, (color indicates mixing by blue yellowing completely to be gently mixed 5-6 times
Uniformly, neutralize complete), until forming the yellow aggegation block of consolidation, it is stored at room temperature 2 minutes.
5), 12000x g are centrifuged 5 minutes, and careful absorption supernatant is added in centrifugal column.12000xg is centrifuged 1 minute, and abandoned stream goes out
Liquid.As supernatant volume is more than 800 μ L, in being segmented into repeatedly adding post, and ibid it is centrifuged, abandons efflux.
6) 650 μ L solution Ws B, 12000xg centrifugation 1 minute, is added, efflux is abandoned.
7), 12000x g are centrifuged 2 minutes, thoroughly the WB of removal residual.
8), centrifugal column is placed in a clean centrifuge tube, 30-50 μ L EB or deionized water (PH is added in the center of post
＞ 7.0) it is stored at room temperature 1 minute.
9), 10000x g are centrifuged 1 minute, and the DNA for eluting is in -20 DEG C of preservations.
7th, digestion verification：Restriction enzyme is selected according to plasmid map and DNA specific fragment restriction enzyme mappings to be cloned
5’：SaL I 3′：EcoR I (typically from double digestion) are verified to positive colony plasmid.
Double digestion reaction system
37 DEG C, warm bath 30 minutes, 1% agarose are set to according to the enzyme activity temperature provided according to company shown in catalogue after mixing
Gel electrophoresis analysis, judge whether fragment is correct according to electrophoretic band size.
8th, it is sequenced：By bacterium colony PCR and the correct clone of plasmid enzyme restriction checking, sequence verification its sequence is finally being carried out just
True property, examining order typically transfers to Nanjing Jin Sirui companies to complete.To sequencing result and known sequence alignment analysis, therefrom
Correct clone is selected, for the structure of next step purpose carrier.
9th, the connection of purpose carrier and the completion of structure
The connection of purpose carrier is that correct genetic fragment and purpose carrier will be sequenced simultaneously by 5 '：SaL I 3′：
The digestion of EcoR I, by DNA glue reclaims, then DNA ligase connection converts Escherichia coli, by bacterium colony PCR and extraction matter
Grain, the method for digestion verification finally obtains the plasmid of the vector gene being connected to.
The connection of a, purpose fragment and expression vector：
Principle：It is attached according to the ratio that purpose fragment and expression vector mol ratio are 3: 1 or 1: 3
T4DNA ligases (1 μ L)+buffer (2 μ L)+17 μ L (purpose fragment+expression vector)
16 DEG C overnight connect
B, conversion：Take E. coli competent to be thawed on ice, the product of purpose fragment and expression vector will be connected with again
It is transformed into E. coli competent, mixes, ice bath 30 minutes, 42 DEG C of heat shocks 90 seconds is immediately placed on 2 minutes on ice；Add 500
μ L non-resistant LB fluid nutrient mediums, 200rpm or 37 DEG C of constant incubator quiescent culture after 60 minutes on 37 DEG C of shaking tables, by bacterium solution
In coating and the solid LB flat boards containing corresponding antibiotic (Spe 50mg/mL)；Its result is observed after 37 DEG C of inversion overnight incubations.
C, bacterium colony PCR are identified
In order to obtain correct positive colony, select the single bacterium colony grown on culture medium at random first and enter performing PCR identification
(general 12 or so), template changes monoclonal bacterium colony into, with sterilized toothpick picking.
The system (from a archaeal dna polymerase EasyTaqMix of TRAN) of bacterium colony PCR
Mix, centrifugation, amplified reaction, reaction condition are carried out by following circulation：
94℃ 5min
35 circulations
1% agarose gel electrophoresis analyzes PCR amplifications, band is correctly chosen bacterium and is added to and contain Spe (50mg/
ML) in the 5mL LB nutrient solutions of antibiotic, 37 DEG C of incubator overnight cultures.
D, plasmid extraction：Plasmid extraction kit from TRAN extracts plasmid
1) bacterium solution of 2mL incubated overnights, is taken, 10000x g are centrifuged 1 minute, remove supernatant.As bacterium solution amount is big, can several times from
The heart is collected.
2) 250 μ L colourless solutions RB (containing RnaseA), are added, concussion suspended bacterial precipitation should not leave small bacterium block.
3) 250 μ L blue solution LB, are added, mixing 4-6 times is leniently spun upside down, thalline is fully cracked, form blue
The bright solution of color, color from half it is bright be changed into bright blueness, indicate cracking completely (no more than 5 minutes).
4) 350 μ L yellow solution NB, are added, (color indicates mixing by blue yellowing completely to be gently mixed 5-6 times
Uniformly, neutralize complete), until forming the yellow aggegation block of consolidation, it is stored at room temperature 2 minutes.
5), 12000x g are centrifuged 5 minutes, and careful absorption supernatant is added in centrifugal column.12000x g are centrifuged 1 minute, abandoned stream
Go out liquid.As supernatant volume is more than 800 μ L, in being segmented into repeatedly adding post, and ibid it is centrifuged, abandons efflux.
6), add 650 μ L solution Ws B, 12000x g to be centrifuged 1 minute, abandon efflux.
7), 12000x g are centrifuged 2 minutes, thoroughly the WB of removal residual.
8), centrifugal column is placed in a clean centrifuge tube, 30-50 μ L EB or deionized water (PH is added in the center of post
＞ 7.0) it is stored at room temperature 1 minute.
9), 10000x g are centrifuged 1 minute, and the DNA for eluting is in -20 DEG C of preservations
E, digestion verification：Continue to select restriction enzyme 5 ' above used：SaL I 3′：EcoR I are to positive colony
Plasmid is verified.
Double digestion reaction system
37 DEG C of constant incubators, warm bath 30 minutes.1% agarose gel electrophoresis detects, chooses banding pattern and correctly protected
Bacterium (40% yellow lid glycerine)
10th, Agrobacterium-mediated Transformation：Electrotransformation converts Agrobacterium
1) Agrobacterium competence, is taken, thawed on ice is placed in, the μ L of plasmid about 5 are added, is placed on ice after mixing 30 minutes.
2), while preparing clean dry electric revolving cup, on ice precooling；
3), electric revolving cup surface is dried, electroporation is put into after the Agrobacterium added with plasmid all being added in electric revolving cups, electricity
Swash conversion；
4), electric revolving cup adds the LB nutrient solutions of 1mL antibiotic-frees, repeatedly pressure-vaccum, in suction 2mL centrifuge tubes, 28 DEG C of shaking tables
200rpm or 28 DEG C of constant incubator renewal cultivation is after 1.5 hours；
5) 500 μ L bacterium solutions, are taken and coats the solid LB media containing antibiotic (Rif50mg/mL and Spe 50mg/mL)
On, 28 DEG C of incubated carton upside down cultures 1-2 days are until monoclonal occurs；
6), choosing positive Agrobacterium single bacterium colony carries out bacterium colony PCR, and the PCR results containing correct purpose band is corresponding
Agrobacterium single bacterium colony carries out expanding numerous (system and reaction condition of bacterium colony PCR are the same with the bacterium colony PCR of Escherichia coli).
11st, Agrobacterium plasmid is extracted：Plasmid extraction kit from TRAN extracts plasmid
1) bacterium solution of 2mL incubated overnights, is taken, 10000x g are centrifuged 1 minute, remove supernatant.As bacterium solution amount is big, can several times from
The heart is collected.
2) 250 μ L colourless solutions RB (containing RnaseA), are added, concussion suspended bacterial precipitation should not leave small bacterium block.
3) 250 μ L blue solution LB, are added, mixing 4-6 times is leniently spun upside down, thalline is fully cracked, form blue
The bright solution of color, color from half it is bright be changed into bright blueness, indicate cracking completely (no more than 5 minutes).
4) 350 μ L yellow solution NB, are added, (color indicates mixing by blue yellowing completely to be gently mixed 5-6 times
Uniformly, neutralize complete), until forming the yellow aggegation block of consolidation, it is stored at room temperature 2 minutes.
5), 12000x g are centrifuged 5 minutes, and careful absorption supernatant is added in centrifugal column.12000x g are centrifuged 1 minute, abandoned stream
Go out liquid.As supernatant volume is more than 800 μ L, in being segmented into repeatedly adding post, and ibid it is centrifuged, abandons efflux.
6) 650 μ L solution Ws B, 12000xg centrifugation 1 minute, is added, efflux is abandoned.
7), 12000x g are centrifuged 2 minutes, thoroughly the WB of removal residual.
8), centrifugal column is placed in a clean centrifuge tube, 30-50 μ LEB or deionized water (PH is added in the center of post
＞ 7.0) it is stored at room temperature 1 minute.
9), 10000x g are centrifuged 1 minute, and the DNA for eluting is in -20 DEG C of preservations
12nd, digestion verification：From restriction enzyme 5 '：SaL I 3′：EcoR I verify to the plasmid for being extracted,
Double digestion reaction system
28 DEG C of constant incubators, warm bath 30 minutes.1% agarose gel electrophoresis detects that analysis result, selector bar band is correct
Agrobacterium preserve glycerol stock (40% blue lid), for converting plant.
The acquisition of embodiment 3, genetically modified plants
The present embodiment obtains transfer-gen plant code name using titbit infestation method
1st, picking has just ensured that the Agrobacterium of bacterium is activated (vigor that activation purpose is to ensure that bacterium), the bacterium that will have been activated
It is added in the 5mL LB nutrient solutions containing antibiotic (50mg/mLRif and 50mg/mLSpe), 28 DEG C of incubator overnight cultures, will
The bacterium solution shaken is put into centrifuge, 5000x g, 10min, abandoning supernatant, add 1mL infect liquid (5% sucrose,
0.02%silwet suspension is dissolved in water), with glue head dropper, gently pressure-vaccum makes bacterium suspend.
2nd, the wild type Col-0 plant for being grown fine and having been bloomed from three boxes, cut off having podded and on plant
The petal of pollination is completed, drawing bacterium solution with glue head dropper drops on the petal do not bloomed, and mark of labelling, and is invaded every 2-3 days
Contaminate once, (the note until all plant all bloom：Infect the previous day every time to water a plant).
3rd, T is collected in time after plant is ripe1For seed, treat that seed drying is sown in for one week or so and contain antibiotic
Resistance solid MQA CK culture dishes on, be first placed into 4 DEG C of refrigerator vernalization three days, then illumination cultivation one week or so, carry out the positive
Seedling T1The screening in generation.
4th, the T grown fine on resistance culture ware is chosen1For plant, move earth culture and support and individual plant label, after after its maturation and
When individual plant collect T2For seed, it is dried for a week after, continuation its single tube is sprinkling upon on resistance culture base, after illumination cultivation a couple of days, choosing
Take and meet the dead T than for 3: 1 living2The foster wait of shifting earth culture is carried out for plant collect T3The plant in generation.By T3Continue to be sprinkling upon for plant anti-
Property culture dish on, choose complete living pure and mild plant and be the seed that can be used as phenotypic analysis.
Embodiment 4, the phenotypic analysis in different elements
The MQA medium components used in this example mainly have
A great number of elements：1MKNO3、1MMgSO4、1MH3PO4、1MCaCl2
Trace element：MS micro (0.5x),
Fe2+Salt：MSFe2+Salt (0.5x)
Mn2+Salt：MSMn2+Salt (0.5x) and 0.5M MES buffer solutions
Carbon source：1% sucrose
A. specific culture medium scheme such as table 1 (100mL)
Note：10 μM of PO43-, 0 μM of K+, 0 μM of Ca2+, 10 μM of NO3-, 0 μM of Mg2+ these gradient add 1.0%
Agarose 1g
1mMCa2+, 1 μM of ABA, 80mMNaCL these gradient add 1.2% agar 1.2g
Another ABA is that temperature is down to hand Wen Shizai and is added after culture medium has gone out bacterium.
Table 1：It is 5.7 (it is 7.0 that 80mMNacl adjusts PH) that culture medium the scheme KOH or BTP of various elements adjust PH
B. planting patterns is as shown in Figure 4
Three repetitions of every kind of gradient, are sealed after finishing with sealed membrane, first in 4 DEG C of refrigerator vernalization three days, then place temperature
It it is 22 DEG C, humidity observes C in 20 days afterwards for the vertical illumination cultivation of culturing room of 40%RH6-6With the phenotype of wild type Col-0,
And root data long and fresh weight and photo acquisition are carried out to it, it can be seen that C6-6Relative to wild type Col-0 in 0 μM of Ca2+There is leaf
The short sensitive phenotype of Huang Er little, root and 1 μM of ABA show as the phenotype that leaf is small, root is short, as shown in the figure (accompanying drawing 2 and accompanying drawing 3)
C. culture dish is put
8 gradients are one group, are divided into three groups
Example：Assuming that eight gradients are respectively with alphabetical A, B, C, D, E, F, G, H
A、B、C、D、E、F、G、H
B、C、D、E、F、G、H、A
C、D、E、F、G、H、A、B
Above example further illustrates present disclosure, but should not be construed as limiting the invention.Do not carrying on the back
In the case of spirit of the invention and essence, the modification or replacement made to the inventive method, step or condition belong to this hair
Bright category.
Claims (7)
1. a kind of Drought-resistant gene of plant Sv4317, its nucleotide sequence is included：
(1) such as SEQ NO：1 or SEQ NO：Sequence shown in 2；
(2) with SEQ NO：1 or SEQ NO：2 DNA sequence dnas for limiting have more than 90% homology, and coding identical function albumen
The sequence of matter.
2. a kind of plant drought GAP-associated protein GAP, its amino acid sequence such as SEQ No：Shown in 3.
3. a kind of Drought-resistant gene of plant, it is characterised in that the plant drought GAP-associated protein GAP described in its coding claim 2.
4. the expression vector of the gene described in claim 1 or 3 is contained.
5. application of the gene described in claim 1 or 3 in plant drought.
6. application according to claim 6, it is characterised in that plant is Sabina vulgaris.
7. improve plant drought resistance method, by described in claim 1 anti-drought gene Sv4317 import host plant cell,
Tissue or individual plants, obtain the plant with drought resistance.
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| CN101078015A (en) * | 2006-05-25 | 2007-11-28 | 中国科学院遗传与发育生物学研究所 | Korshinsk peashrub transcription factor CkAREB and application of the same in adversity resistant plant cultivation |
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| CN101078015A (en) * | 2006-05-25 | 2007-11-28 | 中国科学院遗传与发育生物学研究所 | Korshinsk peashrub transcription factor CkAREB and application of the same in adversity resistant plant cultivation |
| CN101921774A (en) * | 2010-05-24 | 2010-12-22 | 南京大学 | Application of Dnaj-like protein and encoded gene thereof |
| CN102234653B (en) * | 2011-06-29 | 2012-12-12 | 济南大学 | Salt-tolerant and drought-resistant gene TaMYB33 of wheat and coding protein as well as application thereof |
| CN103710355B (en) * | 2013-12-16 | 2015-05-06 | 中国农业科学院生物技术研究所 | Ammopiptanthus mongolicus drought induced related gene, and expression vector and application thereof |
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Address after: 011599 West Hall, F2, Intelligent Manufacturing Industrial Park, Shengle Modern Service Industry Cluster, Horinger County, Hohhot, Inner Mongolia Patentee after: Inner Mongolia Xiaocao digital Ecological Industry Co.,Ltd. Patentee after: Mengcao ecological environment (Group) Co.,Ltd. Address before: 011517 room 2230, enterprise headquarters building, Shengle modern service industry cluster, Shengle economic Park, Helingeer County, Hohhot City, Inner Mongolia Autonomous Region Patentee before: Inner Mongolia mengcao life community big data Co.,Ltd. Patentee before: INNER MONGOLIA MONGOLIAN GRASS ECOLOGICAL ENVIRONMENT (GROUP) Ltd. |
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Effective date of registration: 20230615 Address after: 010070 south side of Shengle Fifth Street, Shengle economic Park, Hohhot City, Inner Mongolia Autonomous Region Patentee after: Mengcao ecological environment (Group) Co.,Ltd. Address before: 011599 West Hall, F2, Intelligent Manufacturing Industrial Park, Shengle Modern Service Industry Cluster, Horinger County, Hohhot, Inner Mongolia Patentee before: Inner Mongolia Xiaocao Digital Ecological Industry Co.,Ltd. Patentee before: Mengcao ecological environment (Group) Co.,Ltd. |
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