CN109628467A - A kind of Chunlan CgWRKY2 gene and its application - Google Patents
A kind of Chunlan CgWRKY2 gene and its application Download PDFInfo
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- CN109628467A CN109628467A CN201910089680.1A CN201910089680A CN109628467A CN 109628467 A CN109628467 A CN 109628467A CN 201910089680 A CN201910089680 A CN 201910089680A CN 109628467 A CN109628467 A CN 109628467A
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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
Abstract
The invention discloses a kind of ChunlansCgWRKY2Encoding gene and its application,CgWRKY2The nucleotide sequence of gene expresses the amino acid sequence of albumen as shown in SEQ ID NO.2 as shown in SEQ ID NO.1.The present invention passes through to Chunlan cultivar ' Song Mei 'CgWRKY2The clone of gene and identification, gene expression analysis, and its function is verified, discovery is overexpressedCgWRKY2The Arabidopsis plant growth and development of gene is slow, and plant is downgraded, and with the early withered equal phenotype for shifting to an earlier date aging of blade, it is seen that the gene will have been widely used in orchid and other plant productions, breeding.
Description
Technical field
The invention belongs to field of plant genetic project technology, and in particular to a kind of ChunlanCgWRKY2Gene and its application.
Background technique
Orchid family (Orchidaceae) is one of maximum section in flowering plant, and the whole world contains more than 25000 kinds, accounts for about all
The 10% of flowering plant.Chunlan (Cymbidium goeringii) belong to small flower pattern Terrestrial orchid type in orchid family Cymbidium, flower
Type is peculiar, pattern is simple and elegant, and the fragrance of a flower is quiet and beautiful, and leaf appearance is graceful, and ornamental values and the economic values are high.Chunlan to growing environment require compared with
Height is highly prone to the influence of the adverse circumstances such as high temperature, low temperature, arid during the growth process, when serious, will lead to gardening ornamental quality
Decline or even plant are dead.Therefore, the molecular mechanism of research plant reply abiotic stress and identification have degeneration-resistant function
The breeding of gene pairs Chunlan and production are of great significance.In plant cell signaling transduction pathway, WRKY transcription factor is considered
It is the key that plant growth and various abiotic stress response hinge, provides important evidence for the genetic improvement of plant.
WRKY transcription factor is one of maximum regulatory protein family in plant, participates in various physiological processes, wherein most prominent
Out be to biology and abiotic stress stress reaction.It has been reported that and shows that plant pair environment stress can be enhanced in WRKY gene
Tolerance.Sunflower HaWRKY76 transgenic plant shows stronger resistance to flooding stress, and yield also increased significantly.AtWRKY25Overexpression enhancing arabidopsis salt tolerance.AtWRKY57Overexpression in rice not only increases the anti-of rice
Drought, and enhance its tolerance to salt and PEG.Therefore, it using technique for gene engineering, clones and obtains from ChunlanCgWRKY2Gene, there are apparent differential expressions under low temperature stress for the gene, therefore willCgWRKY2In gene transferred plant,
Great application prospect.
Summary of the invention
Goal of the invention: for deficiency present in existing breeding technique, the object of the present invention is to provide a kind of ChunlansCgWRKY2Gene.It is a further object of the present invention to provide ChunlansCgWRKY2Application of the gene in orchid breeding.
Technical solution: in order to achieve the above-mentioned object of the invention, The technical solution adopted by the invention is as follows:
A kind of ChunlanCgWRKY2Gene, nucleotide sequence is as shown in SEQ ID NO.1.
The ChunlanCgWRKY2The expression albumen of gene, amino acid sequence is as shown in SEQ ID NO.2.
The ChunlanCgWRKY2Application of the gene in plant production and breeding.
Contain the ChunlanCgWRKY2The carrier of gene.
Contain the ChunlanCgWRKY2The host cell of gene.
The utility model has the advantages that compared with prior art, the present invention passes through to ChunlanCgWRKY2The clone of gene and identification, gene
Expression analysis, verify its function, discovery is overexpressedCgWRKY2The Arabidopsis plant growth and development of gene is slow, and plant is downgraded,
And with the phenotype of the blade aging in advance such as early withered, it is seen that the gene will have extensively in orchid and other plant productions, breeding
Purposes.
Detailed description of the invention
Fig. 1 is ChunlanCgWRKY2The over-express vector figure of gene cloning and building；
Fig. 2 a figure isCgWRKY2Expression in Chunlan is respectively organized, wherein R indicates that root, P indicate that pseudobulb, L indicate
Leaf, F indicate flower；B figure is Chunlan under low temperature stressCgWRKY2The expression of gene；
Fig. 3 a figure is digestion result figure, wherein M:DL2000 Marker；CgWRKY2After being connect with pBI121 with SmaI and
SnabI double digestion；B figure is the screening figure of positive recombinant, wherein M:DL2000 Marker, purpose band size are
2031bp；
Fig. 4 is transgenic Arabidopsis plants PCR result figure, wherein M:DL2000 Marker；CK+: it is sun with vector plasmid DNA
Property control；CK-: wild type DNA is negative control；Water: blank control；
Fig. 5 is to turnCgWRKY2Gene plant figure compared with wild-type Arabidopsis plants plant type, WT in figure, wildtype Arabidopsis thaliana；1-
3, turnCgWRKY2The different strains of gene；
Fig. 6 is to turnCgWRKY2Gene plant and wild type COL Arabidopsis leaf comparison chart；WT in figure, wildtype Arabidopsis thaliana；1-
3, turnCgWRKY2The different strains of gene；
Fig. 7 is to turnCgWRKY2Gene plant and wildtype Arabidopsis thaliana are under low temperature stress, cold stress-related genes expression ratio
To figure.
Specific embodiment
The present invention is described further combined with specific embodiments below.
Embodiment 1
Material used by the present embodiment is Chunlan ' Song Mei ' blade, adopts rear quick-frozen in liquid nitrogen, (- 80 DEG C) of ultra low temperature freezer guarantors
It deposits.
1) extraction of Chunlan blade total serum IgE
It is carried out according to the specification of TaKaRa plant total RNA extraction reagent box, concrete operations are as follows:
The Chunlan blade of Cryopreservation is transferred quickly in the mortar with Liquid nitrogen precooler, with pestle tissue abrasion, therebetween not
It is disconnected that liquid nitrogen is added, until being ground into powder；The sample for being ground into powdery is added to containing 450 μ l Buffer PE's
1.5 mL sterilize in tube, are blown and beaten repeatedly with pipettor until without obvious sediment in lysate；By 12,000 rpm of lysate, 4
DEG C centrifugation 5 minutes；Supernatant is carefully drawn in new 1.5 mL sterilizing tube.1/10 volume of supernatant is added
Buffer NB, Vortex oscillation mixes, 12,000 rpm, 4 DEG C of 5 clocks of centrifugation；Supernatant is carefully drawn to 1.5 new mL
It sterilizes in tube, the Buffer RL of 450 μ L is added, is uniformly mixed solution using liquid-transfering gun；1/2 body of mixed liquor is added
Long-pending dehydrated alcohol, using liquid-transfering gun by solution after mixing, mixed liquor is all transferred to RNA Spin Column immediately
In；12,000 rpm are centrifuged 1 minute, abandon filtrate, RNA Spin Column is put back into 2 ml Collection Tube
In；The Buffer RWA of 500 μ L is added into RNA Spin Column, 12,000 rpm are centrifuged 30 seconds, abandon filter
Liquid；The Buffer RWB of 600 μ L is added into RNA Spin Column, and 12,000 rpm are centrifuged 30 seconds, abandons filtrate；To
50 μ L DNase I reaction solutions are added in the Column film center RNA Spin, are stored at room temperature 15 minutes；To RNA Spin Column
Buffer RWB, 12,000 rpm centrifugation 30 seconds of 350 μ L are added in film center, abandon filtrate；By RNA Spin Column weight
It is newly placed on 2mL Collection Tube, 12,000 rpm are centrifuged 2 minutes；RNA Spin Column is placed in
On the RNase Free Collection Tube of 1.5 mL, it is added 50 μ L's in RNA Spin Column film centre
RNase Free dH2O is stored at room temperature 5 minutes, and 12,000 rpm are centrifuged 2 minutes eluted rnas.Gained RNA is through concentration and purity
- 80 DEG C of refrigerators are stored in after detection to save backup.
It draws 2 μ L RNA to detect using 1% agarose gel electrophoresis, 28S and 18S band is relatively clear as the result is shown, 28S
Band brightness is about twice of 18S, and RNA mass is preferable.RNA purity, OD are detected by micro accounting protein assay260/OD280
It is 2.03, OD260/OD230It is 2.01, integrality is preferable, can be used for reverse transcription.
2) synthesis of the first chain cDNA
Using obtained total serum IgE as template, reverse transcription is carried out using TaKaRa reverse transcription reagent box, is made using Oligo (dT)
For anchor primer, reverse transcription synthesizes the first chain cDNA.Concrete operations are as follows:
The sequence preparating mixture according to following template ribonucleic acid/primer is prepared in centrifuge tube, total amount is 10 μ L: 1 μ g of template,
Oligo (dT) Primer (50 μM) 1 μ L, dNTP Mixture (10mM each) 1 μ L, residual volume RNase-free
ddH2O polishing.It is cooling rapidly on ice after 65 DEG C of heat preservation 5min；The centrifuge tube is centrifuged, is sunken to the mixture in centrifuge tube
Tube bottom.Inverse transcription reaction liquid (20 μ L) is prepared in new centrifuge tube: 10 μ L, 5 × PrimeScript of reaction solution after above-mentioned denaturation
4 μ L, RNase Inhibitor (40U/ μ l) of Buffer 0.5 μ L, PrimeScript RTase (200 U/ μ l) 1 μ L,
RNase Free dH220 μ L of O polishing.It slowly shakes up, in PCR instrument, 30 DEG C of heat preservation 10min, 42 DEG C of heat preservation 30min, 95 DEG C are protected
Warm 5min inactivates enzyme, places on ice, obtains cDNA solution.
3) design of target gene primer and clone
According to existing Chunlan transcript profile sequencing data, other species are utilizedWRKYIt is homologous that related gene sequence carries out Blast
It compares.Using Oligo6.0, Prime5.0 designs corresponding primer, primer sequence are as follows:
CgWRKY2-F:5'-ATGGATGACAGTATATCCATTTT-3',
CgWRKY2-R:5'-TGGATATGGCCCAAGAGATACTC-3'.
Using the first chain of cDNA as template, Chunlan is carried out using PrimerStar Max high fidelity enzymeCgWRKY2Gram of gene
It is grand.PCR amplification system (50 μ L) are as follows: 25 μ lL PrimerStar Max, 2 μ L Forward Primer, 2 μ L Reverse
Primer, 2 μ L Template DNA, 19 μ L ddH2O.PCR program are as follows: reaction condition is 94 DEG C of initial denaturation 3min, 98 DEG C of changes
Property 10s, 60 DEG C of annealing 5s, 72 DEG C of extension 30s, 32 circulation, 72 DEG C of overall elongation 5min, 16 DEG C heat preservation.
PCR after the reaction was completed, takes whole PCR products to detect and cut target fragment by 1.5% agarose gel electrophoresis,
The amplified production of gel purification PCR mesh.Using the DNA gel QIAquick Gel Extraction Kit of Tiangeng company, target fragment purifying is carried out
Recycling, concrete operations are as follows: into adsorption column CA2 (adsorption column is put into collecting pipe) be added 500 μ L equilibrium liquid BL, 12000rpm from
Heart 1min outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；By single goal band from Ago-Gel
In cut, be put into clean centrifuge tube, weigh weight；If it is 0.1g that isometric solution PN(gel is added into blob of viscose,
Volume can be considered 100 μ L, then 100 μ L PN solution are added), 50 DEG C of water-baths are placed, centrifuge tube is mildly constantly spun upside down therebetween,
Until blob of viscose is completely dissolved；Previous step acquired solution is added in an adsorption column CA2, is placed at room temperature for 2min, 12000rpm from
Heart 1min outwells waste liquid in collecting pipe, and adsorption column CA2 is put into collecting pipe；600 μ L rinsing liquids are added into adsorption column CA2
W, 12000rpm are centrifuged 1min, outwell the waste liquid in collecting pipe, adsorption column is put back in collecting pipe；12000rpm is centrifuged 2min,
Rinsing liquid is eliminated as far as possible, and adsorption column is placed in and is placed at room temperature for 5min, is thoroughly dried；Adsorption column is put into a clean centrifuge tube
In, 30 μ L ddH are vacantly added dropwise to adsorbed film middle position2O is stored at room temperature 2min, and it is molten that 12000rpm is centrifuged 2min collection DNA
Liquid.Product after taking 2 μ L recovery purifyings carries out detected through gel electrophoresis using 1% agarose.
4) target fragment is connect with carrier
Cloning vector is the pEASY-Blunt carrier of Quan Shi King Company, is attached reaction, linked system (5 μ L): 4 μ L PCR
Purified product, 1 μ L pEASY-Blunt Vector gently inhale after playing mixing, are placed at room temperature for 5min, centrifuge tube is placed on ice.
5) conversion of connection product
Competent cell Trans5 α bacterial strain is taken out from ultra low temperature freezer, is placed on ice to melt.The overnight connection for drawing 5 μ L produces
Object is added in 100 μ L competent cells；Centrifuge tube is placed in 30 min of ice bath on ice；Water-bath in 42 DEG C of water-baths, heat shock 90
During which s not shake；After be immediately placed on 2 min of ice bath on ice；The Liquid Culture of 800 μ L antibiotic-frees is added in super-clean bench
Base, 37 DEG C, 180 rpm shake 1h recovery；4000 rpm are centrifuged 3 min, suck 800 μ L supernatants；The thallus of precipitating is resuspended, is applied
In LB plate (concentration of Amp is 100 mg/L), 37 DEG C of overnight incubations.
6) screening and verifying of recombinant plasmid
The single colonie of picking overnight growth on the LB solid medium containing antibiotic (Amp), is inoculated into containing same antibiosis
In the LB liquid medium of 750 μ L of element.200 rpm, 37 DEG C are incubated overnight.
PCR amplification system are as follows: 10uL Green TaqMix, 1 μ l M13-F/R, 1 μ L bacterium solution, 7 μ L ddH2O adds to 20
μL。
PCR program are as follows: 94 DEG C of 10min；94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min, 30 cycles；72℃5 min；
16℃forever。
The PCR product for drawing 5 μ L carries out agarose gel electrophoresis detection and analysis.After verifying, by the correct bacterium of stripe size
Liquid sample entrusts Nanjing Genscript Biotechnology Co., Ltd. to be sequenced, and sequencing primer is universal primer M13F/R.Sequencing result exists
It is compared on NCBI.
It is final to determine that clone obtains 1 Chunlan according to the analysis to sequencing resultWRKYEncoding gene is named asCgWRKY2Gene, nucleotide sequence as shown in SEQ ID NO.1,CgWRKY2Gene code length is 2031bp, contains ATG
Initiation codon and TGA terminator codon, wherein ORF overall length is 2031 bp, encodes the protein of 676 amino acid, the albumen
Amino acid sequence is as shown in SEQ ID NO.2.
Embodiment 2
Result of study showsWRKY2Gene has expression (Fig. 2 a) in Chunlan in each histoorgan, but the gene is in the spring
Expression quantity highest in blue blade illustrates that gene function in blade is active.Pass through the blade handled Chunlan low temperature stress
It carries out expression analysis (Fig. 2 b), it was demonstrated thatCgWRKY2 genes play important regulation in the low temperature stress response of Chunlan blade and make
With.
Vegetable material used in the present embodiment be arabidopsis (Arabidopsis thaliana) Col(Columbia) wild
Type seed.
Coli strain used in the present embodiment is Trans5 α；Agrobacterium strains are GV3101, and it is quasi- to be respectively used to conversion
Southern mustard；Plant expression vector used is pBI121 in test.Bacterial strain uses therefor is purchased from Quan Shijin biotech firm and Puli's Si life respectively
Object company.
1)CgWRKY2The building of gene overexpression carrier
Embodiment 1 is obtainedCgWRKY2Gene ORF full length sequence is attached with plant expression vector pBI121, building
Carrier such as Fig. 1.
2) extraction of plasmid:
Middle amount kit specification extraction plasmid is mentioned according to Tiangeng plasmid is small, the specific steps are as follows:
The 10mL bacterium solution being incubated overnight is taken, 12000 rpm are centrifuged 1min, remove supernatant；Take 500 μ L P1 solution (containing RNase
A it) adds in the centrifuge tube there are bacterial sediment, is precipitated using the thorough suspension thalline of vortex instrument；Take 500 μ L P2 solution add to from
In heart pipe, cellular lysate is abundant when mildly spinning upside down, and 700 μ L P3 solution is taken to add in centrifuge tube, mild immediately to turn over up and down
Turn, mix well, after there is white flock precipitate, 12000rpm is centrifuged 10min；500 μ L equilibrium liquid BL are taken to add to adsorption column
In CP4,12000rpm is centrifuged 1min, discards the waste liquid in collecting pipe, adsorption column is put back to collecting pipe, by the supernatant of collection point
It criticizes and is added in Filter column CS, 12000rpm is centrifuged 2min, and adsorption column CP4 carefully is added in the solution by portions collected in collecting pipe
In, 12000rpm is centrifuged 1min, discards the waste liquid in collecting pipe, adsorption column CP4 is put back to collecting pipe；Take 500 μ L protein liquid removals
PD is added in adsorption column CP4, and 12000rpm is centrifuged 1min, discards waste liquid in collecting pipe, adsorption column CP4 is placed back in collection
Pipe；Take 600 μ l rinsing liquid PW(containing dehydrated alcohol) it adds in adsorption column CP4,12000rpm is centrifuged 1min, discards in collecting pipe
Waste liquid, adsorption column CP4 is put back into collecting pipe, 12000rpm is centrifuged 2min, removes rinsing liquid remaining in adsorption column；It will absorption
Column CP4 is moved in new 1.5ml centrifuge tube, and 60 μ L ddH are added to adsorbed film centre2O；Be stored at room temperature 2min, 12000rpm from
Heart 1min, the solution collected in centrifuge tube is plasmid.Plasmid concentration is finally measured, is prepared for experiment in next step.
3) addition of special restriction enzyme site
Using cDNA as template, specific cleavage site is added in the two sides of target gene by PCR method.ChunlanCgWRKY2Base
Because SmaI and SnaBI restriction enzyme site is added in two sides.PCR reaction system, program and used primer are as follows:
PCR amplification system (50 μ L): 25 μ L PrimerStar Max, 2 μ L Forward Primer, 2 μ L Reverse
Primer, 2 μ L Template DNA, 19 μ L ddH2O.PCR program are as follows: reaction condition is 94 DEG C of initial denaturation 3min, 98 DEG C of changes
Property 10s, 60 DEG C of annealing 5s, 72 DEG C of extension 30s, 32 circulation, 72 DEG C of overall elongation 5min, 16 DEG C heat preservation.
Used primer sequence:
CgWRKY2-SmaI-F:5'- CCCGGGATGGATGACAGTATATCCATTTT-3',
CgWRKY2-SnaBI-R:5'- TACGTATGGATATGGCCCAAGAGATACTC-3'.
1.5% agarose gel electrophoresis of obtained PCR product is separated, Tiangeng Ago-Gel DNA reclaim reagent is used
Box carries out recovery purifying, product after the recovery is connect with pBI121 carrier, construction of expression vector.
4) double enzyme digestion reaction
By the pBI121 plasmid of extraction with SmaI and SnaBI digestion 15min under the conditions of 37 DEG C, electrophoresis recycle linear carrier, -20
It DEG C saves backup.Double enzyme digestion reaction system is 20 μ L, 5 × buffer 5 μ L, SmaI 1 μ L, SnaBI of 50 μ L:pBI121 plasmid
1 μ L, ddH2O 23μL.Digestion result is as shown in Figure 3a, wherein M:DL2000 Marker；1:CgWRKY2, it is connect with pBI121
SmaI and SnaBI double digestion is used afterwards.
5) connection reaction
The target gene and carrier pBI121 being recovered to after agarose gel electrophoresis detection digestion, according to detected purity
And concentration, each reagent is added by linked system.Wherein, target fragment molecular number: carrier molecule number=3:1-5:1 connects reactant
System are as follows: linearisation 7 μ L of pBI121 carrier, 3 μ L, 5 × CE II buffer of Insert Fragment, 4 μ l, Exnase II 2 μ l, ddH2O
Up to 20μL.30min is reacted at 37 DEG C, is placed in cooling down on ice.
6) connection product is transferred to Escherichia coli
Product after target fragment is connect with carrier pBI121 is transferred in Escherichia coli Trans5 α competent cell, and method is same
Embodiment 1.
7) identification of recon
Single colonie on picking plate is inoculated into the LB liquid medium containing antibiotic (kanamycins), 37 DEG C, 200 rpm
Shake culture is stayed overnight.Bacterium solution PCR is carried out using target gene overall length primer, with screening positive clone.Positive colony after screening
Nanjing Jin Sirui company is sent to be sequenced.Plasmid is extracted using Tiangeng plasmid extraction kit simultaneously and carries out digestion verification, judges enzyme
Whether consistent cut post-fragment size.It as a result as shown in Figure 3b, is purpose primer PCR as a result, stripe size is 2031 bp.
8) preparation and conversion of Agrobacterium competent cell
The present embodiment prepares Agrobacterium competence using Agrobacterium GV3101, and carry out arabidopsis infects experiment；Agrobacterium sense
By state preparation process are as follows: picking activated Agrobacterium single colonie is inoculated in 5mL LB liquid medium, 28 DEG C, 250
Rpm shakes bacterium culture 20-24 h；2mL bacterium solution is drawn, is inoculated into the triangular flask containing 50mL LB liquid medium, 28 DEG C, 250
Rpm shakes bacterium to OD600Value is 0.8 or so；Bacterium solution after will be enlarged by breeding is placed in 30 min of ice bath on ice, 4 DEG C, 5000 rpm from
5 min of the heart abandons supernatant；0.1 pre-cooled mo1/L CaCl of 10mL is added2Solution, the thallus for the precipitating that sufficiently suspends；4 DEG C,
5000 rpm are centrifuged 5 min, discard supernatant；20 mmo1/L CaCl of 1mL pre-cooling are added2The abundant suspension thalline of solution to get
To GV3101 competent cell to be prepared, be distributed into 100 μ L/ pipe with centrifuge tube, be rapidly added 20% it is sterile sweet
Oil, -80 DEG C of placements save.
The Agrobacterium-mediated Transformation of recon: ice bath melts Agrobacterium competent cell, and 1-5 μ l is recovered after purification
Plasmid is added in the Agrobacterium competence of 200 μ l, is mixed gently, 30 min of ice bath；Use liquid nitrogen flash freezer l min, 37 DEG C of water
Thermal shock 1-5 min in bath, is immediately placed in 1-2 min on ice；Be added 800 μ l be free of any antibiotic LB culture medium, 28
DEG C, 100 rpm recovery 2-4 h；4000 rpm are centrifuged 3 min, sop up partial medium；Residue is mixed well using liquid-transfering gun
Bacterium solution, after be applied to addition 50 mg/L kanamycins and 50 mg/L streptomysins (EHA105) or 100 mg/L celebrating it is mould greatly
On the solid LB training base of plain (GV3101)；30-48 h is cultivated in 28 DEG C of inversions.
The identification of Agrobacterium recon: the single colonie that picking is grown from plating medium is inoculated in containing corresponding antibiosis
In the fluid nutrient medium of element；28 DEG C, 220 rpm overnight incubations；Following primer of being arranged in pairs or groups respectively using 35S-F carries out bacterium solution PCR, draws
Object sequence are as follows:
35S-F:5'-GATAGTGGAAAAGGAAGGTG-3',
35S-CgWRKY2-R:5'-TGGATATGGCCCAAGAGATACTC-3'.
PCR product is detected through 1% agarose gel electrophoresis, and whether identification contains target fragment；The positive colony identified,
After expanding culture, using alkaline lysis method of extracting plasmid, double digestion verifying is carried out；Positive colony after identification is added appropriate sterile sweet
Oil is saved backup in -80 DEG C.
9) conversion of the arabidopsis of mediated by agriculture bacillus
Target gene is transferred in arabidopsis using inflorescence infestation method, concrete operation method are as follows: arabidopsis (col wild type) is kept
Healthy growth state is to blooming；Activation carries the Agrobacterium EHA105 bacterial strain of target gene.Picking single colonie, is inoculated in 5mL
On the LB culture medium of containing kanamycin and streptomysin, 28 DEG C, 250 rpm shake bacterium and just become cloudy to bacterium solution, about 8-10 h；It inhales
1mL bacterium solution is taken, (50mL) is inoculated into triangular flask and shakes 24 h of bacterium, until OD value is about 0.8 or so；By 5000 rpm of bacterium solution in room
Temperature is lower to be centrifuged 5 min, collects thallus after removing supernatant, is suspended with 5% sucrose solution；Before immersion, SilwetL-77, concentration is added
For 0.05% (500 μ l/L), foam is shaken out；The aerial part of arabidopsis is impregnated into 15-30 s, phase in Agrobacterium aaerosol solution
Between shake gently；Dipped arabidopsis is lain low in pallet, is covered with preservative film, masking foil sealing is protected from light, and 4 DEG C, places 24
h；Masking foil is opened, is cultivated under positive elongate member, stops watering when seed maturation.
5% each ingredient of sucrose solution re-suspension liquid is as follows: MS culture medium, adds sucrose 50g/L, MES 0.5g/L, Silwet-
77 500μl /L.(note: pH modulation 5.8 after preparation, bacterium solution centrifugation add SilwetL-77 after being resuspended；Re-suspension liquid and bacterium solution
Conversion relation are as follows: re-suspension liquid dosage: bacterium solution OD* bacterium solution volume=0.8* re-suspension liquid).
10) screening of transgenic plant
The T1 of collection is sterilized for the seed of transgenic arabidopsis with alcohol and mercuric chloride, step are as follows: takes what is obtained in right amount to turn base
Because seed is placed in 1.5mL centrifuge tube, 30 s are impregnated with 75% alcohol；10% sodium hypochlorite sterilizing, 2 min, 30 s；Sterile water
It rinses 3-4 times, replacement is through autoclaved new centrifuge tube after rinsing for the first time；It is suspended with 0.1% agarose solution.
Transgenic arabidopsis seed after sterilizing is seeded in containing antibiotic (50 mg/L of kanamycins and cephalosporin
100 mg/L) 1/2MS solid medium on.22 DEG C, illumination cultivation.About after a week by can be with normal growth on culture medium
In arabidopsis transplanting and soil, continued growth, as a result as shown in Figure 4.
11) detection of transgenic plant
The young leaflet tablet for taking appropriate arabidopsis and transgenic plant extracts DNA, concrete operation step are as follows: will fit using CTAB method
It measures in the 2mL centrifuge tube after blade is placed in sterilization treatment, the CTAB solution of 700 μ l is added, is thoroughly ground with ball milling instrument, 65 DEG C
Stand 10 min；Isometric chloroform: isoamyl alcohol is added, overturn is uniformly mixed it for several times, and 14000 rpm are centrifuged 10 min；
Supernatant is moved in new sterile centrifugation tube, isometric isopropanol is added, be mixed by inversion for several times, is stored at room temperature 2 min,
14000 rpm are centrifuged 10 min, outwell supernatant；70% dehydrated alcohol is added, is washed twice using liquid-transfering gun piping and druming, 14000
Rpm is centrifuged 1min, discards supernatant；Surface liquid is dried up, 20 μ L ddH are added2O dissolution.Take transgenosis and the open country of said extracted
The DNA of raw type arabidopsis, is usedCgWRKY2The specific primer of gene carries out PCR detection.
ChunlanCgWRKY2After genetic transformation arabidopsis, 5 overexpressions are obtained altogetherCgWRKY2Gene arabidopsis strain.With weight
Group plasmid is positive control, and using wild type as negative control, using water as blank control, PCR result is as shown in figure 4, positive control
ForCgWRKY2Genophore PCR as a result, negative control with water be instead of template, WT is using wildtype Arabidopsis thaliana DNA as template.
12) Phenotypic Observation
Acquisition of the difference for transgenic plant: the transgenosis T1 of harvest is sterilized for seed, after screening and culturing, then transplants in nutrition
In soil, 22 DEG C, 16 h illumination/8h dark culturing；Retain the transgenic plant tentatively confirmed after detecting, T1 is harvested after maturation
It for seed, is numbered, obtains T2 generation；It is the same with T1 generation, by T2 for seed it is sterilized after be coated on antibiotic screening training
It supports on base, is placed in 22 DEG C, continuous illumination；10 days or so, survival rate statistics is carried out for seed to the T2 of different numbers, chooses survival
The plant transplanting that ratio is 75% in Nutrition Soil according to 22 DEG C, 16 h illumination/8h dark culturing, and blade is taken to carry out positive inspection
It surveys；Positive T2 continues to number for plant, collects seed, obtains T3 for seed；After Seed sterilization, sieved with screening and culturing medium
Choosing, is placed in continuous illumination culture under light；10 days or so, the T3 of different numbers was observed for plant, all survives and does not occur
Separation is T3 for homozygous plants.
Obtained transgenic line is observed in batches.
(1) it chooses wherein apparent 3 transgenic plants of phenotype to be observed, as a result, it has been found that with wildtype Arabidopsis thaliana phase
Than transgenic Arabidopsis plants growth and development is slow, and plant downgrades (Fig. 5)；When transgenic plant is grown 20 days, blade edge is opened
Begin jaundice symptom occur, blade is locally withered and yellow when growing 25 days, and blade full wafer is withered and yellow (Fig. 6) when growing 30 days
(2) 4 DEG C of low temperature stress processing, detection and low temperature related gene are carried out to transgenic arabidopsisAtCOR47、AtCOR15AWithAtRD29AExpression quantity, see Fig. 7, as a result, it has been found that in wildtype Arabidopsis thaliana and transgenic arabidopsis, 3 genes are coerced in low temperature
Significant up-regulation under compeling, but level-off is significantly higher than wild type in the expression in transgenic line, thus it is speculated that under low temperature stress
TransgenosisCgWRKY2The expression that plant regulates and controls downstream gene participates in low temperature stress；CgWRKY2Overexpression passes through regulationAtCOR47、AtCOR15AAndAtRD29AThe winter resistance of the expression enhancing plant of gene.
The present embodiment is by the Chunlan of overexpressionCgWRKY2The 35S of gene:CgWRKY2It is transferred in model plant arabidopsis, into
Row Phenotypic Observation and analysis.From the results, it was seen that being overexpressed 35S: CgWRKY2Arabidopsis T2 for plant occur growth hair
It is slow to educate, and plant is downgraded, and withered and yellow wait of blade shifts to an earlier date senile symptom；CgWRKY2 overexpression may be by regulating and controlling cold signal dependency basis
CauseAtCOR47、AtCOR15AAndAtRD29AThe winter resistance of the expression enhancing plant of gene.
<120>a kind of Chunlan CgWRKY2 gene and its application
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213>Chunlan Cymbidium goeringii
<400> 1
atggatgaca gtatatccat ttttggagat tgggcagctt caaataccag cccaagaaca 60
tttatatcaa attttttcaa tgaagatact agctcaagac catttcctga tcttggagac 120
agtggacagt taggttttat ggagcgtgag aataagaaaa gaacattgga atcagttcag 180
ttaaatgttc ccaaatcaag cacaagaggg gctttaagtg aaaggatggc agctaggcct 240
ggttttagta cattaacggt caatactagt agaagtggaa cagagaacgt gatctcttca 300
tcttcagaca agcattcttc atgtttgctg ctttcaccag gtgtcagtcc cacggcattg 360
ttggaatctc cagttttcct ttcaaattcc atggcccaac catcgcctac cacagggaaa 420
tttccatttg cacacaccat ggactgtcct cagttttcaa cattagaaac tgacagaagt 480
aatgttcaac agatcgaaga tgttcatcct gaggcattta ccttcccatg gctgggttca 540
aatttctctt tttcatcaca tgttgaaaac aaatttggtt tatccattga tcaaaaacaa 600
tctttagtaa acatgggtgc atctcttcca caagctctga aacaacccgc tggattcaca 660
gaaacatctg aaacaaaaga gagtgcagaa gatgatacga aagctgattc ttttctggca 720
aatgaatgtt caccacctga tgacaatcag aacggagaaa cagatccaaa aggagagttc 780
tcatccatgc ctattagtgc tccagaagat gatggttata actggaggaa gtatgggcag 840
aagcatgtta agggtagtga gttccctcgt agttattata agtgtacttt cttaaattgt 900
ccagtaaaga agaaggttga aagatctctc gagggccatg tcactgaaat aatctacagg 960
ggtgctcaca accaccccaa gccttcttcg agccgcaggt catctacttc gtcttcccac 1020
tctttcagtg atgcacatat ggacaattca gattatcacg tcgttaatgc aaattttgat 1080
ggaaagcttt cattgctaaa cgcacaaaat ggagatgggg gatcagaatg gaggggggat 1140
ggcatggaag ctgtctcttc agcttctgtt tctgaatttg cgacttctaa ttccatgcag 1200
acacatttgt catcagcaat gtctaacgat gaagaggagg atggggcaac tcatggtagt 1260
ttttcaatgg gatgtgatgc agaaggggag gaaatagatt caaagagaag gaaactagat 1320
ttgtgcccaa ttgacatgag tgcagcttca agagccatgc gagagcctcg tgttgtggtt 1380
cagacgacta gcgaggtcga tatcctcgat gatggctacc gatggcgtaa gtatgggcag 1440
aaggttgtca aaggaaatcc aaatccaagg agctactaca agtgcacgaa cccaggatgc 1500
acagtccgca agcatgtcga gagagcatcg catgatctca aatccgtcat caccacatac 1560
gaagggaagc acaaccacga cgtccccgcc accaaaacca gcaaccatca aagctccggt 1620
tcatcgaact gcgtcgcaac tggcatacaa cttcctcata atagcctcca tcgtagggtc 1680
gagcccgttc aagaaggcct catgaggttc gaaaaccacg tgatgattga cccctttgta 1740
ctgccttgta gagagccaca acttggtgca gcagctggat atggtgacta tgctcttgga 1800
attggccacc aggctctggc caaccttggc ataggaggtt taggtccaat gagaccaatg 1860
aggatcccag cttttgcttc agttaatcca tatttggagc accagaggaa ggttggtgga 1920
ggggagccaa aagaagaagt aaggttggag tccgatctgc cagtaatcac ccatgcacct 1980
aaaggttatt atcagtttgc aaacagagta tctcttgggc catatccatg a 2031
<210> 2
<211> 676
<212> PRT
<213>artificial sequence (artiartificial sequence)
<400> 2
Met Ala Ala Ser Ile Ser Ile Pro Gly Ala Thr Ala Ala Ser Ala Thr
1 5 10 15
Ser Pro Ala Thr Pro Ile Ser Ala Pro Pro Ala Gly Ala Thr Ser Ser
20 25 30
Ala Pro Pro Pro Ala Leu Gly Ala Ser Gly Gly Leu Gly Pro Met Gly
35 40 45
Ala Gly Ala Leu Leu Ala Thr Leu Gly Ser Val Gly Leu Ala Val Pro
50 55 60
Leu Ser Ser Thr Ala Gly Ala Leu Ser Gly Ala Met Ala Ala Ala Pro
65 70 75 80
Gly Pro Ser Thr Leu Thr Val Ala Thr Ser Ala Ser Gly Thr Gly Ala
85 90 95
Val Ile Ser Ser Ser Ser Ala Leu His Ser Ser Cys Leu Leu Leu Ser
100 105 110
Pro Gly Val Ser Pro Thr Ala Leu Leu Gly Ser Pro Val Pro Leu Ser
115 120 125
Ala Ser Met Ala Gly Pro Ser Pro Thr Thr Gly Leu Pro Pro Pro Ala
130 135 140
His Thr Met Ala Cys Pro Gly Pro Ser Thr Leu Gly Thr Ala Ala Ser
145 150 155 160
Ala Val Gly Gly Ile Gly Ala Val His Pro Gly Ala Pro Thr Pro Pro
165 170 175
Thr Leu Gly Ser Ala Pro Ser Pro Ser Ser His Val Gly Ala Leu Pro
180 185 190
Gly Leu Ser Ile Ala Gly Leu Gly Ser Leu Val Ala Met Gly Ala Ser
195 200 205
Leu Pro Gly Ala Leu Leu Gly Pro Ala Gly Pro Thr Gly Thr Ser Gly
210 215 220
Thr Leu Gly Ser Ala Gly Ala Ala Thr Leu Ala Ala Ser Pro Leu Ala
225 230 235 240
Ala Gly Cys Ser Pro Pro Ala Ala Ala Gly Ala Gly Gly Thr Ala Pro
245 250 255
Leu Gly Gly Pro Ser Ser Met Pro Ile Ser Ala Pro Gly Ala Ala Gly
260 265 270
Thr Ala Thr Ala Leu Thr Gly Gly Leu His Val Leu Gly Ser Gly Pro
275 280 285
Pro Ala Ser Thr Thr Leu Cys Thr Pro Leu Ala Cys Pro Val Leu Leu
290 295 300
Leu Val Gly Ala Ser Leu Gly Gly His Val Thr Gly Ile Ile Thr Ala
305 310 315 320
Gly Ala His Ala His Pro Leu Pro Ser Ser Ser Ala Ala Ser Ser Thr
325 330 335
Ser Ser Ser His Ser Pro Ser Ala Ala His Met Ala Ala Ser Ala Thr
340 345 350
His Val Val Ala Ala Ala Pro Ala Gly Leu Leu Ser Leu Leu Ala Ala
355 360 365
Gly Ala Gly Ala Gly Gly Ser Gly Thr Ala Gly Ala Gly Met Gly Ala
370 375 380
Val Ser Ser Ala Ser Val Ser Gly Pro Ala Thr Ser Ala Ser Met Gly
385 390 395 400
Thr His Leu Ser Ser Ala Met Ser Ala Ala Gly Gly Gly Ala Gly Ala
405 410 415
Thr His Gly Ser Pro Ser Met Gly Cys Ala Ala Gly Gly Gly Gly Ile
420 425 430
Ala Ser Leu Ala Ala Leu Leu Ala Leu Cys Pro Ile Ala Met Ser Ala
435 440 445
Ala Ser Ala Ala Met Ala Gly Pro Ala Val Val Val Gly Thr Thr Ser
450 455 460
Gly Val Ala Ile Leu Ala Ala Gly Thr Ala Thr Ala Leu Thr Gly Gly
465 470 475 480
Leu Val Val Leu Gly Ala Pro Ala Pro Ala Ser Thr Thr Leu Cys Thr
485 490 495
Ala Pro Gly Cys Thr Val Ala Leu His Val Gly Ala Ala Ser His Ala
500 505 510
Leu Leu Ser Val Ile Thr Thr Thr Gly Gly Leu His Ala His Ala Val
515 520 525
Pro Ala Thr Leu Thr Ser Ala His Gly Ser Ser Gly Ser Ser Ala Cys
530 535 540
Val Ala Thr Gly Ile Gly Leu Pro His Ala Ser Leu His Ala Ala Val
545 550 555 560
Gly Pro Val Gly Gly Gly Leu Met Ala Pro Gly Ala His Val Met Ile
565 570 575
Ala Pro Pro Val Leu Pro Cys Ala Gly Pro Gly Leu Gly Ala Ala Ala
580 585 590
Gly Thr Gly Ala Thr Ala Leu Gly Ile Gly His Gly Ala Leu Ala Ala
595 600 605
Leu Gly Ile Gly Gly Leu Gly Pro Met Ala Pro Met Ala Ile Pro Ala
610 615 620
Pro Ala Ser Val Ala Pro Thr Leu Gly His Gly Ala Leu Val Gly Gly
625 630 635 640
Gly Gly Pro Leu Gly Gly Val Ala Leu Gly Ser Ala Leu Pro Val Ile
645 650 655
Thr His Ala Pro Leu Gly Thr Thr Gly Pro Ala Ala Ala Val Ser Leu
660 665 670
Gly Pro Thr Pro
675
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 3
atggatgaca gtatatccat ttt 23
<210> 4
<211> 23
<212> DNA
<213>artificial sequence (artiartificial sequence)
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tggatatggc ccaagagata ctc 23
<210> 5
<211> 29
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 5
cccgggatgg atgacagtat atccatttt 29
<210> 6
<211> 29
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 6
tacgtatgga tatggcccaa gagatactc 29
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 7
gatagtggaa aaggaaggtg 20
<210> 8
<211> 23
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 8
tggatatggc ccaagagata ctc 23
Claims (5)
1. a kind of ChunlanCgWRKY2Gene, nucleotide sequence is as shown in SEQ ID NO.1.
2. Chunlan described in claim 1CgWRKY2The expression albumen of gene, amino acid sequence is as shown in SEQ ID NO.2.
3. Chunlan described in claim 1CgWRKY2Application of the gene in plant production and breeding.
4. containing Chunlan described in claim 1CgWRKY2The carrier of gene.
5. containing the host cell of Chunlan CgWRKY2 gene described in claim 1.
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| CN111304222A (en) * | 2020-04-16 | 2020-06-19 | 南京林业大学 | Cymbidium CgWRKY11 gene and application thereof |
| CN111304220A (en) * | 2020-04-16 | 2020-06-19 | 南京林业大学 | Cymbidium CgWRKY3 gene and application thereof |
| CN111304221A (en) * | 2020-04-16 | 2020-06-19 | 南京林业大学 | Cymbidium CgWRKY31 gene and application thereof |
| CN111304223A (en) * | 2020-04-16 | 2020-06-19 | 南京林业大学 | Cymbidium CgWRKY24 gene and application thereof |
| CN111424040A (en) * | 2020-04-16 | 2020-07-17 | 南京林业大学 | Cymbidium CgWRKY21 gene and application thereof |
| CN111424039A (en) * | 2020-04-16 | 2020-07-17 | 南京林业大学 | Cymbidium CgWRKY65 gene and application thereof |
| CN111424041A (en) * | 2020-04-16 | 2020-07-17 | 南京林业大学 | Cymbidium CgWRKY49 gene and application thereof |
| CN111424038A (en) * | 2020-04-16 | 2020-07-17 | 南京林业大学 | Cymbidium CgWRKY40 gene and application thereof |
| CN111424037A (en) * | 2020-04-16 | 2020-07-17 | 南京林业大学 | Cymbidium CgWRKY70 gene and application thereof |
| CN111454966A (en) * | 2020-04-16 | 2020-07-28 | 南京林业大学 | Cymbidium CgWRKY4 gene and application thereof |
| CN111304220B (en) * | 2020-04-16 | 2022-04-26 | 南京林业大学 | Cymbidium CgWRKY3 gene and application thereof |
| CN111454966B (en) * | 2020-04-16 | 2022-04-26 | 南京林业大学 | Cymbidium CgWRKY4 gene and application thereof |
| CN111304223B (en) * | 2020-04-16 | 2022-04-26 | 南京林业大学 | Cymbidium CgWRKY24 gene and application thereof |
| CN111424038B (en) * | 2020-04-16 | 2022-04-26 | 南京林业大学 | Cymbidium CgWRKY40 gene and application thereof |
| CN111424040B (en) * | 2020-04-16 | 2022-05-13 | 南京林业大学 | Cymbidium CgWRKY21 gene and application thereof |
| CN111424041B (en) * | 2020-04-16 | 2022-05-27 | 南京林业大学 | Cymbidium CgWRKY49 gene and application thereof |
| CN111424039B (en) * | 2020-04-16 | 2022-05-27 | 南京林业大学 | Cymbidium CgWRKY65 gene and application thereof |
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