CN107674874A - Applications of the gene M YC2 in plant frigostabile ability is improved - Google Patents
Applications of the gene M YC2 in plant frigostabile ability is improved Download PDFInfo
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- CN107674874A CN107674874A CN201711057849.2A CN201711057849A CN107674874A CN 107674874 A CN107674874 A CN 107674874A CN 201711057849 A CN201711057849 A CN 201711057849A CN 107674874 A CN107674874 A CN 107674874A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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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 applications of the gene M YC2 in plant frigostabile ability is improved, the gene M YC2 derives from Columbia ecotype arabidopsis, its cDNA nucleotide sequence such as SEQ ID NO:Shown in 2.The present invention provides genetic resources to cultivate low temperature resistant new variety of plant, has preferable potential using value, to study the molecule mechanism based theoretical of the mechanism of plant response adverse circumstance signal and tolerance adverse environment.
Description
Technical field
The present invention relates to genetic engineering and biology field, specifically, it is resistance in raising plant to be related to gene M YC2
Application in low temperature ability.
Background technology
Due to the immovability of plant, plant grows to environmental change and its sensitivity, when by extraneous non-life
During thing environment stress, the growth of plant is influenceed, limits the yield of crop.In recent years, with the extreme variation of global climate, low temperature
Increasingly becoming a kind of influences extensive abiotic stress, and normal growth and agricultural production to plant cause greatly broken
It is bad.Therefore, the physiological acoustic signals and molecule mechanism of plant responding Chilling stress are studied, find the pass of regulation and control plant low temperature response
Key gene, important theories integration is provided to obtain the acquisition of low temperature resistant plant, has the application of key to providing crop yield
Value.
Molecular biology of plants research at present is more to be carried out with arabidopsis (Arabidopsis thaliana) for model plant
Research, Arabidopsis have that genome is small in crucifer, the advantages such as growth is very fast, be widely used in Plant genetics,
In the research such as cell biology, molecular biology.The molecule responded using arabidopsis as model plant research plant to Chilling stress
Mechanism, the molecular mechanism of plant low temperature response faster can be preferably illustrated, so as to cultivate the low temperature resistant plant of crop for raising
Theoretical foundation is provided.Typically infected or the method for other transgenic methods, Cloning of Genes Related can be carried by Agrobacterium
Body is gone in wildtype Arabidopsis thaliana, obtains the overexpression or gene knockout strain of the gene, then carries out plant tolerance reality again
Detection is tested, studies function of the gene in related adverse circumstance, the function that the gene plays in corresponding adverse circumstance can be learnt, so as to
Excavate the genetic resources of resistance to environment stress.
The content of the invention
It is an object of the invention to provide applications of the gene M YC2 in plant frigostabile ability is improved.
In order to realize the object of the invention, applications of the gene M YC2 provided by the invention in plant frigostabile ability is improved,
The nucleotides sequence of the gene M YC2cDNA is classified as:
i)SEQ ID NO:Nucleotide sequence shown in 2;Or
ii)SEQ ID NO:Nucleotide sequence shown in 2 be substituted, lack and/or increase one or more nucleotides and
Nucleotide sequence with identical function;Or
Iii) under strict conditions with SEQ ID NO:Sequence shown in 2 hybridizes and the nucleotide sequence with identical function,
The stringent condition be in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, it is miscellaneous at 65 DEG C
Hand over, and film is washed with the solution;Or
Iv) and i), ii) or nucleotides of the nucleotide sequence with more than 90% homology and with identical function iii)
Sequence.
MYC2 gene sources are in Columbia ecotype arabidopsis, the numbering in arabidopsis gene group database
AT1G32640.For MYC2 genes by 2179 base compositions, the reading frame of the gene is from 5 ' the 76th to the 1947th alkali in end
Base.The gene reading frame is made up of 1 extron, and reading frame the 1st to the 1872nd bit base is exon sequence.By MYC2
The amino acid sequence of gene code is as shown in Seq ID No.1.It should be appreciated that those skilled in the art can disclose according to the present invention
Amino acid sequence, on the premise of its activity is not influenceed, substitute, lack and/or increase one or several amino acid, obtain institute
State the mutant nucleotide sequence of albumen.
It should be understood that the preferences of the degeneracy and different plant species codon in view of codon, those skilled in the art can
The codon for being adapted to particular species to express is used with as needed.
The present invention also provides the cloning vector of the MYC2 gene orders containing plant frigostabile or its fragment or all kinds of expression
Carrier, the host cell containing the carrier and turn base at the conversion plant cell containing the gene order or its specific fragment
Because of plant.
Plant of the present invention includes monocotyledon and dicotyledon, such as arabidopsis etc..By plant frigostabile
MYC2 genes are overexpressed in plant, are entered using any carrier that foreign gene can be guided to be overexpressed in plant
OK.
The present invention also provides a kind of method for improving plant frigostabile ability, by being overexpressed gene M YC2 in plant,
So as to improve tolerance of the plant to low temperature;
The present invention also provides a kind of method for building low temperature resistant transgenic arabidopsis, and methods described is specially:Extraction is intended
Southern mustard total serum IgE, reverse transcription obtain cDNA, and using cDNA as template, F and R are primer, expand MYC2 genes, amplified production is distinguished
It is building up on plant expression vector, then with the recombinant expression carrier conversion Agrobacterium obtained, is then invaded with the Agrobacterium of conversion
Arabidopsis floral is contaminated, positive transgenic plant is screened, obtains low temperature resistant transgenic arabidopsis.
Wherein, the primers F and R nucleotide sequence are as shown in Seq ID No.3 and 4.
The plant expression vector that the present invention uses is the expression vector with 35S promoter, for example, carrier pGWB17 or
pGWB5。
Foregoing method, preferably described Agrobacterium are GV3101.
In preceding method, the arabidopsis is preferably wild-type Arabidopsis plants, i.e. the arabidopsis with homozygous genotype
Plant.
After the MYC2 gene overexpressions of the present invention, arabidopsis shows as low temperature resistant phenotype.For the ease of to transgenosis
Arabidopsis plant is identified and screened, and used carrier can be processed, and such as adds the alternative mark of plant or tool
Resistant antibiotic marker etc..
MYC2 genes provided by the invention provide genetic resources to cultivate low temperature resistant new variety of plant, have preferably latent
In application value, the molecule mechanism based theoretical of mechanism and tolerance adverse environment for research plant response adverse circumstance signal.
Brief description of the drawings
Fig. 1 is the vegetable protein Western Blot inspections of MYC2 gene overexpression arabidopsis strains in the embodiment of the present invention 2
Survey result;Wherein, A is to turn 35S:MYC2-myc Arabidopsis plants (OE-1), B are to turn 35S:MYC2-GFP Arabidopsis plants (OE-
2)。
Fig. 2 is transgenic arabidopsis OE-1 and the OE-2 table in the embodiment of the present invention 3 under the conditions of non-Cold exposed and Cold exposed
Existing freeze proof phenotype.
Fig. 3 be the embodiment of the present invention 3 under the conditions of non-Cold exposed and Cold exposed, transgenic arabidopsis OE-1's and OE-2
Survival rate.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Molecular Cloning: A Laboratory method (Tsuyoshi Nakagawa et al, Development of
series of gateway binary vectors,pGWBs,for realizing efficient construction
Of fusion genes for plant transformation, 2007), or the bar according to manufacturer's specification suggestion
Part.
PENTR D-TOPO cloning vectors are conventional cloning vector in following examples, commercially available;PGWB17 and pGWB5
Carrier is provided by Chinese Academy of Sciences's heredity and development research institute Li Chuanyou professors laboratory;Arabidopsis kind is Colombia's ecology
Type;The conventional cloning vector of Agrobacterium GV3101 bacterial strains.
Main agents in following examples are:PENTR D-TOPO, gateway molecular cloning related experiment products are purchased
From Invitrogen companies;It is Taq archaeal dna polymerases, T4 ligases, Pyrobest Taq enzymes, KOD, raw purchased from NEB, Toyobo etc.
Thing company;DNTPs is purchased from Genestar companies;The small extraction reagent kit of plasmid and Ago-Gel QIAquick Gel Extraction Kit are public purchased from Axygen
Department;MS culture mediums, agar powder, agarose, ampicillin (Amp), kanamycins (Kan), gentamicin sulphate (Gen), profit
Good fortune puts down antibiotic and sucrose (Glucose), bovine serum albumin(BSA) (BSA), nitrocellulose filter, LB culture mediums etc. such as (Rif)
Purchased from companies such as Sigma, Bio-Rad;Various other chemical reagent used in embodiment are that import or domestic analysis are pure
Reagent.
Primer used in embodiment is synthesized by six directions Hua Da company, and carries out correlative measurement sequence.
The structure of the MYC2 gene overexpression carriers of embodiment 1
To understand the molecular mechanism of plant responding low temperature, from arabidopsis (Arabidopsis thaliana) genome gram
Grand MYC2 genes.Analyzed according to coding region sequence, design primers F and R, the code area of the gene is amplified to come, connected respectively
It is connected on over-express vector pGWB17 and pGWB5.
The primer is:
Sense primer F:5’-CACCATGACTGATTACCGGCTACA-3’(Seq ID No.3)
Anti-sense primer R:5’-ACCGATTTTTGAAATCAAACTTGC-3’(Seq ID No.4)
It is by the specific method that MYC2 genes are connected on carrier pGWB17, pGWB5:Using being connected to pENTR/D-TOPO
Clone on cloning vector, (the recombinant expression carrier of acquisition point is recombinated on pGWB17 and pGWB5 carriers by LP reactions respectively
35S is not named as it:MYC2-myc and 35S:MYC2-GFP), obtain small by the positive colony bacterial plaque after resistance screening, kit
Amount extraction plasmid simultaneously send sequencing, and sequencing correctly shows to be connected into MYC2 genes in pGWB17 and pGWB5 carriers.
The structure of the MYC2 gene overexpression plants of embodiment 2 and detection
PGWB17 and pGWB5 carriers containing MYC2 genes described in embodiment 1 are transformed into Agrobacterium GV3101 bacterium respectively
In strain, then it is transferred to respectively in wild-type Arabidopsis plants, obtains arabidopsis transgenic seedlings.Specific method is:It will contain purposeful
(the μ g/mL of kanamycins 50, the μ g/mL of rifampin 50, celebrating are big mould in the resistant to liquids nutrient solutions of 100mL LB tri- for the Agrobacterium inoculation of carrier
50 μ g/mL of element) in, 28 DEG C of shaken cultivations are stayed overnight, and treat OD600It is worth for 1.0-2.0, with 5000rpm, room temperature centrifugation 10min, collects
Thalline;With 200mL conversion fluids (1/2MS, 5% sucrose, 40 μ L Silwet L-77) suspension thalline;Arabidopsis floral is immersed in
1min in the conversion fluid of Agrobacterium, putting freshness protection package moisturizing and being placed in dark place makes its temperature relatively low, and second day by plant from guarantor
Taken out in fresh bag, put back on illumination cultivation frame normal growth to sowing.
PGWB17 and pGWB5 carriers institute band screening resistant gene is hygromycin, with hygromycin resistance to arabidopsis transgenosis
Seedling is screened, the T of acquisition1The positive seedling that generation has hygromycin resistance carries out individual plant sowing, then to T2It is mould that tide is carried out for seed
The test of plain resistance, select 3/4 it is resistant and remaining 1/4 without resistance strain, illustrate to be connected with purpose base in the strain
The over-express vector of cause is inserted in the form of singly copying.The plant in these strains with hygromycin resistance is removed, then carries out list
Strain sowing, carries out hygromycin resistance screening for seed with the T3 of acquisition, if T4 does not separate for plant, illustrates the transgenosis again
Strain is homozygote, and the homozygote can be used for breeding, Chilling stress processing experiment.
Isolated overexpression strain in the present embodiment.Gained is detected using protein immunoblotting method and is overexpressed strain
System.
1) plant total protein is extracted
Grown on culture dish 12 days seedling are wrapped with masking foil, frozen rapidly standby in liquid nitrogen.It will be planted with liquid nitrogen
Thing material is clayed into power shape, adds protein extract buffer and protease inhibitors, the concussion that is vortexed after 4 DEG C, 13000rpm from
Heart 15min.
2) protein immunoblotting method (Western Blot) detection is overexpressed the protein content in strain
The wild type of equivalent is taken respectively, is overexpressed the vegetable protein of arabidopsis strain, adds 5 × SDS albumen loading buffers
Liquid, 100 DEG C are boiled 5min, after 90V voltages run glue 15min, are changed to 120V voltages and are run glue 1h, 200mA direct current transferring film 2h.Finally
The protein content in strain is overexpressed with Anti-Myc antibody tests.Internal reference is used as using Anti-Actin.
Western Blot testing results are shown in that Fig. 1, A are to turn 35S:MYC2-myc Arabidopsis plants (OE-1), B are to turn 35S:
MYC2-GFP Arabidopsis plants (OE-2), it can be seen that the destination protein expression being overexpressed in strain is stronger.
The method that MYC2 genes are overexpressed in other plants may be referred to the present embodiment progress.
Embodiment 3 is overexpressed the low temperature tolerance ability detection of MYC2 gene plants
The life on culture dish first by the MYC2 gene overexpression strains of gained in wildtype Arabidopsis thaliana seedling and embodiment 2
It is long 12 days, then it is moved into low temperature incubator and carries out freezing processing.For Cold exposed plant, strain is overexpressed in culture dish
After upper growth moves into 4 DEG C of low temperature incubator cultures 3 days after 12 days, enter at line program cooling (1 DEG C/h) to required sub-zero temperature
Manage certain time.More than processing plant dark processing moves into what statistics after 22 DEG C of illumination box cultures 3 days was survived after 24 hours
Number of plant, calculate survival rate.
As a result (Fig. 2 and Fig. 3) is shown, under the conditions of non-Cold exposed and Cold exposed, OE-1 (35S:) and OE-2 MYC2-myc
(35S:MYC2-GFP freeze proof phenotype) is showed.The low temperature tolerance ability that this explanation MYC2 is overexpressed plant is significantly improved.
As can be seen here, tolerance of the plant to low temperature can be significantly improved after arabidopsis MYC2 gene overexpressions.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>Applications of the gene M YC2 in plant frigostabile ability is improved
<130> KHP171116965.7
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 623
<212> PRT
<213>Arabidopsis (Arabidopsis thaliana)
<400> 1
Met Thr Asp Tyr Arg Leu Gln Pro Thr Met Asn Leu Trp Thr Thr Asp
1 5 10 15
Asp Asn Ala Ser Met Met Glu Ala Phe Met Ser Ser Ser Asp Ile Ser
20 25 30
Thr Leu Trp Pro Pro Ala Ser Thr Thr Thr Thr Thr Ala Thr Thr Glu
35 40 45
Thr Thr Pro Thr Pro Ala Met Glu Ile Pro Ala Gln Ala Gly Phe Asn
50 55 60
Gln Glu Thr Leu Gln Gln Arg Leu Gln Ala Leu Ile Glu Gly Thr His
65 70 75 80
Glu Gly Trp Thr Tyr Ala Ile Phe Trp Gln Pro Ser Tyr Asp Phe Ser
85 90 95
Gly Ala Ser Val Leu Gly Trp Gly Asp Gly Tyr Tyr Lys Gly Glu Glu
100 105 110
Asp Lys Ala Asn Pro Arg Arg Arg Ser Ser Ser Pro Pro Phe Ser Thr
115 120 125
Pro Ala Asp Gln Glu Tyr Arg Lys Lys Val Leu Arg Glu Leu Asn Ser
130 135 140
Leu Ile Ser Gly Gly Val Ala Pro Ser Asp Asp Ala Val Asp Glu Glu
145 150 155 160
Val Thr Asp Thr Glu Trp Phe Phe Leu Val Ser Met Thr Gln Ser Phe
165 170 175
Ala Cys Gly Ala Gly Leu Ala Gly Lys Ala Phe Ala Thr Gly Asn Ala
180 185 190
Val Trp Val Ser Gly Ser Asp Gln Leu Ser Gly Ser Gly Cys Glu Arg
195 200 205
Ala Lys Gln Gly Gly Val Phe Gly Met His Thr Ile Ala Cys Ile Pro
210 215 220
Ser Ala Asn Gly Val Val Glu Val Gly Ser Thr Glu Pro Ile Arg Gln
225 230 235 240
Ser Ser Asp Leu Ile Asn Lys Val Arg Ile Leu Phe Asn Phe Asp Gly
245 250 255
Gly Ala Gly Asp Leu Ser Gly Leu Asn Trp Asn Leu Asp Pro Asp Gln
260 265 270
Gly Glu Asn Asp Pro Ser Met Trp Ile Asn Asp Pro Ile Gly Thr Pro
275 280 285
Gly Ser Asn Glu Pro Gly Asn Gly Ala Pro Ser Ser Ser Ser Gln Leu
290 295 300
Phe Ser Lys Ser Ile Gln Phe Glu Asn Gly Ser Ser Ser Thr Ile Thr
305 310 315 320
Glu Asn Pro Asn Leu Asp Pro Thr Pro Ser Pro Val His Ser Gln Thr
325 330 335
Gln Asn Pro Lys Phe Asn Asn Thr Phe Ser Arg Glu Leu Asn Phe Ser
340 345 350
Thr Ser Ser Ser Thr Leu Val Lys Pro Arg Ser Gly Glu Ile Leu Asn
355 360 365
Phe Gly Asp Glu Gly Lys Arg Ser Ser Gly Asn Pro Asp Pro Ser Ser
370 375 380
Tyr Ser Gly Gln Thr Gln Phe Glu Asn Lys Arg Lys Arg Ser Met Val
385 390 395 400
Leu Asn Glu Asp Lys Val Leu Ser Phe Gly Asp Lys Thr Ala Gly Glu
405 410 415
Ser Asp His Ser Asp Leu Glu Ala Ser Val Val Lys Glu Val Ala Val
420 425 430
Glu Lys Arg Pro Lys Lys Arg Gly Arg Lys Pro Ala Asn Gly Arg Glu
435 440 445
Glu Pro Leu Asn His Val Glu Ala Glu Arg Gln Arg Arg Glu Lys Leu
450 455 460
Asn Gln Arg Phe Tyr Ala Leu Arg Ala Val Val Pro Asn Val Ser Lys
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Met Asp Lys Ala Ser Leu Leu Gly Asp Ala Ile Ala Tyr Ile Asn Glu
485 490 495
Leu Lys Ser Lys Val Val Lys Thr Glu Ser Glu Lys Leu Gln Ile Lys
500 505 510
Asn Gln Leu Glu Glu Val Lys Leu Glu Leu Ala Gly Arg Lys Ala Ser
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Ala Ser Gly Gly Asp Met Ser Ser Ser Cys Ser Ser Ile Lys Pro Val
530 535 540
Gly Met Glu Ile Glu Val Lys Ile Ile Gly Trp Asp Ala Met Ile Arg
545 550 555 560
Val Glu Ser Ser Lys Arg Asn His Pro Ala Ala Arg Leu Met Ser Ala
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Leu Met Asp Leu Glu Leu Glu Val Asn His Ala Ser Met Ser Val Val
580 585 590
Asn Asp Leu Met Ile Gln Gln Ala Thr Val Lys Met Gly Phe Arg Ile
595 600 605
Tyr Thr Gln Glu Gln Leu Arg Ala Ser Leu Ile Ser Lys Ile Gly
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<210> 2
<211> 3289
<212> DNA
<213>Arabidopsis (Arabidopsis thaliana)
<400> 2
taagtaatta atctgttata aaaaatatat tctaaaagtt gacaaaacga caaatataaa 60
tcgaaaaaat aggtgaaaag ttatagtaaa gtgtttttcc actgagctca cagttcactc 120
tttggtacaa aagaaacaat cagttaaaat ctaagtcaat cattaaagag cgtgtgatac 180
acgtgcgacc aaacagccgt acgatctcac tcacttccgg tcttaattct taccgcgtcg 240
atccaacagt cgatggggtt aaatcgggat gtgagttcat cgaagcttct ctctctctta 300
caaccatcca cgtttcccaa tgaaatctcg ccacgtaata tccctaacaa caaatttaca 360
cattcaccct caagatcagt tacacatata ataatcgcag acgctctcca tttttctcca 420
ctacgaagac tttctcctat ctctctctct ctcattaaaa acgtgttttt ttttaccggt 480
caccggttta tggaatgact gattaccggc tacaaccaac gatgaatctt tggaccaccg 540
acgacaacgc ttctatgatg gaagctttca tgagctcttc cgatatctca actttatggc 600
ctccggcgtc gacgacaacc acgacggcga cgactgaaac aactccgacg ccggcgatgg 660
agattccggc acaggcggga tttaatcaag agactcttca gcaacgttta caagctttga 720
ttgaaggaac acacgaaggt tggacctacg ctatattctg gcaaccgtcg tatgatttct 780
ccggcgcctc cgtgctcgga tggggagatg gttattacaa aggtgaagaa gataaagcaa 840
acccgagacg gagatcgagt tcgccgccgt tttctactcc ggcggatcag gagtacagga 900
aaaaagtgtt gagagagctt aactcgttga tctccggtgg tgttgctccg tcggatgacg 960
ctgttgatga ggaggtgacg gatacggaat ggtttttctt ggtttcgatg acgcagagct 1020
tcgcttgcgg tgcgggatta gctggtaaag cgtttgcaac gggtaacgcg gtttgggttt 1080
ccgggtcaga tcaattatcc gggtcgggtt gtgaacgggc taagcaagga ggagtgtttg 1140
ggatgcatac tattgcgtgt attccttcgg cgaacggagt tgtggaagtc gggtcaacgg 1200
agccgatccg acagagttcg gaccttatta acaaggttcg aattcttttc aatttcgacg 1260
gcggagctgg agatttatcg ggtcttaatt ggaatcttga cccggatcaa ggtgagaacg 1320
acccgtctat gtggattaat gacccgattg gaacacctgg atctaacgaa ccgggtaacg 1380
gagctccaag ttctagctcc cagctttttt caaagtctat tcagtttgag aacggtagct 1440
caagcacaat aaccgaaaac ccgaatctgg atccgactcc gagtccggtt cattctcaga 1500
cccagaatcc gaaattcaat aacactttct cccgagaact taatttttcg acgtcaagtt 1560
ctactttagt gaaaccaaga tccggcgaga tattaaactt cggcgatgaa ggtaaacgaa 1620
gctccggaaa cccggatcca agttcttatt cgggtcaaac acaattcgaa aacaaaagaa 1680
agaggtcgat ggttttgaac gaagataaag ttctatcatt cggagataaa accgccggag 1740
aatcagatca ctccgatcta gaagcttccg tcgtgaaaga agtagcagta gagaaacgtc 1800
caaagaaacg aggaagaaag ccagcaaacg gtagagaaga gccactaaac cacgtcgaag 1860
cagagagaca aagacgcgag aaactaaacc aaagattcta cgcgttacga gcggttgtac 1920
caaacgtttc aaaaatggat aaagcttcgt tactcggtga cgcaatcgct tacatcaacg 1980
agcttaaatc caaagtagtc aaaacagagt cagagaaact ccaaatcaag aaccagctcg 2040
aggaagtgaa actcgagctc gccggaagaa aagcgagtgc tagtggagga gatatgtcgt 2100
cttcgtgttc ttcgattaaa ccggtgggga tggagattga agtgaagata attggttggg 2160
acgcaatgat tagagttgaa tctagtaaga ggaatcatcc ggcggcgagg ttgatgtcgg 2220
cgttgatgga tttggagttg gaagtgaatc acgcgagtat gtcggtggtt aacgatttga 2280
tgattcaaca agcgacggtg aagatgggtt ttaggatcta tacgcaagaa cagctcagag 2340
caagtttgat ttcaaaaatc ggttaaaagg gtgtgttttg ggaagtttag aaagttatgg 2400
ggtcaaatca taattaattc gttttagtgg cttcagtaat tttgtagatt ttagttttgt 2460
aagaaaaaaa tcttaaaata gagcgacaag tttcttcttt tgctctatgt ttgagtctgt 2520
atcgttttat tgttgtatct cctcaatgag taaacttgta tatattgata tgagtaatat 2580
gagttagtta ctgaaaaagt taatatttct acacaatttc atatgataaa aaagaaaaag 2640
aaatcgagta taaatggtct aaaataagag actaatggtc atgatcactg attttgtaca 2700
tatggtcaaa aatgtctctc ctaacacatc aaacttagga agagagacaa atcttaagta 2760
agttaagtcg agagggagac catatgcaca gttttcttaa gtcagtgtga taatgtttcc 2820
tcttgtctaa tcaataagac acaacacaca taaaccttag tataatacta catggatctt 2880
cgaatacaca gacagagtca cagacaagaa aaagcaaatc acttgctttt gtcttgatca 2940
tttccaatat ctacaaaaat agaaatcttg aagagattat tgaatctgaa atctgctttg 3000
tttgtgcatt ctcttcctcg atcacctcgc aagatcctct agcaacctga ccaatcctca 3060
agaatcaagt tttcttcttt ctagcttcag ctttccaatc tcgacgatgc ggccttatct 3120
tccgtttaac cgactcacaa gaactatctt gagccttgtt cataatcaaa tgagctcttc 3180
tagcttcttt tttccaattt ctatcgcaat aaacccgccg cacaaagttc tcaacataac 3240
cactaggtcc attaaactct agtccctcgt cttcttcttc ttcttcttc 3289
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
caccatgact gattaccggc taca 24
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
accgattttt gaaatcaaac ttgc 24
Claims (7)
1. applications of the gene M YC2 in plant frigostabile ability is improved, it is characterised in that the nucleosides of the gene M YC2cDNA
Acid sequence is:
i)SEQ ID NO:Nucleotide sequence shown in 2;Or
ii)SEQ ID NO:Nucleotide sequence shown in 2 is substituted, lacks and/or increased one or more nucleotides and has
The nucleotide sequence of identical function;Or
Iii) under strict conditions with SEQ ID NO:Sequence shown in 2 hybridizes and the nucleotide sequence with identical function, described
Stringent condition is in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, is hybridized at 65 DEG C, and
Film is washed with the solution;Or
Iv) and i), ii) or nucleotides sequence of the nucleotide sequence with more than 90% homology and with identical function iii)
Row.
2. application according to claim 1, it is characterised in that the plant includes monocotyledon and dicotyledon.
3. application according to claim 2, it is characterised in that the plant includes arabidopsis.
A kind of 4. method for improving plant frigostabile ability, it is characterised in that by being overexpressed gene M YC2 in plant, so as to
Improve tolerance of the plant to low temperature;
The definition of the gene M YC2 is the same as described in claim 1.
5. the method for the low temperature resistant transgenic arabidopsis of structure, it is characterised in that methods described is specially:It is total to extract arabidopsis
RNA, reverse transcription obtain cDNA, and using cDNA as template, F and R are primer, expand MYC2 genes, amplified production is building up to respectively
On plant expression vector, then with the recombinant expression carrier conversion Agrobacterium obtained, then plan south is infected with the Agrobacterium of conversion
Mustard inflorescence, positive transgenic plant is screened, obtains low temperature resistant transgenic arabidopsis;
Wherein, the primers F and R nucleotide sequence are as shown in Seq ID No.3 and 4.
6. according to the method for claim 5, it is characterised in that the plant expression vector be selected from carrier pGWB17 or
pGWB5。
7. the method according to claim 5 or 6, it is characterised in that the Agrobacterium is GV3101.
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CN118185957A (en) * | 2024-05-16 | 2024-06-14 | 湖南工程学院 | PgMYC2 gene for increasing PPD type ginsenoside content in ginseng cells and application thereof |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CN113337519A (en) * | 2021-06-03 | 2021-09-03 | 浙江农林大学 | Application of different copies of BrMYC2/3/4 gene in plant growth |
CN113337519B (en) * | 2021-06-03 | 2022-04-05 | 浙江农林大学 | Application of different copies of BrMYC2/3/4 gene in plant growth |
CN114480424A (en) * | 2021-06-03 | 2022-05-13 | 浙江农林大学 | Application of BrMYC2 gene overexpression in plant growth |
CN114480424B (en) * | 2021-06-03 | 2023-07-21 | 浙江农林大学 | Application of BrMYC2 gene overexpression in plant growth |
CN114561396A (en) * | 2022-01-21 | 2022-05-31 | 兰州大学 | Tyrannospora rex heat-resistant gene ZxDPB3-1 and application thereof in cultivation of heat-resistant crops |
CN114561396B (en) * | 2022-01-21 | 2023-10-13 | 兰州大学 | Bawang heat-resistant gene ZxDPB3-1 and application thereof in cultivation of heat-resistant crops |
CN118185957A (en) * | 2024-05-16 | 2024-06-14 | 湖南工程学院 | PgMYC2 gene for increasing PPD type ginsenoside content in ginseng cells and application thereof |
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