CN107674874A - Applications of the gene M YC2 in plant frigostabile ability is improved - Google Patents

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

Applications of the gene M YC2 in plant frigostabile ability is improved

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

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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.