CN108624567A

CN108624567A - The application of plant EBF1 albumen and its encoding gene in building low temperature resistant plant

Info

Publication number: CN108624567A
Application number: CN201710158161.7A
Authority: CN
Inventors: 杨淑华; 施怡婷; 姜博晨; 张晓燕; 郭红卫
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2018-10-09

Abstract

The present invention relates to genetic engineering and molecular biology fields, specifically disclose the application of plant EBF1 albumen and its encoding gene in building low temperature resistant plant.The present invention is by the study found that plant frigostabile ability can be significantly improved by being overexpressed EBF1 genes in plant.This new opplication of EBF1 albumen and its encoding gene provided by the invention, new germ plasm resource is provided to cultivate low temperature resistant new variety of plant, certain theoretical foundation has been established simultaneously for research plant low temperature response molecular mechanism and raising plant stress-resistance Journal of Sex Research.

Description

The application of plant EBF1 albumen and its encoding gene in building low temperature resistant plant

Technical field

The present invention relates to genetic engineering and molecular biology fields, are related to plant EBF1 albumen and its encoding gene and are building Application in low temperature resistant plant.

Background technology

Due to the immovability of plant, the growth and development of plant is to environmental change and its sensitivity, when by extraneous non-life When object environment stress, the growth of plant is influenced, limits the yield of crop.In recent years, with the extreme variation of global climate, low temperature Increasingly becoming a kind of influencing 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, the pass of regulation and control plant low temperature response is found Key gene, the acquisition to obtain low temperature resistant plant provide important theories integration, have crucial application to providing crop yield Value.

Molecular biology of plants research at present is mostly carried out with arabidopsis (Arabidopsis thaliana) for model plant The advantages such as research, Arabidopsis have genome small in crucifer, and growth is very fast, be widely used in Plant genetics, In the researchs such as cell biology, molecular biology.The molecule that plant responds Chilling stress is studied by model plant of arabidopsis Mechanism faster can preferably illustrate the molecular mechanism of plant low temperature response, to cultivate the low temperature resistant plant of crop for raising Theoretical foundation is provided.It is generally 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, function of the gene in related adverse circumstance is studied, can learn the function that the gene plays in corresponding adverse circumstance, to Excavate the genetic resources of resistance to environment stress.

Invention content

In order to solve problems in the prior art, the object of the present invention is to provide plant EBF1 albumen and its encoding gene in structure Build the application in low temperature resistant plant.

In order to realize the object of the invention, technical scheme is as follows：

Present invention firstly provides application of the EBF1 albumen in building low temperature resistant plant, specially：By improving EBF1 Expression quantity of the albumen in plant, obtains low temperature resistant genetically modified plants；

Wherein, the amino acid sequence of EBF1 albumen is：

I) amino acid sequence shown in SEQ ID No.1；Or

Ii) by SEQ ID No.1 by one or several amino acid residues substitution and/or lack and or add and with SEQ ID No.1 amino acid sequences with the same function.

Further the present invention provides EBF1 genes (encoding gene of aforementioned EBF1 albumen) to build low temperature resistant plant by In application, specially：By being overexpressed EBF1 genes in plant, low temperature resistant genetically modified plants are obtained；

Wherein, the nucleotides sequence of EBF1 genes is classified as：

I) nucleotide sequence shown in SEQ ID No.2；

Ii) nucleotide sequence shown in SEQ ID No.2 be substituted, lack and/or increase one or more nucleotide and Express the nucleotide sequence of identical function protein；Or

Iii) under strict conditions with sequence hybridizes shown in SEQ ID No.2 nucleotide sequence；

For EBF1 genes shown in SEQ ID No.2 by 3158 base compositions, the reading frame of the gene is from 5 ' ends the 257th Position is to the 2924th bit base.The gene reading frame is made of 2 exons and 1 introne, the 1st to the 25th alkali of reading frame Base, the 397th to the 2258th bit base is exon sequence, remaining is its intron sequences.EBF1 gene sources are in brother's rival Sub- Arabidopsis thaliana ecotype, the number in arabidopsis gene group database is AT2G25490.

By the amino acid sequence of EBF1 gene codes as shown in SEQ ID No.1.It should be appreciated that those skilled in the art can According to amino acid sequence disclosed by the invention, do not influence its it is active under the premise of, substitution, missing and/or increase by one or several A amino acid obtains the mutant nucleotide sequence of the albumen.

It should be understood that in view of the degeneracy of codon and the preferences of different plant species codon, those skilled in the art can The codon for being suitble to particular species expression is used with as needed.

In aforementioned applications, the plant includes monocotyledon and dicotyledon, such as arabidopsis etc..Plant is resistance to low Warm EBF1 genes are overexpressed in plant, including the use of any carrier that foreign gene can be guided to be overexpressed in plant into Row.

The present invention also provides a kind of methods building low temperature resistant transgenic arabidopsis, include the following steps：

1) arabidopsis total serum IgE is extracted, reverse transcription obtains cDNA, and using cDNA as template, F and R are primer, expand EBF1 bases Amplified production is building up to the expression vector for being suitble to gene expression in plants with 35S promoter, such as pC-TAPa (Vector maps by cause See Fig. 5), the recombinant expression carrier 35S-EBF1-TAP of acquisition；

The expression vector contains attR1::Cm^r::ccdB::AttR2 sequences can be inserted by molecular cloning Gateway technologies Enter target gene；

2) Agrobacterium GV3101 is converted with 35S-EBF1-TAP, then infects arabidopsis floral using the Agrobacterium of conversion, Obtain low temperature resistant transgenic arabidopsis seedling.

Wherein, the nucleotide sequence of primers F described in step 1) and R are as shown in SEQ ID No.3 and 4.The arabidopsis Preferably wild-type Arabidopsis plants, the i.e. Arabidopsis plant with homozygous genotype.

After the 35S-EBF1-TAP gene overexpressions of the present invention, arabidopsis shows as low temperature resistant phenotype.For the ease of Transgenic Arabidopsis plants are identified and are screened, used carrier can be processed, it is alternative that plant is such as added Label or resistant antibiotic marker etc..

By the gene EBF1 to protein kinase in coded plant, (full name is EIN3-BINDING F BOX to the present invention PROTEIN 1) research, it is found that the transfer-gen plant for being overexpressed the gene has apparent low temperature resistant table compared with WT lines Type.

The beneficial effects of the present invention are：

EBF1 albumen and its encoding gene provided by the invention provide genetic resources to cultivate low temperature resistant new variety of plant, To study the mechanism of plant response adverse circumstance signal and being resistant to the molecule mechanism based theoretical of adverse environment.

Description of the drawings

Fig. 1 is 35S:EBF1-TAP (software Vector NTI Suite 8.0) sequencing result.

Fig. 2 is the EBF1 protein expression figures that strain is overexpressed in the embodiment of the present invention 2.

Fig. 3 is that low-temperature treatment plant recovery situation after strain Cold exposed and non-Cold exposed is overexpressed in the embodiment of the present invention 3 Photo.

Fig. 4 is that low-temperature treatment survival rate of plant is united after being overexpressed strain Cold exposed and non-Cold exposed in the embodiment of the present invention 3 Meter figure.

Fig. 5 is expression vector pC-TAPa structure collection of illustrative plates.

Specific implementation mode

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as gateway Molecular Cloning: A Laboratories method (Federico Katzen et al, Single Step BP/LR combined Gateway reactions, 2013), or according to the condition of manufacturer's specification suggestion.

TOPO TA gateway entry vectors are common cloning vector in following embodiment, commercially available；PC-TAPa is carried Body is provided by southern University of Science and Technology Guo Hongwei professors laboratory；Arabidopsis kind is Columbia ecotype；Agrobacterium The common cloning vector of GV3101 bacterial strains.

Main agents in following embodiment are：Gateway molecular cloning related experiment products are purchased from Invitrogen Company；Taq archaeal dna polymerases, Pyrobest Taq enzymes, KOD, are purchased from the biotech firms such as NEB, Toyobo at T4 ligases；dNTPs Purchased from Genestar companies；The small extraction reagent kit of plasmid and Ago-Gel QIAquick Gel Extraction Kit are public purchased from Shanghai JaRa bioengineering Department；MS culture mediums, agar powder, agarose, ampicillin (Amp), kanamycins (Kan), gentamicin sulphate (Gen), profit Good fortune puts down antibiotic and Glucose, BSA, nitrocellulose filter, LB Medium etc. such as (Rif) purchased from Sigma, Bio-Rad etc. Company；Various other chemical reagent used in embodiment are import or domestic analytical reagents.

Primer used in embodiment is synthesized by six directions Hua Da company, and carries out correlative measurement sequence.

The structure of 1 EBF1 gene overexpression carriers of embodiment

It is analyzed according to the coding region sequence of EBF1 genes (number in arabidopsis gene group database is AT2G25490), Specific primer F and R are designed, the code area of the gene is amplified to come, is connected to on expression vector pC-TAPa.

Primer used is：

Sense primer F：

5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGTCTCAGATCTTTAGTTTTGCCG-3’(SEQ ID No.3)；

Downstream primer R：

5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGGAGAGGATGTCACATTTGTAAAGA-3’(SEQ ID No.4)。

It is by the specific method that EBF1 genes are connected on carrier pC-TAPa：Using being connected to gateway entry vectors TOPO TA are obtained by what BP and LP reacted through the positive colony bacterial plaque after resistance screening, and kit extracts plasmid simultaneously in a small amount Hua Da gene sequencing is sent to be sequenced.

Sequencing correctly shows to be connected into EBF1 coding region sequences in pC-TAPa carriers, as a result such as Fig. 1.

The structure of 2 EBF1 gene overexpression plants of embodiment and detection

PC-TAPa carriers containing EBF1 genes described in embodiment 1 are transformed into Agrobacterium GV3101 bacterial strains, then are transferred to In wild-type Arabidopsis plants, arabidopsis transgenic seedlings are obtained.Specific method is：By the Agrobacterium inoculation containing purposeful carrier In tri- resistant to liquids culture solutions of 100mL LB, 50 μ g/mL of kanamycins (Kan), 50 μ g/mL of rifampin (Rif), gentamicin (Gen) 50 μ g/mL) in, 28 DEG C of shaken cultivations are stayed overnight, and wait for OD₆₀₀Value is 1.0-2.0, and with 4000rpm, room temperature centrifuges 15min, receives Collect thalline；With 200mL conversion fluids (1/2MS, 5% sucrose, 40 μ L Silwet L-77) suspension thalline；Arabidopsis floral is impregnated The 1min in the conversion fluid of Agrobacterium, putting on freshness protection package moisturizing and being placed in dark place keeps its temperature relatively low, second day by plant from It is taken out in freshness protection package, puts back on illumination cultivation frame normal growth to sowing.

PC-TAPa carriers institute band screening resistant gene is gentamicin, with gentamicin resistance to arabidopsis transgenosis children Seedling is screened, the T of acquisition₁In generation, there is the positive seedling of gentamicin resistance to carry out single plant sowing, then to T₂For seed celebrate big The test of chloramphenicol resistance selects that 3/4 is resistant and the strain of remaining 1/4 not no resistance, illustrates to be connected with purpose in the strain The over-express vector of gene is inserted into the form of singly copying.Plant with gentamicin resistance in these strains is removed, then into Row single plant sowing carries out gentamicin resistance screening for seed with the T3 of acquisition, if T4 is not detached for plant, illustrates this turn Gene strain is homozygote, which 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, rapid jelly is spare in liquid nitrogen.It will be planted with liquid nitrogen Object material is clayed into power shape, and protein extract buffer and protease inhibitors is added, in 4 DEG C after the concussion that is vortexed, 13000rpm from Heart 15min.

2) protein immunoblotting method detection is overexpressed the protein content in strain

The wild type of equivalent is taken respectively, is overexpressed the vegetable protein of arabidopsis strain, and 5 × SDS albumen loading buffers are added 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 It is overexpressed the protein content in strain with Anti-Myc antibody tests.Anti-Actin is as internal reference.

Testing result is shown in Fig. 2, it can be seen that the destination protein expression being overexpressed in strain is stronger.

The method that EBF1 genes are overexpressed in other plants can refer to the present embodiment and carry out.

Embodiment 3 is overexpressed the low temperature tolerance ability detection of EBF1 gene plants

EBF1 gene overexpression the strains OE-1 and OE-2 of gained in wildtype Arabidopsis thaliana seedling and embodiment 2 are existed first It is grown 12 days on culture dish, is then moved into low temperature incubator and carries out freezing processing.For Cold exposed plant, it is overexpressed strain Be after moving into 4 DEG C of low temperature incubator cultures after OE-1 and OE-2 are grown 12 days on culture dish 3 days, into line program cooling (1 DEG C/ H) certain time is handled to required sub-zero temperature.The above processing plant dark processing moves into 22 DEG C of illumination box trainings after 24 hours The number of plant of survival is counted after supporting 3 days, calculates survival rate.

As a result (Fig. 3 and Fig. 4) is shown, under the conditions of non-Cold exposed and Cold exposed, OE-1 and OE-2 show freeze proof table Type.This illustrates that the low temperature tolerance ability of EBF1 overexpression plant is significantly improved.

It can be seen that arabidopsis E3 ligases EBF1 can significantly improve tolerance of the plant to low temperature after being overexpressed.

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 made some modifications or improvements, 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

Application of the 1.EBF1 albumen in building low temperature resistant plant, which is characterized in that by improving EBF1 albumen in plant Expression quantity obtains low temperature resistant genetically modified plants；

Wherein, the amino acid sequence of EBF1 albumen is：

I) amino acid sequence shown in SEQ ID No.1；Or

Ii it) by the substitution of one or several amino acid residues and/or is lacked and ored add and and SEQ by SEQ ID No.1 ID No.1 amino acid sequences with the same function.
2. application according to claim 1, which is characterized in that the plant includes monocotyledon and dicotyledon.
3. application according to claim 2, which is characterized in that the plant includes but not limited to arabidopsis.
Application of the 4.EBF1 genes in building low temperature resistant plant, which is characterized in that by being overexpressed EBF1 genes in plant, Obtain low temperature resistant genetically modified plants；

Wherein, the nucleotides sequence of EBF1 genes is classified as：

I) nucleotide sequence shown in SEQ ID No.2；

Ii) nucleotide sequence shown in SEQ ID No.2 is substituted, lacks and/or increases one or more nucleotide and expression The nucleotide sequence of identical function protein；

Iii) under strict conditions with sequence hybridizes shown in SEQ ID No.2 nucleotide sequence.
5. application according to claim 4, which is characterized in that the plant includes monocotyledon and dicotyledon.
6. application according to claim 5, which is characterized in that the plant includes but not limited to arabidopsis.
7. a kind of method building low temperature resistant transgenic arabidopsis, which is characterized in that include the following steps：

1) arabidopsis total serum IgE is extracted, reverse transcription obtains cDNA, and using cDNA as template, F and R are primer, expand EBF1 genes, will Amplified production is building up on the plant gene expression vector with 35S promoter, the recombinant expression carrier of acquisition；

2) recombinant expression carrier is infected to arabidopsis floral by the way of agriculture bacillus mediated, obtains the quasi- south of low temperature resistant transgenosis Mustard plant；

Wherein, the nucleotide sequence of primers F described in step 1) and R are as shown in SEQ ID No.3 and SEQ ID No.4.
8. a kind of method building low temperature resistant transgenic arabidopsis, which is characterized in that include the following steps：

1) nucleotide sequence shown in SEQ ID No.2 is building up on expression vector pC-TAPa, the recombinant expression carrier of acquisition 35S-EBF1-TAP；

2) recombinant expression carrier 35S-EBF1-TAP by the way of agriculture bacillus mediated is infected into arabidopsis floral, obtained low temperature resistant Transgenic Arabidopsis plants.