CN113173981B

CN113173981B - Phyllostachys pubescens PeDREB3 gene and application thereof in plant cold resistance regulation

Info

Publication number: CN113173981B
Application number: CN202110604100.5A
Authority: CN
Inventors: 李雪平; 宋笑龙; 李夷骞
Original assignee: International Center for Bamboo and Rattan
Current assignee: International Center for Bamboo and Rattan
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2022-05-17
Anticipated expiration: 2041-05-31
Also published as: CN113173981A

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a moso bamboo PeDREB3 gene and application thereof in plant cold resistance regulation. The invention discloses a moso bamboo PeDREB3 gene and a biological function thereof. The sequences of the Phyllostachys pubescens PeDREB3 protein and the coding gene thereof are respectively shown in SEQ ID No.1 and SEQ ID No. 2. The invention clones the PeDREB3 gene from the moso bamboo for the first time and verifies the biological function by over-expressing the gene in arabidopsis thaliana. The PeDREB3 gene has the function of regulating and controlling the cold resistance of plants, can provide powerful support for the construction of transgenic moso bamboos and provides valuable candidate genes for molecular breeding of moso bamboos.

Description

Phyllostachys pubescens PeDREB3 gene and application thereof in plant cold resistance regulation

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a moso bamboo PeDREB3 gene and application thereof in plant cold resistance regulation.

Background

Phyllostachys edulis is a scattered bamboo species of Phyllostachys (Phyllostachys) of the Bambusoideae (Bambusoideae) of the Gramineae (Gramminales) and mostly prefers warm and humid climates, and is mainly geographically distributed in the south. Although the introduction work of large-scale 'south bamboo north shift' is carried out in 50-60 years of the 20 th century, some scattered bamboos with strong resistance are introduced to the regions north of the yellow river watershed, and the bamboo introduction work is more successful, the temperature is always a main limiting factor for the difficult further popularization and application of bamboo plants in the regions north of the yellow river watershed in China. Therefore, it is necessary to improve bamboo species and cultivate new varieties of bamboos with strong cold resistance, which is an important way to expand the distribution range of bamboos. The research on the functions of the related genes in the moso bamboo body under the cold condition is a molecular basis for new species cultivation, and has important practical significance for revealing the cold-resistant molecular mechanism of the bamboo, breaking through the limitation of conventional breeding and accelerating the breeding process of the bamboo.

Disclosure of Invention

The invention aims to provide a moso bamboo PeDREB3 gene and application thereof.

In order to realize the purpose of the invention, the invention provides the following technical scheme:

in a first aspect, the present invention provides a moso bamboo PeDREB3 gene, the amino acid sequence of which is as follows (1) or (2):

(1) as shown in SEQ ID NO. 1;

(2) the amino acid sequence shown as SEQ ID NO.1 is the amino acid sequence which is derived from the (1) and has the same function after one or more amino acids are substituted, deleted or added. The nucleotide sequence of the Phyllostachys pubescens PeDREB3 gene is shown as SEQ ID NO. 1.

The invention adopts the following method to clone and obtain the PeDREB3 gene:

(1) extracting total RNA from the bamboo leaf sheet material, and reversely transcribing the extracted total RNA into cDNA. In the invention, the total RNA of the moso bamboo is extracted by adopting a method for extracting the total RNA of cells commonly used in the field, and the Trizol method is specifically adopted in the embodiment of the invention.

(2) After extracting the total RNA of the moso bamboo, carrying out reverse transcription on the total RNA to synthesize cDNA. In the invention, the cDNA is synthesized by adopting a conventional cDNA synthesis method in the field without other special requirements; in the examples of the present invention, cDNA synthesis was carried out using the AMR reverse transcription kit from Baobioengineering (Dalian) Co., Ltd.

(3) After obtaining the cDNA, PCR amplification of the PeDREB3 gene was performed to obtain the target fragment.

(4) After PCR amplification is carried out to obtain a target fragment, sequencing the target fragment to obtain a PeDREB3 gene; after PCR amplification, the target fragment is preferably purified, and the purification method is not particularly limited, and may be performed using a DNA purification kit known to those skilled in the art.

(5) After the purification is completed, the purified target fragment is preferably connected to a pGEM-TEAsy vector, introduced into Escherichia coli DH5 alpha competent cells, and sequenced after being verified to be a positive clone by colony PCR.

In a second aspect, the invention provides a coding gene of the moso bamboo PeDREB3 protein.

Preferably, the nucleotide sequence of the coding gene of the phyllostachys pubescens PeDREB3 protein is shown as SEQID No. 2.

In a third aspect, the present invention provides a biomaterial containing a gene encoding the phyllostachys pubescens peddreb 3 protein, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or non-regenerable plant cells or tissues.

In a fourth aspect, the invention provides the use of a Phyllostachys pubescens PeDREB3 protein, a Phyllostachys pubescens PeDREB3 gene or a biological material containing the gene in regulation and control of cold resistance of plants.

The plants of the invention include, but are not limited to, Arabidopsis thaliana and Phyllostachys pubescens.

Preferably, the plant cold resistance is regulated and controlled by increasing the expression level of the pelteobagrus pubescens PeDREB3 gene in the plant, so that the cold resistance of the plant is increased.

In a fifth aspect, the invention provides application of a moso bamboo PeDREB3 protein, a moso bamboo PeDREB3 gene or a biological material containing the gene in preparation of transgenic plants.

Preferably, the transgenic plant is a transgenic plant with increased cold resistance.

In a sixth aspect, the invention provides the use of a Phyllostachys pubescens PeDREB3 protein, a Phyllostachys pubescens PeDREB3 gene or a biological material containing the gene in plant breeding.

Preferably, the breeding objective is to increase the stress tolerance (preferably cold tolerance) of a plant. The PeDREB3 gene participates in the cold stress response of the moso bamboo, and the cold stress can induce the expression of the gene.

In a seventh aspect, the present invention provides a method for improving cold resistance of a plant, comprising: the method comprises the following steps of (1) overexpressing a phyllostachys pubescens PeDREB3 gene in a plant by using a genetic engineering means;

wherein the over-expression mode is selected from one or more of the following combinations of (1) to (5):

(1) by introducing a plasmid containing the gene;

(2) by increasing the copy number of the gene on the plant chromosome;

(3) by altering the promoter sequence of said gene on the plant chromosome;

(4) by operably linking a strong promoter to the gene;

(5) by introducing an enhancer;

the plant includes Arabidopsis thaliana and Phyllostachys pubescens.

In the present invention, the expression vector carrying the target gene can be introduced into plant cells by using conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, and the like.

The method specifically comprises the following steps: the moso bamboo PeDREB3 gene is transferred into an arabidopsis plant to obtain a transgenic plant over-expressing the moso bamboo PeDREB3 gene;

preferably, the Phyllostachys pubescens PeDREB3 gene is constructed on a plant expression vector pCAMBIA2300, agrobacterium is transformed, then an arabidopsis inflorescence is impregnated, and a transgenic plant is screened.

In one embodiment of the invention, the construction method of the plant expression vector pCAMBIA2300-PeDREB3 is as follows:

according to the added restriction enzyme cutting sites, EcoRI and HindIII are used for carrying out double restriction on the PeDREB3-Teasy positive recombinant plasmid and the expression vector pCAMBIA 2300. And (3) carrying out agarose gel detection on the enzyme digestion product, then recovering the enzyme digestion product, and connecting the PeDREB3 gene fragment into a pCAMBIA2300 vector by using T4DNA ligase to construct an expression vector pCAMBIA2300-PeDREB 3.

The preparation method of the transgenic arabidopsis is as follows:

the recombinant expression vector is transformed into agrobacterium GV3101 by a freeze-thaw method, and is transformed into Arabidopsis by a floral dip method. Harvested seeds were sown on 1/2MS medium with 50mg/L kanamycin (Kan) for selection. Extracting RNA from arabidopsis thaliana leaves of T1 generation, carrying out reverse transcription to obtain cDNA, and carrying out PCR identification by using a primer PeDREB3-F, PeDREB 3-R.

In an eighth aspect, the present invention provides the use of a transgenic plant obtained according to the above method in plant breeding.

Breeding methods described above include, but are not limited to, transgenic, hybrid, backcross, selfing, or asexual propagation.

The invention has the beneficial effects that: the invention clones the moso bamboo PeDREB3 gene for the first time and reveals the biological function of the moso bamboo PeDREB3 gene, and the influence of PeDREB3 on the cold resistance of transgenic arabidopsis thaliana is investigated by constructing a PeDREB3 gene expression vector and heterologously transforming arabidopsis thaliana, and the cold resistance of arabidopsis thaliana over-expressing PeDREB3 is found to be obviously improved. The invention provides a powerful tool for moso bamboo transgenic research and provides a valuable candidate gene for moso bamboo resistance molecule breeding.

Drawings

FIG. 1 is an electrophoresis diagram (A) of PCR products of the gene clone PeDREB3 and an electrophoresis diagram (B) of enzyme digestion products of PCAMBIA2300-PeDREB3 in example 2 of the present invention; wherein, Lane 1 in A is PeDREB3PCR product, Lane 1 in B is PCAMBIA2300-PeDREB3 enzyme digestion product, M is DNA Marker, DNA Marker in A is DL2000, and DNAMmarker in B is DL 15000.

FIG. 2 shows the relative expression level of the PeDREB3 gene in the salt-stressed bamboo roots and leaves in example 4 of the present invention.

FIG. 3 shows the relative expression level of the PeDREB3 gene in the low temperature stressed bamboo roots and leaves in example 4 of the present invention.

FIG. 4 shows the changes of MDA content, CAT, POD and SOD enzyme activities of PeDREB3 transgenic Arabidopsis thaliana and wild Arabidopsis thaliana under low temperature stress at 4 ℃ in example 5 of the present invention, wherein CK is Arabidopsis thaliana under normal growth condition at 25 ℃ of incubator, WT is wild Arabidopsis thaliana plant, and OE-1, OE-5 and OE-7 are different PeDREB3 transgenic Arabidopsis thaliana plants respectively.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 cloning of the Phyllostachys pubescens PeDREB3 Gene

The method comprises the steps of taking moso bamboo leaves as a material, extracting total RNA of the leaves according to the instructions of a Trizol RNA extraction kit (Tiangen Biochemical technology Co., Ltd.), taking 1ng of RNA, carrying out reverse transcription according to the reverse transcription kit to obtain cDNA, and digesting a cDNA product by using RNase.

The PeDREB3 gene was amplified by PCR using Oligo7 software designed primers based on the Phyllostachys pubescens genome database (http:// www.forestrylab.org/db/PhePacBio/ExtractSeq/phe/index. php) and detected by electrophoresis (FIG. 1A).

The primer sequences are as follows:

an upstream primer PeDREB 3-F:

5′-GGAATTCATGGCGAAGAACTACCCGGCCG-3′；

the downstream primer PeDREB 3-R:

5′-CCGAAGCTTTTATGCATCAGCAAAC-3′。

the polymerase chain reaction conditions were as follows:

20 μ L reaction: 10 XPCR Buffer 2.0 uL, 2.5mM dNTP Mix 2 uL, upstream primer 1.0 uL, downstream primer 1.0 uL, cDNA template 2.0 uL, LA Taq DNApolymerse 0.2 uL, ddH₂O 11.8μL。

PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 59 ℃ for 30 s; extension at 72 ℃ for 1min for 35 cycles; 10min at 72 ℃; storing at 4 ℃.

And connecting the recovered product to a pGEM-T Easy vector, transforming DH5 alpha competent cells, selecting positive clones to perform plaque PCR detection, and sequencing the positive clones (Shanghai Bioengineering Co., Ltd.) to obtain an accurate sequencing result. The nucleotide sequence of the PeDREB3 gene is shown as SEQ ID NO.2, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 1.

Example 2 construction of plant expression vector pCAMBIA2300-PeDREB3

pGEM-T Easy connected with the PeDREB3 fragment is cut by EcoRI and HindIII and the recombinant plasmid is constructed by the expression vector pCAMBIA2300-CaMV35S (Promega corporation, USA) cut by EcoRI and HindIII, and the cutting system is as follows (50 muL):

enzyme digestion is carried out for 4h at 37 ℃; the product was subjected to agarose gel electrophoresis, and the large fragment of plasmid pCAMBIA2300-CaMV35S and the small fragment of PeDREB3 were recovered using a gel recovery kit (Axygen). The two recovered products were ligated using T4DNA ligase in the following ligation reaction (20. mu.L):

the ligation reaction was carried out overnight at 4 ℃ and the ligation products were all transformed into DH 5. alpha. competent cells. Overnight culture at 37 ℃, selecting single clone, carrying out colony PCR verification, carrying out amplification culture, extracting plasmid pCAMBIA2300-PeDREB3, carrying out sequencing and enzyme digestion verification (B in figure 1), and successfully constructing plant recombinant expression plasmid pCAMBIA2300-PeDREB 3.

Example 3 transformation of plant expression vector pCAMBIA2300-PeDREB3 Arabidopsis thaliana 1 and transformation of Agrobacterium GV3101 Strain by Freeze-thawing method

Adding 1ng of recombinant expression vector plasmid pCAMBIA2300-PeDREB3 into 100 mu L of competent cells GV3101, carrying out ice bath for 10min, quickly freezing the competent cells in liquid nitrogen for 5min, quickly transferring the competent cells into a 37 ℃ constant-temperature water bath kettle for 5min, then placing the competent cells on ice for 5min, adding 600 mu L of LB liquid culture medium into a centrifugal tube, carrying out shake culture in a 28 ℃ shaking table for 2-3h, and recovering the thalli. 60 μ L of the bacterial solution was applied to YEP solid medium containing kanamycin (50mg/mL) and rifampicin (50mg/mL) resistance, and the resulting suspension was plated upside down on a shaker at 28 ℃ for about 2-3 days until white colonies grew. After the monoclonal colony is picked and subjected to PCR detection, positive clone is selected and cultured by shaking.

2. Arabidopsis inflorescence dip-dyeing

The above-mentioned positive clones were inoculated into 10mL of YEP (50. mu.g/mL rifampicin + 100. mu.g/mL kanamycin) liquid medium, shake-cultured at 28 ℃ for 12 hours in an incubator (160rpm), 2mL of the culture was transferred to 200mL of YEP (50. mu.g/mL rifampicin + 100. mu.g/mL kanamycin) and subjected to mass culture, shake-cultured at 28 ℃ for 12 hours in an incubator (160rpm), and the OD of the culture concentration was OD₆₀₀To 1.8-2.2. mu.g/mL, 50mL of the culture was centrifuged at 5000rpm for 5min at 4 ℃ in a 50mL centrifuge tube, and the resulting mixture was vigorously suspended in a transformation medium (MS 2.2g, 5% sucrose, pH adjusted to 5.8, mixed with 0.2% Silwet L-77) and the precipitate was diluted to 1.0. mu.g/mL. Approximately 200mL of the DNA was prepared for dip-staining Arabidopsis thaliana. Soaking the overground part of the just flowering arabidopsis into the transformation liquid for 3min, wrapping the plant with a preservative film, carrying out dark culture for 12-16h, removing the preservative film, placing the plant in an incubator for culture, and waiting for harvesting seeds.

3. Screening of homozygotes

Seeds of T0 generation Arabidopsis thaliana are placed in a centrifuge tube, sterilized by adding 1mL of 70% alcohol for 5min, sterilized by using 1mL of 2.6% sodium hypochlorite solution for 10min, and then washed 5 times by using sterile water. Uniformly sowing seeds on a screening culture medium with 1/2MS +100mg/L kanamycin, purifying at 4 ℃ for 2 days, putting the seeds into an artificial climate incubator for culturing until 4 cotyledons grow out, transplanting green and normally-growing positive plants into soil for culturing, collecting T1 generation seeds by dividing into single plants after maturation, screening T1 generation seedlings by using the same method, counting the proportion of positive plants and non-positive plants of each strain of T1 generation, transplanting the positive plants of the strains with the proportion of 3:1 into the soil for culturing, and obtaining T2 generation seeds. T2 seedlings are screened by the same method to obtain T3 seeds.

4. PCR identification of positive plants

Extracting positive arabidopsis thaliana leaf RNA, carrying out reverse transcription to obtain cDNA, carrying out PCR identification by using a primer PeDREB3-F, PeDREB3-R, and finding that positive plants all contain PeDREB3, which indicates that the PeDREB3 is successfully transferred into arabidopsis thaliana.

Example 4 analysis of Phyllostachys pubescens PeDREB3 Gene expression level under salt and Low temperature stress

1. Material treatment

The moso bamboo seeds are collected in a Guangxi Zhuang autonomous region, are placed in a constant-temperature illumination incubator, the day and night temperature is 25 ℃/18 ℃, the photoperiod is 16h/8 h/dark, are cultured for about three months, high-salt stress and low-temperature stress are respectively simulated by 200mM NaCl at the low temperature of 4 ℃, young and tender roots and leaves at the same parts after treatment for 0h, 1h, 3h, 6h, 12h and 24h are respectively taken and are rapidly frozen in liquid nitrogen, and are frozen and preserved at the temperature of minus 80 ℃.

2. Synthesis of cDNA template

Extracting total RNA from root and leaf of Phyllostachys pubescens seedling with Trizol reagent, removing genomic DNA with DNase I (TIANDZ) without RNase, and measuring A with ultraviolet spectrophotometer₂₆₀And A₂₈₀The ratio and the RNA concentration are detected by 1 percent agarose gel electrophoresis, the first strand of cDNA is synthesized by utilizing an AMR reverse transcription kit of bioengineering (Dalian) Co., Ltd, and the synthesized product is stored in a refrigerator at the temperature of-20 ℃.

3. Real-time fluorescent quantitative PCR

And detecting the expression condition of the target gene by real-time fluorescent quantitative PCR (qRT-PCR). The TIP41 gene served as an internal reference gene.

The primer sequences are as follows:

TIP41-F：5'-AAAATCATTGTAGGCCATTGTCG-3'，

TIP41-R：5'-ACTAAATTAAGCCAGCGGGAGTG-3'；

PeDREB3 5-F:5'-TCAAGAACCCGAGCAAGACG-3'；

PeDREB3 5-R:5'-CGTAGGAGCCGAGCCAGAT-3'。

the 10 μ L reaction was as follows:

reaction procedure: 1min at 94 ℃; 94 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 10s, 45 cycles.

Other reaction parameters are system default settings, 3 biological replicates are set for each reaction, and Roche is used

480 Analyzer data, utilize 2^-ΔΔCTThe method analyzes 3 times of biological experimental data.

4. Results and analysis of the experiments

After salt and low-temperature stress treatment, the expression conditions of the PeDREB3 gene in the roots and leaves of the moso bamboo seedlings are respectively detected, and the results are shown in fig. 2 and fig. 3: after low temperature and salt treatment, the expression level of PeDREB3 in the roots and leaves showed a tendency of rising and falling, and the expression level in the roots was higher than that in the leaves. Under the low-temperature treatment, the expression level of the PeDREB3 in the roots and the leaves reaches the highest 12h after the treatment, and is respectively 26 times and 105 times of that before the treatment. Under salt stress, the expression level of PeDREB3 in leaves is not greatly changed in general, but shows a remarkable rising trend in roots, and reaches 16.6 times of that before treatment after 1h of treatment.

Example 5 analysis of Low temperature resistance of transgenic Arabidopsis thaliana having PeDREB3 Gene

Carrying out low-temperature stress treatment at 4 ℃ on seedlings of the T3 generation of the transgenic PeDREB3 gene arabidopsis thaliana and wild seedlings, and carrying out physiological index detection. Under normal growth conditions, the MDA content and the three enzyme activities of the transgenic arabidopsis thaliana and the wild arabidopsis thaliana have no significant difference. After low-temperature treatment, the activities of CAT and POD of the transgenic arabidopsis are higher than those of wild type and have obvious difference; although the difference of the SOD enzyme activities of the transgenic arabidopsis thaliana and the wild arabidopsis thaliana is not obvious, the SOD enzyme activity of the transgenic arabidopsis thaliana is higher than that of the wild arabidopsis thaliana on the whole; the MDA content is obviously increased after treatment, and the wild type Arabidopsis is obviously higher than the transgenic type (figure 4).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. The moso bamboo PeDREB3 protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The gene encoding the phyllostachys pubescens PeDREB3 protein of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is as shown in SEQ ID No. 2.

4. Biomaterial containing the gene according to claim 2 or 3, characterized in that it is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a non-regenerable plant cell or tissue.

5. Use of the protein of claim 1 or the gene of claim 2 or 3 or the biomaterial of claim 4 for the regulation of cold resistance in arabidopsis thaliana or moso bamboo.

6. Use of the gene according to claim 2 or 3 or the biological material according to claim 4 for the preparation of transgenic plants.

7. A method of improving cold resistance in a plant, comprising: overexpressing the gene of claim 2 or 3 in a plant by genetic engineering means;

the mode of overexpression is selected from one or more of the following combinations of (1) to (5):

(1) by introducing a plasmid containing the gene;

(2) by increasing the copy number of the gene on the plant chromosome;

(3) by altering the promoter sequence of said gene on the plant chromosome;

(4) by operably linking a strong promoter to the gene;

(5) by introducing an enhancer;

the plant is Arabidopsis thaliana or Phyllostachys pubescens.

8. The method as claimed in claim 7, wherein the Phyllostachys pubescens PeDREB3 gene is transferred into Arabidopsis thaliana plant to obtain transgenic plant over expressing Phyllostachys pubescens PeDREB3 gene.

9. The method as claimed in claim 8, wherein the Phyllostachys pubescens PeDREB3 gene is constructed on a plant expression vector pCAMBIA2300, Agrobacterium is transformed, and then Arabidopsis thaliana inflorescence is impregnated to screen transgenic plants.

10. Use of a transgenic plant obtained by the method of any one of claims 7 to 9 in plant breeding.