CN114907463A

CN114907463A - Sirber cotton gene GthCBF4 interaction protein and screening method thereof

Info

Publication number: CN114907463A
Application number: CN202210515337.0A
Authority: CN
Inventors: 韩江萍; 蔡小彦; 候宇清; 周忠丽; 许艳超; 王恒; 郑杰; 王玉红; 刘方
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-08-16

Abstract

The invention discloses a Sirper cotton gene GthCBF4 interaction protein, which comprises SRC2, RD22, GAPC2 and FBA 2. The invention also provides a screening method of the Thurber cotton gene GthCBF4 interaction protein, a bait expression vector pGBKT7-GthCBF4 of the Thurber cotton gene GthCBF4 is constructed, and screening of the Thurber cotton gene GthCBF4 interaction protein is carried out in a yeast library by using a method of a Mating screening library. The invention applies high-throughput sequencing to a yeast two-hybrid technology, screens interactive proteins in a yeast library by using a method of Mating screening library, screens out proteins SRC2, RD22 and GAPC2 which can interact with GthCBF4, has biological functions of participating in abiotic stress, responding to hormone regulation, inhibiting chlorophyll degradation, participating in a protein ubiquitination process, transferase activity, oxidoreductase activity and the like, and lays a foundation for deeply researching a molecular regulation network of GthCBF4 participating in stress response and an action mechanism thereof.

Description

Sirber cotton gene GthCBF4 interaction protein and screening method thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a Thurber cotton gene GthCBF4 interaction protein and a screening method thereof.

Background

Cotton is one of the most important economic crops in the world, provides main natural raw materials for the textile industry for human beings, and is also an important reserve material for the life and economic development of people. At present, more than 80 countries are planted, and the planting area in China reaches 530 ten thousand hectares throughout the year. With the shift of the main cotton production area in China to the inland cotton area in northwest of Xinjiang, low temperature becomes an important factor restricting the cotton production in China.

The cotton originates from tropical and subtropical regions, belongs to non-cold domesticated crops, needs enough heat conditions for normal growth and is very sensitive to low-temperature stress. After the cotton is subjected to low temperature for a period of time, intracellular ice and intercellular ice are formed, mechanical damage is caused to plant cells, and even the plant is dead. Cold stress can cause the plant growth to be affected and the maturing rate to be obviously reduced. The main performance is as follows: leaf wilting, leaf spot injury, tissue softening, and reproductive organ damage. Finally, the crop is reduced in large area and even completely harvested. Siper cotton is the only cotton seed distributed in temperate climate of the wild species of the cotton D genome, and has relatively bad living environment due to the special geographical position and changeable climate conditions, and has higher genetic diversity and better stress resistance potential after long-term natural selection.

CBF belongs to AP2/ERF (APETALA2/ethylene stress protein) family transcription factor, also called DREB (dehydration stress incorporation protein), and plays an important role in plant response to low-temperature stress. The CBF gene has low expression level under the condition of normal temperature, but the expression level is rapidly increased within a few minutes under the stimulation of low temperature, and the CBF gene is restored to the original level after a few hours. Researches on cold-resistant genes and regulatory networks in crops such as arabidopsis thaliana and rice have been reported, and a CBF pathway is the most main low-temperature response pathway, wherein genes such as ICE1, CBF and COR are the most main regulatory genes. In this pathway, ICE1 is degraded through ubiquitination pathway mediated by HOS1(High oxidative expression 1), and when plants are stressed at low temperature, HOS1 leads ICE1 to be degraded through interaction with ICE1, and expression of CBF and its downstream genes is inhibited, so that cold tolerance of plants is regulated. The GthCBF4(CBF4) gene is a homologous gene of CBF/DREB1, and the CBF4 gene over-expresses Arabidopsis thaliana, so that the expression of downstream genes containing CRE/DRE elements in a promoter region is activated, and the cold resistance and the drought resistance of transgenic plants are improved. These all indicate that plants have complex signal transduction pathways in drought, high salt and low temperature stress response, which are not completely independent of various stress signal transduction pathways, and are connected together by some common members to form a complex signal transduction network. Therefore, the research on the interacting protein of the gene GthCBF4 of seperb cotton has important significance on the breeding of cotton with drought resistance, salt resistance and low-temperature stress resistance.

Disclosure of Invention

The invention aims to provide a Thurber cotton gene GthCBF4 interaction protein and a screening method thereof, screen out the Thurber cotton gene GthCBF4 interaction proteins SRC2, RD22 and GAPC2, and lay a foundation for deeply researching a molecular regulation network of the Thurber cotton GthCBF4 participating in stress response and an action mechanism thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a Sirper cotton gene GthCBF4 interacting protein, the gene ID of the Sirper cotton gene GthCBF4 is EVM0024003.1, the Sirper cotton gene GthCBF4 interacting protein comprises SRC2, RD22, GAPC2 and FBA2, the amino acid sequence of the SRC2 is shown in SEQ ID No. 1; the amino acid sequence of the RD22 is shown as SEQ ID No. 2; the amino acid sequence of the GAPC2 is shown as SEQ ID No. 3; the amino acid sequence of FBA2 is shown in SEQ ID No. 4.

The invention also provides a screening method of the Thurber cotton gene GthCBF4 interaction protein, a target fragment of the Thurber cotton gene GthCBF4 is amplified by PCR, a bait expression vector pGBKT7-GthCBF4 of the Thurber cotton gene GthCBF4 is constructed, and screening of the Thurber cotton gene GthCBF4 interaction protein is carried out in a yeast library by using a method of using a Mating screening library.

Preferably, the nucleotide sequence of the target fragment of the Seebeck cotton gene GthCBF4 is shown in SEQ ID No. 7.

Further preferably, the primer sequences for amplifying the target fragment are shown as SEQ ID No.5 and SEQ ID No. 6.

Further preferably, the medium in which the Sesberg cotton gene GthCBF4 interactin is screened in a yeast library is SD/-Trp, SD/-Leu-Trp, SD/-His-Leu-Trp/X- α -gal/AbA and SD/-His-Leu-Trp-Ade/X- α -gal/AbA deficient solid medium.

The invention has the following beneficial effects: the invention constructs a recombinant yeast decoy expression vector pGBKT7-GthCBF4 of a gene GthCBF4 of sepher cotton, and screens and analyzes interaction protein of the gene GthCBF4 of septer cotton by a yeast two-hybrid technology. The invention applies high-throughput sequencing to a yeast two-hybrid technology, screens interacting proteins in a yeast library by using a method of Mating screening library, screens out proteins SRC2, RD22, GAPC2 and FBA2 which can interact with GthCBF4, has biological functions of participating in abiotic stress, responding to hormone regulation, inhibiting chlorophyll degradation, participating in a protein ubiquitination process, transferase activity, oxidoreductase activity and the like, and lays a foundation for deeply researching a molecular regulation network of GthCBF4 participating in stress response and an action mechanism thereof.

Drawings

FIG. 1 is a schematic diagram of the cloning of the GthCBF4 gene of Gossypium september in example 1;

FIG. 2 is a graph showing the results of toxicity test and self-activation test of the recombinant yeast decoy expression vector pGBKT7-GthCBF4 in example 2;

FIG. 3 is a graph showing the results of Mating efficiency tests on the hybridization of the bait vector pGBKT7-GthCBF4 and a yeast library in example 3;

FIG. 4 is a schematic diagram of the screening of positive clones and the detection of PCR agarose gel electrophoresis of a part of the colonies of positive clones in example 4;

FIG. 5 is the schematic diagram of the interaction between the gene GthCBF4 and GthFBA2 and GthRD22 in example 5.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments.

Siper cotton seedlings were provided by the Cotton research institute of Chinese academy of agricultural sciences; the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) Co., Ltd; plasmids pGBKT7 and pGBKT7-53 were purchased from Shanghai Europe and Yi biomedical science and technology; coli DH 5. alpha. competent cells were purchased from Otsugaceae Biotechnology Ltd; the SanPrep column type DNA glue recovery kit and the SanPrep column type plasmid DNA small extraction kit are purchased from Shanghai biological engineering Co., Ltd; Y2H Gold yeast competent cells were purchased from Shanghai Diego Biotechnology, Inc.

Example 1 construction of the decoy expression vector pGBKT7-GthCBF4 of the Serber Cotton Gene GthCBF4

Under normal conditions, when the seedling of Siseph cotton grows to the three-leaf one-heart stage, taking a proper amount of samples of roots, stems and leaves of the Siseph cotton seedling, extracting total RNA of the samples by using an RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit, carrying out reverse transcription to obtain cDNA as a template, carrying out PCR amplification by using SEQ ID No.4 and SEQ ID No.5 as primers to obtain a target fragment of a gene GthCBF4, carrying out electrophoresis detection on 1.2% agarose gel, cutting the gel, and then recovering the target fragment by using a SanPrep column type DNA gel recovery kit, wherein the nucleotide sequence of the target fragment of the gene GthCBF4 is shown as SEQ ID No. 6. The yeast decoy expression vector pGBKT7 is digested by restriction enzymes Nde I and BamH I, and the vector fragment is recovered. The recovered product is ligated with a yeast bait vector fragment, and the ligation product is transformed into E.coli DH5 alpha competent cells. And (3) selecting the monoclone to perform colony PCR identification, sending the positive monoclone to Shanghai biological engineering Limited company to perform sequencing, and verifying the correctness of the sequence. After the positive clone with correct sequencing is subjected to amplification culture, a plasmid is extracted by using a SanPrep column type plasmid DNA small-scale extraction kit. The recombinant yeast decoy expression vector pGBKT7-GthCBF4 of the sepher cotton gene GthCBF4 is constructed.

The construction schematic diagram of the expression vector pGBKT7-GthCBF4 is shown in FIG. 1-A; the schematic diagram of RNA quality detection of Sirber cotton leaf tissue is shown in FIG. 1-B. Agarose gel electrophoresis of PCR amplification product of Thurber cotton GthCBF4 gene is shown in FIG. 1-C; agarose gel electrophoresis of the monoclonal PCR amplification product of E.coli transformed with the expression vector pGBKT7-GthCBF4 of Thurber's cotton GthCBF4 is shown in FIG. 1-D.

EXAMPLE 2 toxicity and self-activation testing of bait carriers

On the basis of example 1, plasmids pGBKT7-53 and pGBKT7-T were used as positive controls, and pGBKT7-Lam and pGBKT7-T were used as negative controls. 100 μ L Y2H Gold yeast competent cells were thawed on ice and the reactions were constructed as in Table 1. Sequentially adding 2-5ng corresponding plasmid, 10 μ L Carrier DNA (salmon sperm DNA, 95-100 deg.C for 5min, ice for 3min, and repeating once more), 500 μ L PEG/LiAC, sucking, beating several times, and mixing. 30min in 30 deg.C water bath (turning 6-8 times for mixing) and 15min in 42 deg.C water bath (turning 6-8 times for mixing) respectively. 4000 r.min ^-1 Centrifuging for 1min, discarding supernatant, resuspending with 1ml YPD Plus liquid culture medium, recovering and culturing at 30 deg.C for 1h by shaking table, 4000 r.min ^-1 Centrifugation was carried out for 1min, the supernatant was discarded, 400. mu.L of ddH2O was used to resuspend the cells, 100. mu.L of the suspension was plated on SD/-Trp-deficient solid medium, and the cells were cultured at 30 ℃ for 3-5 days to observe the growth of colonies. Picking single clone on SD/-Trp defect type solid culture medium in YPDA liquid culture medium, at 30 deg.C 200 r.min ^-1 Culturing for 20-24h, and adding dropwise into solution containing 0, 100, 200, 300, 400 and 500 ng/mL ^-1 And culturing the AbA on SD/-Trp/-Leu/X-alpha-gal and SD/-Trp/X-alpha-gal deficient solid culture medium at 30 ℃ for 3-5d, observing the growth condition of plaques, and judging whether the recombinant decoy vector can activate the expression of the AbAr reporter gene. The results are shown in FIG. 2.

TABLE 1 toxicity and self-activation test reaction Table

Table 1 Toxicity and self-activation test response table

As shown in FIG. 2, FIG. 2-A is a toxicity test of the bait expression vector pGBKT7-GthCBF 4. Growth of yeast competent cells Y2HGold, in SD/-Trp medium, transformed with empty-load (pGBKT7) and recombinant yeast bait expression vector pGBKT7-GthCBF 4. FIG. 2-B is a self-activation assay of the bait expression vector pGBKT7-GthCBF 4. Negative control (pGBKT7-Lam + pGADT7-T), positive control (pGBKT7-53+ pGADT7-T) and recombinant yeast decoy expression vector pGBKT7-GthCBF4 transformed yeast competent cells Y2HGold, growth on SD/-Trp/-Leu/X- α -gal and SD/-Trp/X- α -gal deficient solid media containing different concentrations of AbA.

The results show that: the yeast containing the bait vector was of the same monoclonal size as the yeast carrying the empty vector pGBKT7, indicating that the recombinant bait vector did not affect the normal growth of yeast and was not toxic to yeast (FIG. 2-A). The yeast transformed with the bait vector pGBKT7-GthCBF4 can normally grow on SD/-Trp/X-alpha-gal/AbA defective solid culture medium, and can activate an X-alpha-gal reporter gene to grow a blue clone, which indicates that the yeast has self-activation activity; however, yeast activity decreased significantly with increasing concentration of AbA, when AbA concentration reached 400 and 500 ng-mL ^-1 In this case, the yeast with the bait transfer vector failed to grow normally on SD/-Trp/X-. alpha. -gal/AbA deficient solid medium, indicating that the activity of CBF4 was inhibited by AbA. Therefore, the composition may contain 400 ng/mL ^-1 SD deficient medium at AbA concentration is a screening library medium.

Example 3 screening of interacting proteins by Mating

On the basis of example 2, single colonies 2-3mm in size containing Bait plasmid on SD/-Trp-deficient solid medium were picked and cloned into SD/-Trp liquid medium, and cultured at 30 ℃ for 16-20h until OD600 reached 0.8. 1000g were centrifuged for 5min, and the supernatant was discarded before resuspension of the cells in 5ml SD/-Trp broth. 1ml of AD library bacterial solution and 45ml (containing 50 ug. m) were addedL ^- ¹ Kan ⁺ ) 2 XYPDA liquid medium, at 30 ℃ 65 r.min ^-1 Culturing for 12 h. Observing the hybrid bacteria solution under 40 × optical microscope to show binders in Mickey head or clover shape, centrifuging at 1000g for 10min, and adding 50mL (containing 50 ug. mL) ^-1 Kan ⁺ ) The bacterial cells were suspended in a 0.5 XYPDA liquid medium. Centrifuging again at 1000g for 10min, and centrifuging with 10mL (containing 50 ug. mL) ^-1 Kan ⁺ ) The cells were resuspended in 0.5 × YPDA liquid medium, and the total volume was measured. A small amount of the culture solution was diluted 10-fold, 100-fold, 1000-fold and 10000-fold, and applied to SD/-Trp, SD/-Leu and SD/-Leu-Trp defective solid media, respectively. The rest mixed bacterial liquid is coated on SD/-His-Leu-Trp/X-alpha-gal/AbA defective solid culture medium, and cultured for 3-5d at 30 ℃ for primary screening. Counting the number of colonies on each colony plate, and calculating the matting efficiency. And picking blue clones on the SD/-His-Leu-Trp/X-alpha-gal/AbA solid defective culture medium, transferring to the SD/-His-Leu-Trp-Ade/X-alpha-gal/AbA solid defective culture medium with higher screening strength, and carrying out secondary screening.

The results of testing the hybridization Mating efficiency of the bait vector pGBKT7-GthCBF4 and the yeast library in this example are shown in FIG. 3. FIG. 3-A: bait cells were hybridized with the yeast library for 12h, and binders of Mickey or clover were observed under a 40 Xoptical microscope. FIG. 3-B: number of clones on SD/-Trp/-Leu deficient solid medium. FIG. 3-C: number of clones on SD/-Leu deficient solid medium.

The results show that: after 12h hybridization of the bait vector pGBKT7-GthCBF4 with the yeast library, many Mickel-shaped binders were observed under a 40 Xlight microscope (FIG. 3-A). The number of the screened clones is 1.356 multiplied by 107cfu/ml, the mating efficiency is about 8.4 percent, and the requirements of subsequent experiments are met.

The number of clones on the SD/-Trp/-Leu deficient solid medium was 12 (FIG. 3-B)

The number of clones on SD/-Leu deficient solid medium was 143 (FIG. 3-C)

The number of clones screened was diploid (cfu/ml). times.resuspension volume (ml): 12 × 11.3 × 10 × 10000 ═ 1.356 × 107cfu/ml

MATING efficiency [ (No.cfu/ml of diptides)/(No.cfu/ml of limiting partner) ]. times.100%. approximately.8.4%

Example 4 screening of Positive clones and bioinformatic analysis

Blue clones on SD/-His-Leu-Trp-Ade/X-alpha-gal/AbA solid defective culture medium in example 3 were picked, yeast colony PCR was performed, PCR products were subjected to 1.2% agarose gel electrophoresis detection, single colonies were picked and sent to the company for sequencing, BLASTx alignment analysis was performed on the obtained Gene sequences in a Cotton FGD (https:// cottonfg. org /) database to obtain corresponding Gene information, and then Gene Ontology (GO) annotation was performed using Uniprot (https:// www.uniprot.org /) website. The GO annotation of the GthCBF4 gene interaction candidate protein is detailed in Table 2. The positive clone screening and the PCR agarose gel electrophoresis detection of a part of the positive clone colonies are shown in the attached figure 4.

TABLE 2 GO annotation of GthCBF4 gene interaction candidate proteins

Table 2 GO annotations ofGthCBF4 gene interaction candidateproteins

Example 5 Yeast two-hybrid Point-to-Point interaction validation

Reactions were constructed according to Table 3 on the basis of example 3, and were co-transformed into Y2H Gold yeast competent cells, spread on SD/-Leu-Trp deficient solid medium, cultured at 30 ℃ for 3-5d, single clones with a diameter of 3-4mm were picked up on SD/-Leu-Trp deficient solid medium, diluted 100-fold and 1000-fold, spotted uniformly on SD/-Leu-Trp/X- α -gal/AbA, SD/-His-Leu-Trp/X- α -gal/AbA and SD/-His-Leu-Trp-Ade/X- α -gal/A deficient solid medium, and cultured at 30 ℃ for 3-5d to observe colony growth. The results are shown in FIG. 5.

TABLE 3 Point-to-point verification reaction Table

Table 3 Reactiontable ofpoint-to-pointverification

The schematic diagram of the interaction between the GthCBF4 gene and GthFBA2 and GthRD22 is shown in FIG. 5.

The results show that: the positive clone can activate an AbA reporter gene, and the AbA reporter gene normally grows on SD/-Leu-Trp/X-alpha-gal/AbA, SD/-His-Leu-Trp/X-alpha-gal/AbA and SD/-His-Leu-Trp-Ade/X-alpha-gal/AbA defective solid culture media and can activate the expression of X-alpha-gal to turn yeast blue, and the negative control cannot grow on corresponding culture media, thereby indicating that the plasmid cotransformation is successful. pGBKT7-GthCBF4, pGADT 7-GthFBA 2 and pGADT7-GthRD22 co-transform yeast competent Y2HGold can normally grow on SD/-Leu-Trp/X-alpha-gal/AbA defective solid culture medium and make the yeast blue, the blue color is still very deep after the thallus is diluted, and the same situation is also realized on SD/-His-Leu-Trp/X-alpha-gal/AbA and SD/-His-Leu-Trp-Ade/X-alpha-gal/AbA defective solid culture medium with higher screening strength, which indicates that the interaction relationship exists between the two genes of GthFBA2 and GthRD22 and the GthCBF4 gene.

The present specification and figures are to be regarded as illustrative rather than restrictive, and it is intended that all such alterations and modifications that fall within the true spirit and scope of the invention, and that all such modifications and variations are included within the scope of the invention as determined by the appended claims without the use of inventive faculty.

Sequence listing

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actgatcgtc cggtgaattt ttccgatgac tatgtgatgt tagcttcgag ttatccaaag 120

aggcgagctg ggaggaagaa gttccgggag actcgacacc cggtgtaccg tggagttcgc 180

cggaggaatc ccgggaagtg ggtttctgaa gtgagggagc ctaataagaa gtcgaggatt 240

tggcttggaa ctttcccgac ggcggatatg gcggcgcgtg ctcacgacgt ggcagctata 300

gcactgagag ggaagtcagc ttgtttgaac ttcgctgact cagcttggaa tcttccggtc 360

ccggcttctt ccgacccaaa ggatatccaa aagacggtgg cggaggtggc ggagactttc 420

agaacggctg agcattcgag cgggaattct agaaacgatg caaagagaag tgaaaacacg 480

gagatggaga aagggtttta cttggacgaa gaagcgttgt ttgggacaca aagattttgg 540

gcaaatatgg ctgccggtat gatgatgtca cctcctcgtt ccggtcatga cggaggatgg 600

gaagaacatg aagtcgatga ttatgtacct ttatggagtt attctattta a 651

Claims

1. The Thurber cotton gene GthCBF4 interaction protein, wherein the gene ID of the Thurber cotton gene GthCBF4 is EVM0024003.1, the Thurber cotton gene GthCBF4 interaction protein comprises SRC2, RD22, GAPC2 and FBA2, and the amino acid sequence of SRC2 is shown as SEQ ID No. 1; the amino acid sequence of the RD22 is shown as SEQ ID No. 2; the amino acid sequence of the GAPC2 is shown in SEQ ID No. 3; the amino acid sequence of FBA2 is shown in SEQ ID No. 4.

2. A method for screening the interacting protein of the Thurber cotton gene GthCBF4 is characterized in that a target fragment of the Thurber cotton gene GthCBF4 is amplified by PCR, a bait expression vector pGBKT7-GthCBF4 of the Thurber cotton gene GthCBF4 is constructed, and the screening of the Thurber cotton gene GthCBF4 interacting protein is carried out in a yeast library by using a method of using a Mating screening library.

3. The Thurber cotton gene GthCBF4 interaction protein according to claim 2, wherein the nucleotide sequence of the target fragment of the Thurber cotton gene GthCBF4 is shown in SEQ ID No. 7.

4. The Gossypium september gene GthCBF4 interacting protein according to claim 3, wherein the primer sequences for amplifying the target fragment are shown in SEQ ID No.5 and SEQ ID No. 6.

5. The screening method according to claim 4, wherein the culture medium in which the Seebeck cotton gene GthCBF4 interacting protein is screened in the yeast library is SD/-Trp, SD/-Leu-Trp, SD/-His-Leu-Trp/X- α -gal/AbA and SD/-His-Leu-Trp-Ade/X- α -gal/AbA deficient solid medium.