Pyrus betulaefolia PbeMYB2 protein and application of pyrus betulaefolia PbeMYB2 protein in regulation and control of plant drought resistance

Abstract

The invention discloses a pyrus betulaefolia PbeMYB2 protein and application of the pyrus betulaefolia PbeMYB2 protein in regulation and control of plant drought resistance. According to the invention, an MYB transcription factor MYB2 is separated and cloned from pyrus betulaefolia. A nucleotide sequence of a corresponding coding gene is shown in a sequence table SEQ ID NO.2; the gene can quickly respond to dehydration, salt, ABA and low-temperature stress, PbeMYB2 is transferred into tobacco for functional verification, it is found that under drought stress, the survival rate, chlorophyll content and proline content of a PbeMYB2 transgenic tobacco strain are remarkably higher than those of a wild control, the water loss rate and electrolyte permeability are remarkably lower than those of the wild control, active oxygen accumulation is remarkably reduced, and the PbeMYB2 transgenic tobacco strain is remarkably superior to that of the wild control. And the expression of 16 stress resistance related genes can be induced. The PbeMYB2 protein obtained by the invention plays an important role in regulating and controlling a plurality of stress-resistant functional genes in a plant drought resisting process; in addition, the invention also helps to understand a molecular mechanism of plant MYB transcription factor regulation and control of drought resistance, and provides a new gene resource for research on improvement of plant drought resistance by genetic engineering means.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, relates to the research of drought resistance-related proteins, and particularly relates to a Pyrus betula PbeMYB2 protein and an application thereof in regulating plant drought resistance. Background Art

[0002] my country is the world's most important origin center of Pyrus plants, with abundant Pyrus resources, including cultivated species such as Pyrus pyrifolia Nakai., Pyrus bretschneideri Rehd., and Pyrus ussuriensis Maxim., as well as wild species such as Pyrus betulaefolia Bunge., which is often used as rootstock. In recent years, although the pear industry has made a series of major progress, it is often subject to environmental stresses such as low temperature, drought, and soil salinity, which has hindered the rapid development of my country's pear industry. Therefore, how to efficiently and quickly improve the stress resistance of pears has become a problem that breeders urgently need to solve. With the development of biotechnology and tissue culture technology, the use of plant genetic engineering to cultivate resistant varieties has broad application prospects in pear stress resistance breeding.

[0003] In order to cope with various abiotic stresses, plants have formed an effective defense mechanism in the long-term evolution process. In this process, many stress resistance genes are activated to protect plants from damage caused by osmotic stress, oxidative stress and ion toxicity (Bartels and Sunkar, 2005). The expression of these functions is mostly regulated by specific transcription factors, such as the MYB transcription factor family (Hu et al., 2008). MYB transcription factors are widely present in animals, plants and bacteria (Lippold et al., 2009). They contain one, two or three incomplete repeats (51-53 amino acids) in their DNA binding domains. According to the number of these repeats, these MYB transcription factors can be divided into three subfamilies, R2R3 type MYB, R1R2R3 type MYB and MYB (Katiyar et al., 2012). In plants, R2R3 MYB transcription factors are the most common (Stracke et al., 2001), with 126 and 109 R2R3 MYB transcription factors in Arabidopsis and rice, respectively (Yanhui et al., 2006). In recent years, several studies have shown that R2R3 MYB transcription factors are involved in plant responses to abiotic stresses, such as: in Arabidopsis, under drought and salt stress, AtMYC2 and AtMYB2 act as transcription factors to regulate the expression of genes in the ABA signaling pathway (Abe et al., 2003); AtMYB102 acts as a key hub to integrate Arabidopsis responses to osmotic stress and the ABA signaling pathway (Denekamp and Smeekens, 2003). Other studies have found that three Arabidopsis MYB proteins, AtMYB44, AtMYB60 and AtMYB61, are involved in regulating the response of plant stomata to drought stress (Cominelli et al., 2005; Liang et al., 2005; Jung et al., 2008); AtMYB41 gene also has a short transcriptional response to osmotic stress (Lippold et al., 2009); and AtMYB15 acts as a negative regulator to downregulate the expression of some low temperature response genes (Agarwal et al., 2006a). In rice, there are also multiple R2R3 type MYB transcription factors involved in the response to abiotic stress, for example: overexpression of rice OsMYB4 enhances the resistance of transgenic Arabidopsis to low temperature (Vannini et al., 2004); overexpression of OsMYB2 greatly enhances the resistance of rice to cold, dehydration and salt (Yang et al., 2012). These studies all indicate that plant MYB transcription factors can indeed respond to abiotic stresses and play different roles in them.

[0004] Although several MYB transcription factors have been cloned in plants and their roles in abiotic stress have been identified, most of these studies have focused on model plants and food crops, and there are few reports on MYB transcription factors in fruit trees. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a Pyrus betulaefolia Bunge protein. The present invention isolates the R2R3 type MYB protein PbeMYB2 from Pyrus betulaefolia Bunge. The present invention also verifies the role of the gene encoded by it in regulating plant drought resistance. The gene is transformed into tobacco by Agrobacterium-mediated method. The survival rate, chlorophyll and proline content of the obtained transgenic plants under drought stress are significantly higher than those of the wild control, and the active oxygen accumulation, water loss rate and electrolyte permeability are significantly lower than those of the wild control, and the expression of 16 stress resistance-related genes is induced. This provides a new idea for the study of how to use genetic engineering technology to improve the salt tolerance of fruit trees, and is also of great significance for understanding the molecular mechanism of MYB2 regulating drought resistance.

[0006] The present invention is achieved through the following technical solutions:

[0007] A Pyrus betula PbeMYB2 protein is isolated from pear and is a R2R3 type MYB protein with drought resistance, named PbeMYB2, and its sequence is shown in SEQ ID NO.1 in the sequence table.

[0008] A further improvement of the present invention is:

[0009] A PbeMYB2 gene, the nucleotide sequence of the gene is shown in the sequence list SEQ ID NO.2, and the amino acid sequence is shown in the sequence list SEQ ID NO.1.

[0010] A further improvement of the present invention is:

[0011] A gene cloning primer, wherein the cloning primer is the full-length cDNA sequence of the gene PbeMYB2 according to claim 2 cloned by using PCR technology.

[0012] Furthermore, the nucleotide sequence of the homology arm primer pair of the PCR is as follows:

[0013] Forward primer: 5'-gcccttgctcaccatggatccATGGATATTAATAATGTTCATGATCAGATG-3', i.e. SEQ ID NO. 3;

[0014] Reverse primer: 5'-gagaacacgggggactctagaTTAGTCATCACTAAGCTGCTGTTGC-3', i.e. SEQ ID NO.4.

[0015] Furthermore, the present invention also provides a plant overexpression vector, wherein the vector is a transgenic plant obtained by transforming the PbeMYB2 gene into tobacco using an Agrobacterium-mediated genetic transformation method. Biological function analysis shows that the cloned gene of the present invention has drought resistance, plays an active role in cleaning up active oxygen in plants, and is also involved in regulating the expression of multiple stress resistance-related genes.

[0016] Furthermore, the present invention uses qRT-PCR technology to analyze the expression pattern of the PbeMYB2 gene under different abiotic stress treatments. PbeMYB2 can respond to a variety of abiotic stresses, among which the response to dehydration, ABA and salt stress is stronger, but the response to low temperature stress is weaker, indicating that the PbeMYB2 gene is a stress resistance-related gene.

[0017] The nucleotide sequences of the qRT-PCR primer pairs are as follows:

[0018] Forward primer 2: 5'-CAGGTGGTCCAAGATAGCCG-3', i.e., SEQ ID NO. 5;

[0019] Reverse primer 2: 5'-CTCGGACTCAGATGTCGTAG-3', i.e. SEQ ID NO.6.

[0020] A further improvement of the present invention is:

[0021] Use of biological materials related to the Pyrus betulaeformis PbeMYB2 protein, comprising the above-mentioned protein, gene, cloning primers and expression vector, in regulating plant drought resistance, wherein the related biological materials further comprise any one of the following 1) to 4):

[0022] 1) A DNA molecule whose coding region is shown in SEQ ID No. 2;

[0023] 2) an expression cassette containing the nucleic acid molecule described in 1);

[0024] 3) a recombinant vector containing the nucleic acid molecule described in 1), or a recombinant vector containing the expression cassette described in 2);

[0025] 4) A recombinant microorganism containing the nucleic acid molecule described in 1), or a recombinant microorganism containing the expression cassette described in 2), or a recombinant microorganism containing the recombinant vector described in 3).

[0026] The beneficial effects of the present invention are:

[0027] Pyrus betulifolia Bunge is an excellent germplasm resource, which is widely used as a rootstock in the production of pear trees and has strong tolerance to low temperature, drought and salinity. The present invention isolates and clones a MYB transcription factor MYB2 from it. The amino acid sequence of the protein is shown in the sequence table SEQ ID NO.1, and the nucleotide sequence of the corresponding coding gene is shown in the sequence table SEQ ID NO.2; PbeMYB2 contains an open reading frame of 873bp, encoding 290 amino acids, an isoelectric point of 5.1, and a molecular weight of 32.8kDa.

[0028] The protein has a typical R2R3 domain, is located in the cell nucleus, and has transcriptional activation activity. The gene described in the present invention can quickly respond to dehydration, salt, ABA and low temperature stresses. PbeMYB2 was transferred into tobacco for functional verification. It was found that the survival rate, chlorophyll and proline content of the transgenic tobacco strains overexpressing PbeMYB2 under drought stress were significantly higher than those of the wild control, the water loss rate and electrolyte permeability were significantly lower than those of the wild control, the accumulation of active oxygen was significantly reduced, and the expression of 16 stress resistance-related genes could be induced. It shows that the PbeMYB2 protein obtained by the present invention belongs to the R2R3 type MYB transcription factor, which plays an important role in the process of plant drought resistance by regulating multiple stress resistance functional genes; in addition, the present invention also helps to understand the molecular mechanism of plant MYB transcription factors regulating drought resistance, and provides new gene resources for the research on improving plant drought resistance by genetic engineering means. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The amino acid sequence alignment results of pear protein PbeMYB2 with European sweet cherry PaMYB108 (XP_021812709.1), apple MdMYB108 (XP_008343185.2), peach PpMYB108 (XP_007221521.1), apricot PdMYB2 (BBG98572.1) and cannabis CsMYB78 (XP_030482260.2) are shown in Figure 1. PbeMYB2 contains two MYB characteristic conserved domains (R2 domain and R3 domain, respectively marked with underscores).

[0030] Figure 2 The expression characteristics of PbeMYB2 gene under different abiotic stress treatments are analyzed. A is the expression characteristics of PbeMYB2 at 0h, 6h, 12h, 24h and 48h of low temperature stress (4℃); B is ABA (100μmol·L -1) treatment for 0h, 1h, 2h, 4h and 8h; C is the expression characteristics of PbeMYB2 at 0h, 0.5h, 1h, 3h and 6h of dehydration stress; D is the expression characteristics of PbeMYB2 at salt stress (200mmol·L -1 The expression characteristics of PbeMYB2 at 0h, 6h, 12h, 24h and 48h of NaCl. The leaves of Pyrus betulaeformis with uniform and robust growth for 45 days were used as test materials. At least 60 seedlings were used for each treatment. The samples were randomly taken and treated with liquid nitrogen and stored at -80℃.

[0031] Figure 3 The subcellular localization of PbeMYB2 protein in tobacco leaves. AD is the subcellular localization of the empty control, and EH is the subcellular localization of PbeMYB2.

[0032] Figure 4 Analysis of the transcriptional activation activity of PbeMYB2 protein.

[0033] Figure 5 For positive identification of transgenic tobacco lines with PbeMYB2 gene. A is identification of tobacco T0 generation positive plants by specific primers; B is identification of overexpression level of PbeMYB2 in T2 generation positive plants.

[0034] Figure 6 This is a drought resistance phenotype analysis of 30-day overexpression tobacco lines. A is a 30-day-old wild-type (WT) and overexpression PbeMYB2 tobacco seedlings; B, wild-type (WT) and overexpression PbeMYB2 tobacco seedlings after 15 days of continuous drought; C, wild-type (WT) and overexpression PbeMYB2 tobacco seedlings after 7 days of watering; D, survival rate of wild-type (WT) and overexpression PbeMYB2 tobacco seedlings after 7 days of watering.

[0035] Figure 7 The dehydration phenotype and water loss rate analysis of transgenic tobacco plants with PbeMYB2 gene. A is the phenotype of each strain before and after 9 hours of natural dehydration; B is the water loss rate analysis.

[0036] Figure 8 Drought resistance analysis of 60-day-old overexpressed PbeMYB2. A is the phenotype of 60-day-old wild-type and overexpressed PbeMYB2 tobacco seedlings before, during and after drought recovery; B, electrolyte exudation rate of wild-type and overexpressed PbeMYB2 tobacco seedlings after 12 days of drought; C, chlorophyll content of wild-type and overexpressed PbeMYB2 tobacco seedlings after 12 days of drought; D, proline content of wild-type and overexpressed PbeMYB2 tobacco seedlings after 12 days of drought.

[0037] Fig. 9 DAB and NBT chemical staining to analyze H2O2 and O2 before and after drought －Accumulation in leaves of transgenic lines and controls. A, DAB staining before drought treatment; B, DAB staining after drought treatment; C, NBT staining before drought treatment; D, NBT staining after drought treatment.

[0038] Fig.10 The expression levels of stress-resistance-related genes in the PbeMYB2 overexpressing lines before and after drought stress. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with specific embodiments.

[0040] Example 1: Cloning of the full-length cDNA of the pear PbeMYB2 gene

[0041] The Arabidopsis thaliana AtMYB2 sequence was submitted to the pear genome server for blast, and the nucleotide sequence with the highest score was selected and submitted to Pfam to verify the conserved protein domain. The homology arm primer pairs for amplification of the sequence were designed using CE Design 4.0.

[0042] Using 45-day-old Pyrus betulaeformis as the test material, total RNA was extracted and reverse transcribed, and the obtained first-strand cDNA was used to amplify the full-length cDNA sequence of the PbeMYB2 gene. Total RNA was extracted using a magnetic bead total RNA extraction kit (BG0051-S, purchased from Chongqing Baoguang Biotechnology Co., Ltd.), and 1 μg of RNA sample was treated with ExonScript RT Mix (purchased from Chongqing Baoguang Biotechnology Co., Ltd.) at 37°C for 10 minutes, 55°C for 15 minutes, and 85°C for 5 minutes to obtain the first-strand cDNA.

[0043] The full-length cDNA sequence of the PbeMYB2 gene was amplified using PCR technology. The primer pair for the homology arm of the PbeMYB2 gene was: forward primer 5'-gcccttgctcaccatggatccATGGATATTAATAATGTTCATGATCAGATG-3' (corresponding to SEQ ID NO.3); reverse primer: 5'-gagaacacgggggactctagaTTAGTCATCACTAAGCTGCTGTTGC-3' (corresponding to SEQ ID NO.4); the PCR reaction system and reaction procedure are shown in the following table:

[0044] Table 1 PCR amplification reaction system

[0045]

[0046] Table 2 PCR reaction program

[0047]

[0048] After the PCR is completed, the PCR product is subjected to 1% agarose gel electrophoresis and then recovered using a gel recovery kit (purchased from Shanghai Bioengineering Co., Ltd.). The recovery steps refer to the instruction manual. The recovered purified PCR product is ligated with the gel recovery product of the 35S-GFP vector plasmid that has been double-digested with Xba I and BamHI (purchased from Thermo Fisher Scientific). The ligation reaction system includes: 4.5 μL of recovered purified PCR product, 0.5 μL of vector gel recovery and 5.0 μL of 2×Seamless Cloning Mix (purchased from Beijing Qingke Xinye Biotechnology Co., Ltd.). Ligation is carried out at 50°C for 25 minutes. Take 10 μL of the ligation product and transform Escherichia coli DH5α using the heat shock method. In the presence of 100 mg·L -1 Positive clones were screened on a kanamycin LB solid plate, and 5 positive clones were selected for sequencing (completed by Jinweizhi Company). The sequencing results showed that the length of the target fragment amplified by the present invention was 873 bp, and its nucleotide sequence was shown in SEQ ID NO.2. Through sequence comparison analysis, it was determined that the sequence was the target gene required by the present invention, and this gene was named PbeMYB2.

[0049] The PbeMYB2 gene encodes 290 amino acids, with an isoelectric point of 5.1 and a calculated molecular weight of 32.8 kDa. Amino acid sequence alignment results showed that the PbeMYB2 protein contained two conserved domains, R2 and R3 ( Figure 1 ), these domains are unique to MYB proteins and are highly conserved among the MYB proteins reported in studies, proving that PbeMYB2 is a MYB-type transcription factor.

[0050] Example 2: Analysis of expression characteristics of PbeMYB2 gene under different abiotic stress treatments, subcellular localization analysis and transcriptional activation activity analysis of PbeMYB2 protein

[0051] 1. Analysis of expression characteristics of PbeMYB2 gene under different abiotic stress treatments

[0052] To analyze the effects of different abiotic stresses on the expression of PbeMYB2, qRT-PCR was used to detect the changes in the relative expression of PbMYB2 gene during different abiotic stress treatments ( Figure 2 ), indicating that the PbeMYB2 gene responds to a variety of abiotic stresses. Based on the sequenced PbeMYB2 gene sequence, specific expression primers (SEQ ID NO.5 and SEQ ID NO.6) were designed using Primer 6.0 to analyze gene tissue expression and expression patterns under a variety of abiotic stresses.

[0053] The nucleotide sequences of the qRT-PCR primer pair are: forward primer 2: 5'-CAGGTGGTCCAAGATAGCCG-3' (corresponding to SEQ ID NO.5); reverse primer 2: 5'-CTCGGACTCAGATGTCGTAG-3' (corresponding to SEQ ID NO.6). The internal reference gene primer is PbTublin-F / R. Fluorescence quantitative PCR LightCycler 480 (Roche, USA) was used to analyze the expression characteristics of the PbeMYB2 gene. The reaction procedure is (Table 3):

[0054] Table 3qRT-PCR reaction procedure

[0055]

[0056] The detection of each sample was repeated 4 times in parallel, and the relative expression was calculated using 2 -ΔΔCt Quantitative data calculations were performed.

[0057] 2. Subcellular localization analysis of PbeMYB2 protein

[0058] Through fluorescent fusion protein labeling technology, the subcellular localization of PbeMYB2 protein in tobacco leaves was explored. The PbeMYB2 gene was introduced into tobacco leaves using Agrobacterium for expression, and the subcellular localization experiment of the protein was carried out.

[0059] Place the tobacco leaves to be infected under a fluorescent lamp for 2 hours, and select Nicotiana benthamiana with stretched and shiny leaves, similar plant size and similar leaf size for use. Pick 35S-PbeMYb2-GFP Agrobacterium and 35S-GFP Agrobacterium (empty control) monoclonal clones in 5mL LB liquid culture medium (containing 25μg / mL rifampicin Rif and 50μg / mL kanamycin Kan), activate and culture overnight at 28°C and 220rpm / min; take 1mL of the above two Agrobacterium liquids and transfer them to 25mL LB liquid culture medium (containing Kan and Rif) for overnight culture. Centrifuge the above two culture solutions separately, 4000rpm / min, 10min, remove the supernatant; resuspend the bacteria with 10mM MgCl2 (containing 100μMAs) suspension, adjust OD 600Tobacco plants with good growth conditions were selected, and 1 mL of bacterial solution was injected into the lower epidermis of tobacco leaves using a needleless syringe for transient expression analysis. After being cultured in an incubator for 48 h, DAPI (4,6-diamidino-2-phenylindoledihydrochloride) was used to mark the nucleus. Then, the successfully infected area on the tobacco leaves was selected, placed on a clean slide, covered with a coverslip, and GFP fluorescence was observed under a laser confocal microscope (Zeiss LSM 710, Germany). Figure 3 ).

[0060] 3. Analysis of transcriptional activation activity of PbeMYB2 protein

[0061] The plasmids of the fusion expression vector pMN6-PbeMYB2, the internal reference vector pRNL and the reporter vector pGLL were co-transfected into Arabidopsis leaf protoplasts by the PEG-mediated method, and the transcriptional activation activity of PbMYB2 was verified using a transient analysis system.

[0062] Two restriction sites, Sma I and Sac I, were added to both ends of the cloned PbeMYB2 gene for PCR amplification. The gel-recovered product was ligated with the pMD18-T vector to obtain the recombinant vector pMD18-T. S / S -PbeMYB2, and use Sma I and SacI to double-digest the recombinant vector and the plasmid of the transcription activation expression vector pMN6. The gel recovery products of the two were connected to obtain the fusion expression vector pMN6-PbeMYB2. Use a plasmid extraction kit to extract pMN6-PbeMYB2, pRNL, and pGLL plasmids, and determine their concentrations. Use enzymatic hydrolysis (cellulase and pectinase mixture) to separate protoplasts from Arabidopsis leaves. Mix pMN6-PbeMYB2, pRNL and pGLL plasmids in a ratio of 1:1:1. Mix 10μL of plasmid DNA (containing 5μg pMN6-PbeMYB2, 5μg pRNL and 10μg pGLL) with 100μL Arabidopsis leaf protoplasts (about 10 5 Add 110 μL of PEG-Ca transfection solution (40% PEG 4000, 200 mmol·L -1 Mannitol, 100mmol·L -1CaCl2). Incubate the mixture at room temperature for 20-30 min. Dilute the PEG-DNA-protoplast mixture with W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl solution, 2 mM MES at pH 5.7) and mix gently. As a control group, pMN6 and pRNL plasmids were mixed in a 1:1 ratio with a total plasmid amount of 10-20 μg and transformed into Arabidopsis protoplasts. The transformed protoplasts were incubated in W5 medium for 16-24 h at 23-25 ​​°C in the dark. Use the Dual Luciferase Assay Kit (Promega) according to the instructions of the kit. Collect the protoplasts, centrifuge (500g, 5min), and discard the supernatant. Add lysis buffer, mix well, and lyse the cells. Centrifuge (12,000g, 1min), and take the supernatant for luciferase activity determination. According to the instructions of the kit, add the lysate to a 96-well plate. First, the fluorescence signal of Firefly luciferase was measured, and then the fluorescence signal of Renilla luciferase was measured. According to the formula in the manual, the transcriptional activation activity of PbeMYB2 was analyzed ( Figure 4 ).

[0063] Example 3: Construction of plant overexpression vector, genetic transformation of tobacco and identification of transgenic positive and overexpression strains

[0064] 1. Construction of plant overexpression vector and Agrobacterium transformation

[0065] The plasmid was extracted from the correctly sequenced PbeMYB2 Escherichia coli, and double enzyme digestion was used to identify whether the extracted plasmid contained the expected target fragment, and the integrity of the plasmid was confirmed. After passing the inspection, it was transformed into Agrobacterium tumefaciens (GV3101) competent cells.

[0066] 20 μL of fresh bacterial solution was added to 50 mL of LB (containing 50 μg / mL Kan) liquid medium and cultured in a shaker at 37°C at 220 rpm for 10 h. After the bacterial solution was fully expanded, the PbeMYB2 plasmid was extracted using a plasmid DNA small-scale extraction kit (purchased from Beijing Qingke Biotechnology Co., Ltd.). The extraction method was referred to the kit instructions:

[0067] (1) Take 2 mL of overnight culture solution and centrifuge at 12,000 rpm for 1 min to collect the cells and remove the supernatant as much as possible;

[0068] (2) Add 250 μL Buffer PA to resuspend the bacterial pellet and vortex until no obvious bacterial clumps remain;

[0069] (3) Add 250 μL Buffer PB and gently invert the tube eight times to fully lyse the bacteria;

[0070] (4) Add 350 μL Buffer PC, gently invert up and down 8 times to mix thoroughly, and centrifuge at 12,000 rpm for 10 min;

[0071] (5) Carefully aspirate the supernatant and transfer it to the adsorption column (do not aspirate the precipitate). Centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and return the adsorption column to the empty collection tube.

[0072] (6) Add 500 μL Buffer PWA to the adsorption column, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid;

[0073] (7) Add 600 μL Buffer PWB to the adsorption column and centrifuge at 12,000 rpm for 30 s to discard the waste liquid;

[0074] (8) Repeat step 7;

[0075] (9) Place the adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 1 min;

[0076] (10) Remove the adsorption column and place it in a clean 1.5 mL centrifuge tube. Incubate at 25°C for 1 min to allow the residual ethanol to evaporate. Add 35 μL of Eluent to the middle of the adsorption membrane, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0077] After extracting the recombinant plasmid of 35S-PbeMYB2-GFP, it was transformed into Agrobacterium (GV3101) competent cells using the freeze-thaw method.

[0078] (1) Pipette 10 μL PbeMYB2 plasmid and add it to 200 μL Agrobacterium competent cells, mix gently and place on ice for 30 min.

[0079] (2) After quick freezing in liquid nitrogen for 5 min, immediately place in a 37°C water bath for 5 min and then quickly place on ice for 5 min.

[0080] (3) Add 500 μL of LB antibiotic-free liquid culture medium and culture in a shaker at 220 rpm at 28°C for 3 h.

[0081] (4) Centrifuge at 4000 rpm for 5 min, discard part of the supernatant, retain about 200 μL, mix with pipette tip, and evenly spread on LB solid plate (where Rif final concentration is 25 ng / μL, Kan final concentration is 50 ng / μL), and invert and culture in the dark at 28°C for 2 days.

[0082] 2. Genetic transformation of tobacco

[0083] The PbeMYB2 gene was transferred into tobacco plants using Agrobacterium-mediated leaf disc transformation technology and was stably expressed in the tobacco genome.

[0084] (1) The first fully expanded leaf of a healthy, non-transgenic tobacco plant was selected, and the surface was disinfected and the explant tissue was cut (cut into squares with a size of approximately 0.5×0.5 cm). The explant tissue was obtained and placed with the upper surface of the leaf facing downward on MS medium (MS+0.3 mg / L NAA+2 mg / L 6-BA) and pre-cultured at 28°C in the dark for 2 days.

[0085] (2) Use the tip of the gun to dip into the fresh PbeMYB2 Agrobacterium positive bacterial liquid, streak it on LB (containing kan, Rif) solid medium (Rif final concentration 25 ng / μL, Kana final concentration 50 ng / μL), and place it in a 28°C incubator for dark culture for 2 days; pick the monoclonal Agrobacterium on the above medium, streak it on LB (containing kan, Rif) solid medium, and place it in a 28°C incubator for dark culture for 2 days; scrape the streaked plaque, add liquid MS medium, place it in a 28°C shaker for shaking (200 rpm / min), and wait until the bacterial liquid concentration reaches OD 600 =0.5, the culture was terminated, the bacterial solution was collected in a 50 mL sterile centrifuge tube, centrifuged at 4000 rpm and 25°C for 10 min, the supernatant was discarded, and the bacterial pellet was resuspended in 40 mL MS liquid culture medium for later use.

[0086] (3) The tobacco leaf disc pre-cultured for 2 days was resuspended in the Agrobacterium bacterial solution and fully infected for 10 minutes. The leaf disc was then placed on sterile filter paper, and the excess bacterial solution on the leaf surface was absorbed. The leaf disc was placed on MS solid culture medium (MS + 0.3 mg / L NAA + 2 mg / L 6-BA) and co-cultured at 25°C in the dark for 3 days.

[0087] (4) After co-cultivation for 3 days, tobacco leaf discs were soaked in an aqueous solution containing 500 mg / L Cef for 5 min, washed twice, and then rinsed three times with sterile water. The leaf discs were placed on sterile filter paper to absorb excess water on the leaf surface. The leaf discs were inoculated on MS solid culture medium (MS + 500 mg / L Cef + 50 mg / L Kan) for resistance screening and selected at (28°C, light, 16 h) / (25°C, dark, 10 h). The culture medium was replaced every 10 days.

[0088] (5) When the adventitious buds on the screening medium grow to about 1 cm, they are cut off and transferred to MS rooting medium (1 / 2MS + 500 mg / L Cef + 50 mg / L Kan) for further growth. After the root system of the plant is well developed, the root medium is washed off and the plant is placed in tap water for 1 week to harden the seedlings until new roots grow. The plant is then transferred to a greenhouse for further cultivation.

[0089] 3. Identification of positive and overexpressing transgenic lines

[0090] According to the above method, transgenic tobacco strains were obtained, and genomic DNA was extracted from the leaves of the transgenic tobacco strains using a plant genomic DNA extraction kit (purchased from Beijing Qingke Biotechnology Co., Ltd.), and PCR amplification was performed using PbeMYB2 gene specific primers (SEQ ID NO.3 and SEQ ID NO.4) to verify the transgenic insertion (the method refers to the gene cloning step in Example 1), and the wild tobacco leaf DNA was used as a control. The positive strains that can amplify the target band are then analyzed by RT-PCR. The method of RNA extraction and cDNA synthesis from the leaves of transgenic tobacco plants is the same as in Example 1, and PCR amplification is performed using PbeMYB2 specific expression primers (SEQ ID NO.5 and SEQ ID NO.6), and NtUbiquitin is used as an internal reference gene to screen out overexpression strains.

[0091] The RT-PCR amplification reaction system and procedure are shown in Table 4 and Table 5:

[0092] Table 4 RT-PCR amplification reaction system (50 μL)

[0093]

[0094]

[0095] Table 5 RT-PCR amplification reaction program

[0096]

[0097] The PCR amplification products and RT-PCR products were detected by agarose gel electrophoresis to determine the positive (1-8) and overexpression lines (OE3 and OE6) ( Figure 5 A and 5B).

[0098] Example 4: Drought resistance identification of overexpressing transgenic tobacco lines

[0099] 1. Analysis of drought resistance of 30-day-old transgenic tobacco lines under drought stress

[0100] To evaluate the drought resistance of the transgenic line PbeMYB2 under drought stress, 30-day-old transgenic tobacco (OE3 and OE6 lines) and wild-type (WT) tobacco seedlings were used as experimental materials, and drought treatment and dehydration treatment were used to detect the drought tolerance of each line.

[0101] (1) Drought treatment of seedlings. Transgenic and control tobacco plants were watered normally for 3 days, then watered for 15 days. After returning to normal watering for 7 days, the survival rate was calculated. Figure 6 ). The phenotype of the transgenic lines was stronger than that of the wild-type control 30 days after drought stress, and the survival rate was significantly higher than that of the wild-type control.

[0102] (2) Seedling dehydration treatment. The roots of the 30-day-old transgenic seedlings and control seedlings were removed and immediately placed in a room temperature environment for dehydration with an air humidity of 40-45%. The fresh weight of the seedlings was measured every 0.5 h during the treatment process. The material before dehydration was used as the control. The fresh weight data of the seedlings at 0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 7 h, and 9 h after treatment were selected for water loss analysis ( Figure 7 ). The phenotype of the transgenic line 30 days after dehydration stress was better than that of the wild control, and the water loss rate was significantly lower than that of the wild control ( Figure 7 ).

[0103] 2. Analysis of drought resistance of transgenic tobacco lines under drought stress for 60 days

[0104] (1) 60-day seedling drought treatment

[0105] After 3 days of normal watering, transgenic and control tobacco plants were subjected to drought treatment without watering for 12 days, and then normal watering was resumed for 10 days to observe phenotypic changes ( Figure 8 A) The phenotype of the transgenic lines was stronger than that of the wild-type control 60 days after drought stress.

[0106] (2) Physiological index measurement

[0107] The leaves of transgenic seedlings and control seedlings at 60 days old and 10 days after drought treatment were selected to determine the electrolyte exudation rate using the osmotic conductivity method, and the chlorophyll and proline contents in the tested leaves were determined according to the kit.

[0108] Determination of leaf electrolyte exudation rate. Take an appropriate amount of leaves and put them into a 50mL centrifuge tube filled with 20mL deionized water, place it in a shaker and shake for 2h, and then use an electrolyte exudation rate meter to measure its initial electrolyte exudation rate (C1). After the sample is boiled in boiling water for 10min to maximize the permeability of leaf cell ions, it is cooled to room temperature and its electrolyte exudation rate C2 is measured. The relative electrolyte exudation rate (C%) = 100×C1 / C2. After 60d of drought stress, the electrolyte exudation rate of the transgenic line was significantly lower than that of the wild control ( Figure 8 B).

[0109] Chlorophyll content detection: Take fresh plant leaves, wash them with distilled water, then dry the surface water, remove the midrib, weigh about 0.1g, cut them into pieces and put them in a mortar.

[0110] Add 1 mL of distilled water and 10 mg of reagent 1, grind thoroughly under weak light conditions, and transfer to a 10 mL test tube.

[0111] Rinse the mortar with the extraction solution, transfer all the rinse solution into a 10mL test tube, make up to 10mL with the extraction solution, and extract in the dark for 3 hours. The extraction is complete when the color of the tissue residue at the bottom is close to white. If the tissue residue has not completely turned white, continue to extract until the color of the tissue residue is close to white.

[0112] Preheat the UV spectrophotometer for 30 minutes, adjust the wavelength to 645nm and 663nm, and zero the UV spectrophotometer with the extract. Take 200μL of the upper extract in a micro-glass cuvette and measure the absorbance at 663nm and 645nm, which are recorded as A and B respectively. 663 and A 645 .

[0113] Chlorophyll a content (mg / g mass) = (21.2 × A 663 -4.48×A 645 )×Vt×F÷W÷1000=0.01×(21.2

[0114] ×A 663 -4.48×A 645 )×F÷W

[0115] Chlorophyll b content (mg / g mass) = (38.2 × A 645 -7.8×A 663 )×Vt×F÷W÷1000=0.01×(38.2

[0116] ×A 645 -7.8×A 663 )×F÷W

[0117] Total chlorophyll content (mg / g mass) = (33.7 × A 645 +13.4×A 663 )×Vt×F÷W÷1000=0.01×(33.7

[0118] ×A 645 +13.4×A 663 )×F÷W

[0119] Vt: extraction volume, 10 mL; F: dilution factor; W: sample mass, g.

[0120] The chlorophyll content of the transgenic lines was significantly higher than that of the wild-type control ( Figure 8 C).

[0121] Proline (Pro) content detection: Weigh 0.1g of leaves and grind them, add 1mL of extracting solution and homogenize them in an ice bath; then place them in a boiling water bath for 10min of shaking extraction, centrifuge at 10000g at room temperature for 10min, take the supernatant, cool it and test it.

[0122] The spectrophotometer was preheated for 30 min, the wavelength was adjusted to 520 nm, and the spectrophotometer was zeroed with distilled water.

[0123] Treatment of standards: Dilute the standards with distilled water to 40, 20, 10, 8, 4, 2, 1, and 0.5 μg / mL.

[0124] Mix the reagents according to the operating table in the manual, cover the lid tightly after mixing, wrap the sealing film, place in a boiling water bath and keep warm for 30 minutes, shake once every 10 minutes, and after cooling, draw 0.2mL into a cuvette for colorimetric comparison at a wavelength of 520nm, record the absorbance values ​​of A test tube, A standard tube, and A blank tube, and calculate ΔA = A test tube - A blank tube, ΔA standard = A standard tube - A blank tube. The blank tube only needs to be done 1 to 2 times.

[0125] A standard curve was drawn with the concentration of the standard solution as the abscissa and the ΔA standard as the ordinate to obtain a linear regression equation y=kx+b. Substituting ΔA into the equation, x (μg / mL) was obtained.

[0126] Calculated according to tissue mass: Pro content (μg / g mass) = x*Vt / W

[0127] Vt: volume of extraction solution added, 1 mL; W: sample mass, g.

[0128] The proline content of the transgenic lines was significantly higher than that of the wild-type control ( Figure 8 D).

[0129] Example 5: Analysis of the accumulation level of active oxygen in overexpressing transgenic tobacco lines and analysis of the expression of stress-related genes in overexpressing transgenic tobacco lines before and after drought stress

[0130] 1. Analysis of reactive oxygen accumulation levels in overexpressing transgenic tobacco lines

[0131] The changes in the levels of reactive oxygen species (ROS) in transgenic PbeMYB2 tobacco lines under drought stress were identified by histochemical staining. DAB and NBT staining were used to detect hydrogen peroxide (H2O2) and superoxide anions (O2 －) accumulation to evaluate the antioxidant capacity of transgenic lines under drought stress ( Fig. 9 ).

[0132] (1) DAB staining was used to analyze the accumulation of hydrogen peroxide (H2O2) in transgenic and control leaves. Leaves of 30-day transgenic and control tobacco plants were selected before and 10 days after drought treatment. The leaves of the plants to be tested were immersed in 1 mg / mL DAB staining solution for 8 hours, and then the green color in the leaves was decolorized with anhydrous ethanol to detect the accumulation of H2O2 in the leaves. There was no difference in DAB staining of the leaves of the tested lines during drought stress treatment ( Fig. 9 A), after drought stress treatment, the staining range and degree of the overexpressed PbeMYB2 transgenic lines were significantly lighter than those of the wild-type control ( Fig. 9 B), indicating that less H2O2 accumulated in the leaves.

[0133] (2) NBT staining method for analysis of superoxide anions (O2 － ) in transgenic and control leaves. Leaves of 30-day transgenic and control tobacco plants before and 10 days after drought treatment were selected. The leaves of the plants to be tested were immersed in a staining solution containing 1 mg / mL NBT for 2 hours, and then the green color in the leaves was decolorized with anhydrous ethanol to detect the O2 － Under drought stress treatment, there was no difference in NBT staining of leaves of each tested strain ( Fig. 9 C), after drought stress treatment, the staining range and degree of the overexpressed PbeMYB2 transgenic lines were significantly lighter than those of the wild-type control ( Fig. 9 D), indicating that O2 accumulated in the leaves － less.

[0134] 2. Analysis of the expression levels of stress resistance-related genes in overexpressing transgenic tobacco lines before and after drought stress

[0135] To gain a deeper understanding of the molecular mechanism of drought resistance in transgenic tobacco overexpressing PbeMYB2, 16 genes related to scavenging reactive oxygen species (ROS) or stress response, including NtADC (NP_001312119.1), NtAPX (U15933.1), NtCAT (U93244.1), NtSOD (AB093097), NtERD1 (Gu144573), NtERF5 (AY655738), NtERD10 The expression levels of A(AB049335), NtLEA5(AF053076), NtLEA14(XP_009621630), NtNCED1(HM068892), NtP5CS(HM854026), NtCOR15(MF152621.1), NtRD17(XP_016502582.1), NtRD22(NP_197943.1), NtRD29A(TC115477 in DFCI database), and NtDREB1(EU727155.1) before and after drought treatment were analyzed.

[0136] RNA extraction and cDNA synthesis from tobacco leaves were performed according to the method of Example 1. Fluorescence quantitative PCR (qRT-PCR) was used to detect the difference in expression of all tested genes in transgenic and control leaves before and after drought treatment. The PCR program, system, and internal reference primers were consistent with those in Example 2 of the data analysis method. The study found that overexpression of PbeMYB2 induced the expression of these 16 stress resistance-related genes before and after drought treatment ( Fig.10 ).

[0137] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A Pyrus betula PbeMYB2 protein, characterized in that The protein is a drought-resistant R2R3 type MYB protein isolated from pear, named PbeMYB2, and its sequence is shown in SEQ ID NO.1 in the sequence table.

2. A PbeMYB2 gene, characterized in that The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.2, and the amino acid sequence is shown in the sequence listing SEQ ID NO.

1.

3. A gene cloning primer, characterized in that: The cloning primer is the full-length cDNA sequence of the gene PbeMYB2 according to claim 2 cloned using PCR technology.

4. A gene cloning primer according to claim 3, characterized in that: The nucleotide sequence of the homology arm primer pair of the PCR is as follows: Forward primer: 5'-gcccttgctcaccatggatccATGGATATTAATAATGTTCATGATCAGATG-3', i.e. SEQ ID NO. 3; Reverse primer: 5'-gagaacacgggggactctagaTTAGTCATCACTAAGCTGCTGTTGC-3', i.e. SEQ ID NO.

4.

5. A plant overexpression vector, characterized in that: The PbeMYB2 gene was transformed into tobacco using the Agrobacterium-mediated genetic transformation method to obtain transgenic plants.

6. Use of the biological material related to the Pyrus betulaeformis PbeMYB2 protein according to claims 1 to 5 in regulating plant drought resistance, characterized in that: The related biological material further comprises any one of the following 1) to 4): 1) A DNA molecule whose coding region is shown in SEQ ID No. 2; 2) an expression cassette containing the nucleic acid molecule described in 1); 3) a recombinant vector containing the nucleic acid molecule described in 1), or a recombinant vector containing the expression cassette described in 2); 4) A recombinant microorganism containing the nucleic acid molecule described in 1), or a recombinant microorganism containing the expression cassette described in 2), or a recombinant microorganism containing the recombinant vector described in 3).

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