Rice heat tolerance related gene DREB2B and its encoded protein and application

Abstract

The application relates to the field of plant genetic engineering, and specifically provides a rice heat tolerance related gene DREB2B and an encoded protein and application thereof. The CDS sequence of the rice DREB2B gene is shown as SEQ ID NO. 2, the gene encodes an AP2 / ERF transcription factor, and transgenic knockout and overexpression experiments prove that the gene positively regulates rice heat tolerance. Meanwhile, the application identifies that the expression of DREB2B can be transactivated HsfA3 in response to high-temperature stress. The application analyzes the mechanism of the gene in regulating rice heat tolerance, DREB2B and lays a solid theoretical foundation for future site-directed genetic improvement of rice heat tolerance by means of molecular design.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant genetic engineering, and relates to a rice heat tolerance related gene DREB2B and an encoded protein thereof and application. BACKGROUND

[0002] Under the global warming environment, the problem of high-temperature heat damage to rice is becoming increasingly serious. When the environmental temperature rises by 1℃, the average yield of rice decreases by 3.2%, and the quality of rice deteriorates and the growth is damaged. In the middle and lower reaches of the Yangtze River in China, high-temperature heat damage to early rice and medium rice occurs easily from June to August every year. With the intensification of the greenhouse effect year by year, the frequency, duration, scope and maximum temperature of high-temperature heat damage are all on the rise, which poses a serious threat to rice production. In view of this problem, it is of great significance to explore and cultivate rice heat-resistant gene sites, deeply analyze the molecular mechanism of rice heat tolerance, and apply it to the cultivation of new rice varieties resistant to high temperature to resist high-temperature heat damage to rice, so as to ensure national food security.

[0003] DREB transcription factors belong to the AP2 / ERF transcription factor family, can specifically bind to C-repeat / DRE elements, thereby activating the expression of stress response genes such as low temperature and drought, and play an important role in plant tolerance to abiotic stress. At present, a plurality of DREB homologous genes have been isolated in rice. Among them, OsDREB1A can activate the expression of calcium ion channel protein gene OsCNGC9 , promote the increase of calcium ion concentration in cytoplasm to regulate the cold tolerance of rice; OsDREB1C / E / G All of the three genes can participate in the regulation of ROS removal under cold stress, and can also respond to high temperature, drought and salt stress to different degrees. OsDREB6 positively regulates the tolerance of rice to osmotic stress, high salt stress and cold stress by affecting the accumulation of osmotic substances and ROS. OsDREB2B can interact with OsSRO1c to form protein aggregates, directly respond to low temperature and dynamically change, and regulate the expression of key cold-resistant genes including COLD1 However, there is no relevant report on the correlation between OsDREB2B and rice heat tolerance.

[0004] Therefore, it is of great significance to explore and identify heat-resistant genes, analyze the genetic basis of rice heat tolerance, and combine biotechnology to carry out molecular design breeding of heat tolerance, so as to fundamentally solve the problem of high-temperature heat damage to rice, accelerate the breeding of heat-resistant rice varieties, promote the process of commercial rice breeding, and effectively solve the problem of lack of heat-resistant varieties in production. Improving the heat tolerance of rice by genetic means is not only the most economical and effective solution, but also has important significance for ensuring the quality and high yield and stable yield of rice, and has wide application value in the field of rice molecular breeding. SUMMARY

[0005] The technical problem to be solved by this invention is how to regulate the heat tolerance of plants. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0007] The first aspect of this invention provides the application of genes overexpressing the DREB2B protein, wherein the application is any of the following:

[0008] A1) Application in improving the heat resistance of rice;

[0009] A2) Application in the preparation of products that improve the heat resistance of rice;

[0010] A3) Application in cultivating heat-resistant rice;

[0011] A4) Application in the preparation of products for cultivating heat-resistant rice;

[0012] A5) Application in heat-resistant breeding of rice or improvement of heat-resistant germplasm resources of rice;

[0013] A6) In activating rice HsfA3 Applications in gene transcription, the aforementioned HsfA3 The CDS sequence of the gene is shown in SEQ ID NO. 4;

[0014] The amino acid sequence of the DREB2B protein is shown in SEQ ID NO.3.

[0015] A second aspect of the present invention provides the application of biomaterials related to the DREB2B protein, wherein the application is any of the following:

[0016] D1) Application in improving the heat resistance of rice;

[0017] D2) Application in the preparation of products that improve the heat resistance of rice;

[0018] Application of D3 in the cultivation of heat-resistant rice;

[0019] D4) Application in the preparation of products for cultivating heat-resistant rice;

[0020] D5) Application in heat-resistant breeding of rice or improvement of heat-resistant germplasm resources of rice;

[0021] D6) In activating rice HsfA3 Applications in gene transcription, the aforementioned HsfA3 The CDS sequence of the gene is shown in SEQ ID NO. 4;

[0022] The biomaterial is any one of the following E1) to E3):

[0023] E1) An expression cassette containing a nucleic acid molecule encoding the DREB2B protein;

[0024] E2) A recombinant vector containing a nucleic acid molecule encoding the DREB2B protein;

[0025] E3) A recombinant microorganism containing a nucleic acid molecule encoding the DREB2B protein, or a recombinant microorganism containing the expression cassette described in E1), or a recombinant microorganism containing the recombinant vector described in E2), wherein the microorganism is Agrobacterium;

[0026] The nucleotide sequence of the nucleic acid molecule encoding the DREB2B protein is shown in SEQ ID NO. 2.

[0027] A third aspect of the present invention provides a method for cultivating heat-resistant plants, the method comprising overexpressing a gene for the DREB2B protein in a plant to obtain a plant with enhanced heat resistance, the plant being rice.

[0028] In the method described above, the gene that overexpresses the DREB2B protein in plants is obtained by using transgenic technology to increase the expression level of the gene encoding the DREB2B protein in rice.

[0029] In the method described above, the expression level of the gene encoding the DREB2B protein in rice is increased by introducing a plant expression vector that integrates the nucleic acid molecule shown in SEQ ID NO. 2 into the target plant using transgenic technology.

[0030] In this method, plants with improved heat resistance are obtained by overexpressing the DREB2B protein gene in the plant, thereby activating downstream... HsfA3 This is achieved through gene expression.

[0031] A fourth aspect of this invention provides a plant heat tolerance regulation module, the module comprising an upstream DREB2B transcription factor and downstream target genes directly regulated by it. HsfA3 The composition, in which DREB2B is combined with HsfA3 To activate the C-repeat / DRE element in the starter sub-region HsfA3 The expression.

[0032] The specific technical steps for implementing this invention are as follows:

[0033] I. DREB2B positively regulates rice heat resistance

[0034] This invention uses CRISPR / Cas9 technology to construct DREB2B By knocking out the vector and transforming it into the Wuyunjing 31 (W31) background, homozygous transgenic knockout lines were obtained.dreb2b Figure 1 DREB2B DREB2B-OE DREB2B DREB2B dreb2b DREB2B-OE DEEB2B Figure 1 Figure 1 Figure 1 DREB2B Figure 1 DREB2B

[0035] II. DREB2B activates the transcription of to response to high temperature HsfA3

[0036] To further explore the molecular mechanism of DREB2B transcription factor regulating the heat tolerance of rice, the inventors screened the downstream target gene of DREB2B transcription, which is HsfA3 . The transcription level of the gene in the DREB2B-OE and dreb2b strain is significantly increased and decreased, respectively; after 43℃ high temperature treatment, its expression is further increased, but the significant difference still exists between the DREB2B-OE and dreb2b strain (Fig. 1A). It has been shown in the existing research that DREB2B can specifically bind to C-repeat / DRE element (A / GCCGAC) to regulate stress response genes. The inventors divided the promoter of Figure 2 into 5 segments, connected to pLacZi vector, and then co-transferred into yeast strain EGY48 with pB42AD-DREB2B to carry out yeast one-hybrid experiment. The results show that the promoter segments containing C-repeat / DRE element in HsfA3 can be combined by DREB2B, while the other segments cannot (Fig. 1B). HsfA3 Figure 2 ​​​​​​​​​​​​​​​​​(B in the text). Then treated at high temperature. DREB2B-GFP Chromatin was extracted from the / W31 overexpression line, and ChIP-qPCR was performed. EMSA results further confirmed that DREB2B can bind to... HsfA3 The P5 and P6 regions of the gene promoter ( Figure 2 C in Figure 2 (D in the text). In summary, this indicates that the DREB2B transcription factor can directly bind to... HsfA3 The C-repeat / DRE element (A / GCCGAC) in the promoter region is activated and its transcription is resumed in response to high temperature stress.

[0037] three, HsfA3 Positive regulation of rice heat resistance

[0038] Previous experimental results have confirmed DREB2B It can positively regulate the rice's tolerance to high temperatures. HsfA3 As a target gene for its direct transcriptional activation, does it also possess similar thermostable functions? The present inventors constructed a model in the wild-type W31 background. HsfA3 Knockout and overexpression transgenic genetic materials were used to obtain genotype-homozygous and phenotype-stable lines. These lines were then subjected to high-temperature treatment at 43℃, followed by a 7-day recovery period at 28℃, during which survival rates were statistically analyzed. Results showed that wild-type W31... hsfa3 and HsfA3-OE The survival rates of the strains were approximately 16.13%, 11.46%, and 80.21%, respectively. HsfA3 The overexpression of the gene significantly enhanced the heat resistance of the strain. Figure 3 A in Figure 3 (B in the text); and HsfA3 In their corresponding knockout and overexpression transgenic lines, the expression levels were downregulated and upregulated, respectively. Figure 3 (C in the middle).

[0039] The detection of SOD activity and H2O2 content showed that HsfA3-OE The SOD activity in the strain was significantly higher than that in W31 both before and after high temperature, while the H2O2 content was significantly lower than that in W31 both before and after high temperature. Figure 3 D in Figure 3 (E in the original text). Furthermore... hsfa3 The SOD activity of all strains was significantly lower than that of W31 after high temperature, while the H2O2 content was significantly higher than that of W31 after high temperature. Figure 3 D in Figure 3 E in the text). Analysis of NBT and DAB staining results in the leaves showed that... HsfA3-OE The strain showed fewer and lighter blue spots after high-temperature treatment, indicating a higher accumulation of H2O2 and O2. - The content is low; while hsfa3The strain has more blue spots and deeper staining after high temperature, and accumulates more H2O2 and O2 - content is higher Figure 3 , F). In summary, HsfA3 can reduce the accumulation of endogenous ROS and alleviate oxidative damage to cells, and positively regulate the heat tolerance of rice.

[0040] Five、 HsfA3 located DREB2B downstream of the genetics of rice heat tolerance

[0041] To clarify the function and relationship of DREB2B and HsfA3 in heat tolerance regulation, the inventors constructed genetic combination materials of the two and detected their heat tolerance. Compared with W31 (15.63%), OsDREB2B (82.29%) and OsDREB2B (80.21%) all showed significant heat tolerance phenotype; while OsDREB2B (7.29%), OsDREB2B (11.46%) and OsDREB2B (0) showed significant heat intolerance phenotype. In addition, the survival rate of OsDREB2B (72.92%) was significantly higher than that of OsDREB2B (7.29%), indicating that overexpression of OsDREB2B can partially restore the heat tolerance lost by the loss of function of OsDREB2B. In addition, the survival rate of OsDREB2B (17.71%) was between that of OsDREB2B (82.29%) and OsDREB2B (11.46%), showing a more biased OsDREB2B. The above results show that OsDREB2B is located downstream of the genetics of rice heat tolerance and regulates rice heat tolerance on the same pathway. DREB2B-OE HsfA3-OE dreb2b hsfa3 dreb2b / hsfa3 Figure 4 dreb2b / HsfA3-OE dreb2b HsfA3 DREB2B Figure 4 DREB2B-OE / hsfa3 DREB2B-OE hsfa3 hsfa3 Figure 4 HsfA3 DREB2B

[0042] The present application identifies a gene that positively regulates the heat tolerance of rice DREB2B , which encodes an AP2 / ERF transcription factor that can respond to high temperature stress, and the transcription level and protein enrichment amount significantly increase under high temperature conditions; overexpression of the gene in rice can significantly improve the heat tolerance of the plant, and when the gene is deleted, it shows high temperature sensitivity. The present application clarifies the molecular mechanism of DREB2B regulating the heat tolerance of rice by activating downstream genes HsfA3 . The present application reveals HsfA3 ​​​​​​​​​​​​​​​​​​The rice heat tolerance can be positively regulated by reducing the accumulation of endogenous ROS to alleviate the oxidative damage of cells. The present application provides new gene resources and theoretical guidance for rice heat tolerance improvement and molecular design breeding, helps to breed new rice varieties resistant to high temperature, improves the yield of crops, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 DREB2B is positively regulated by high temperature to regulate rice heat tolerance: Figure 1 A in the formula is DREB2B Gene knockout target site and mutation type of knockout strain; Figure 1 B and C in the formula are wild type W31, dreb2b and DREB2B Heat tolerance analysis of OE strain; Figure 1 D in the formula is the expression level of dreb2b and DREB2B OE strain before and after high temperature treatment; DREB2B Figure 1 E and F in the formula are the changes of transcription level (E) and DREB2B protein enrichment (F) over time under 43 DEG C high temperature treatment. DREB2B

[0044] DREB2B transactivates the expression of HsfA3 to respond to high temperature: Figure 2 A in the formula is the transcription level of Figure 2 and dreb2b strain before and after high temperature treatment; DREB2B-OE B, C and D in the formula are respectively yeast single hybridization, ChIP-qPCR and EMSA to confirm that DREB2B can bind HsfA3 promoter region. Figure 2 HsfA3 DREB2B positively regulates rice heat tolerance:

[0045] A and B in the formula are heat tolerance analysis of Figure 3 and HsfA3 OE strain; Figure 3 C in the formula is the expression level of hsfa3 and HsfA3 OE strain; Figure 3 D and F in the formula are SOD activity (D) and H2O2 content (E) detection of hsfa3 and HsfA3 strain before and after high temperature, and NBT and DAB staining of leaf (F). HsfA3 Figure 3 hsfa3 HsfA3-OE

[0046] Figure 4 DREB2B transactivates the expression of HsfA3 to respond to high temperature:​​​​​DREB2B and HsfA3 Heat tolerance analysis of genetic materials: Figure 4 A, B in W31, dreb2b , DREB2B -OE, hsfa3 , HsfA3 -OE, DREB2B -OE / hsfa3 , dreb2b / HsfA3 -OE and dreb2b / hsfa3 Heat tolerance analysis of transgenic lines. DETAILED DESCRIPTION

[0047] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0048] Example 1: High temperature treatment of rice seedlings

[0049] Rice seeds were soaked and germinated normally, and seeds with uniform growth were selected and sown on water culture plates in an incubator. Yoshida nutrient solution (Coolboy, NSP1040-1kg) was used for culture, and the nutrient solution was replaced every two days. The conventional culture conditions were temperature 28℃, relative humidity 60%, 14 h light / 10 h darkness. High temperature treatment was 43℃, relative humidity 80%, relative humidity 80%, 14 h light / 10 h darkness. The three-leaf stage water culture seedlings were treated with high temperature (43℃, RH > 80%) for about 40 h, and then recovered for 7 days (28℃, RH = 60%), and the survival rate was calculated to judge the high temperature tolerance of the material.

[0050] Example 2: Construction of vector

[0051] (1) Knockout vector: According to the website http: / / skl.scau.edu.cn / home / , the knockout target site was selected, and the CRISPR / CAS9 technology was used to construct the knockout vector of DREB2B and HsfA3 gene, and the corresponding background parent was transformed by Agrobacterium-mediated method to obtain homozygous knockout transgenic lines;

[0052] (2) Overexpression vector: The full-length CDS sequence of DREB2B and HsfA3 gene in W31 was amplified DREB2B The CDS sequence of the gene is shown in SEQ ID NO. 2, HsfA3The gene CDS (as shown in SEQ ID NO. 4) is linked to... pUbi::GFP In the vector, construct a recombinant vector. pUbi::DREB2B-GFP and pUbi::HsfA3-GFP Agrobacterium-mediated transformation of parents with different backgrounds yielded homozygous overexpression transgenic lines.

[0053] (3) Yeast single-hybrid (Y1H) vector: amplification of W31 DREB2B The full-length CDS sequence of the gene is ligated into pB42AD Vector; amplification HsfA3 The promoter sequences of the genes are respectively linked into pLacZi Carrier.

[0054] (4) Prokaryotic expression vector: amplification of W31 DREB2B The full-length CDS sequence of the gene was constructed into pET28a The DREB2B-His fusion protein was induced and purified by transforming Escherichia coli BL21(DE3) expression strain into a prokaryotic expression vector.

[0055] Example 3: Yeast one-hybrid (Y1H)

[0056] pB42AD-DREB2B and pLacZi-HsfA3 1 μg of each plasmid was co-transformed into yeast strain EGY48 (see the instruction manual, Coolaber, SK2401-200T for specific transformation steps). After transformation, the cells were evenly spread onto duplex-deficient (SD-Trp-Ura) solid medium and incubated at 28°C for 3-5 days. Single colonies were picked and mixed with 50 μL of sterile water, and serially diluted 10, 100, and 1000 times. The yeast cultures of different dilutions were spotted onto SD-Trp-Ura duplex-deficient (glucose-free, with 1% raffinose and 2% galactose added) solid medium and incubated at 28°C for 3-5 days. Then, X-Gal (80 mg / L) was added to the colonies, and the colony color development was observed.

[0057] Example 4: Chromatin-immunoprecipitation (ChIP)

[0058] (1) Formaldehyde cross-linking: Take 3-5 g of rice leaves and put them into a 50 mL centrifuge tube, add 36 mL of pre-cooled Extraction buffer 1 (0.4 M Sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 0.15 % Triton X-100, 1 mM PMSF), 1 mL of 37% formaldehyde (final concentration 1%), and vacuum at 4°C for 20-30 min. Add 2.5 mL of 2 M glycine solution (final concentration 125 mM) to the centrifuge tube, and vacuum for 5-10 min to terminate cross-linking. Pour off the solution, wash the sample with an appropriate amount of double-distilled water for 3-5 times, and freeze the sample in liquid nitrogen.

[0059] (2) Chromatin extraction: Take 3-5 g crosslinked sample, grind in liquid nitrogen. Transfer the ground sample to a 50 mL centrifuge tube, add 30 mL Extraction buffer 1, vortex to mix. Filter the solution through a filter cloth into a new 50 mL centrifuge tube, centrifuge at 4°C, 4 000 rpm for 20 min, discard the supernatant. Resuspend the pellet with 20 mL Extraction buffer 2 (10 mM Tris-HCl pH 8.0, 0.25 M Sucrose, 10 mM MgCl2, 0.15% Triton X-100, 1 mM PMSF) solution, mix by gently blowing. Centrifuge at 4°C, 10 000 rpm for 20 min, discard the supernatant. Wash the pellet with Extraction buffer 2 once again (the pellet should be white or light green, if it is dark green, it means there is too much impurity, you can add 1 mL Extraction buffer 2 again to wash), resuspend the pellet with 8 mL Extraction buffer 3 (10 mM Tris-HCl pH 8.0, 1.7 M Sucrose, 2 mM MgCl2, 0.15% Triton X-100, 1 mM PMSF). Take another 50 mL centrifuge tube, add 8 mL Extraction buffer 3, carefully add the resuspended pellet solution to the top of the solution, centrifuge at 4°C, 10 000 rpm for 10 min, discard the supernatant. Resuspend the pellet with 1 mL nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF), incubate on ice for 30 min, and the leaf chromatin solution is obtained. Divide the chromatin solution into two 1.5 mL centrifuge tubes, sonicate on ice, centrifuge at 4°C, 12 000 rpm for 15 min, take the supernatant. Combine the supernatants of the two tubes, take 100 μL as a control (input), dilute the remaining sample with 3-5 volumes of ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 1 mM PMSF).

[0060] (3) Immunoprecipitation: Take 100 μΐ of Protein A / G magnetic beads (Invitrogen, 10001D and 10003D) in a 2.0 mL centrifuge tube, and wash the magnetic beads with 1 mL of ChIP dilution buffer. Add the diluted chromatin sample, and pre-hybridize at 4°C for 30-60 min. Take the pre-hybridized supernatant through a magnetic stand, and add 1 μg of the antibody of the target protein, and incubate overnight at 4°C. Add 100 μΐ of Protein A / G magnetic beads again, and incubate at 4°C for 60 min, and discard the supernatant. Wash the magnetic beads with 1 mL of low-salt solution (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), high-salt solution (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), lithium chloride solution (0.25 M LiCl, 1% deoxycholate sodium, 1% NP40, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at 4°C, 5 min each time. Add 250 μΐ of ChIP elution buffer, vortex to mix, and stand for 5 min, and take the supernatant; add 250 μΐ of ChIP elution buffer (100 mM NaHCO3, 1% SDS) again, vortex to mix, and take the supernatant; and combine all the supernatants to about 500 μΐ. Add 20 μΐ of 5 M NaCl to the immunoprecipitated sample and the input group (400 μΐ of ChIP elution buffer is added to the input sample) respectively, and de-crosslink at 65°C for 6 h. After de-crosslinking, purify the immunoprecipitated sample through a DNA purification kit, and the input sample does not need to be purified.

[0061] (4) ChIP qRT-PCR: Design fluorescent quantitative primers in the promoter region of the target gene, and cover the full-length sequence of the promoter as much as possible. Take the purified sample and the input sample as templates, and detect the enrichment amount of each fragment of the promoter through qPCR.

[0062] Example 5: Prokaryotic protein expression and purification

[0063] (1) Protein induction: The prokaryotic expression vector was transformed into BL21(DE3) expression strain, and the plate was cultured at 37°C for 12-16 h. A single colony was selected and inoculated into 5 mL of LB liquid medium (with the addition of the corresponding antibiotic), and cultured at 37°C, 200 rpm overnight. According to the ratio of 1:100, it was inoculated into 100 mL of LB liquid medium, and cultured at 37°C, 200 rpm until the OD600 was 0.4-0.6. 50 μL of IPTG (Isopropyl-beta-D-thiogalactopyranoside, 1M) was added to the culture medium to a final concentration of 0.5 mM to induce protein expression, and cultured at 18°C, 120 rpm for 16-18 h.

[0064] (2) Ultrasonic disruption: After protein induction, 1 mL of bacterial solution was taken to detect protein expression. The remaining bacterial solution was centrifuged at 4°C, 12000 rpm for 10 min to collect the bacterial body, and the supernatant was discarded. 5 mL of pre-cooled PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.4) and protease inhibitors (PMSF, 1:100) were added to the precipitate, and vortexed to mix. The bacterial body was disrupted by ultrasonic disrupter, with a working power of 300 W, working for 3 seconds and stopping for 3 seconds, and ultrasonic disruption for 20-30 min (the solution of soluble protein after disruption was clear). Centrifugation at 4°C, 12000 rpm for 10 min, and the supernatant was filtered through a microporous filter (0.45 μm). An appropriate amount of PBS was added to the precipitate and vortexed to mix. 1 mL of filtered supernatant and precipitate solution was taken to detect ultrasonic disruption.

[0065] (3) Protein purification: An appropriate amount of Ni-NTA purification medium (full form gold, P2218) was added to the chromatography column, and then 10 times the medium volume of equilibration buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0) was added. The chromatography column was equilibrated by natural flow under gravity, and repeated 3-5 times. The filtered supernatant was added to the chromatography column, and the effluent was collected and repeated. 1 mL of effluent was taken for detection. 10 times the column volume of equilibration buffer was added to wash the chromatography medium, and the filtrate was discarded, and repeated 3-5 times. 2 mL of imidazole eluent (300 mM NaCl, 50 mM NaH2PO4, 10 mM Tris-Hcl, 100 mM imidazole, pH 8.0) was added to elute His protein, and repeated once. The eluent was the purified target protein solution, and the protein was stored at -80°C.

[0066] (4) Take a small amount of purified protein and each component in the purification process, add an appropriate amount of protein loading buffer, SDS-PAGE electrophoresis to detect the protein purification concentration.

[0067] Example 6: Gel migration experiment (EMSA, Electrophoretic Mobility Shift Assay)

[0068] Locking HsfA3 The C-repeat / DRE elements in the promoter region of the gene were extended by about 15 bp to both ends, and two biotin-labeled probes HsfA3-P5 and HsfA3-P6 were synthesized, and two other biotin-labeled mutant probes were obtained by replacing the C-repeat / DRE element (A / GCCGAC) with AAAAAA. Take 100 ng of purified protein and 0.5 ng of labeled probe (use unlabeled probe as competitive probe) to prepare a 20 μL reaction system, incubate at 25°C for 20-30 min, and allow the protein to fully bind to the probe. After the reaction is completed, 5 μL of 5 x Non-reducing Loding Buffer is added and mixed well. Prepare 8% non-denaturing polyacrylamide gel electrophoresis, and the gel is pre-electrophoresed at a constant voltage of 100 V for 30 min. Load, electrophorese at a constant voltage of 100 V for 60 min. After electrophoresis, transfer to membrane at a constant voltage of 100 V for 30-60 min, cross-link the nylon membrane with an ultraviolet cross-linker, then perform blocking, washing, developing and photographing steps, and the detailed steps refer to the EMSA kit (Biuntian, GS009).

[0069] The above only lists several specific embodiments of the present application. It is necessary to point out that the present application is not limited to the above embodiments, and all modifications that can be directly derived or inferred by those skilled in the art from the disclosed content of the present application should be considered as falling within the scope of protection of the present application.

Claims

1. Use of a gene overexpressing a DREB2B protein, characterized in that, The application is any of the following: A1) application in improving heat tolerance of rice; A2) application in preparing a product for improving heat tolerance of rice; A3) application in breeding heat-tolerant rice; A4) application in preparing a product for breeding heat-tolerant rice; A5) application in heat-tolerant breeding of rice or improvement of heat-tolerant germplasm resources of rice; The amino acid sequence of the DREB2B protein is shown in SEQ ID NO.

3.

2. Use of a biological material related to the DREB2B protein as described in claim 1, characterized in that, The application is any of the following: D1) application in improving heat tolerance of rice; D2) application in preparing a product for improving heat tolerance of rice; D3) application in breeding heat-tolerant rice; D4) application in preparing a product for breeding heat-tolerant rice; D5) application in heat-tolerant breeding of rice or improvement of heat-tolerant germplasm resources of rice; The biological material is any of the following E1) to E3): E1) an expression cassette containing a nucleic acid molecule encoding a DREB2B protein; E2) a recombinant vector containing a nucleic acid molecule encoding a DREB2B protein; E3) a recombinant microorganism containing a nucleic acid molecule encoding a DREB2B protein, or a recombinant microorganism containing the expression cassette of E1), or a recombinant microorganism containing the recombinant vector of E2), the microorganism being Agrobacterium; The amino acid sequence of the DREB2B protein is shown in SEQ ID NO. 3, and the nucleotide sequence of the nucleic acid molecule encoding the DREB2B protein is shown in SEQ ID NO.

2.

3. A method of breeding heat tolerant plants, characterized by, The method comprises overexpressing a gene of a DREB2B protein in a plant to obtain a plant with improved heat tolerance, the plant being rice, and the amino acid sequence of the DREB2B protein is shown in SEQ ID NO.

3.

4. The method of claim 3, wherein, The gene of the DREB2B protein overexpressed in the plant is to increase the expression amount of the coding gene of the DREB2B protein in rice by using transgenic technology.

5. The method of claim 4, wherein, The increase of the expression amount of the coding gene of the DREB2B protein in rice by using transgenic technology is by introducing a plant expression vector integrated with the nucleic acid molecule shown in SEQ ID NO. 2 into the target plant.

6. The method of claim 3, wherein, The plants exhibited improved heat resistance were obtained by overexpressing the DREB2B protein gene in the plants, thereby activating downstream... HsfA3 This is achieved through gene expression; HsfA3 The CDS sequence of the gene is shown in SEQ ID NO.4.

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