Application of OsBBX29 gene in regulation and control of high-temperature stress resistance of rice

Abstract

The invention provides application of an OsBBX29 gene in regulation and control of high-temperature stress resistance of rice. The nucleotide sequence of the OsBBX29 gene is shown as SEQ ID NO.1, the CDS sequence of the OsBBX29 gene is shown as SEQ ID NO.2, and the amino acid sequence of the coded protein is shown as SEQ ID NO.3. Through molecular means and construction of rice OsBBX29 overexpression and knockout mutant plants, it is found that the OsBBX29 gene can positively regulate and control rice heat resistance, seedling stage high temperature stress resistance can be remarkably enhanced through overexpression of the OsBBX29 gene in rice, and the OsBBX29 knockout mutant plants show reduced high temperature stress resistance compared with wild type plants.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to application of the OsBBX29 gene in regulating high temperature stress resistance of rice. Background Art

[0002] Rice is one of the world's major food crops and occupies an important position in agricultural production. Increasing food production is a key strategy to ensure food security. High temperatures caused by climate change have a significant impact on the growth and development of major food crops. Heat stress has a significant negative impact on the entire growth cycle of rice, severely limiting the improvement of rice yield and quality. In response to high temperature stress, plants use different regulatory networks to regulate important genes, physiological and biochemical adaptability. Some high temperature genes have been reported. However, a deeper understanding of the complex regulation of rice response to high temperature stress still requires further exploration of genes related to heat stress resistance and in-depth analysis of their mechanisms. This is of great significance for the breeding of new excellent rice varieties resistant to high temperature stress.

[0003] B-box (BBX) proteins are plant-specific zinc-finger transcription factors. In rice, the BBX family comprises 30 members, which can be divided into five structural types based on the number of B-box domains and the presence of CCT domains. OsBBX29 contains only two B-box domains and belongs to subfamily IV, but its involvement in rice's high-temperature stress response has not been investigated. Summary of the Invention

[0004] The present invention provides a new use of the OsBBX29 gene in regulating the high temperature stress resistance of rice, and provides a theoretical basis for the breeding and genetic improvement of high temperature stress-resistant rice varieties.

[0005] The specific technical solutions adopted in the present invention are as follows: Application of the OsBBX29 gene in regulating high temperature stress resistance in rice, wherein the nucleotide sequence of the OsBBX29 gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3; Furthermore, the regulation includes: overexpressing the OsBBX29 gene to improve the high temperature stress resistance of rice, and knocking out the OsBBX29 gene to reduce the high temperature stress resistance of rice.

[0006] A method for improving rice's resistance to high temperature stress by manipulating the expression of the OsBBX29 gene through genetic engineering comprises the following steps: overexpressing the OsBBX29 gene in rice by transducing the OsBBX29 gene into rice, thereby improving the rice's resistance to high temperature stress.

[0007] A method for reducing rice's resistance to high temperature stress by manipulating the expression of the OsBBX29 gene through genetic engineering comprises the following steps: knocking out the OsBBX29 gene in rice through the CRISPR / Cas9 gene editing method, thereby reducing the rice's resistance to high temperature stress.

[0008] Use of the OsBBX29 gene in cultivating transgenic rice with improved or reduced resistance to high temperature stress, wherein the nucleotide sequence of the OsBBX29 gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3; Furthermore, the applications include: application of overexpressing the OsBBX29 gene in cultivating transgenic rice with improved resistance to high temperature stress, and application of knocking out the OsBBX29 gene in cultivating transgenic rice with reduced resistance to high temperature stress.

[0009] A method for cultivating transgenic rice with improved resistance to high temperature stress by manipulating the expression of the OsBBX29 gene through genetic engineering comprises the following steps: overexpressing the OsBBX29 gene in rice to cultivate transgenic rice with improved resistance to high temperature stress.

[0010] A method for cultivating transgenic rice with reduced resistance to high temperature stress by manipulating the expression of the OsBBX29 gene through genetic engineering comprises the following steps: knocking out the OsBBX29 gene in rice to cultivate transgenic rice with reduced resistance to high temperature stress.

[0011] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses molecular means and constructs rice OsBBX29 overexpression and knockout mutant plants to find that the OsBBX29 gene can positively regulate rice's resistance to high temperature stress. Overexpression of the OsBBX29 gene in rice can significantly enhance high temperature stress resistance at the seedling stage, while OsBBX29 knockout mutant plants show reduced high temperature stress resistance compared with the wild type.

[0012] (2) The present invention also confirmed through some physiological and biochemical analyses that the OsBBX29 gene may enhance the resistance of rice to high temperature stress by reducing the degree of cell membrane damage during the rice seedling stage.

[0013] (3) The present invention verified through subcellular localization and yeast autoactivation experiments that OsBBX29 is distributed in both the nucleus and cytoplasm, but has no transcriptional autoactivation ability, and inferred that it may not function in the form of a transcription factor.

[0014] (4) The present invention identifies the function of the OsBBX29 gene in the response of rice to high temperature stress, provides a candidate gene for the breeding of rice varieties resistant to high temperature stress, and provides a theoretical and practical basis for the genetic improvement of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Analysis of the changes in OsBBX29-Ⅳ family expression in the wild type under high temperature stress treatment for different time periods; each group was repeated three times, and the internal reference gene was Actin1.

[0016] Figure 2 :A is the structure and mutation information of the OsBBX29 gene, and the SG region is the target site for gene editing; B is the sequencing peak map comparison of the two mutant strains osbbx29-1 and osbbx29-2.

[0017] Figure 3 : A shows the phenotypes of wild-type ZH11 and osbbx29 mutant rice (osbbx29-1 and osbbx29-2) before and after high temperature stress treatment; B shows the survival rate statistics of wild-type ZH11 and osbbx29 mutant rice (osbbx29-1 and osbbx29-2) 7 days after recovery treatment. The significance analysis method was Student's t-test. Error bars represent mean ± SE. **** indicates P value less than 0.001.

[0018] Figure 4 A shows relative electrical conductivity in leaves of wild-type ZH11 and osbbx29 mutant rice (osbbx29-1 and osbbx29-2) before and after heat stress. B shows malondialdehyde (MDA) content in leaves of wild-type ZH11 and osbbx29 mutant rice (osbbx29-1 and osbbx29-2) before and after heat stress. Significance analysis was performed using the Tukey test. Error bars represent mean ± SE. Different letters indicate significant differences with P values ​​less than 0.05. CK: before heat stress; Heat: after heat stress.

[0019] Figure 5 :A is the expression level identification of OsBBX29-OE overexpressing plants, Relative expression is the relative expression level of the gene, each group was repeated three times, and the internal reference gene was Actin1; B is the phenotype of wild-type ZH11 and OsBBX29 overexpressing lines (OE9-5 and OE18-2) before and after high temperature stress treatment; C is the survival rate statistics of wild-type ZH11 and OsBBX29 overexpressing lines (OE9-5 and OE18-2) after 7 days of recovery treatment, the significance analysis method was Student's t-test, error bars represent mean ± SE, *** indicates P value less than 0.001.

[0020] Figure 6 A shows relative electrical conductivity measurements of leaves in wild-type ZH11 and OsBBX29-overexpressing rice lines (OE9-5 and OE18-2) before and after high-temperature stress. B shows malondialdehyde (MDA) content measurements of leaves in wild-type ZH11 and OsBBX29-overexpressing rice lines (OE9-5 and OE18-2) before and after high-temperature stress. Significance analysis was performed using the Tukey test. Error bars represent mean ± SE. Different letters indicate significant differences with P values ​​less than 0.05. CK: before high-temperature stress; Heat: after high-temperature stress.

[0021] Figure 7 : Subcellular localization map of OsBBX29; OsBBX29-EGFP is enhanced green fluorescent protein; RFP-H2B is nuclear marker red fluorescent protein; Chloroplasts is chloroplast; Bright field is bright field; Merged is superimposed field; scale bar 50 μm.

[0022] Figure 8 :OsBBX29 transcriptional activation verification diagram; BK-53+AD-T is the positive control; BK-Lam+AD-T is the negative control; BK-OsEIL5+AD is the experimental group; SD / -Trp / -Leu is the double-deficient culture medium; SD / -Trp / -Leu / -His / -Ade is the quadruple-deficient culture medium; each group is divided into three concentration gradients (10 0 , 10 -1 , 10 -2 ). DETAILED DESCRIPTION

[0023] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0026] In the present invention, the nucleotide sequence of the OsBBX29 gene is shown in SEQ ID NO.1, the nucleotide sequence of the coding region of the OsBBX29 gene, i.e., the CDS, is shown in SEQ ID NO.2, and the amino acid sequence of the OsBBX29 protein encoded by the OsBBX29 gene is shown in SEQ ID NO.3. SEQ ID NO.1: GTTTCTCTCGAGTTCCTCCCTCCTCGCTCGCCACAATCTCTCTCCCGGCTTCGTTCCTCCT CTCGCCCCGCGACTCCCGCTCTGCTCCTCCTCGTCGCCGCCGCCGCCGCCGCCGCCGCC GCCGCCGCCGCTGTGTGTACGACTACGACGGCGAAGAGCCAGGCGGGGAGGACCGATC CGCCGGAGGAGGAGGAGGAGGGAGTGGGGGGGCGGGGAGATGGGGGAGGGGGGGCG ATGACAGCGGGCGCGGCTCGGCGCGGGCGGGATGCGGACGATCTGCGACGTGTGCGAG AGCGCGCCGGCGGTGCTCTTCTGCGTGGCCGACGAGGCCGCGCTCTGCCGGTCCTGCG ACGAGAAGGTGCCCCCTCTCTTCAACCCTGCCTTCGTCCTCTGCTCTACAACGCGCACG CGTCGGAGTCCGCTTGCCGAATCGTAGATCGGGTTATGCGATGCTTCAATTGCGGTCGCT TGTTTCACGCTGGGCGATCGGGTTGTTTGATTGTGCAATTAAGATCTCACGAGATGGTTT ACTACGCTCGAATTAGCGTGTGGATCTGCGTGTTTCTTTTTTGGGTACAGAAAGCCATTT AATTTTGACTGCGAATCAACTGCGTACTGCTCACTGGCCTGGAGCAATTCATTCATTAAG GCCTTTTTTTTTCATTCGTTGAGTATTGTGGACTTATGACCAAACGAAGTTATGGTATAA TAGGATGCCAGCGTGCATTGATAGTGAACTAACTGTAGTGCTTGTTTGGGTAGTAACCGAG AAATTGCCTTATGGGGTATCCACTTATTTCTTGTCTGACTTCAAAATGAGGCCTATTGCCT AGAGAATTCCACTTGAAATTAAAGCTGGCCACTTTAGAACTGGAAAACAAAGCATAAT TCTATGATGTTGTTATCGAAGGAGGCACTTTTACAGCATAAAACATACATAATAAGTTCAA ATAAGTACTTGAATCCAGCAATTGGGTTACTTCGGAACGGGATTTCTTAGAAGATCACAC TACCCTGAGTTCTATATCTAGCTTGGTGGTCGTTAGACACCTCCAGGCATCATCATTCATG GCCATATGTGGGACAGTGCGAGATGAGCTTGTCGTGATTTTGCCATTTTGGTTGGAATTT CTGTTAACCTTATTGGCTATGTGTAATACTTAAGTCGATCATTGTTCTGTAGAAGTAATGAT TGAATTTCCCTTGTTGAAATTCTTCCAATAGTAAAGAAAATAGTTAGTGATAGATTTTGCACT TGTGTTTACACGATTTTTCAGTCCATATGTTTACTGTTATTGTAGCCAAGATTATGCTTCT GCCTCACTTTAATTGCAGAATGCAGAGTCTGGAATTGCAGATTTCTAGAGTTAATGAATA CCCCCTCCGTCCCAAAAAAAGTCCAATCCTGAACACATTCGTGTCCAGATTCGTAGCTA GGGTTTGCTTTTTTATGGGACAGAGTAGTATGTCTTATACTATGTTACTGTACATGATTCCA GTCGCTACCTTCAATTGCTAATAAAAACATACATGAGAGCATCCTTTACTCATTCTTTAGC TGCTCAACTAGTTTTGCGCTCCGTAACCAATGCTATGACACTGGTTTCTTTGGTTTTTTAT AATATTTGGAACTGATGACATGATTAAAAATCATCAGTACGTGCAAAAGTGCAACCAGGT TGTAGATGAAAGTTCCAACCGGAAATGGAATTGTTGTATGTGGGTGGCCCCATCCCTAAT GAGGCCAAACATTATGAAACATCCTTCGTGGGCAGATCTTTTGGTCAAATATCCCATTGT GCTCTCACATTCTTATATTATTATTGATTTACCCATTAAAACATTGTACGTCTATTCTGTTTTA CGACTATGTTTTGAGCAGTTAAGTTCTCTTCCAACTATGAAAGGTCATCTTTTCTGGTTCT TAGTCCTTACTTATGTCTTTATGTCAGTTGAGCACCTGTTTTAATGAGTTAATAAGGTTTGT CAGTAATGGATCTGCTGAAGAACACGATGTATGCTGATTATGTGCAGGCAACATAAGAAA TCAGATACTGTTAATAAAGCTGACATTTTTGCATGGATTTGTTTACTGAAAGTTTGCCAGA TTGGTTGTTGTACTCTGTATGTTAGAGATGCATATCCATATTCAGCTCTATGAGAACTTCG ATCAACAAAAATTACTCGCTCCATTGATTGTTATAAGATGTATTTTGGTTTGAAGAGGCCAA ACTTTGTAGATTTTGACCAACAATCAGTCTATACAAGTTATTTTAGTAGCTGTGCAATATC CTTCATGTGGTATCATTAGCATGAAGAAAAACAAATATTACAGCTTTGGTCTCAATAACC CATTAAATATTCATGCTGCATTTTCTGATATTTCTTCCAATGTTGTGATGCAGGTGCATATG TGTAACAAGCTTGCTAGGCGGCACGTGAGAGTTGGGCTTGCAGACCCTAATAAAGTTCA ACGCTGTGATATATGTGAAAATGCCCCCGGTATTCTTTCTTATTTATGAATAGTTTCTTATC ATATTTTGCCTGTTAAAGCTTGTTGTGATTATGTCCTGCTGTCCATGCAGCCTTCTTCTATT GCGAGATAGATGGTACATCACTTTGCCTTAGTTGTGATATGACTGTTCATGTTGGTGGGA AACGAACCCATGGAAGATACCTGCTCCTAAGGCAACGGGTTGAAGTAGGTTATTGTTTA GAAACCAACTTCGTATTGTAATCTCACGCCTTATTTTCTAGTGCTTGAAGTTTGAAATTTT GATTAGTTTCCAGGAGATAAACCAGGTCATATGGATGATGTTGCTATGCAACAGAAAGAT CCTGAAAACCGGACGGATCAAAAGAAGGCCCCTCACTCAGTAACAAAGGAGCAAATGG CAAACCATCATAATGTGTCTGATGATCCAGCCTCAGATGGCAACTGCGATGACCAGGGTA ACATCGATTCCAAAATGATTGATCTTAATATGAGACCCGTCCGTACTCATGGACAAGGTT CAAACTCACAGGTACATCACATTCTCCATGTTAATGCAATGGGCTCATCTATTATTACCTT TACCGATCACTCACATTTTCCTCTCTGGTCTATGCTCTTCCCTTACTGATGACAAACCAGA CTCAGGGCGTGGATGTTAGCGTCAACAATCATGATTCTCCAGGAGTGGTGCCAACATGT AATTTCGAACGAGAAGCCAACAAATAAATACCGTAACACCAGGATGTACATTTCTTCGG TTGCTGCTATATAGCAGTAGCTCTCCGGGAGGTTGGATTAGCTTTGGTCTTCAGCCAGGC AGAATATGTGTTATTACATGGTCCTCTTGTGTCGCTAGTAGTATGATTCTAGTAGAGCTGA AGTTGAACGAGTTCATGTCAACTCGGCATGCCATATGCCGTGGACGCTATTTATCGTCGT GTACTCGTGTTGTGGTTGTAAGACTGGTGTCACATGGGCCATACGGGAAGCACAGTGGG CTGTCACTTGTGCTTTTGTGGATTGCTGCTGTGCGCTATTAGAAACTGTTCAAAAGATTA ATAGACCCATCATTTTTTTTCGGTGTATATCCAAATGTTGCTTC SEQ ID NO.2: ATGCGGACGATCTGCGACGTGTGCGAGAGCGCGCCGGCGGTGCTCTTCTGCGTGGCCG ACGAGGCCGCGCTCTGCCGGTCCTGCGACGAGAAGGTGCATATGTGTAACAAGCTTGCT AGGCGGCACGTGAGAGTTGGGCTTGCAGACCCTAATAAAGTTCAACGCTGTGATATATG TGAAAATGCCCCCGCCTTCTTCTATTGCGAGATAGATGGTACATCACTTTGCCTTAGTTGT GATATGACTGTTCATGTTGGTGGGAAACGAACCCATGGAAGATACCTGCTCCTAAGGCA ACGGGTTGAATTTCCAGGAGATAAACCAGGTCATATGGATGATGTTGCTATGCAACAGAA AGATCCTGAAAACCGGACGGATCAAAAGAAGGCCCCTCACTCAGTAACAAAGGAGCAA ATGGCAAACCATCATAATGTGTCTGATGATCCAGCCTCAGATGGCAACTGCGATGACCAG GGTAACATCGATTCCAAAATGATTGATCTTAATATGAGACCCGTCCGTACTCATGGACAA GGTTCAAACTCACAGACTCAGGGCGTGGATGTTAGCGTCAACAATCATGATTCTCCAGG AGTGGTGCCAACATGTAATTTCGAACGAGAAGCCAACAAATAASEQ ID NO.3: MRTICDVCESAPAVLFCVADEAALCRSCDEKVHMCNKLARRHVRVGLADPNKVQRCDICE NAPAFFYCEIDGTSLCLSCDMTVHVGGKRTHGRYLLLRQRVEFPGDKPGHMDDVAMQQK DPENRTDQKKAPHSVTKEQMANHHNVSDDPASDGNCDDQGNIDSKMIDLNMRPVRTHGQ GSNSQTQGVDVSVNNHDSPGVVPTCNFEREANK Example 1: Expression analysis of OsBBX-IV gene in response to high temperature treatment by qRT-PCR To investigate the response of OsBBX-Ⅳ gene family members to high temperature stress, two-week-old ZH11 wild-type rice seedlings were selected under normal conditions and subjected to high temperature stress at 45°C. Samples were collected at 0, 1, 3, 6, 9, and 12 hours after stress treatment and quickly frozen in liquid nitrogen. RNA was extracted from the samples and converted into cDNA through reverse transcription. qRT-PCR was performed using a real-time fluorescence quantitative PCR instrument (BIO-RAD, CFX96). The results were exported and analyzed using 2 -ΔΔCt Relative quantitative data analysis was performed using the real-time fluorescence quantitative PCR method (real-time fluorescence quantitative PCR was repeated three times, the internal reference gene was Actin1, and the primer sequences were shown in Table 1).

[0027] Table 1 Primer sequences qRT-PCR analysis showed that ( Figure 1 ), all ten genes showed varying degrees of induced upregulation, with significant increases reaching peak levels after 9 or 12 hours of high temperature stress. OsBBX29 showed the greatest upregulation, reaching approximately 85-fold, followed by OsBBX4 and OsBBX14, each with a 40-fold increase. The remaining seven genes only reached a maximum expression level of approximately 25-fold. These results indicate that within the OsBBX-IV subfamily, OsBBX29 is the most responsive to high temperature stress, suggesting that it may play an important role in heat stress in rice.

[0028] Example 2: Acquisition of OsBBX29 functional knockout mutants and overexpression mutants and analysis of gene editing I. Acquisition of OsBBX29 functional knockout mutants In order to explore whether knocking out the OsBBX29 gene would reduce the high temperature stress resistance of rice, the OsBBX29 functional knockout mutant, osbbx29 mutant seeds (number: 323-21774) were purchased directly from the rice mutant germplasm resource bank constructed using CRISPR / Cas9 by BioGene Technology (Jiangsu) Co., Ltd. The mutant was based on the wild type ZH11 as the background material, and the target sequence was AGATGGCAACTGCGATGTACCAGG. After germination, the seeds were cultured to the 3-leaf 1-heart stage, and the seedling genomic DNA was extracted. The mutation pattern was analyzed and confirmed by PCR amplification (primers are shown in Table 2) and sequencing. The structure of the OsBBX29 gene and the editing site are shown in Figure 2. Figure 2 As shown in A, Figure 2The sequencing peaks shown in B show that the osbbx29-1 mutant has a T insertion, while the osbbx29-2 mutant has a G deletion. Both mutations lead to premature termination of the coding sequence of the OsBBX29 gene. After removing the Cas9 vector, homozygous plants were propagated for subsequent experiments.

[0029] Table 2 Primername Forward(5'-3') Reverse(5'-3') 21774 GATCCTGAAAACCGGACGGA GGCACCACTCCTGGAGAATC 2. Obtaining OsBBX29 Overexpressing Plants The full-length CDS sequence of the OsBBX29 gene was amplified using the cDNA of ZH11 (primer sequences, OsBBX29-OE-F:ttacttctgttgcaacATGCGGACGATCTGCGACGTGTGCG, OsBBX29-OE-R:gtggtctttgtaatcTTATTTGTTGGCTTCTCGTTCG, lowercase letters indicate homologous sequences). The amplified gene fragment was then ligated into the overexpression vector pBWA(V)HU by homologous recombination, and the expression of the OsBBX29 gene was driven by the maize Ubiqutin promoter. The successfully constructed and correctly sequenced recombinant plasmid was transformed into Agrobacterium strain EHA105 and infected the callus tissue of wild-type ZH11. Hygromycin resistance was used for screening, and the expression level of OsBBX29 in the transgenic lines was detected by qRT-PCR.

[0030] The specific method is as follows: Use real-time fluorescence quantitative PCR instrument (BIO-RAD) to detect, export the results after completion, and use 2 -ΔΔCt Relative quantitative data analysis was performed using the real-time fluorescence quantitative PCR method (real-time fluorescence quantitative PCR was repeated three times, and the internal reference gene was Actin1).

[0031] The results are as follows Figure 5 As shown in A, among the eight independent overexpression lines, the expression of the OsBBX29 gene was significantly upregulated in OE 9-5 and OE 18-2, and these two lines were selected for subsequent studies.

[0032] Example 3: Analysis of phenotypes and physiological indicators of OsBBX29 transgenic rice under high temperature treatment 1. Phenotypic Statistics Seedlings of wild-type Zhonghua 11 (ZH11), osbbx29 mutants (osbbx29-1 and osbbx29-2), and OE9-5 and OE18-2, two strains with higher OsBBX29 expression, were subjected to a high-temperature stress treatment at 45°C, 60% relative humidity, and 14 hours of light and 10 hours of darkness. Seedlings were removed from the heat stress treatment until phenotypic changes appeared, and the duration of the stress treatment was adjusted based on the growth status of each batch. Phenotypic photographs of the plants were taken before and after the heat stress treatment. After the heat stress treatment, the plants were allowed to recover for 7 days at 28°C, 60% relative humidity, and 14 hours of light and 10 hours of darkness. Survival was then assessed by photographing the plants, and the emergence of new leaves was used to determine survival.

[0033] 2. Analysis of Physiological Indicators The specific method for determining relative conductivity is: 1) Before and after high temperature stress treatment, select leaves from the same part of each strain, wash them with tap water, and then rinse them with deionized water three times; 2) Use filter paper to absorb the moisture on the leaf surface, cut the leaf into segments of appropriate length (avoiding the main vein), and quickly weigh three fresh samples, each weighing 2 g; 3) Place equal amounts of sample into conical flasks containing 20 ml of deionized water, seal with sealing film, and soak at room temperature for 12 hours; 4) Use a conductivity meter to measure the conductivity of the extract (R1). Heat in a boiling water bath for 30 minutes, cool to room temperature, shake well, and measure the conductivity of the extract again (R2). Relative conductivity = R1 / R2 x 100%.

[0034] Specific determination method of malondialdehyde (MDA) content: Malondialdehyde (MDA) content was determined using a plant malondialdehyde test kit (purchased from Nanjing Jiancheng Bioengineering Institute). The specific method is as follows: 1) Sample Pretreatment: Select rice plants approximately two weeks old and, before and after heat stress treatment, mince leaves from the same part of the plant tissue. Accurately weigh the plant tissue and add 9 volumes of extract (10-fold dilution of Reagent V) at a ratio of 1:9 (weight to volume). Homogenize the minced leaves in an ice-water bath, centrifuge at 3500-4000 rpm for 10 minutes, and collect the supernatant for analysis.

[0035] 2) Add reagents to the centrifuge tube in sequence according to the following table Table 3 MDA content determination system Mix well with a vortex mixer, place in a water bath at 95°C or above for 20 min, cool in running water, and measure the absorbance using a spectrophotometer at a wavelength of 530 nm.

[0036] 3) Calculation formula: Note: C 标准 is the concentration of the standard, 10 nmol / mL; W is the weight of the plant tissue (g); V 提 is the total amount of extract added (mL).

[0037] The results showed that after 7 days of high temperature stress treatment, the survival rate of the osbbx29 mutant was significantly lower than that of the wild type ZH11 ( Figure 3 ); After high temperature stress treatment, the MDA content and relative conductivity of the leaves of the osbbx29 mutant were significantly higher than those of the wild type ZH11 ( Figure 4 ), showing lower resistance to high temperature stress; and after 7 days of recovery treatment after high temperature stress treatment, the survival rate of OsBBX29 overexpressing plants was higher than that of wild type ZH11 ( Figure 5 ); After high temperature stress treatment, the MDA content and relative conductivity of leaves of OsBBX29 overexpressing plants were lower than those of wild type ZH11 ( Figure 6 ), the membrane damage after high temperature stress was less, showing stronger resistance to high temperature stress.

[0038] These results indicate that the OsBBX29 gene can positively regulate high temperature stress resistance in rice.

[0039] Example 4: OsBBX29 subcellular localization The fusion protein of OsBBX29 and EGFP was constructed for subcellular localization. The CDS sequence of OsBBX29 with the termination codon removed was amplified using the cDNA of ZH11 (primer sequence, OsBBX29-EGFP-F:ATA GGATCC ATGCGGACGATCTGCGAC,OsBBX29-EGFP-R:ATA GTCGAC The amplified gene fragment was then ligated into an empty EGFP subcellular localization vector by enzyme digestion and ligation. The 35s promoter was used to drive the expression of OsBBX29-EGFP. The successfully constructed and correctly sequenced recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101 and injected into transgenic tobacco stably expressing RFP-H2B (a fusion of red fluorescent protein and the known nuclear localization protein H2B) for transient expression.

[0040] The specific steps are as follows: 1) In a clean bench, inoculate a single colony of Agrobacterium containing the target plasmid into 5 mL of autoclaved LB liquid medium (containing antibiotics Rif, Kan, and Gent) and incubate overnight at 28°C in a shaker at 200 rpm. 2) Pipette 100 μL of the overnight culture solution and inoculate it into 5 mL of LB liquid medium (containing antibiotics Rif, Kan, and Gent) for expansion. Incubate the culture overnight at 28°C in a shaker at 200 rpm. 3) Centrifuge at room temperature for 10 min at 5000 rpm, discard the supernatant, resuspend the cells in staining solution (10 mM MgCl2 + 10 mM MES + 150 μM AS), adjust the resuspension to OD600 = 1.0 using a spectrophotometer, and then let it stand at room temperature for 2-3 h; 4) Use a 1 mL medical syringe (without the needle) to inject the bacterial solution into the underside of the tobacco leaf; 5) 24-48 hours after the injection, cut tobacco leaves of appropriate size to prepare pressed discs, and observe the GFP and RFP fluorescence signals using a laser confocal microscope (Leica).

[0041] Results: The GFP green fluorescence signal of the OsBBX29-EGFP fusion protein overlapped with the RFP red fluorescence signal localized in the cell nucleus. Green fluorescence was also observed in the cytoplasm, indicating that OsBBX29 was localized in both the nucleus and cytoplasm.

[0042] Example 5: Detection of OsBBX29 transcriptional self-activation ability The full-length CDS sequence of OsBBX29 was amplified using the cDNA of ZH11 (primer sequence, BK-BBX29-F:CG GAAT TC ATGCGGACGATCTGCGAC,BK-BBX29-R:CG GGATCC The amplified gene fragment was then ligated into the bait vector pG BKT7 via restriction enzyme digestion and ligation. The constructed vector and pGADT7 were co-transformed into competent yeast strain AH109 cells using the PEG / LiAc method. A positive control, co-transformation with BK-53 + AD-T, and a negative control, co-transformation with BK-Lam + AD-T, were also established. After transformation, the cells were plated onto SD / -Trp-Leu-deficient medium and incubated upside down at 30°C in a constant-temperature incubator for 2-3 days. Selected colonies were then transferred to SD / -Trp-Leu-His-Ade-deficient medium and the growth of the co-transformed yeast on this medium was observed to test whether the protein exhibited self-activation ability.

[0043] Results: Yeast co-transfected with BK-OsBBX29 and AD empty vector could not grow normally in SD / -Leu-Trp-His-Ade medium, indicating that OsBBX29 has no transcriptional self-activation ability. Therefore, we infer that OsBBX29 may not function as a transcription factor, but may need to act through protein interaction.

Claims

1. OsBBX29 The application of the gene in regulating high temperature stress resistance in rice is characterized by: described OsBBX29 The nucleotide sequence of the gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that: The regulation includes: overexpression OsBBX29 Genes that improve rice resistance to high temperature stress, and knockout OsBBX29 Genes reduce resistance to high temperature stress in rice.

3. A method of genetically engineering the OsBBX29 A method for improving rice resistance to high temperature stress by gene expression, characterized in that: The following steps are involved: By OsBBX29 Gene transfer into rice OsBBX29 The gene is overexpressed in rice, thereby improving the rice's resistance to high temperature stress.

4. A method of genetically engineering the OsBBX29 A method for reducing high temperature stress resistance in rice by gene expression, characterized in that: The following steps are involved: CRISPR / Cas9 gene editing method was used to modify the OsBBX29 Gene knockout reduces the resistance of rice to high temperature stress.

5. OsBBX29 The use of a gene in cultivating transgenic rice with improved or reduced resistance to high temperature stress is characterized by: described OsBBX29 The nucleotide sequence of the gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

3.

6. The use according to claim 5, characterized in that: The applications include: overexpression OsBBX29 Application of genes in breeding transgenic rice with improved resistance to high temperature stress, and knockout OsBBX29 Application of genes in breeding transgenic rice with reduced resistance to high temperature stress.

7. A method of genetically engineering the OsBBX29 A method for cultivating transgenic rice with improved resistance to high temperature stress by gene expression, characterized in that: The following steps are involved: Overexpression in rice OsBBX29 Gene, to cultivate transgenic rice with improved resistance to high temperature stress.

8. A method of genetically engineering the OsBBX29 A method for cultivating transgenic rice with reduced resistance to high temperature stress by gene expression, characterized in that: The following steps are involved: Knockout in rice OsBBX29 Gene, to cultivate transgenic rice with reduced resistance to high temperature stress.

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