Brassica napus heat-resistant gene and application thereof

Abstract

The invention discloses a brassica napus heat-resistant gene and application thereof, the nucleotide sequence of the brassica napus heat-resistant gene is shown as SEQ ID NO: 1, and the amino acid sequence of protein coded by the gene is shown as SEQ ID NO: 2. Through deep research, the gene is successfully cloned, the important role of the gene in the heat resistance of the brassica napus is disclosed, and a key gene resource is provided for cultivating a heat-resistant brassica napus variety. According to the invention, the expression of the BnaA01. HSFA1a gene is regulated and controlled through a genetic engineering means, so that the heat resistance of the brassica napus is remarkably improved, a new way is opened up for cultivating an excellent brassica napus variety adapting to a high-temperature environment, and the method has important significance for guaranteeing the stable development of the brassica napus industry and the national food safety.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering and agricultural biotechnology, and relates to a heat-resistant gene of Brassica napus BnaA01.HSFA1a Isolation and cloning methods of the strain, and its application in improving the heat tolerance of Brassica napus. Background Art

[0002] Brassica napus is one of my country's major oilseed crops, widely cultivated in areas such as the Yangtze River Basin. It exhibits excellent seed yield, oil content, and disease resistance. However, as the greenhouse effect intensifies, extreme high temperatures are becoming more frequent and lasting longer, which has a significant negative impact on the growth and development of Brassica napus, leading to a significant decline in rapeseed yield and quality, seriously threatening national food security. Therefore, identifying genes associated with heat stress and cultivating heat-tolerant Brassica napus varieties are of vital practical significance.

[0003] Plant heat shock transcription factors play a key role in transcriptional regulation within cells. They can recognize and specifically bind to conserved heat shock element (HSE) motifs within the promoter regions of heat shock protein genes, thereby regulating the transcription and expression of heat shock protein genes and improving plant stress resistance. In 1990, Scharf isolated three HSFs family genes from tomato using DNA ligand screening. Members of the HSFA subfamily possess an activation domain (AHA) that directly participates in the regulation of downstream genes, while members of the HSFB and HSFC subfamilies lack this domain.

[0004] The HSFA subfamily plays a key role in plant response to heat shock. Transcriptome data analysis showed that more than 65% of genes whose expression was upregulated under high temperature stress depended on HSFA1 family members. Studies have shown that in the process of high temperature signal transduction and heat tolerance acquisition, the four members of HSFA1 are upstream of other heat shock genes and act together as major regulatory factors. Under high temperature stress, HSFA1a It can be positively or negatively regulated by proteins that interact with it, thereby regulating other heat shock transcription factors and enhancing the heat tolerance of plants. For example, in tomatoes, HSFA1a The gene will be activated under high temperature stress, and its overexpression can improve the heat tolerance of pollen. HSFA1a Mutations can lead to a significant reduction in pollen resistance. HSFA1a Overexpression also significantly upregulated the expression of HSFA2 and HSFA3 in transgenic tomato anthers, thereby regulating the expression of downstream HSP70 and HSP90 genes. HSFA1a This indicates that HSFA1aThe enhanced heat tolerance of tomato pollen may be achieved through the induction and regulation of heat shock proteins. In Arabidopsis, all heat shock transcription factors can be induced, with HSFA1 members being the most prominent. HSFA1a Overexpression of HSFA1 can not only enhance the heat tolerance of plants, but also improve their salt tolerance, hypoxia tolerance, drought tolerance, high light intensity tolerance and other stress resistance capabilities. However, the various members of Arabidopsis HSFA1 can interact with each other, and a single mutation will not reduce heat tolerance.

[0005] At present, although some plants HSFA1a There has been some research on genes in Brassica napus. BnaA01.HSFA1a There are no detailed reports on the heat-resistance function, expression pattern and regulatory mechanism of genes. BnaA01.HSFA1a Genes have important theoretical and practical value for breeding heat-resistant Brassica napus varieties and ensuring stable rapeseed yield and quality. Summary of the Invention

[0006] The present invention aims to provide a heat-resistant gene for Brassica napus BnaA01.HSFA1a The nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 2. Through in-depth research, the present invention successfully cloned this gene and revealed its important role in the heat tolerance of Brassica napus, providing a key gene resource for breeding heat-tolerant Brassica napus varieties.

[0007] In addition, the present invention also relates to a recombinant vector comprising BnaA01.HSFA1a The gene can be used to overexpress or interfere with the expression of the gene in Brassica napus.

[0008] Furthermore, the overexpression vector comprises a promoter sequence and a nucleotide sequence shown in SEQ ID NO: 1; the interference vector comprises a BnaA01.HSFA1a RNAi sequence of the gene.

[0009] The present invention also provides a clone BnaA01.HSFA1a The genetic method comprises the following steps: A. Total RNA was extracted from Brassica napus leaves and reverse transcribed to obtain cDNA.

[0010] B. Using the cDNA as a template, PCR amplification was performed using specific primers to obtain a gene fragment containing SEQ ID NO: 1. The specific primer sequences are as follows: Forward primer: 5'-GCCAGAATTCATGATGGATGGTGTAACCGG-3', Reverse primer: 5'-TAGAGTCGACTCACAGTTGCTTTGT-3', C. Recover and purify the PCR amplification product, and perform sequencing verification.

[0011] The present invention further provides a method for improving the heat tolerance of Brassica napus by Agrobacterium-mediated BnaA01.HSFA1a The gene is introduced into Brassica napus plants, causing them to overexpress the gene, thereby enhancing the plants' tolerance to high temperature stress.

[0012] The present invention also provides the BnaA01.HSFA1a Application of a gene in regulating plant heat tolerance, comprising enhancing the plant's tolerance to high temperature stress by overexpressing the gene.

[0013] The present invention also provides the BnaA01.HSFA1a Application of genes in preparing heat-tolerant rapeseed varieties.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The heat-resistant gene of Brassica napus was cloned for the first time BnaA01.HSFA1a , and verified its important function in improving the heat tolerance of Brassica napus, providing new genetic resources and theoretical basis for the heat tolerance breeding of Brassica napus.

[0015] Provides simple and efficient BnaA01.HSFA1a Gene cloning methods facilitate the widespread dissemination and utilization of the gene in subsequent research and applications.

[0016] Controlled by genetic engineering BnaA01.HSFA1a The expression of the gene has significantly improved the heat tolerance of Brassica napus, opened up a new path for cultivating excellent rapeseed varieties that can adapt to high temperature environments, and is of great significance to ensuring the stable development of the rapeseed industry and national food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a diagram of DNA molecular identification of overexpressing Brassica napus plants and screening of positive plants. Figure 1 In the figure, A: DNA molecular identification; B: positive plant identification (scale bar = 2 cm); C: expression level identification of overexpression lines; ** indicates extremely significant differences at the P < 0.01 level.

[0019] Figure 2 It is a diagram showing the DNA molecular identification and interference effect of Brassica napus plants. Figure 2 In the figure, A: schematic diagram of interference vector; B: DNA molecular identification; C: identification of interference expression level; ** indicates significant difference at the P<0.01 level.

[0020] Figure 3 This is the observation diagram of the high temperature stress phenotype of overexpressing Brassica napus seeds. Figure 3 Figure 2: Figure 2. A-D: Phenotypes of Brassica napus seeds 7 days after treatment with CK, 3 h, 4 h, and 5 h of high temperature stress (scale bar = 2 cm); E: Schematic diagram of the seed sowing area; F: Diagram of the high temperature treatment process; G: Statistical diagram of the number of seeds with green leaves, n = 30, repeated 3 times; ** indicates extremely significant differences at the P < 0.01 level.

[0021] Figure 4 This is a diagram of the phenotypic changes in overexpressing Brassica napus seedlings after high temperature stress. Figure 4 Middle, AB: Phenotypes of 7-day-old overexpressing Brassica napus seedlings under high temperature stress, → represents the changes in cotyledons after high temperature stress; CF: Observation of cotyledons of 7-day-old overexpressing Brassica napus seedlings (scale bar = 2 cm).

[0022] Figure 5 This is a diagram showing the phenotypic changes of Brassica napus seedlings after being subjected to high temperature stress. Figure 5 Middle, AB: Phenotypes of 7-day-old disturbed Brassica napus seedlings after high temperature stress, → represents the changes in cotyledons after high temperature stress; CF: Observation of cotyledons of 7-day-old disturbed Brassica napus seedlings (scale bar = 2 cm).

[0023] Figure 6 This is a comparison of the phenotypes before and after high temperature stress treatment of rapeseed seedlings after overexpression and interference. Figure 6 Middle, AC: overexpression rapeseed before and after high temperature stress treatment and recovery phenotype; DF: interference rapeseed before and after high temperature stress treatment and recovery phenotype (scale bar = 2 cm).

[0024] Figure 7 These are the NBT and DAB staining results of overexpression and interference Brassica napus leaves. Figure 7 Middle, A: NBT and DAB staining of overexpression line; B: NBT and DAB staining of interference line (scale bar = 1 cm).

[0025] Figure 8 This is a graph showing the determination of chlorophyll content, malondialdehyde content, soluble sugar content and proline content in transgenic Brassica napus after heat stress. Figure 8In the figure, AB: determination of chlorophyll a and b contents; C: determination of chlorophyll content; DF: determination of malondialdehyde content, soluble sugar content, and proline content; ** indicates extremely significant differences at the P < 0.01 level.

[0026] Figure 9 yes BnaA01.HSFA1a Observation of the dyeing of Brassica napus pollen before and after high temperature treatment. Figure 9 Middle, AH: staining of overexpression lines before and after high temperature treatment; IP: staining of interference lines before and after high temperature treatment (scale bar = 100 μm).

[0027] Figure 10 yes BnaA01.HSFA1a Statistical chart of pollen vitality of Brassica napus. Figure 10 In the figure, A: Statistics of pollen viability of each line before high temperature stress; B: Statistics of pollen viability of each line after high temperature stress, ** indicates extremely significant differences at the P < 0.01 level. DETAILED DESCRIPTION

[0028] The following describes the details in conjunction with specific embodiments.

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0030] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0031] Example 1 This embodiment describes BnaA01.HSFA1a genes, and BnaA01.HSFA1a Gene cloning.

[0032] BnaA01.HSFA1a The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0033] According to the download BnA01.HSFA1aThe CDS sequence of SEQ ID NO: 1 and the selected DsRed overexpression vector were used to design primers using SnapGene software. The CDS sequence amplification primers are as follows: BnaA01.HSFA1a -F: 5'-GCCAGAATTCATGATGGATGGTGTAACCGG-3', BnaA01.HSFA1a -R: 5'-TAGAGTCGACTCACAGTTGCTTTGT-3'; DsRed-F: 5'-TGGAGAGGACACTAGTGGATCC-3', DsRed-R: 5'-CATGCGATCATAGGCGTCTC-3'.

[0034] This example describes in detail the heat-resistant gene of Brassica napus BnaA01.HSFA1a The cloning process is divided into three parts: CDS sequence amplification, promoter sequence amplification and RNAi interference fragment amplification.

[0035] 1. CDS sequence amplification PCR amplification was performed using the Brassica napus ZS11 cDNA template provided by the research group and the above primers.

[0036] The PCR reaction system is: template 1 μL, BnaA01.HSFA1a- F 1 μL, BnaA01,HSFA1a- R 1 μL, 2×T8 High-Fidelity Master Mix 12.5 μL, ddH2O, make up to 25 μL.

[0037] The PCR reaction program was as follows: initial denaturation (95°C) for 1 min, deformation (95°C) for 30 s, annealing (56°C) for 30 s, extension (72°C) for 2 min, final extension (72°C) for 10 min, and cooling (12°C) for 30 min.

[0038] The annealing temperature and extension time are appropriately adjusted according to the melting curves of different primers and the size of the target fragment to be amplified.

[0039] 2. Promoter sequence amplification The extracted BnA01.HSFA1a Primers were designed using SnapGene software based on the 1500 bp promoter sequence upstream of the gene and the selected pCAMBIA1305.1 promoter vector as shown below: BnaA01.HSFA1a –1305-F1: 5'-GCCGGAATTCGAGGCTGATTATGCATCTTAACCGC-3', BnaA01.HSFA1a –1305-R1: 5', -GAGCCATGGCTTGTACGGAGAGAGAGAGAGGG -3', 1305.1-F: 5'-GAGCCATGGCTTGTACGGAGAGAGAGAGGG-3', 1305.1-R: 5′-GTAAAACGACGGCCAGT-3′.

[0040] The Brassica napus ZS11 DNA provided by the research group was used as the amplification template and PCR amplification was performed according to the above primers.

[0041] The PCR reaction system is: template 1 μL, BnaA01.HSFA1a –1305-F1 1 μL, BnaA01.HSFA1a –1305-R1 1 μL, 2×T8 High-Fidelity Master Mix 12.5 μL, ddH2O, make up to 25 μL.

[0042] The PCR reaction procedure was the same as described above, where the annealing temperature was adjusted according to the melting curve of the primers.

[0043] 3. RNAi interference fragment amplification choose BnA01.HSFA1a and its copy members BnC01.HSFA1a The homologous fragments of the gene were used as interference fragments 1 to 3, combined with the RNAi interference vector pFGC5941M, and primers were designed using SnapGene software as shown below: BnaA01.HSFA1a –RNAi-F1: 5'-ATTACCATGGTTGTTCGCCGGGCTCCAC-3', BnaA01.HSFA1a –RNAi-R1: 5'-GCCGACGTCATTTACCGGCGGAGACTCAAAAAGC-3', BnaA01.HSFA1a –RNAi-F2: 5'-TCCGGATCCACCGGCGGAGACTCAAAAAGC-3', BnaA01.HSFA1a –RNAi-R2: 5'-GCCTCTAGATTGTTCGCCGGGCTCCAC-3', PAP12-F: 5'-CAACTTTATTTCGTATCCTCATTCGGTCT-3', PAP12-R: 5'-AGACCGAATGAGGATACGAAATAAAGTTG-3', OCS-R: 5'-CGCAATTGTTTGTTATTGTGGCGCTCTA-3'.

[0044] Interference fragment 1 is BnaA01.HSFA1a –RNAi-F1 / BnaA01.HSFA1a –RNAi-R1 matching fragment, interference fragment 2 is BnaA01.HSFA1a –RNAi-F2 / BnaA01.HSFA1a – The fragment matching RNAi-R2, interference fragment 3 is the fragment matching PAP12-F / PAP12-R.

[0045] The Brassica napus ZS11 cDNA provided by the research group was used as the amplification template, and PCR amplification was performed according to the above primers to amplify the forward and reverse fragments simultaneously.

[0046] The PCR reaction system is: template 1 μL, BnaA01.HSFA1a -RNAi-F1 / R1 1 μL, BnaA01.HSFA1a -RNAi-F2 / R2 1 μL, 2×T8 High-Fidelity Master Mix 12.5 μL, ddH2O 9.5 μL.

[0047] The PCR reaction procedure was the same as above.

[0048] Select both BnaA01.HSFA1a –RNAi-F2 and BnaA01.HSFA1a –RNAi-R2 used the above PCR amplification reaction system and procedures to amplify the reverse fragment and finally performed gel recovery.

[0049] Example 2 This example describes in detail the construction process of the blunt-ended vector. The specific steps are as follows: 1. Fragment ligation The fragments recovered above were ligated to the blunt-ended vectors. The ligation system was: Gel recovery product: 5 μL, pBM20S Vector: 1 μL, 10× Toposmart: 1 μL, Add ddH2O to make up to 10 μL.

[0050] Add the above reagents to the EP tube and mix gently with a pipette. Place the EP tube in the PCR instrument, close the PCR hot cover, and set the program to: 25°C, reaction time 30 min.

[0051] 2. Transformation of E. coli with ligation products After the program is completed, the ligation product in the EP tube is transformed into the competent E. coli Trans-T10. The specific steps are as follows: Take a tube of Trans-T10 competent E. coli cells and place it on ice until it thaws to a mixture of ice and water. Immediately add the ligation product to the competent cells and mix gently. Place the mixture in an ice bath for 30 minutes. Preheat a water bath to 42°C. After the ice bath, quickly place the EP tube in a water bath and heat shock at 42°C for 45 seconds. Immediately return the EP tube to ice and let it rest for 2 minutes.

[0052] 3. Bacterial culture and screening Aseptically add 800 μL of antibiotic-free LB medium to an EP tube. Place the EP tube in a 37°C shaker at 200 rpm for 1.5 hours to recover the cells. Centrifuge at 5000 rpm for 4 minutes to collect the cells. Carefully discard 650 μL of the supernatant and resuspend the remaining cells using a pipette. Spread the entire resuspended cell mass onto LB solid medium supplemented with kanamycin (Kan). Invert the plate in a 37°C incubator and incubate overnight.

[0053] 4. Monoclonal screening and bacterial testing Pick a single colony and inoculate it into 800 μL of LB liquid medium containing kanamycin. Incubate the medium in a 37°C shaker at 200 rpm for 12 hours. Perform PCR detection using the universal primers M13-F and M13-R, using the cultured bacterial liquid as a template.

[0054] PCR bacterial detection reaction system: 1 μL of bacterial solution, 0.5 μL of M13-F, 0.5 μL of M13-R, 5 μL of 2×Taq Mix, and ddH2O to make up to 10 μL.

[0055] 5. Sequencing and Plasmid Extraction The PCR products were sequenced and compared with the reference sequences using MAGA11.0 software. The bacterial suspension with successful sequencing results was inoculated into LB medium with corresponding resistance at a ratio of 1:200 and cultured overnight. The plasmid was extracted according to the operating instructions of the column mini plasmid kit of Beijing Tiangen Company.

[0056] Specific steps for plasmid extraction: In a 2 mL centrifuge tube, centrifuge at 12,000 rpm for 2 minutes to collect the cells. Repeat the centrifugation 2-4 times to ensure that the cells are fully pelleted. Add 250 μL of Buffer P1 containing RNase and mix thoroughly by pipetting. Add 250 μL of Buffer P2 and rapidly invert the tube 6-8 times to lyse the cells until the liquid is transparent. Add 350 μL of Buffer P3, ensuring that the interval between the addition of P2 and P3 does not exceed 5 minutes. Immediately and gently invert the tube 6-8 times to mix until a clearly visible white flocculent mass appears. Centrifuge at 12,000 rpm for 10 minutes. Add 500 μL of equilibration buffer BL to the adsorption column CP3 and centrifuge at 12,000 rpm for 1 minute. Discard the recovery buffer from the collection tube. Add the supernatant from the centrifugation to the adsorption column CP3 and centrifuge at 12,000 rpm for 1 minute. Discard the recovery buffer from the collection tube. Add 600 μL of rinse buffer PW to the adsorption column CP3 and centrifuge at 12,000 rpm for 1 minute. Discard the recovered solution in the collection tube and repeat the rinse. Finally, centrifuge at 12,000 rpm for 2 minutes to remove any residual liquid. Place the adsorption column CP3 in a new 1.5 mL centrifuge tube and let it stand at room temperature for 5 minutes to evaporate. Add 40 μL of preheated ddH2O and let it stand at room temperature for 2 minutes to fully dissolve the plasmid. Centrifuge at 12,000 rpm for 1 minute to collect the plasmid.

[0057] 6. Plasmid Detection and Storage Check the plasmid concentration to ensure it is greater than 1000 ng / μL. Check the plasmid quality by agarose gel electrophoresis. If a single, bright band appears on the electrophoresis pattern, the plasmid is of acceptable quality. Store the qualified plasmid at -20°C for subsequent use.

[0058] This blunt-end vector construction process is suitable for effectively connecting the target gene fragment with the blunt-end vector, and through transformation, screening and verification steps, obtaining E. coli clones containing recombinant plasmids, providing a basis for subsequent gene function research and application.

[0059] Example 3 This example describes in detail the process of genetic transformation of Brassica napus and positive identification and screening of transgenic plants. The specific steps are as follows: 1. Seed disinfection and cultivation Select plump, healthy J9709 Brassica napus seeds and place approximately 60 seeds in a 5 mL centrifuge tube. Aseptically sterilize the tubes in a clean bench using 75% anhydrous ethanol for 90 seconds, followed by 50% 84 disinfectant for 210 seconds. Manually shake the tubes during sterilization. Rinse the seeds 3-5 times with sterile water to remove as much residual disinfectant as possible.

[0060] 2. Dark Cultivation of Seeds Use sterilized tweezers to evenly distribute the seeds (approximately 30 seeds) on the M0 solid medium. Place the glass dish containing the medium in a sterile seeding box and seal it with parafilm. Place the seeding box in a dark environment and incubate for 7 days.

[0061] 3. Agrobacterium Transformation and Cultivation Agrobacterium containing the recombinant plasmid of the overexpression vector and the interference vector was cultured in YEB liquid medium containing kanamycin (Kan) and rifampicin (Rif) until the OD600 value of the bacterial liquid reached about 0.4.

[0062] 4. Agrobacterium infection Under aseptic conditions, pipette 2 mL of Agrobacterium solution into a sterile 2 mL centrifuge tube and centrifuge at 5000 rpm for 1 minute to collect the cells. Resuspend the cells in 2 mL of DM solution. Add 18 mL of DM solution to a sterile glass dish. Remove the cotyledons from dark-cultured rapeseed seedlings and cut the hypocotyls into approximately 1 cm long segments. Place these segments into the glass dish containing the resuspended bacterial solution. Add another 2 mL of the resuspended bacterial solution to fully infect the explants. Keep the infiltration time to 10 minutes. Begin aspirating the infiltration solution 2 minutes before the end of the infiltration period.

[0063] 5. Explant Culture and Subculture Evenly place the infected explants onto M1 medium and incubate them in the dark in a plant tissue culture room, upside down, for 1-2 days (maximum 2 days). If the explants are observed to be free of bacterial infection or expelling bacteria, subculture the explants onto M2 medium under 16 h light / 8 h dark conditions at 22°C for 20 days.

[0064] 6. Callus Subculture and Bud Induction After M2 culture is complete, the growing callus is subcultured onto M3 medium and hormones are used to induce bud formation. The subculture conditions for M3 are the same as for M2, but the subculture process needs to be repeated 3-5 times to ensure that all buds produce new leaves.

[0065] 7. Rooting and transplanting Once the explants grow new leaves on M3 medium, they are subcultured into glass bottles containing M4 medium and sealed until roots develop. When the hydroponic plants have developed a well-developed root system and have 3-4 leaves, they are transplanted into an artificial climate incubator for further growth.

[0066] 8. Positive Identification After the transplanted plants have grown for one week, positive identification is performed. BnaA01.HSFA1a The DNA of plants transformed with overexpression and interference vectors was extracted using the SDS method.

[0067] The PCR positive identification primers for overexpression plants are: DsRed-F and DsRed-R. At the same time, the vector red light is used to mark, and the red fluorescent protein excitation gun LUYOR-3415CG is used to screen positive seedlings and record them by taking photos to further identify positive plants.

[0068] The PCR positive identification primers for interference plants are: BnaA01.HSFA1a- RNAi-F1 and PAP12-R, BnaA01.HSFA1a- RNAi-F2 and OCS-R.

[0069] 9. Fluorescence quantitative PCR analysis Leaves from different lines of T1 generation plants that were transgenic and positive for overexpression vectors and interference vectors that had grown for one week were selected for RNA extraction and reverse transcription to obtain cDNA, followed by fluorescence quantitative PCR analysis to detect the expression level of the target gene in the plants.

[0070] Overexpression vector BnaA01G0088300ZS -Bn-F and BnaA01G0088300ZS -Bn-R primers for identification.

[0071] Interference carrier BnaA01G0088300ZS -Bn-F and BnaA01G0088300ZS -Bn-R primers and BnaC01G0107300ZS -Bn-F and BnaC01G0107300ZS -Bn-R primers for identification.

[0072] Through the above steps, the genetic transformation of Brassica napus and the screening and identification of positive transgenic plants are completed, providing transgenic materials for subsequent heat tolerance research and variety breeding.

[0073] Embodiment 4: This embodiment describes BnaA01.HSFA1a Expression analysis and heat tolerance analysis results of transgenic plants.

[0074] In this example, 21 strains were obtained through DNA molecular identification (Figure 1A). BnaA01.HSFA1a We found that the positive plants of Brassica napus overexpressed BnaA01.HSFA1a Transgenic rapeseed seedlings can emit bright red light under a fluorescent gun, which is consistent with the characteristics of DsRed red light carrier (Figure 1B). BnaA01.HSFA1a The expression level of overexpressing rapeseed lines was found BnaA01.HSFA1a The expression level was significantly increased, and the three strains with the highest expression levels were selected. BnOE#1, BnOE#2, and BnOE#3 Conduct follow-up experimental research (Figure 1C). Figure 1 is the heat-resistant gene of Brassica napus BnaA01.HSFA1a Figure 1 shows the results of DNA molecular identification, positive plant screening, and expression level identification of overexpression lines.

[0075] Figure 1 A in the figure is DNA molecular identification: the electrophoresis pattern of the PCR amplification product is shown by agarose gel electrophoresis. M represents the DNA molecular weight standard (marker), J9709 is the control group, and Yang represents the positive control. The results showed that the overexpression plants showed the same BnaA01.HSFA1a The specific band corresponding to the gene (about 750 bp) indicated that positive plants were successfully screened.

[0076] Figure 1 Figure B is the positive plant screening: the plants were screened using a red fluorescent protein excitation gun. BnaA01.HSFA1a Transgenic positive rapeseed seedlings can emit bright red light under a fluorescence gun, which is consistent with the characteristics of the DsRed red light vector, further confirming the successful screening of overexpression plants.

[0077] Figure 1 Middle C is the expression level identification of overexpression lines: Real-time fluorescence quantitative PCR was used to identify the expression level of overexpression lines. BnaA01.HSFA1a Gene expression levels were measured. The results showed that the relative expression levels of the overexpressing strains (BnOE#1 to BnOE#6) were significantly higher than those of the control strain, J9709. Among them, BnOE#1, BnOE#2, and BnOE#3 showed the highest expression levels, indicating significant upregulation of gene expression in these strains. Therefore, these strains were selected for subsequent experimental studies.

[0078] A total of 41 strains were screened through DNA molecular identification (Figure 2B). BnaA01.HSFA1a Interference with positive plants of Brassica napus. Based on the insertion fragment of the interference vector (A in Figure 2), simultaneous identification of the interference lines BnaA01.HSFA1a and BnaC01.HSFA1a The quantitative results showed that BnaA01.HSFA1a The interference effect is greater than BnaC01.HSFA1a , selected three strains with interference expression levels of more than 60%, RNAi#1, RNAi#2, and RNAi#3 , and conduct subsequent experimental studies (Figure 2C). Figure 2 shows BnaA01.HSFA1a Related identification results of interference with Brassica napus plants and information on interference vectors. Figure 2 middle: A: Schematic diagram of interference vector: The diagram clearly shows the structural composition of the interference vector, including 35S promoter, RNAi- BnaA01.HSFA1a 、BnRAP212、 BnaA01.HSFA1a and OCS terminator, and marked the restriction endonuclease sites between each element, such as Nco I, Aat II, BamH I and Xba I, reflecting the construction details and design principles of the interference vector, providing key vector information for subsequent experiments.

[0079] B: DNA molecular identification: Agarose gel electrophoresis results showed that 41 strains were successfully screened through PCR amplification BnaA01.HSFA1a Interference with positive Brassica napus plants. M represents DNA molecular weight standard (marker), and J9709 is the control group.

[0080] C: Identification of interference expression level: Real-time fluorescence quantitative PCR detection results showed that BnaA01.HSFA1a and BnaC01.HSFA1a expression levels. Quantitative results showed BnaA01.HSFA1a The interference effect of RNAi#1 was significantly better than that of BnaC01.HSFA1a. Based on this, three strains (RNAi#1, RNAi#2 and RNAi#3) with an interference efficiency of more than 60% were selected for subsequent experimental studies.

[0081] This example will be obtained from the above experiment BnaA01.HSFA1aOverexpression rapeseed lines BnOE#1, BnOE#2 and BnOE#3 The T1 seeds were subjected to 48℃ high temperature stress germination experiment. The experimental results showed that: When untreated (Figure 3A): Untreated control J9709 and overexpression strain seeds were evenly sown on filter paper. All seeds germinated normally and grew in the same manner, with no significant differences.

[0082] After 3-4 hours of high temperature treatment (BC in Figure 3): the germination of the control J9709 seeds was significantly inhibited, the germination rate was significantly reduced, and the seedlings grew slowly; while the germination of the overexpression strain seeds was almost uninhibited, the germination rate remained at a high level, and the seedlings grew well.

[0083] After 5 hours of high temperature treatment (Figure 3D): the germination rate of the control J9709 was further significantly reduced, and seed germination was severely inhibited; in contrast, the number of germinated seeds of the overexpression line remained at a high level, showing strong heat resistance.

[0084] In addition, the experiment also counted the number of seeds that germinated and grew green leaves under different treatment times (Figure 3, G). The results showed: The number of green leaves of the control J9709 decreased significantly after high temperature treatment, indicating that high temperature stress caused great damage to its seed germination and seedling growth.

[0085] The number of green leaves grown from seeds of overexpression lines BnOE#1, BnOE#2 and BnOE#3 under high temperature stress was significantly higher than that of the control, indicating that overexpression BnaA01.HSFA1a The gene can significantly enhance the heat tolerance of seeds and improve their germination ability in high temperatures.

[0086] These phenotypic observations and data are consistent with BnaA01.HSFA1a The results of the study on overexpression of Arabidopsis thaliana were consistent, proving that BnaA01.HSFA1a The gene also plays a positive role in regulating seed heat tolerance in Brassica napus.

[0087] It should be noted that due to the limited number of seeds harvested from the interference strains, this experiment failed to conduct relevant high-temperature stress experiments on the interference strains, and therefore it is impossible to provide data on the response of the interference strains to high-temperature stress at the seed stage.

[0088] This example further explores BnaA01.HSFA1aThe response of gene-overexpressing and gene-interference rapeseed lines to high temperature stress was investigated. Seeds of the overexpressing and gene-interference rapeseed lines, as well as the control J9709, were sown in MS medium. After 7 days, healthy seedlings with consistent growth were selected and subjected to a high-temperature treatment of 44°C for 1 hour. The culture was then allowed to recover for 7 days, and the survival of each line was observed. The results are as follows: Overexpression lines: As shown in Figure 4 AB, BnaA01.HSFA1a Overexpression lines (BnOE#1, BnOE#2, and BnOE#3) were less affected by high temperature stress, with only slight damage on the edges of the cotyledons, but they continued to grow and develop, indicating that overexpression BnaA01.HSFA1a The gene significantly enhanced the tolerance of transgenic rapeseed to heat stress.

[0089] Control J9709: Figure 4 CF shows that the cotyledons of the control J9709 seedlings showed severe yellowing after high temperature stress and eventually died completely, indicating that the control plants had poor tolerance to high temperature stress.

[0090] Experiments have shown that overexpression BnaA01.HSFA1a The rapeseed lines with the gene showed enhanced tolerance to high temperature stress, while the control lines were severely affected. BnaA01.HSFA1a The important role of genes in improving heat tolerance in Brassica napus.

[0091] This example further explores BnaA01.HSFA1a The responses of the interference strain and the control J9709 under high temperature stress. BnaA01.HSFA1a Seeds of the interference strains RNAi#1, RNAi#2, RNAi#3 and the control J9709 were sown in MS medium. After 7 days, healthy seedlings with consistent growth were selected and subjected to high temperature treatment at 44°C for 1 hour. The culture was then restored for 7 days, and the survival of each strain was observed.

[0092] After 1 hour of high temperature stress at 44℃, BnaA01.HSFA1a Interference strain RNAi#1, RNAi#2, and RNAi#3 The seedlings of J9709 and the control J9709 showed severe yellowing and death. Specifically, the leaves turned yellow and withered, the growth stagnated, and eventually died. There was no significant difference (AB in Figure 5). It is speculated that the high temperature of 44℃ may cause the BnaA01.HSFA1a Interference strain RNAi#1, RNAi#2, and RNAi#3 The stress levels of the control J9709 and the control J9709 exceeded the tolerance limit of the plants, resulting in both plants showing severe damage in appearance, with no significant difference (Figure 5 CF). This indicates that under such high-intensity high-temperature stress, BnaA01.HSFA1a Whether the gene is expressed or not has little effect on the survival of the plant, and it is difficult for the plant to survive under such extreme conditions.

[0093] In addition, to further determine BnaA01.HSFA1a To investigate the regulatory function of genes in heat tolerance of Brassica napus, we harvested T1 generation overexpression, interference seeds and control J9709 and sowed them on filter paper. After 10 days of growth, we selected positive rapeseed seedlings and J9709 with consistent growth and transplanted them into soil. We watered them regularly and quantitatively. After culturing for 2 weeks, we placed them in a high temperature of 44°C for 10 hours, returned them to room temperature, and placed them in an artificial climate incubator for normal culture for 2 days to observe the phenotype. BnaA01.HSFA1a Overexpression lines BnOE#1 、 BnOE#2 and BnOE#3 Before high temperature treatment, there were no significant differences in growth and development between the J9709 and control strains. However, after high temperature treatment, the leaves of the J9709 strains wilted more significantly than those of the overexpressing strains. After returning to room temperature, the overexpressing rapeseed strains survived, while the control strains did not (Figure 6A). BnaA01.HSFA1a Interference strain RNAi#1 、 RNAi#2 and RNAi#3 There was no significant difference in the growth and development of the plants before high temperature treatment between the interference strains and the control J9709. However, after high temperature treatment, the leaves of both the interference strains and the control wilted. After returning to room temperature, BnaA01.HSFA1a Interference strain RNAi#1 、 RNAi#2 and RNAi#3 All of the J9709 cells and the control J9709 died and could not recover (Figure 6B). BnaA01.HSFA1a Phenotypic observations of the seedlings and high temperature treatment of the overexpression and interference Brassica napus lines and the control J9709 further demonstrated that BnaA01.HSFA1a Positive regulation of heat stress tolerance in Brassica napus.

[0094] The rapeseed leaves of overexpression, interference and J9709 were selected 4 hours after high temperature stress and stained with DAB and NBT dyes. The results showed that the control line and the overexpression line BnOE#1 、 BnOE#2 and BnOE#3 The number of spots on the leaves is greater and the coloration is significantly darker (Figure 7A). BnaA01.HSFA1a The H2O2 and O2- content accumulated by overexpressing rapeseed was lower than that of the control. BnaA01.HSFA1a Interference strain RNAi#1 、 RNAi#2 and RNAi#3 Compared with the control, the number of spots on the leaves of J9070 was slightly more and the coloration was slightly darker (Figure 7B). BnaA01.HSFA1aThe H2O2 and O2- contents accumulated by the interference strain were higher than those in the control. BnaA01.HSFA1a Overexpressing rapeseed is less damaged by high temperature stress than BnaA01.HSFA1a Interference plants and control J9709, and BnaA01.HSFA1a The high temperature stress damage to the disturbed plants was greater than that of the control J9709.

[0095] After high temperature stress BnaA01.HSFA1a Overexpression of Brassica napus lines BnOE#1 、 BnOE#2 、 BnOE#3 The chlorophyll a content, chlorophyll b content, and chlorophyll content were significantly higher than those of the control J9709 and the interference strain. RNAi#1 、 RNAi#2 、 RNAi#3 The chlorophyll a content, chlorophyll b content, and chlorophyll content of J9709 leaves were significantly lower than those of the control J9709 (Figure 8, AC). BnaA01.HSFA1a Overexpression of Brassica napus lines BnOE#1, BnOE#2, BnOE#3 The MDA content accumulated in leaves was significantly lower than that in the control J9709 and interference strains, while the interference strains RNAi#1, RNAi#2, RNAi#3 The malondialdehyde content in the leaves was significantly higher than that in the control J9709 (D in Figure 8). BnaA01.HSFA1a Overexpression of rapeseed [[ID=б6]]BnOE#1, BnOE#2, BnOE#3 The accumulated soluble sugar and free proline contents were higher than those of the control and interference strains, while the interference strain RNAi#1, RNAi#2, RNAi#3 The accumulated soluble sugar and free proline contents in the leaves were significantly lower than those in the control J9709 (EF in Figure 8). BnaA01.HSFA1a Phenotypic observation of overexpression and interference Brassica napus seedlings and analysis of phenotypic and physiological and biochemical changes during the seedling stage showed that BnaA01.HSFA1a Overexpression of the gene may enhance the heat tolerance of Brassica napus plants after heat stress treatment by reducing the accumulation of the harmful substance MDA and increasing the content of osmotic substances such as soluble sugar and free proline.

[0096] Rapeseed is susceptible to high temperature stress during flowering. BnaA01.HSFA1a To investigate the heat resistance of Brassica napus pollen, we collected overexpression, interference and control J9709 Brassica napus pollen and performed heat resistance tests. BnaA01.HSFA1a The pollen grains of overexpression, interference and control J9709 were all stained dark red before high temperature stress treatment, indicating that the pollen viability of the three was basically the same before treatment. However, after high temperature stress treatment, some pollen grains of control J9709 were stained light red, while the pollen grains of overexpression line BnOE#1, BnOE#2 andBnOE#3 can still be dyed crimson ( Figure 9 AH in the middle), which shows that the pollen vigor of the overexpression line is stronger than that of the control and is less affected, while the interference line RNAi#1, RNAi#2, RNAi#3 Most pollen grains have turned light pink, and their vitality has decreased further (IP in Figure 9). BnaA01.HSFA1a Pollen viability of overexpression, interference and control J9709 rapeseed lines before and after high temperature stress. BnaA01.HSFA1a The pollen viability of overexpression, interference and control J9709 before treatment was basically the same, reaching more than 90% ( Figure 10 (A). After high temperature treatment, J9709 and BnaA01.HSFA1a Interference strain RNAi#1, RNAi#2, RNAi#3 The pollen activity of BnaA01.HSFA1a Overexpression lines BnOE#1, BnOE#2, BnOE#3 Even with a decrease of about 30%, pollen viability can still be maintained above 65% (Figure 10B).

[0097] In summary, the results show that BnaA01.HSFA1a Overexpression lines can enhance the heat tolerance of Brassica napus pollen.

[0098] Through the above implementation, BnaA01.HSFA1a It should be noted that there may be some incorrect or unclear notations in the original text, such as the "б6" in the translation of ID=66 which might be a typo. Please double-check the original text for accuracy. The gene can be widely used in the heat-resistant breeding of Brassica napus, improving the growth and production capacity of rapeseed in high temperature environments, and providing technical support for ensuring the stable development of the rapeseed industry.

[0099] In the description of the present invention, it should be understood that "-" and "~" represent a range between two values, and the range includes the endpoints. For example, "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0100] In the description of the present invention, the term "and / or" is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0101] In the description of the invention, the numerical values ​​of time, temperature, ratio and mass involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0102] In the description of the present invention, the term "about" or "approximately" is used to express the approximate value of a numerical value or range, allowing a certain error to ensure the flexibility and practicality of the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0103] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A heat-resistant gene of Brassica napus, characterized in that: The heat-resistant gene of Brassica napus is BnaA01.HSFA1a , the nucleotide sequence is shown in SEQ ID NO:

1.

2. A protein encoded by the gene according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that Containing the claim 1 BnaA01.HSFA1a The gene is expressed in a vector for overexpressing or interfering with the expression of the gene in Brassica napus.

4. The recombinant vector according to claim 3, characterized in that The overexpression vector comprises a promoter sequence and a nucleotide sequence shown in SEQ ID NO: 1; the interference vector comprises a promoter sequence and a nucleotide sequence shown in SEQ ID NO: 1; BnaA01.HSFA1a RNAi sequence of the gene.

5. A method for cloning the gene according to claim 1, comprising the following steps: A. Extract total RNA from Brassica napus leaves and obtain cDNA through reverse transcription; B. using the cDNA as a template and using specific primers to perform PCR amplification to obtain a gene fragment comprising SEQ ID NO: 1; C. Recover and purify the PCR amplification product, and perform sequencing verification.

6. The method according to claim 5, characterized in that The specific primers described in step B are: BnaA01.HSFA1a–F: 5′-GCCAGAATTCATGATGGATGGTGTAACCGG-3′; BnaA01.HSFA1a–R: 5′-TAGAGTCGACTCACAGTTGCTTTGT-3′.

7. A method for improving the heat tolerance of Brassica napus, characterized in that: Including the claim 1 BnaA01.HSFA1a The gene is introduced into Brassica napus plants to overexpress the gene.

8. The method according to claim 7, characterized in that The gene is transformed into Brassica napus by Agrobacterium-mediated method, and the Agrobacterium carries the recombinant vector according to claim 3 or 4.

9. Use of the gene according to claim 1 in regulating plant heat tolerance, characterized in that: The application includes enhancing the tolerance of plants to high temperature stress by overexpressing the gene.

10. Use of the gene according to claim 1 in preparing heat-resistant rapeseed varieties.

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