Application of blueberry VcETR-1 gene in regulation and control of high temperature stress resistance of plants

Abstract

The invention discloses application of a blueberry VcETR-1 gene in regulation of high temperature stress resistance of plants, and relates to the technical field of plant genetic engineering. According to the application of the blueberry VcETR-1 gene disclosed by the invention in regulating and controlling the high temperature stress resistance of the plant, the nucleotide sequence of the blueberry VcETR-1 gene is as shown in SEQ ID NO. 1. The gene can improve the stress resistance of a plant at a high temperature of 45 DEG C through overexpression, and can regulate the response of the plant to 200 [mu] mol / L of exogenous salicylic acid. Experiments show that 10-20% of leaves of the transgenic plant are still good in state after high-temperature treatment, and physiological indexes such as antioxidant enzyme activity and the like are better. The invention further provides a related application method and a primer pair for cloning the gene, the blank of research on the heat-resistant function of the blueberry ethylene receptor gene is filled, and an important basis and technical support are provided for cultivating new varieties of high-temperature-resistant plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the application of the blueberry VcETR-1 gene in regulating the plant's ability to resist high temperature stress. Background Technology

[0002] With the intensification of global warming and the increasing frequency of extreme heat events, plant growth and development face severe challenges. High-temperature stress can damage plant cell membrane systems, reduce photosynthetic efficiency, and accumulate reactive oxygen species, leading to growth stagnation, reduced yields, and even death. Blueberries, as an important economic fruit tree, are highly sensitive to temperature throughout their growth cycle. Southern highbush blueberry varieties, in particular, are prone to problems such as yellowing leaves and stunted fruit development under high summer temperatures, severely hindering industry development. Therefore, identifying key genes in plants that resist high-temperature stress and improving crop heat resistance through molecular breeding techniques has become a research hotspot in the agricultural field.

[0003] Plants have developed complex stress regulation mechanisms over long-term evolution, among which the ethylene signaling pathway plays a crucial role in responding to abiotic stresses. Ethylene receptors (ETRs), as initiators of ethylene signaling, participate in plant growth, development, and stress responses by sensing ethylene signals and regulating downstream gene expression. Previous studies have shown that ETR family genes are involved in high-temperature stress responses in model plants such as Arabidopsis and rice, but their function in blueberry remains unclear.

[0004] Currently, research on blueberry heat resistance mainly focuses on physiological regulation, such as alleviating heat damage through exogenous spraying of salicylic acid (SA), but this method has limitations such as high cost and short time-limited effectiveness. At the molecular level, although some gene families related to stress resistance (such as HSP and bZIP) have been identified, cloning, functional verification, and application research of ethylene receptor genes remain lacking. In addition, the blueberry genetic transformation system is not yet mature, and the lack of efficient gene editing or overexpression technologies restricts the application and transformation of heat-resistant genes. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a blueberry heat-resistant gene VcETR-1. Another technical problem to be solved by the present invention is to provide an expression protein of the blueberry heat-resistant gene VcETR-1. A further technical problem to be solved by the present invention is to provide an application of the blueberry heat-resistant gene VcETR-1 for regulating the blueberry's resistance to high-temperature stress and its response to exogenous SA.

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

[0007] Application of the blueberry VcETR-1 gene in regulating the plant's resistance to high temperature stress, the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the high temperature is 45°C.

[0009] Furthermore, the plant includes blueberries.

[0010] Application of the blueberry VcETR-1 gene in regulating the plant's response to exogenous salicylic acid, the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.1.

[0011] Furthermore, the concentration of the exogenous salicylic acid is 200 μmol / L.

[0012] Application of the expressed protein of the blueberry VcETR-1 gene in regulating the plant's resistance to high temperature stress, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] The application of the expressed protein of the blueberry VcETR-1 gene in regulating the plant's response to exogenous salicylic acid, the amino acid sequence of which is shown in SEQ ID NO.2.

[0014] A method for improving the resistance of plants to high temperature stress includes introducing the blueberry VcETR-1 gene into plants to overexpress the blueberry VcETR-1 gene in the plants; the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.1.

[0015] A method for regulating the plant’s response to exogenous salicylic acid includes introducing the blueberry VcETR-1 gene into the plant to overexpress the blueberry VcETR-1 gene in the plant, wherein the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.1.

[0016] A primer pair for cloning the blueberry heat-resistant gene VcETR-1, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0017] The sequences of the primer pair are as follows:

[0018] VcETR-1-F: 5'-ATGTTGAAGAAATTAGCATCTG-3',

[0019] VcETR-1-R: 5'-CATGACTTTGTTTGCCTGG-3'.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1) This invention is the first to clone the blueberry heat-resistant gene VcETR-1, whose nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. This invention transforms the blueberry VcETR-1 gene into blueberries, effectively regulating the blueberry's resistance to stress under high-temperature environments (e.g., 45℃). Results from examples show that after high-temperature treatment, blueberry lines transgenic with the VcETR-1 gene still retained 10-20% of their leaves in good condition compared to the control group (CK), indicating that overexpression of this gene can enhance the plant's adaptability to high-temperature environments. Under high-temperature stress, the activities of catalase (CAT) and peroxidase (POD) in transgenic plants were significantly increased. Although the activity of superoxide dismutase (SOD) decreased, the overall antioxidant system could still more efficiently scavenge reactive oxygen species (e.g., H2O2) and reduce malondialdehyde (MDA) accumulation, thereby alleviating cell membrane lipid peroxidation damage and maintaining cell structural stability.

[0022] 2) This invention introduces the VcETR-1 gene into blueberries, effectively regulating the blueberry's response to exogenous salicylic acid (SA, 200 μmol / L). Results from the examples show that after SA treatment, the POD activity of the transgenic plants further increased, and under combined high-temperature and SA treatment, the MDA content did not show a significant increase compared to the control group, indicating that this gene can synergistically enhance the plant's adaptability to combined stress. Under SA treatment, although the CAT activity of the transgenic plants decreased slightly compared to the high-temperature treatment group, it was still higher than the control group, and the increase in H2O2 content was controllable, indicating that it can balance the salicylic acid-induced physiological response through gene regulation, avoiding secondary damage to the plant caused by excessive stress. Attached Figure Description

[0023] Figure 1 Motif analysis diagram of key heat resistance genes in blueberries and their family members (A); Conserved domain analysis diagram of key heat resistance gene VcbZIP in blueberries and its family members (B);

[0024] Figure 2 Sequence alignment diagram of VcETR family members, key heat-resistant genes in blueberries;

[0025] Figure 3 Phylogenetic tree diagram of the VcbZIP family;

[0026] Figure 4 A graph showing gene expression levels in different tissues and developmental stages of blueberries;

[0027] Figure 5 A graph showing the expression levels of heat stress-related genes in positive seedlings of transgenic blueberries;

[0028] Figure 6Figure showing phenotypic changes in transgenic blueberries after high temperature and exogenous SA treatment;

[0029] Figure 7 This figure shows the expression of the VcETR-1 gene in transgenic blueberry lines after high temperature and exogenous SA treatment. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0031] The blueberry material used for gene cloning in this application was collected from healthy, uniform two-year-old leaves of the southern highbush blueberry variety 'O'Neal' from the Blueberry and Blackberry Research Base of the Institute of Botany, Chinese Academy of Sciences, Lishui District, Nanjing City, Jiangsu Province (119°03′E, 31°35′N). The leaves were brought back in liquid nitrogen and stored at -80°C.

[0032] The quantitative sample materials of different blueberry tissues used in this application were collected from healthy 'O'Neill' variety plants cultivated in the greenhouse of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The tissue culture seedlings were collected from the tissue culture laboratory of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, and were one month old, of the 'Tianhou' variety.

[0033] Example 1

[0034] 1. Cloning and sequence analysis of the blueberry VcETR-1 gene

[0035] The open reading frame (ORF) of the candidate gene VcETR-1 (gene-Vadar_002881) was obtained using BioXM 2.6 software. It was corrected using the existing three-generation full-length transcript set. Primers for candidate gene cloning were designed using Oligo 6.0 software, and the ORF sequence was cloned using the high-fidelity PCR enzyme Prime STAR Max DNA Polymerase from TaKaRa. The 50 μL PCR reaction system consisted of: 25 μL Primer Star Max, 1 μL each of the preceding and following primers, 1 μL cDNA template, and 22 μL ddH2O. The PCR reaction program was: 98℃ for 3 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 15 s, 35 cycles; 72℃ for 3 min, followed by incubation at 4℃. After amplification, the PCR products were detected by 1% agarose gel electrophoresis, and bands matching the predicted target gene amplification product length were excised.

[0036] The primer sequences are shown below:

[0037] VcETR-1-F: 5'-ATGTTGAAGAAATTAGCATCTG-3',

[0038] VcETR-1-R: 5'-CATGACTTTGTTTGCCTGG-3';

[0039] M13 General-F:5'-TGTAAAACGACGGCCAGT-3',

[0040] M13 General-R:5'-CAGGAAACAGCTATGACC-3'.

[0041] The excised electrophoresis gel was purified using the BioTeKe Rapid Agarose Gel DNA Recovery Kit. The purified product was then ligated to a vector according to the instructions of the pClone007 Blunt Vector Kit (TSINGKE), and transformed into competent *E. coli* cells. After a brief recovery period, the cells were plated onto ampicillin-resistant LB medium. After overnight incubation, single colonies were randomly selected and transferred into ampicillin-resistant LB medium, and incubated on a shaker for 8 hours (37°C, 200 rpm). Colony PCR was performed using 2×T5 Super PCR Mix (Colony) (TSINGKE). Positive colonies were sent to Nanjing Qingke Biotechnology Co., Ltd. for first-generation sequencing verification. After sequencing, plasmids were extracted from correctly aligned *E. coli* colonies using a plasmid extraction kit (BioTeKe) and stored at -20°C for subsequent experiments.

[0042] The final sequencing yielded the nucleotide sequence of the blueberry heat-resistant gene VcETR-1, as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein, as shown in SEQ ID NO.2.

[0043] 2. Analysis of the physicochemical properties of proteins

[0044] To further investigate whether similar mechanisms of action exist among related family members, three VcETR family gene members—gene-Vadar_002881 (VcETR-1), gene-Vadar_005286 (VcETR-2), and gene-Vadar_033228 (VcETR-3)—were screened from blueberry leaf transcriptome data. The minimum free energy (MFE) and center-of-mass secondary structure of the mRNAs of the three VcETR gene family members were analyzed using RNAfold software. The results showed that VcETR-1 had the highest MFE (-758.26 kcal / mol), while VcETR-2 had the lowest (-804.12 kcal / mol). Furthermore, the MFE, center-of-mass structure, and thermodynamic set of the three VcETRs differed. The NetPhos online tool was used to predict potential phosphorylation sites, and the NetNGlyc-1.0 online tool was used to predict protein glycosylation sites. The results showed that VcETR-1 had 70 phosphorylation sites and 5 glycosylation modification sites with probabilities of 0.6728, 0.6052, 0.5541, 0.4928, and 0.4892, respectively; VcETR-2 had 76 phosphorylation sites and 1 glycosylation modification site with a probability of 0.5306; and VcETR-3 had 70 phosphorylation sites and 4 glycosylation modification sites with probabilities of 0.6686, 0.5637, 0.4929, and 0.4891, respectively.

[0045] The physicochemical properties of the protein were analyzed using the ProtParam online tool (https: / / web.expasy.org / protparam / ); subcellular localization analysis was performed using the online tool Wolf Psort (https: / / wolfpsort.hgc.jp); secondary structure analysis was conducted using the online tool SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsaautomat.pl?page=npsa_sopma.html); and a three-dimensional model was constructed and validated using SWISS-MODEL (https: / / swissmodel.expasy.org / ). Conserved motifs were predicted using the online tool MEME (http: / / meme-suite.org / ) with default parameters and a motif count of 10; conserved domain analysis was performed using NCBI's CD search (https: / / www.ncbi.nlm.nih.gov / Structure / bwrpsb / bwrpsb.cgi). Protein phosphorylation sites were predicted using the NetPhos online tool (https: / / services.healthtech.dtu.dk / service.php?NetPhos-3.1), and protein glycosylation sites were predicted using the NetNGlyc-1.0 online tool (http: / / www.cbs.dtu.dk / services / NetNGlyc / ).

[0046] The physicochemical properties analysis of the proteins showed that the relative molecular weights (MW) of the VcETR family genes were 84.7, 97.9, and 84.7 kDa, respectively, with theoretical isoelectric points (PI) ranging from 7.04 to 8.03 and instability coefficients ranging from 35.7 to 44.16. Subcellular localization showed that all VcETR family genes were located in the endoplasmic reticulum, indicating that they may have similar functions.

[0047] The results are as follows Figure 1 As shown, the VcETR-1 and VcETR-3 sequences have high similarity, both containing 20 motifs, while the VcETR-2 sequence contains only 13 motifs. Figure 1 The three genes in the VcETR family simultaneously contain the Utp11, Myb_CC_LHEQLE, and REC domains. Figure 1 (B).

[0048] 3. Phylogenetic tree analysis

[0049] The gene sequences were compared with gene sequences from other species in GeneBank using the BLAST tool in NCBI, and the top 20 genes with the highest similarity were selected (if the total number of gene sequences was less than 20, all were selected). Multiple alignments of the gene sequences were performed using DNAMAN 6 software. The 20 protein sequences with the highest similarity to the target gene (FDH) were found in the NCBI database. The Clustal W function in MEGA11 software was used to remove sequences with low alignment quality. Then, a phylogenetic tree was constructed using MEGA 11 software with a neighbor-joining (NJ) method and a bootstrap test run of 1000 times.

[0050] The results are as follows Figure 2 As shown, the GAF superfamily and REC superfamily are the main structural domains in the VcETR family genes.

[0051] The results are as follows Figure 3 As shown, VcETR-1 and VcETR-3 are most closely related to ETR 2 (Rhododendron vialii), while they are more distantly related to ETR 2-like-2 and ETR-2-like-3 (Actinidia eriantha). VcETR-2 is most closely related to ETR 2 (Theobroma cacao) and ETR 2 (Durio zibethinus), while it is more distantly related to ETR 2-like-2 and ETR-2-like-3 (A. eriantha).

[0052] 4. Detection of expression levels of key genes in different tissues of blueberries

[0053] Quantitative samples of different blueberry tissues were selected from healthy 'O'Neill' variety plants cultivated in the greenhouse of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. Samples were taken from different tissue parts of the blueberry plant, including stems (stem tip 1, stem segment 2), leaves (different time periods: April, May, June, July, August, September, and October), flowers (different developmental stages), and fruits (green fruits, green-to-red fruits, red fruits, red-to-purple fruits, and purple fruits). After flash freezing in liquid nitrogen, the samples were stored at -80℃ for subsequent RNA extraction. Total RNA was extracted from the samples according to the instructions of the Bioteke Plant (Beijing Bioteke Co., Ltd.; Cat#RP3302) general plant total RNA extraction kit. Reverse transcription of the total RNA from each sample was performed using the Evo M-MLV reverse transcription premix kit, and the cDNA from the samples was preserved. Gene-specific primers were designed using Primer Premier 5.0, with the blueberry GADPH gene as an internal reference gene, and 2... -ΔΔCt Gene expression levels were calculated using RT-qPCR. TB Green Premix Taq II (Tli RNASEH Plus) (TaKaRa, Dalian, China) was used. The 15 μL reaction mixture contained 7.5 μL TB Green Premix Taq II fluorescent dye, 1 μL cDNA template, 0.6 μL each of forward and reverse primers, and 5.3 μL ddH2O. The PCR amplification program was set on a QuantStudio 3 (ABI, Thermo Fisher Scientific, USA). The program was: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 45 cycles; 95℃ for 15 s; 60℃ for 60 s; 95℃ for 1 s.

[0054] The results are as follows Figure 4 As shown, VcETR-1 expression was highest in the first stage of fruit development (green fruit). The expression trend of VcETR-1 in blueberry leaves at different developmental stages was similar, with the highest expression level in July and the second highest in August. Furthermore, the expression trend of VcETR-1 in blueberry stem segments consistently showed an increase in expression level with increasing lignification. In summary, VcETR-1 expression was highest in leaves, followed by stem segments.

[0055] Example 2

[0056] 1. Constructing an overexpression vector for the blueberry heat-resistant gene VcETR-1

[0057] The VcETR-1 gene overexpression vector was constructed using the plant binary expression vector pCAMBIA1303 (resistance kan). Agrobacterium competent cells stored at -80℃ were removed and thawed on ice. 50 μL of competent cells and 10 μL of plasmid were added to a centrifuge tube, gently mixed, and incubated on ice for 5 min. After the ice bath, the centrifuge tube was flash-frozen in liquid nitrogen for 5 min, then incubated in water at 37℃ for 5 min, followed by another 5 min on ice. 700 μL of LB liquid medium was added to the centrifuge tube, and the tube was incubated at 28℃ and 200 rpm for 2 h. The tube was centrifuged at 4000 rpm for 5 min, and the supernatant was removed using a pipette. The remaining colony precipitate and culture were gently mixed. Finally, on a clean bench, the culture was evenly spread onto LB solid medium using a sterilized and cooled spreader. The culture dish was inverted and incubated overnight at 28℃. Colonies were observed after 48 h. Single colonies were selected for PCR verification. Finally, the transformed bacterial culture containing the positive plasmid was stored at -80°C for subsequent genetic transformation experiments.

[0058] 2. Genetically transformed blueberries

[0059] Blueberry tissue culture seedlings were cut into 1cm segments and inoculated into a pre-culture medium, then cultured in the dark for 2 days. The bacterial culture was then shaken at 28℃ and 200rpm until the OD concentration reached approximately 0.6. The culture was centrifuged at 3000rpm for 15 minutes, the supernatant was discarded, and Agrobacterium suspension was added and the mixture resuspended. The pre-cultured blueberry stem segments were then placed in the Agrobacterium resuspension and inoculated at normal pressure for 10 minutes. The inoculated stem segments were then inoculated into a co-culture medium and cultured at 25℃ under light for 4 days. After co-culture, the stem segments were placed in a sterilization solution for 8 minutes, with occasional shaking to effectively remove Agrobacterium. The sterilized stem segments were then inoculated into a selection medium and cultured at 25℃ in the dark for 10 days. Finally, they were cultured under light for 50 days, and the stem growth was observed and recorded. The condition of blueberry stem segments during the pre-culture, co-culture, and screening culture stages of genetic transformation was observed and recorded. Blueberry 'Tianhou' plants of the same period with no vector / no genetic transformation were used as the control group. The six VcETR-1 genotype blueberry plants obtained were tested for positive seedlings.

[0060] 2. Quantitative detection of transgenic plants

[0061] Total RNA was extracted from blueberry transgenic plants according to the instructions of the Bioteke Geneporation (Beijing Bioteke Co., Ltd.; Cat#RP3302) universal plant total RNA extraction kit. The total RNA was reverse transcribed using the Evo M-MLV reverse transcription premix kit, and the cDNA was preserved. Gene-specific primers were designed using Primer Premier 5.0, with the blueberry GADPH gene as an internal reference gene, to analyze the expression of key genes. -ΔΔCt Methods: Gene expression levels were calculated. RT-qPCR was performed using TB Green Premix Taq II (Tli RNASEH Plus) (TaKaRa, Dalian, China). The 15 μL reaction mixture contained 7.5 μL TB Green Premix Taq II fluorescent dye, 1 μL cDNA template, 0.6 μL each of forward and reverse primers, and 5.3 μL ddH2O. Gene expression levels in transgenic blueberries were analyzed, and blueberries with high VcETR-1 expression were screened. Based on blueberry transcriptome data, 10 genes significantly upregulated in response to heat stress were selected, including ATPase, bZIP-2, bZIP-3, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, and WRKY. The expression of these heat stress-related genes in blueberry lines with high VcETR-1 expression was analyzed. Primer sequences are shown below:

[0062] VcETR-1-qRT-F: 5'-CCCTCCCGTCACTTCGTTCC-3',

[0063] VcETR-1-qRT-R: 5'-GCGACAATCAACGGCCAGGA-3';

[0064] GADPH-qRT-F: 5'-GGTTATCAATGATAGGTTTGGCA-3',

[0065] GADPH-qRT-R: 5'-CAGTCCTTGCTTGATGGACC-3';

[0066] ATPase-qRT-F: 5'-AATGCCGGACACGCACAGTT-3',

[0067] ATPase-qRT-R: 5'-TGAGCCCGTCGGATCACCTT-3';

[0068] bZIP-2-qRT-F: 5'-GGCATATCACCACAACCCGCAT-3',

[0069] bZIP-2-qRT-R:5’-CCCTTGCACTTTCCCTTTGGCT-3’;

[0070] bZIP-3-qRT-F:5’-TGCGTTTGGTCACGCACCAT-3’,

[0071] bZIP-3-qRT-R:5’-TTGCCGAAGCATGGCCGATT-3’;

[0072] HSF A-3-qRT-F:5’-GGCCAGACGAGTGGGACTCA-3’,

[0073] HSF A-3-qRT-R:5’-GCCTCTGCTCTGCTGCTTGG-3’;

[0074] HSP20-1-qRT-F:5’-CGGGACATTGCTCCGTTCCC-3’,

[0075] HSP20-1-qRT-R:5’-CCGGCGTTTCCTTCCAGTCC-3’;

[0076] HSP20-2-qRT-F:5’-ACTCGCGTGGACTGGAAGGA-3’,

[0077] HSP20-2-qRT-R:5’-GCGGTGCCACGTATCGTTCT-3’;

[0078] HSP20-3-qRT-F:5’-CGACGAGGCAACGACAGCTT-3’,

[0079] HSP20-3-qRT-R:5’-CCGGCGTTTCCTTCCAGTCC-3’;

[0080] HSF C-1-qRT-F:5’-CCGCCGTCGGTTATGAGCAG-3’,

[0081] HSF C-1-qRT-R:5’-CACCATGGCTGTGCCGGAAA-3’;

[0082] HSP70-qRT-F:5’-CCGCGTTCAGCTGTGTAGGG-3’,

[0083] HSP70-qRT-R:5’-CGCCAATTAGGCGCTCGGTA-3’;

[0084] WRKY-qRT-F: 5'-AGCCGAAATTCGCTTTTCCAGACAA-3',

[0085] WRKY-qRT-R: 5'-TGGACTGGCGTTGCACTTGTT-3'.

[0086] The results are as follows Figure 5 As shown, three transgenic blueberry lines with high VcETR-1 expression levels (ETR-2, ETR-3, and ETR-5) were obtained, with the highest VcETR-1 gene expression level (20.19) in the ETR-5 line. The expression levels of six heat stress-related genes—ATPase, HSF A-3, HSP20-2, HSP20-3, HSF C-1, and HSP70—were all higher in the CK group than in the VcETR-1 transgenic blueberry. The expression levels of bZIP-3 and WRKY in the VcDELLA-1 transgenic blueberry were significantly higher than in the CK.

[0087] 3. Phenotypic observation and physiological index determination of transgenic lines under high temperature stress

[0088] The phenotypes of VcETR-1-overexpressing blueberry lines (FDH-8, FDH-9, and FDH-10) subjected to 45℃ high-temperature stress and 200 μmol / L exogenous SA treatment for 24 h were observed and recorded. Changes in leaf SPAD values ​​and nitrogen content under high-temperature and SA treatment conditions were analyzed. SPAD values ​​and nitrogen content in leaves were measured using a SPAD-502plus instrument. Propylene glycol (MDA) content was determined using the thiobarbituric acid (TBA) method; superoxide dismutase (SOD) activity was determined using the nitrocyanophthalein tetrazolium method; and hydrogen peroxide (H2O2) content and catalase (CAT) and peroxidase (POD) activities were determined using kits from Nanjing Jiancheng Biotechnology Co., Ltd.

[0089] The results are as follows Figure 6 As shown, after 24 hours of exogenous SA treatment, the phenotypes of blueberry plants did not change significantly. However, after high-temperature treatment, both VcETR-1 transgenic plants and CK plants showed varying degrees of yellowing, withering, and water shortage in their leaves. Compared to CK plants, 10-20% of the leaves of VcETR-1 transgenic plants were still in good condition.

[0090] The results are shown in Table 1. High temperature and SA treatments significantly reduced SOD activity in both CK and VcETR-1 blueberries, with no significant difference in SOD activity between the high temperature and SA treatment groups. High temperature and SA treatments significantly reduced CAT activity in CK, while significantly increasing CAT activity in VcETR-1 blueberries. Furthermore, the SA treatment group showed a significantly lower CAT activity in VcETR-1 blueberries compared to the high temperature treatment group. High temperature and SA treatments significantly increased POD activity in both CK and VcETR-1 blueberries, with a significantly higher POD activity in the SA treatment group compared to the high temperature treatment group. High temperature and SA treatments also significantly increased MDA content in both CK and VcETR-1 blueberries. Compared to the high-temperature treatment group, the MDA content in the SA treatment group was significantly increased. In VcETR-1 blueberries, there was no significant difference in MDA content between the high-temperature and SA treatment groups, but the MDA content in the SA treatment group was significantly decreased compared to the high-temperature treatment group. After high-temperature and SA treatments, the H2O2 content in both CK and VcETR-1 blueberries significantly increased, and the H2O2 content in the SA treatment group was significantly increased compared to the high-temperature treatment group. Therefore, under high-temperature and SA treatments, the SOD activity of all transgenic blueberry lines significantly decreased, while the POD activity, CAT activity, MDA content, and H2O2 content significantly increased.

[0091] Table 1. Changes in antioxidant physiological indicators of transgenic blueberries after high temperature and exogenous SA treatment.

[0092]

[0093] Note: Lowercase letters indicate significance test results of p < 0.05, and uppercase letters indicate significance test results of p < 0.01.

[0094] 4. Expression of the VcETR-1 gene after high temperature and exogenous SA treatment

[0095] Gene quantification analysis was performed on blueberry leaf samples from various strains treated with high temperature and exogenous SA for 0, 6, 12, and 24 hours.

[0096] The results are as follows Figure 7As shown, under high-temperature treatment, the expression level of the VcETR-1 gene in CK increased with increasing treatment time; under SA treatment, the expression level of the VcETR-1 gene in CK reached its maximum at 6 h and then decreased. The expression trends of the VcETR-1 gene were consistent among different VcETR-1 transgenic blueberry lines. In ETR-2, ETR-3, and ETR-5 blueberry lines, the expression level reached its maximum at 6 h of high-temperature treatment and then decreased; in SA treatment, it reached its maximum at 12 h and then decreased. Specifically, after 6 h of high-temperature treatment, the VcETR-1 gene expression level in ETR-5 reached as high as 254.3, which was 133.8 times that in CK at the same time point; after 12 h of SA treatment, the VcETR-1 gene expression level in ETR-5 reached as high as 123.5, which was 3.5 times that in CK at the same time point.

[0097] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of the blueberry VcETR-1 gene in regulating the plant's resistance to high temperature stress, wherein the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The high temperature is 45°C.

3. The application according to claim 1, characterized in that, The plant includes blueberries.

4. Application of the blueberry VcETR-1 gene in regulating the plant's response to exogenous salicylic acid, wherein the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, The concentration of the exogenous salicylic acid is 200 μmol / L.

6. Application of the expression protein of the blueberry VcETR-1 gene in regulating the plant's resistance to high temperature stress, wherein the amino acid sequence of the expression protein is shown in SEQ ID NO.

2.

7. Application of the expression protein of the blueberry VcETR-1 gene in regulating the plant's response to exogenous salicylic acid, the amino acid sequence of which is shown in SEQ ID NO.

2.

8. A method for improving the resistance of plants to high-temperature stress, characterized in that, This includes introducing the blueberry VcETR-1 gene into plants to overexpress the blueberry VcETR-1 gene in the plants; the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.

1.

9. A method for regulating the plant's response to exogenous salicylic acid, characterized in that, This includes introducing the blueberry VcETR-1 gene into plants to overexpress the blueberry VcETR-1 gene in plants, wherein the nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.

1.

10. A primer pair for cloning the blueberry heat-resistant gene VcETR-1, characterized in that, The nucleotide sequence of the blueberry VcETR-1 gene is shown in SEQ ID NO.1; The sequences of the primer pair are as follows: VcETR-1-F: 5'-ATGTTGAAGAAATTAGCATCTG-3', VcETR-1-R: 5'-CATGACTTTGTTTGCCTGG-3'.

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