Blueberry VcbZIP-1 gene as well as expression protein and application thereof

Abstract

The invention discloses a blueberry VcbZIP-1 gene as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the blueberry VcbZIP-1 gene disclosed by the invention is as shown in SEQ ID NO. 1, and the amino acid sequence of the expression protein of the blueberry VcbZIP-1 gene is as shown in SEQ ID NO. 2. The result of the embodiment shows that the blueberry VcbZIP-1 gene can promote the improvement of the high temperature stress resistance (45 DEG C) of plants, and the heat resistance of transgenic arabidopsis thaliana and blueberry strains is obviously superior to that of wild types; meanwhile, the response capability of plants to exogenous SA can be regulated and controlled, and after 200 mu mol / LSA treatment, the antioxidant activity of transgenic arabidopsis thaliana and blueberry strains is remarkably enhanced. Effective gene resources are provided for heat resistance improvement of plants, and important theoretical and practical significance is achieved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a blueberry VcbZIP-1 gene, its expressed protein, and its applications. Background Technology

[0002] With the intensification of global climate change and the frequent occurrence of extreme heat events, plant growth and development face severe challenges. High-temperature stress can damage plant cell membrane systems, denature proteins, and reduce photosynthetic efficiency. It also triggers a large accumulation of reactive oxygen species (ROS), causing oxidative damage and ultimately leading to reduced crop yield and deterioration in quality. Therefore, identifying genes related to plant resistance to high-temperature stress, elucidating their molecular mechanisms of stress resistance, and improving plant heat resistance through genetic engineering have become research hotspots in the field of agricultural biotechnology.

[0003] Blueberries (Vaccinium spp.), as a newly emerging fruit tree with high nutritional value, are rich in anthocyanins, vitamins, and other active ingredients, and market demand continues to grow. However, most blueberry varieties originated in temperate regions and are sensitive to high temperatures. Sustained high temperatures in summer easily lead to yellowing leaves, stunted shoot growth, and poor fruit development, severely restricting the large-scale development of the blueberry industry in high-temperature areas. Currently, research on blueberry resistance to heat stress mainly focuses on physiological responses, such as changes in antioxidant enzyme activity and the accumulation of osmotic regulatory substances. However, the discovery and functional verification of their intrinsic molecular regulatory mechanisms, especially key stress-resistance genes, remain relatively scarce, resulting in a lack of effective genetic resources for improving heat-resistant varieties.

[0004] Transcription factors play a central regulatory role in plant stress responses, among which the bZIP (basic leucine zipper) family has attracted widespread attention due to its involvement in stress signal transduction and the regulation of stress-resistance gene expression. Previous studies have shown that plant bZIP transcription factors can enhance plant tolerance to high-temperature stress by binding to cis-acting elements in the promoters of downstream stress-resistance genes, thereby activating the expression of related genes such as heat shock proteins (HSPs) and antioxidant enzymes. For example, some bZIP members in Arabidopsis thaliana significantly improve the plant's heat resistance by regulating heat stress response pathways. However, the function of bZIP genes in different plants exhibits species specificity. The heat-resistance function of bZIP family members in blueberry remains unclear, especially regarding the cloning, expression patterns, and specific mechanisms of action of key members in resisting high-temperature stress.

[0005] In current technologies, the application of heat-resistant genes in plants is mostly concentrated on model plants or major crops. There is insufficient development of heat-resistant gene resources for specialty fruit trees such as blueberries, and a lack of functional genes that can be directly used for genetic improvement. Therefore, isolating and identifying bZIP genes with heat-resistant functions from blueberries, clarifying their regulatory mechanisms, and applying them to improve plant heat resistance is of significant theoretical and practical importance for enhancing the high-temperature adaptability of blueberries and other crops and ensuring the sustainable development of the industry. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a blueberry VcbZIP-1 gene. Another technical problem to be solved by the present invention is to provide an expression protein of the blueberry VcbZIP-1 gene. A further technical problem to be solved by the present invention is to provide an application of the blueberry VcbZIP-1 gene for regulating the blueberry's resistance to high-temperature stress and its response to exogenous SA.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A blueberry VcbZIP-1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The amino acid sequence of the expressed protein of the blueberry VcbZIP-1 gene is shown in SEQ ID NO.2.

[0010] Recombinant vector containing the blueberry VcbZIP-1 gene and host cell.

[0011] A primer pair for cloning the blueberry VcbZIP-1 gene, the sequence of which is:

[0012] VcbZIP-1-F: 5'-ATGCTTTCATCAGATGTTGCA-3',

[0013] VcbZIP-1-R: 5'-GTGAAGCAGATTAGTGGAAGT-3'.

[0014] Application of the blueberry VcbZIP-1 gene in regulating plant resistance to high temperature stress.

[0015] Furthermore, the high temperature is 45°C.

[0016] Furthermore, the plant is blueberry or Arabidopsis thaliana.

[0017] Application of the blueberry VcbZIP-1 gene in regulating the plant's response to exogenous SA.

[0018] Furthermore, the concentration of the exogenous SA is 200 μmol / L.

[0019] Furthermore, the plant is blueberry or Arabidopsis thaliana.

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

[0021] 1) This invention discloses for the first time the nucleotide sequence of the blueberry VcbZIP-1 gene (as shown in SEQ ID NO.1) and the amino acid sequence of its expressed protein (as shown in SEQ ID NO.2). Furthermore, it provides specific primer pairs for cloning this gene, enabling efficient and accurate cloning of the target gene and facilitating related research.

[0022] 2) In this invention, the blueberry VcbZIP-1 gene was transferred into Arabidopsis thaliana. Three Arabidopsis lines with high VcbZIP-1 expression (bzip-3, bzip-5, and bzip-6) were selected. Under 45℃ high-temperature treatment, the expression of heat stress-related genes was significantly upregulated. The expression level of the ERECTA gene in the bzip-6 line was approximately 480 times that of the wild-type (WT). After 24 hours of high-temperature treatment, the SPAD value of WT plant leaves decreased by 24.8%, and the N content decreased by 23.3%. In contrast, among the transgenic lines, the SPAD value of bzip-3 decreased by 23.1%, and the N content decreased by 20.2%. Although the decrease was slightly higher in some lines, combined with the phenotypic characteristics, its heat resistance was superior to that of WT. Furthermore, changes in indicators such as POD activity and MDA content in transgenic Arabidopsis after high-temperature treatment indicate that its antioxidant system is better able to cope with the damage caused by high temperature. For example, the POD activity of the bzip-heat group was 493.46±22.47 U / g fresh weight, which was higher than that of the WT-heat group (368.80±9.76 U / g fresh weight).

[0023] 3) In this invention, the VcbZIP-1 gene was transferred into blueberries. Two transgenic blueberry lines (bZIP-3 and bZIP-4) expressing high levels of VcbZIP-1, after treatment at 45℃, still showed 10-20% better leaf condition compared to the control group (CK). Physiologically, the CAT activity in the bZIP-heat group was 24.32±2.71 U / g fresh weight, higher than the 21.21±1.30 U / g fresh weight in the CK-heat group, indicating stronger hydrogen peroxide scavenging ability and better stress resistance under high temperatures.

[0024] 4) In this invention, after treating transgenic Arabidopsis thaliana lines with exogenous SA (200 μmol / L), the SOD activity of the transgenic Arabidopsis thaliana was significantly increased. The SOD activity of the bzip-SA group was 469.03 ± 0.68 U / g fresh weight, which was higher than that of the WT-SA group (457.29 ± 2.03 U / g fresh weight), indicating that its antioxidant capacity was enhanced under SA-mediated. At the same time, after 6 h of SA treatment, the expression level of VcbZIP-1 gene in bzip-6 reached 553.5, which was 138.2 times that in WT at the same time point, demonstrating the strong response of the gene to SA.

[0025] 5) In this invention, after treating transgenic blueberry lines with exogenous SA (200 μmol / L), the POD activity of transgenic blueberries was significantly enhanced. The POD activity of the bZIP-SA group was 146.00±12.79 U / g fresh weight, which was much higher than that of the CK-SA group (67.99±4.34 U / g fresh weight). Moreover, after 6 h of SA treatment, the expression level of VcbZIP-1 gene in bZIP-4 was as high as 167.3, which was 111.5 times that in CK at the same time. This indicates that the gene can effectively regulate the response of blueberries to SA and enhance their stress-related physiological activities. Attached Figure Description

[0026] 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);

[0027] Figure 2 Phylogenetic tree diagram of the VcbZIP family;

[0028] Figure 3 A graph showing gene expression levels in different tissues and developmental stages of blueberries;

[0029] Figure 4 The figure shows the results of the expression analysis of heat stress-related genes in transgenic Arabidopsis thaliana.

[0030] Figure 5 Phenotypic diagram of transgenic Arabidopsis thaliana under high temperature treatment (A), phenotypic diagram of transgenic Arabidopsis thaliana under exogenous SA treatment (B), and diagram showing the changes in SPAD and nitrogen content in leaves of transgenic Arabidopsis thaliana (C).

[0031] Figure 6 This figure shows the expression of the VcbZIP-1 gene in transgenic Arabidopsis thaliana lines after high temperature and exogenous SA treatment.

[0032] Figure 7 A graph showing the expression levels of heat stress-related genes in positive seedlings of transgenic blueberries;

[0033] Figure 8This figure shows the expression of the VcbZIP-1 gene in transgenic blueberry lines after high temperature and exogenous SA treatment. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] The quantitative sample materials of different blueberry tissues used in this application were collected from healthy 'O'Neill' cultivars cultivated in the greenhouse of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. Tissue culture seedlings were collected from the tissue culture laboratory of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, with a seedling age of one month, and the variety was 'Tianhou'. The Arabidopsis thaliana was a laboratory-preserved Colombian wild type.

[0037] Example 1

[0038] 1. Cloning and sequence analysis of the blueberry VcbZIP-1 gene

[0039] The open reading frame (ORF) of the candidate gene VcbZIP-1 (gene-Vadar_025320) was obtained using BioXM 2.6 software. It was corrected using the existing three-generation full-length transcriptome 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.

[0040] The primer sequences are shown below:

[0041] VcbZIP-1-F: 5'-ATGCTTTCATCAGATGTTGCA-3',

[0042] VcbZIP-1-R: 5'-GTGAAGCAGATTAGTGGAAGT-3';

[0043] M13 General-F:5'-TGTAAAACGACGGCCAGT-3',

[0044] M13 General-R:5'-CAGGAAACAGCTATGACC-3'.

[0045] 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.

[0046] The final sequencing yielded the nucleotide sequence of the blueberry VcbZIP-1 gene, as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein, as shown in SEQ ID NO.2.

[0047] 2. Analysis of the physicochemical properties of proteins

[0048] To further investigate whether similar mechanisms of action exist among its related family members, 32 VcbZIP family gene members were screened from blueberry leaf transcriptome data. The minimum free energy (MFE) and centroid secondary structure of the mRNAs of these 32 VcbZIP gene family members were analyzed using RNAfold software. The results showed that VcbZIP-5 had the highest total free energy (-121.80), while VcbZIP-26 had the lowest (-776.60).

[0049] 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 / ).

[0050] The physicochemical properties analysis of the proteins showed that the relative molecular weight of the VcbZIP gene family ranged from 22.8 to 94.9 kDa, the theoretical isoelectric point ranged from 4.80 to 9.83, and the instability coefficient ranged from 38.86 to 71.40. Subcellular localization showed that most genes in the VcbZIP gene family were located in the nucleus, but VcbZIP-29 was located in the cytoplasm.

[0051] The results are as follows Figure 1 As shown, the results indicate that all genes in the VcbZIP family contain the structural motif EKADPKRQRRMJKNRESARRSRERKQAYVQELE. Figure 1 (A); 29 genes in the VcbZIP family simultaneously contain the bZIP domain, and 5 genes simultaneously contain the DOG1 domain (A). Figure 1 Of the proteins translated from the VcbZIP family genes, α-helices account for 17.66-65.98%, β-sheets account for 0-11.08%, and random coils account for 29.91-75.54%.

[0052] 3. Phylogenetic tree analysis

[0053] Using existing blueberry heat-resistant transcriptome data from our research group, we investigated the phylogenetic relationships of bZIPs and performed phylogenetic tree analysis on 32 VcbZIP family genes in the blueberry transcriptome and bZIP family gene sequences in Arabidopsis and strawberry.

[0054] The results are as follows Figure 2 As shown, the 32 VcbZIP genes belong to 11 subfamilies, and VcbZIP-1 belongs to the bZIP S subfamily.

[0055] 4. Detection of expression levels of key genes in different tissues of blueberries

[0056] 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.

[0057] The results are as follows Figure 3As shown, VcbZIP-1 expression was highest in the fourth stage of fruit development; VcbZIP-1 expression was highest in blueberry leaves in June, followed by September. In summary, the expression levels of VcbZIP-1 in leaves and stem segments were much higher than those in fruit.

[0058] Example 2

[0059] 1. Constructing a blueberry VcbZIP-1 gene overexpression vector

[0060] The bZIP 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 centrifuge tubes, gently mixed, and incubated on ice for 5 min. After the ice bath, the centrifuge tubes were 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 tubes, and the tubes were incubated at 28℃ and 200 rpm for 2 h. The tubes were 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 dishes were 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.

[0061] 2. Genetic transformation of Arabidopsis thaliana

[0062] Before infection, select robust Arabidopsis thaliana plants in full bloom, water them thoroughly, and remove the pods. Immerse the inflorescences in the resuspension solution, gently shake, and remove after 1 minute. Shake off excess resuspension solution from the Arabidopsis plants, wrap them with plastic wrap, place them on their sides in a tray, and incubate in the dark for 24 hours. Then remove the plastic wrap and incubate upright for 7 days before a second infection. Once the Arabidopsis thaliana has matured, collect all mature seeds, designated as the T0 generation. Take a small amount of T0 generation transgenic Arabidopsis thaliana seeds, sterilize and disinfect them, and sow them in MS solid medium containing 35 mg / L Kan. Select robust Arabidopsis thaliana seedlings and transplant them into the medium for further cultivation. Once the Arabidopsis thaliana has matured, collect all seeds, designated as the T1 generation. Use the same method to screen for resistance in Arabidopsis thaliana and collect seeds, designated as the T2 generation. The same method was used to screen for resistance in Arabidopsis thaliana, and T3 generation seeds were obtained. The T3 generation seeds were then sown, and their growth was observed and recorded.

[0063] 3. Quantitative detection of transgenic plants

[0064] Total RNA was extracted from Arabidopsis transgenic plants according to the instructions of the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novizan Biotechnology Co., Ltd.; RC411). 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 Arabidopsis AtUBC gene (GenBank: AT5g25760) 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 system 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 Arabidopsis plants were analyzed, and Arabidopsis lines with high VcbZIP-1 expression were screened. Literature review on Arabidopsis responses to heat stress revealed that McMYB52, CHIP, SIL, HSF-16, ERECTA, 13MBF-1L, and ZmCIPH play important roles in Arabidopsis heat stress response. The expression of these heat stress-related genes in Arabidopsis lines with high VcbZIP-1 expression was analyzed. Primer sequences are shown below.

[0065] VcbZIP-1-qRT-F: 5'-CCGGAGGTCTCGGATGCGTA-3',

[0066] VcbZIP-1-qRT-R: 5'-GGCTCCCTAAGCGGAGGTCA-3';

[0067] AtUBC-qRT-F: 5'-CTGCGACTCAGGGAATCTTCTAA-3',

[0068] AtUBC-qRT-R: 5'-TTGTGCCATGAATTGAACCC-3';

[0069] McMYB52-qRT-F:

[0070] 5'-TCTCTGTCGACTCTAGAGGATCCATGTGTACCAGAGGTCAC-3',

[0071] McMYB52-qRT-R:

[0072] 5’-AACGATCGGGGAAATTCGAGCTCTTAGGTAGAGTTTCCGAC-3’;

[0073] CHIP-qRT-F: 5’-ACTGAAAACATCGGCAACAAC-3’,

[0074] CHIP-qRT-R: 5’-ATGTGAGGCACCACTTGAATC-3’;

[0075] SIL-qRT-F: 5’-GGTGGTTTGTGTTTTGGGGC-3’,

[0076] SIL-qRT-R: 5’-AGTCGAGTCACTTTGCAGGC-3’;

[0077] HSF-16-qRT-F: 5’-CCCTACGTTTCTGTATCAGCTACT-3’,

[0078] HSF-16-qRT-R: 5’-CCTCTTCTTCCTTCATATTCCTCT-3’;

[0079] ERECTA-qRT-F: 5’-CTGCTCCACTTCCATCTAC-3’,

[0080] ERECTA-qRT-R: 5’-ACTGCCTCATCACTCACT-3’;

[0081] 13MBF-1L-qRT-F: 5’-CAATGAGAAGCCTCAAGTGATCCA-3’,

[0082] 13MBF-1L-qRT-R: 5’-GTTTTTACCGACAAGGACAAAGCT-3’;

[0083] ZmCIPKHT-qRT-F: 5’-CCATAACCAGTCACGTCAAAA-3’,

[0084] ZmCIPKHT-qRT-R: 5’-GGTAACCTGAAACACAATTCCG-3’.

[0085] The results are as Figure 4As shown, based on the gene expression results of different Arabidopsis thaliana lines with different genotypes, three high-expression lines of VcbZIP-1 (bzip-3, bzip-5, and bzip-6) were selected from high to low expression levels. Among them, the expression level of bzip-6 was the highest, at 306.8. These three high-expression Arabidopsis thaliana lines were used for subsequent 45℃ high-temperature experiments. Compared with WT Arabidopsis thaliana, heat stress-related genes were significantly upregulated in VcbZIP-1 transgenic Arabidopsis thaliana. The expression levels of McMYB52, CHIP, SIL, HSF-16, ERECTA, 13MBF-1L, and ZmCIPH were all the highest in the bzip-6 line. The expression level of the ERECTA gene in the bzip-6 line was approximately 480 times that in the WT line.

[0086] 4. Phenotypic observation and physiological index determination of transgenic lines under high temperature stress

[0087] Arabidopsis thaliana lines (bzip-3, bzip-5, and bzip-6) expressing VcbZIP-1 were subjected to high-temperature stress at 45℃ and treatment with 200 μmol / L exogenous SA. Plant phenotypes at 0 h, 6 h, and 24 h after high-temperature and exogenous SA treatment were observed and recorded. Changes in SPAD values ​​and N content in leaves at 0 h and 24 h after high-temperature treatment 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 nitrocyanin tetrazolium method; and hydrogen peroxide (H2O2) content and catalase (CAT) and peroxidase (POD) activities were determined using kits from Nanjing Jiancheng Biotechnology Co., Ltd.

[0088] The results are as follows Figure 5 As shown, after 6 hours of high-temperature treatment, compared to WT Arabidopsis, bzip-3, bzip-5, and bzip-6 plants began to show obvious yellowing and dehydration of leaves. After 24 hours of high-temperature treatment, both VcbZIP-1 and WT plants showed varying degrees of leaf yellowing, wilting, and dehydration, with bzip-3, bzip-5, and bzip-6 showing greater yellowing than WT. Figure 5 (A). Under SA treatment, there were no significant changes in any Arabidopsis lines (A). Figure 5The results of SPAD values ​​and N content changes in leaves of different Arabidopsis thaliana strains showed that after 24 hours of high-temperature treatment, both SPAD values ​​and N content in the leaves of VcbZIP-1 transgenic plants and WT plants decreased. Specifically, the SPAD value of WT plant leaves decreased by 24.8%, and the N content decreased by 23.3%; the SPAD values ​​of leaves in different strains of VcbZIP-1 transgenic Arabidopsis thaliana decreased by 23.1%, 29.2%, and 34.7%, respectively, and the N content decreased by 20.2%, 26.8%, and 35.5%, respectively. Figure 5 (C).

[0089] The results are shown in Table 1. High-temperature treatment decreased SOD activity in both WT and bZIP Arabidopsis leaves, while exogenous SA treatment increased SOD activity. The changes in CAT activity were the opposite of SOD; high-temperature treatment increased CAT activity in both WT and bZIP Arabidopsis, while exogenous SA treatment decreased it. Both high-temperature and exogenous SA treatments increased POD activity, MDA content, and H2O2 content in WT and bZIP Arabidopsis. The POD activity and MDA content in the high-temperature treatment were significantly higher than those in the exogenous SA treatment, while the H2O2 content in the high-temperature treatment was significantly lower than that in the exogenous SA treatment.

[0090] Table 1. Changes in antioxidant physiological parameters of transgenic Arabidopsis thaliana after high temperature and exogenous SA treatment.

[0091]

[0092] Note: Lowercase letters indicate significance test results of p < 0.05, and uppercase letters indicate significance test results of p < 0.01.

[0093] 5. Expression of the VcbZIP-1 gene after high temperature and exogenous SA treatment

[0094] Gene quantification analysis was performed on leaf samples of various Arabidopsis thaliana strains treated with high temperature and exogenous SA for 0, 6, 12, and 24 hours.

[0095] The results are as follows Figure 6As shown, under high-temperature treatment, the expression level of VcbZIP-1 gene in WT increased at 6 h, decreased at 12 h, and reached its maximum at 24 h. Under exogenous SA treatment, the expression level of VcbZIP-1 gene in WT increased with increasing treatment time. The expression trend of VcbZIP-1 gene was consistent among different VcbZIP-1 transgenic Arabidopsis lines. The expression level of VcbZIP-1 gene reached its maximum at 6 h of high-temperature and SA treatment in bzip-3, bzip-5, and bzip-6 lines, and then decreased with the extension of treatment time. Among them, after 6 h of high-temperature treatment, the expression level of VcbZIP-1 gene in bzip-5 reached as high as 2671.7, which was 867.2 times that in WT at the same time. After 6 h of SA treatment, the expression level of VcbZIP-1 gene in bzip-6 reached as high as 553.5, which was 138.2 times that in WT at the same time. In summary, the expression patterns of the VcbZIP-1 gene under high temperature and SA treatments are similar. Specifically, the expression level of the VcbZIP-1 gene in WT plants under both high temperature and SA treatments is continuously increased. Under both high temperature and SA treatments, the expression level of the VcbZIP-1 gene increases within 6 hours and then decreases.

[0096] Example 3

[0097] 1. Genetically transformed blueberries

[0098] 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 four VcbZIP-1 genotype blueberry plants obtained were tested for positive seedlings.

[0099] 2. Quantitative detection of transgenic plants

[0100] 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 VcbZIP-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 VcbZIP-1 expression was analyzed. Primer sequences are shown below:

[0101] VcbZIP-1-qRT-F: 5'-CCGGAGGTCTCGGATGCGTA-3',

[0102] VcbZIP-1-qRT-R: 5'-GGCTCCCTAAGCGGAGGTCA-3';

[0103] GADPH-qRT-F: 5'-CTGCGACTCAGGGAATCTTCTAA-3',

[0104] GADPH-qRT-R: 5'-TTGTGCCATGAATTGAACCC-3';

[0105] ATPase-qRT-F: 5'-AATGCCGGACACGCACAGTT-3',

[0106] ATPase-qRT-R: 5'-TGAGCCCGTCGGATCACCTT-3';

[0107] bZIP-2-qRT-F:5’-GGCATATCACCACAACCCGCAT-3’,

[0108] bZIP-2-qRT-R:5’-CCCTTGCACTTTCCCTTTGGCT-3’;

[0109] bZIP-3-qRT-F:5’-TGCGTTTGGTCACGCACCAT-3’,

[0110] bZIP-3-qRT-R:5’-TTGCCGAAGCATGGCCGATT-3’;

[0111] HSF A-3-qRT-F:5’-GGCCAGACGAGTGGGACTCA-3’,

[0112] HSF A-3-qRT-R:5’-GCCTCTGCTCTGCTGCTTGG-3’;

[0113] HSP20-1-qRT-F:5’-CGGGACATTGCTCCGTTCCC-3’,

[0114] HSP20-1-qRT-R:5’-CCGGCGTTTCCTTCCAGTCC-3’;

[0115] HSP20-2-qRT-F:5’-ACTCGCGTGGACTGGAAGGA-3’,

[0116] HSP20-2-qRT-R:5’-GCGGTGCCACGTATCGTTCT-3’;

[0117] HSP20-3-qRT-F:5’-CGACGAGGCAACGACAGCTT-3’,

[0118] HSP20-3-qRT-R:5’-CCGGCGTTTCCTTCCAGTCC-3’;

[0119] HSF C-1-qRT-F:5’-CCGCCGTCGGTTATGAGCAG-3’,

[0120] HSF C-1-qRT-R:5’-CACCATGGCTGTGCCGGAAA-3’;

[0121] HSP70-qRT-F:5’-CCGCGTTCAGCTGTGTAGGG-3’,

[0122] HSP70-qRT-R: 5'-CGCCAATTAGGCGCTCGGTA-3';

[0123] WRKY-qRT-F: 5'-AGCCGAAATTCGCTTTTCCAGACAA-3',

[0124] WRKY-qRT-R: 5'-TGGACTGGCGTTGCACTTGTT-3'.

[0125] The results are as follows Figure 7 As shown, two transgenic blueberry lines with high VcbZIP-1 expression levels (bZIP-3 and bZIP-4) were obtained, with the bZIP-4 line exhibiting the highest VcbZIP-1 gene expression level (2.06). Based on the gene expression levels of different transgenic blueberry lines within each genotype, from highest to lowest, two high-expression VcbZIP-1 blueberry lines (bZIP-3 and bZIP-4) were selected for subsequent 45℃ high-temperature experiments. The expression levels of heat stress-related genes ATPase, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, and WRKY in transgenic blueberries were all lower than in the control group (CK); while the expression levels of bZIP-2 and bZIP-3 in transgenic blueberries were higher than in the control group (CK).

[0126] 3. Phenotypic observation and physiological index determination of transgenic lines under high temperature stress

[0127] Blueberry lines (bZIP-3 and bZIP-4) expressing VcbZIP-1 were subjected to high-temperature stress at 45℃ and treatment with 200 μmol / L exogenous SA. Plant phenotypes at 0h, 6h, and 24h after high-temperature and exogenous SA treatment were observed and recorded. Changes in SPAD values ​​and N content in leaves at 0h and 24h after high-temperature treatment 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 method (TBA); superoxide dismutase (SOD) activity was determined using the nitrocyanotetrazole method; and hydrogen peroxide (H2O2) content and catalase (CAT) and peroxidase (POD) activities were determined using kits from Nanjing Jiancheng Biotechnology Co., Ltd.

[0128] The results are shown in Table 2. After 24 hours of exogenous SA treatment, no significant changes were observed in the phenotypes of the blueberry plants. However, after high-temperature treatment, both the VcbZIP-1 transgenic plants and the CK plants exhibited varying degrees of leaf yellowing, wilting, and dehydration. However, compared to the CK plants, 10-20% of the leaves in the VcbZIP-1 transgenic plants remained in good condition. Analysis of the antioxidant enzyme activities and MDA and H2O2 contents in the leaves under high-temperature and SA treatments showed that SOD activity was significantly reduced in both CK and VcbZIP-1 blueberries after high-temperature and SA treatments. Compared to the high-temperature treatment group of VcbZIP-1 blueberries, the SOD activity in the SA treatment group was significantly reduced. After high-temperature and SA treatments, CAT activity was significantly reduced in CK, while CAT activity was significantly increased in VcbZIP-1 blueberries. In VcbZIP-1 blueberries, CAT activity was significantly increased in the SA treatment group compared to the high-temperature treatment group. After high temperature and SA treatment, the POD activity in both CK and VcbZIP-1 blueberries significantly increased, and the POD activity in the SA treatment group was significantly increased compared to the high temperature treatment group. After high temperature and SA treatment, the MDA content in both CK and VcbZIP-1 blueberries significantly increased, and the MDA content in the SA treatment group was significantly increased compared to the high temperature treatment group. After high temperature and SA treatment, the H2O2 content in both CK and VcbZIP-1 blueberries significantly increased. Therefore, under high temperature and SA treatment, the SOD activity of all transgenic blueberry lines significantly decreased, while the POD activity, CAT activity, MDA content, and H2O2 content significantly increased.

[0129] Table 2. Changes in antioxidant physiological indicators of transgenic blueberries after high temperature and exogenous SA treatment.

[0130]

[0131]

[0132] Note: Lowercase letters indicate significance test results of p < 0.05, and uppercase letters indicate significance test results of p < 0.01.

[0133] 4. Expression of the VcbZIP-1 gene after high temperature and exogenous SA treatment

[0134] 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.

[0135] The results are as follows Figure 8As shown, under high-temperature treatment, the expression level of the VcbZIP-1 gene in CK first increased, reaching a maximum at 12 h, and then decreased. Under SA treatment, the expression level of the VcbZIP-1 gene in CK increased with the extension of treatment time. The expression trend of the VcbZIP-1 gene was consistent among different VcbZIP-1 transgenic blueberry lines. In the bZIP-3 and bZIP-4 blueberry lines, the expression level reached its maximum after 6 h of high-temperature and SA treatment, and then decreased with the extension of treatment time. Specifically, after 6 h of high-temperature treatment, the VcbZIP-1 gene expression level in bZIP-4 reached 101.5, which was 26.7 times that in CK at the same time. After 6 h of SA treatment, the VcbZIP-1 gene expression level in bZIP-4 reached 167.3, which was 111.5 times that in CK at the same time. In conclusion, the expression level of the VcbZIP-1 gene in transgenic blueberries showed a consistent trend under high-temperature and SA treatments.

[0136] 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. A blueberry VcbZIP-1 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expression protein of the blueberry VcbZIP-1 gene according to claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A recombinant vector and host cell containing the blueberry VcbZIP-1 gene as described in claim 1.

4. A primer pair for cloning the blueberry VcbZIP-1 gene of claim 1, characterized in that, The sequences of the primer pair are as follows: VcbZIP-1-F: 5'-ATGCTTTCATCAGATGTTGCA-3', VcbZIP-1-R: 5'-GTGAAGCAGATTAGTGGAAGT-3'.

5. The application of the blueberry VcbZIP-1 gene as described in claim 1 in regulating the plant's resistance to high-temperature stress.

6. The application according to claim 6, characterized in that, The high temperature is 45°C.

7. The application according to claim 5, characterized in that, The plant in question is either blueberry or Arabidopsis thaliana.

8. The application of the blueberry VcbZIP-1 gene as described in claim 1 in regulating the plant's ability to respond to exogenous SA.

9. The application according to claim 6, characterized in that, The concentration of the exogenous SA was 200 μmol / L.

10. The application according to claim 8, characterized in that, The plant in question is either blueberry or Arabidopsis thaliana.

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