Blueberry high-temperature-resistant gene VcFDH-1 as well as expression protein and application thereof

Abstract

The invention discloses a blueberry high-temperature-resistant gene VcFDH-1 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 high-temperature-resistant gene VcFDH-1 disclosed by the invention is as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the blueberry high-temperature-resistant gene VcFDH-1 is as shown in SEQ ID NO.2. The blueberry VcFDH-1 gene is transferred into blueberries, and the result shows that after a VcFDH-1 gene transferred blueberry strain is subjected to high-temperature treatment, the damage such as leaf wilting and withering is obviously reduced; the stress resistance of the blueberries in a high-temperature environment is enhanced; 200 [mu] mol / L exogenous SA treatment can be responded, and the high temperature stress relieving effect of the blueberries through the exogenous SA is enhanced; the expression of a heat stress related gene can be regulated and controlled. The invention provides a theoretical basis for the research of a blueberry heat resistance molecular mechanism, and also provides a key gene resource for the cultivation of a new variety of heat-resistant blueberries through genetic engineering.

Description

Technical Field

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

[0002] Blueberries, as a newly emerging fruit tree with both high nutritional and economic value, are extremely sensitive to environmental temperature during their growth and development. In recent years, global warming has led to frequent extreme heat events. In southern regions, prolonged high temperatures in summer often cause blueberry plants to suffer from leaf wilting, stunted fruit development, and reduced yields, severely restricting the large-scale development of the blueberry industry. Among these varieties, southern highbush blueberries such as 'O'Neill,' while widely cultivated due to their excellent fruit quality, have relatively weak heat tolerance and are prone to physiological metabolic disorders in environments above 35℃, becoming a key bottleneck limiting their cultivation expansion.

[0003] Plants have developed complex high-temperature response mechanisms through long-term evolution, initiating defense pathways such as antioxidant systems and heat shock protein synthesis by regulating the expression of specific genes. Formate dehydrogenase (FDH), a key enzyme involved in energy metabolism and stress response, plays an important role in plant responses to oxidative stress and the maintenance of cellular homeostasis. Previous studies have shown that members of the FDH gene family participate in high-temperature stress signal transduction in model plants such as Arabidopsis and rice, but their function in blueberry remains unclear, particularly regarding the heat tolerance regulation mechanism mediated by FDH, which lacks systematic research.

[0004] Currently, improving the heat tolerance of blueberries mainly relies on traditional breeding methods, but these methods have limitations such as long cycles, complex genetic backgrounds, and low improvement efficiency. Genetic engineering technology provides an efficient pathway for targeted improvement of crop resistance; by cloning key stress-resistance genes and achieving their precise expression, new stress-resistant varieties can be rapidly bred. However, as a woody plant, blueberry's genetic transformation system is still imperfect, and it lacks functionally validated key heat-resistance genes, resulting in slow progress in related molecular breeding research.

[0005] Furthermore, the physiological damage caused by the accumulation of reactive oxygen species (ROS) in blueberries under high-temperature stress, leading to membrane lipid peroxidation and protein denaturation, is the core reason for the decline in their heat resistance. Existing research largely focuses on the alleviating effects of exogenous spraying of antioxidants (such as salicylic acid), while the underlying molecular regulatory network, especially how key genes respond to high-temperature stress by regulating downstream targets such as antioxidant enzyme systems and heat shock proteins, remains unclear.

[0006] Therefore, identifying key heat-resistant genes in blueberries and analyzing their expression patterns and regulatory mechanisms under high-temperature stress can not only provide a theoretical basis for understanding the molecular basis of blueberry heat resistance, but also provide key gene resources and technical support for breeding new heat-resistant blueberry varieties through genetic engineering, which is of great significance for promoting the sustainable development of the blueberry industry. Summary of the Invention

[0007] 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 VcFDH-1. Another technical problem to be solved by the present invention is to provide an expression protein of the blueberry heat-resistant gene VcFDH-1. A further technical problem to be solved by the present invention is to provide an application of the blueberry heat-resistant gene VcFDH-1 for regulating the blueberry's resistance to high-temperature stress and its response to exogenous SA.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A heat-resistant gene for blueberries, VcFDH-1, has the nucleotide sequence shown in SEQ ID NO.1.

[0010] The amino acid sequence of the protein expressing the blueberry heat-resistant gene VcFDH-1 is shown in SEQ ID NO.2.

[0011] Recombinant expression vector containing the blueberry heat-resistant gene VcFDH-1 and host cells.

[0012] Application of the blueberry heat-resistant gene VcFDH-1 in regulating the blueberry's resistance to stress under high-temperature conditions.

[0013] Furthermore, the high-temperature environment temperature is 45°C.

[0014] Application of the blueberry heat-resistant gene VcFDH-1 in regulating the blueberry's response to exogenous SA.

[0015] Furthermore, the concentration of the exogenous SA is 200 μmol / L.

[0016] A primer pair for cloning the blueberry heat-resistant gene VcFDH-1, the sequence of which is:

[0017] VcFDH-1-F: 5'-ATGGCGATGGTGATGAAGC-3',

[0018] VcFDH-1-R: 5'-ACGGTACTGGGGAGCTAGT-3'.

[0019] Application of the blueberry heat-tolerant gene VcFDH-1 in regulating the expression of heat stress-related genes.

[0020] Furthermore, the heat stress-related genes include ATPase, bZIP-2, bZIP-3, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, and WRKY.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) This invention is the first to clone the blueberry heat-resistant gene VcFDH-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 VcFDH-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 transfected with the VcFDH-1 gene still have 10-20% of their leaves remaining in good condition compared to the control group, indicating that this gene can significantly improve the blueberry's tolerance to high temperatures and reduce damage such as leaf wilting and death caused by high temperatures.

[0023] 2) This invention introduces the VcFDH-1 gene into blueberries, which effectively regulates the blueberry's response to exogenous salicylic acid (SA, concentration 200 μmol / L). Results from the examples show that after treatment with exogenous SA, the SOD activity in the SA-treated group significantly increased, and the MDA content significantly decreased compared to the high-temperature treatment group, indicating that this gene can enhance the effect of exogenous SA in alleviating high-temperature stress in blueberries.

[0024] 3) This invention transforms the VcFDH-1 gene into blueberries, which can regulate the expression of various heat stress-related genes, including ATPase, bZIP-2, bZIP-3, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, and WRKY. Results from the examples show that in the FDH-8 line transformed with the VcFDH-1 gene, the expression level of the HSP20-1 gene is approximately 100 times that of the control group. Attached Figure Description

[0025] 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);

[0026] Figure 2 Sequence alignment diagram of VcFDH family members, key heat-resistant genes in blueberries;

[0027] Figure 3 Phylogenetic tree diagram of the VcbZIP family;

[0028] Figure 4 A graph showing gene expression levels in different tissues and developmental stages of blueberries;

[0029] Figure 5 A graph showing the expression levels of heat stress-related genes in positive seedlings of transgenic blueberries;

[0030] Figure 6 This figure shows the expression of the VcFDH-1 gene in transgenic blueberry lines after high temperature and exogenous SA treatment. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Example 1

[0035] 1. Cloning and sequence analysis of the blueberry VcFDH-1 gene

[0036] The open reading frame (ORF) of the candidate gene VcFDH-1 (gene-Vadar_021194) was obtained using BioXM 2.6 software analysis. 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-speed, 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.

[0037] The primer sequences are shown below:

[0038] VcFDH-1-F: 5'-ATGGCGATGGTGATGAAGC-3',

[0039] VcFDH-1-R: 5'-ACGGTACTGGGGAGCTAGT-3';

[0040] M13 General-F:5'-TGTAAAACGACGGCCAGT-3',

[0041] M13 General-R:5'-CAGGAAACAGCTATGACC-3'.

[0042] 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.

[0043] The final sequencing yielded the nucleotide sequence of the blueberry heat-resistant gene VcFDH-1, as shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein, as shown in SEQ ID NO.2.

[0044] 2. Analysis of the physicochemical properties of proteins

[0045] To further investigate whether similar mechanisms of action exist among related family members, three VcFDH family gene members—gene-Vadar_021194 (VcFDH-1), gene-Vadar_030653 (VcFDH-2), and gene-Vadar_027852 (VcFDH-3)—were screened from blueberry leaf transcriptome data. The minimum free energy (MFE) and centroid secondary structure of the mRNAs of these three VcFDH gene family members were analyzed using RNAfold software. The results showed that VcFDH-1 had the highest total free energy (MFE) at -360.94, while VcFDH-2 had the lowest at -469.68. The NetPhos online tool was used to predict potential phosphorylation sites, and the NetNGlyc-1.0 online tool was used to predict glycosylation sites. The results revealed that VcFDH-1 had 25 phosphorylation sites and 1 glycosylation modification site with a probability of 0.7644.

[0046] 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 / ).

[0047] The physicochemical properties analysis of the proteins showed that the relative molecular weights of the VcFDH family genes were 43.0, 55.2, and 41.9 kDa, respectively, with theoretical isoelectric points ranging from 6.64 to 8.57 and instability coefficients ranging from 26.92 to 32.88. Subcellular localization showed that VcFDH-1 and VcFDH-2 were located in mitochondria, while VcFDH-3 was located in chloroplasts.

[0048] The results are as follows Figure 1 As shown, VcFDH-1 contains 11 motifs, VcFDH-2 contains 13 motifs, and VcFDH-3 contains 9 motifs. Seven of these motifs are shared across the VcFDH gene family (motifs 1, 2, 3, 4, 5, 6, and 8). Conserved domains of genes related to the VcFDH gene family were analyzed using NCBI's CD searc online tool. The results showed that all three genes in the VcFDH family contain the PNL03139 domain, and the RPA_2b-aaRSs_OBF_like domain is specific to the FDH-2 sequence.

[0049] 3. Phylogenetic tree analysis

[0050] 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 MEGA11 software with a neighbor-joining (NJ) bootstrap test run of 1000 times.

[0051] The results are as follows Figure 2 As shown, PLN03139superfamily and RPA_2b-aaRSs_ORF_like superfamily are the main structural domains in the VcFDH family genes.

[0052] The results are as follows Figure 3 As shown, VcFDH-1 is most closely related to VcFDH-1 (Red Horse Silver Flower R. vialii), while it is more distantly related to FDH-2 (Cornus florida) and FDH-3 (Cornus florida); VcFDH-2 and VcFDH-3 are most closely related to FDH-1 (Red Horse Silver Flower R. vialii), while they are more distantly related to FDH (Heveabrasiliensis).

[0053] 4. Detection of expression levels of key genes in different tissues of blueberries

[0054] 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.

[0055] The results are as follows Figure 4 As shown, VcFDH-1 expression levels were higher in both green and red blueberries; the highest expression level of VcFDH-1 was observed in blueberry leaves in September, followed by June. In summary, the expression levels of VcFDH-1 did not differ significantly across tissue stages.

[0056] Example 2

[0057] 1. Constructing an overexpression vector for the blueberry heat-resistant gene VcFDH-1

[0058] The FDH 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.

[0059] 2. Genetically transformed blueberries

[0060] 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' of the same period with no vector / no genetic transformation was used as the control group. The 10 VcFDH-1 genotype blueberry plants obtained were tested for positive seedlings.

[0061] 2. Quantitative detection of transgenic plants

[0062] 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 VcFDH-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 VcFDH-1 expression was analyzed. Primer sequences are shown below:

[0063] VcFDH-1-qRT-F:5’-ACGCCATTCCATCCGGCCTA-3’,

[0064] VcFDH-1-qRT-R:5’-ATCTTCCGCGACCGAGACCA-3’;

[0065] GADPH-qRT-F:5’-GGTTATCAATGATAGGTTTGGCA-3’,

[0066] GADPH-qRT-R:5’-CAGTCCTTGCTTGATGGACC-3’;

[0067] ATPase-qRT-F:5’-AATGCCGGACACGCACAGTT-3’,

[0068] ATPase-qRT-R:5’-TGAGCCCGTCGGATCACCTT-3’;

[0069] bZIP-2-qRT-F:5’-GGCATATCACCACAACCCGCAT-3’,

[0070] bZIP-2-qRT-R:5’-CCCTTGCACTTTCCCTTTGGCT-3’;

[0071] bZIP-3-qRT-F:5’-TGCGTTTGGTCACGCACCAT-3’,

[0072] bZIP-3-qRT-R:5’-TTGCCGAAGCATGGCCGATT-3’;

[0073] HSF A-3-qRT-F:5’-GGCCAGACGAGTGGGACTCA-3’,

[0074] HSF A-3-qRT-R:5’-GCCTCTGCTCTGCTGCTTGG-3’;

[0075] HSP20-1-qRT-F:5’-CGGGACATTGCTCCGTTCCC-3’,

[0076] HSP20-1-qRT-R:5’-CCGGCGTTTCCTTCCAGTCC-3’;

[0077] HSP20-2-qRT-F:5’-ACTCGCGTGGACTGGAAGGA-3’,

[0078] HSP20-2-qRT-R: 5'-GCGGTGCCACGTATCGTTCT-3';

[0079] HSP20-3-qRT-F: 5'-CGACGAGGCAACGACAGCTT-3',

[0080] HSP20-3-qRT-R: 5'-CCGGCGTTTCCTTCCAGTCC-3';

[0081] HSF C-1-qRT-F: 5'-CCGCCGTCGGTTATGAGCAG-3',

[0082] HSF C-1-qRT-R: 5'-CACCATGGCTGTGCCGGAAA-3';

[0083] HSP70-qRT-F: 5'-CCGCGTTCAGCTGTGTAGGG-3',

[0084] HSP70-qRT-R: 5'-CGCCAATTAGGCGCTCGGTA-3';

[0085] WRKY-qRT-F: 5'-AGCCGAAATTCGCTTTTCCAGACAA-3',

[0086] WRKY-qRT-R: 5'-TGGACTGGCGTTGCACTTGTT-3'.

[0087] The results are as follows Figure 5 As shown, six transgenic blueberry lines with high VcFDH-1 expression levels (FDH-2, FDH-4, FDH-6, FDH-8, FDH-9, and FDH-10) were obtained, with the highest VcFDH-1 gene expression level (241.91) in the FDH-8 line. Three high-VcFDH-1 expression blueberry lines (FDH-8, FDH-9, and FDH-10) were screened for subsequent 45℃ high-temperature experiments. The expression levels of heat stress-related genes bZIP-2, HSP20-1, and WRKY in transgenic blueberries were significantly higher than those in the control group (CK). For example, the HSP20-1 gene expression level in the FDH-8 line was approximately 100 times that in the CK line.

[0088] 3. Phenotypic observation and physiological index determination of transgenic lines under high temperature stress

[0089] The phenotypes of blueberry lines (FDH-8, FDH-9, and FDH-10) expressing VcFDH-1 were observed and recorded after 24 hours of high-temperature stress at 45℃ and treatment with 200 μmol / L exogenous SA. 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 nitrocyanocyanate tetrazolium method; and hydrogen peroxide (H2O2) content and catalase (CAT) and peroxidase (POD) activities were determined using kits from Nanjing Jiancheng Biotechnology Co., Ltd.

[0090] The results are shown in Table 1. 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 VcFDH-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 VcFDH-1 transgenic plants remained in good condition. After high-temperature and SA treatment, the SOD activity in both CK and VcFDH-1 blueberries significantly decreased; compared to the high-temperature treatment group of VcFDH-1 blueberries, the SOD activity in the SA treatment group significantly increased. After high-temperature and SA treatment, the CAT activity in CK significantly decreased, while the CAT activity in VcFDH-1 blueberries significantly increased. Furthermore, compared to the high-temperature treatment group, the CAT activity in the SA treatment group significantly decreased, while in VcFDH-1 blueberries, the CAT activity in the SA treatment group significantly increased compared to the high-temperature treatment group. After high-temperature and SA treatment, the POD activity in both CK and VcFDH-1 blueberries significantly increased. After high temperature and SA treatment, the MDA content in both CK and VcFDH-1 blueberries increased significantly. Furthermore, in VcFDH-1 blueberries, the MDA content in the SA treatment group was significantly lower than that in the high temperature treatment group. Similarly, after high temperature and SA treatment, the H2O2 content in both CK and VcFDH-1 blueberries increased significantly, and the H2O2 content in the SA treatment group was significantly higher than that in the high temperature treatment group. This indicates that under high temperature and SA treatment, the SOD activity of all transgenic blueberry lines decreased significantly, while the POD activity, CAT activity, MDA content, and H2O2 content all increased significantly.

[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 VcFDH-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 6 As shown, under high temperature and SA treatment conditions, the expression level of the VcFDH-1 gene in the CK increased, reaching a maximum at 12 h, and then decreased. The expression trends of the VcFDH-1 gene were consistent among different VcFDH-1 transgenic blueberry lines. In the FDH-8, FDH-9, and FDH-10 blueberry lines, the expression level first decreased after high temperature treatment, reaching a minimum at 6 h, then increased, reaching a maximum at 12 h; after SA treatment, it first decreased, reaching a minimum at 12 h, and then increased. Specifically, after 12 h of high temperature treatment, the VcFDH-1 gene expression level in FDH-8 reached as high as 677.8, which was 4.4 times that in the CK at the same time point; after 24 h of SA treatment, the VcFDH-1 gene expression level in FDH-8 reached as high as 575.5, which was 14.1 times that in the 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. A blueberry heat-resistant gene VcFDH-1, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expression protein of the blueberry heat-resistant gene VcFDH-1 as described in claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A recombinant expression vector containing the blueberry heat-resistant gene VcFDH-1 as described in claim 1, and a host cell.

4. The application of the blueberry heat-resistant gene VcFDH-1 as described in claim 1 in regulating the stress resistance of blueberries under high-temperature conditions.

5. The application according to claim 4, characterized in that, The high-temperature environment temperature is 45℃.

6. The application of the blueberry heat-resistant gene VcFDH-1 as described in claim 1 in regulating the blueberry's response to exogenous SA.

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

8. A primer pair for cloning the blueberry heat-resistant gene VcFDH-1 as described in claim 1, characterized in that, The sequences of the primer pair are as follows: VcFDH-1-F: 5'-ATGGCGATGGTGATGAAGC-3', VcFDH-1-R: 5'-ACGGTACTGGGGAGCTAGT-3'.

9. The application of the blueberry heat-resistant gene VcFDH-1 as described in claim 1 in regulating the expression of heat stress-related genes.

10. The application according to claim 9, characterized in that, The heat stress-related genes include ATPase, bZIP-2, bZIP-3, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, and WRKY.

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