Application of blueberry VcDELLA-1 gene in improving high-temperature stress resistance of plants

Abstract

The invention discloses application of a blueberry VcDELLA-1 gene in improving high temperature stress resistance of plants, and belongs to the field of plant genetic engineering. Based on blueberry heat stress transcriptome data analysis, it is found that only VcDELLA-1 in a DELLA family contains a DLPYAD motif, the expression quantity of the gene in a salicylic acid treatment group is remarkably higher than that of a control group, and it is prompted that the gene is the blueberry heat-resistant key gene. Under high temperature and salicylic acid treatment, VcDELLA-1 expression in VcDELLA-1 transgenic arabidopsis thaliana and blueberry plants is remarkably up-regulated, and the curling and shrinking degree of leaves is relatively light; the activity of superoxide dismutase is remarkably reduced, the activity of peroxidase and the content of malondialdehyde and hydrogen peroxide are remarkably increased, and a large number of heat stress related genes are expressed. The VcDELLA-1 gene improves the high-temperature resistance of plants by regulating and controlling the expression of heat stress related genes, and provides important gene resources for cultivating new varieties of high-temperature-resistant plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of the blueberry VcDELLA-1 gene in improving the plant's resistance to high temperature stress. Background Technology

[0002] Plants frequently face the threat of high-temperature stress during their growth and development. Global warming has led to more frequent extreme heat events, and high-temperature stress has become one of the major abiotic stress factors limiting crop yield and quality. High-temperature stress causes a series of physiological and biochemical changes in plants, including the accumulation of reactive oxygen species, protein denaturation and inactivation, and membrane system damage, which can even lead to plant death in severe cases. Therefore, identifying and understanding key heat-resistant genes in plants and elucidating their molecular mechanisms is of great significance for breeding new heat-resistant crop varieties through genetic engineering.

[0003] DELLA proteins are key negative regulators in the gibberellin (GA) signaling pathway and belong to a subfamily of the GRAS transcription factor family. DELLA proteins are named for their highly conserved N-terminal DELLA domain, which is the core region for GA signal sensing and transmission. Studies have shown that DELLA proteins not only participate in regulating plant growth and development processes, such as seed germination, stem elongation, flowering time, and fruit development, but also play an important role in plant responses to various abiotic stresses. For example, DELLA proteins (GAI, RGA, RGL1, RGL2, RGL3) in Arabidopsis thaliana are involved in regulating responses to low-temperature stress, drought stress, and salt stress; overexpression of specific DELLA family members can enhance plant tolerance to stress. However, functional studies of DELLA proteins in plant heat tolerance are relatively limited, especially in woody plants, where systematic reports are lacking.

[0004] Blueberries (Vaccinium spp.) are a small berry fruit with high economic value, and their fruits are rich in anthocyanins, vitamins, and various bioactive components, making them popular with consumers. However, blueberries are quite sensitive to high-temperature stress; high summer temperatures often lead to leaf scorch, reduced photosynthesis, and decreased fruit quality, severely hindering the sustainable development of the blueberry industry. Currently, research on the discovery and functional verification of heat-resistant genes in blueberries is still in its early stages, and gene resources available for heat-resistant breeding are relatively scarce. The existence of the DELLA family of genes in blueberries and their role in regulating heat tolerance in blueberries have not yet been reported.

[0005] Therefore, cloning the DELLA family genes from blueberries and exploring their function in improving plant heat stress tolerance not only enriches plant heat tolerance gene resources but also provides a theoretical basis and gene resources for improving the heat tolerance of blueberries and other crops through genetic engineering. This invention addresses the above needs by cloning the VcDELLA-1 gene from blueberries for the first time and verifying its function in improving plant heat tolerance. Summary of the Invention

[0006] This invention addresses the problems in existing technologies such as the scarcity of plant heat-resistant gene resources, the unclear function of blueberry DELLA family genes, and the lack of effective technical means to improve the heat resistance of blueberries. It provides an application of the blueberry VcDELLA-1 gene in improving the plant's ability to withstand heat stress.

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

[0008] The application of the blueberry VcDELLA-1 gene in improving the plant's tolerance to high temperature stress includes: overexpressing the blueberry VcDELLA-1 gene in plants, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0009] In some embodiments, the application includes the following steps:

[0010] S1: Construct a plant expression vector for the VcDELLA-1 gene;

[0011] S2: Transform the plant expression vector of the VcDELLA-1 gene into plant tissues or cells;

[0012] S3: Cultivate and screen transgenic plants with improved tolerance to high-temperature stress;

[0013] The plant in question is either blueberry or Arabidopsis thaliana.

[0014] In some embodiments, the method for constructing the plant expression vector of the VcDELLA-1 gene specifically involves inserting the expression cassette of the blueberry VcDELLA-1 gene into the vector backbone, wherein the vector backbone contains a plant selective marker gene and / or a terminator sequence.

[0015] In some embodiments, the blueberry VcDELLA-1 gene expression cassette is constructed by linking the blueberry VcDELLA-1 gene to a plant functional promoter to form an expression cassette.

[0016] In some embodiments, the plant functional promoter is the cauliflower mosaic virus 35S promoter.

[0017] In some embodiments, the transformation is performed using an Agrobacterium-mediated genetic transformation method.

[0018] In some embodiments, the improvement in plant tolerance to high-temperature stress is manifested in that, under high-temperature stress conditions, the transgenic plant, compared with the wild-type plant, possesses at least one characteristic selected from the group consisting of:

[0019] (a) The degree of leaf damage is reduced;

[0020] (b) Increased superoxide dismutase activity;

[0021] (c) Increased catalase activity;

[0022] (d) Increased peroxidase activity;

[0023] (e) Malondialdehyde content decreased;

[0024] (f) The expression levels of heat stress-related genes are upregulated.

[0025] In some embodiments, the heat stress-related genes are selected from at least one of the following genes: McMYB52, CHIP, SIL, HSF16, ERECTA, ZmCIPH, bZIP-2, bZIP-3, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, WRKY.

[0026] The use of any of the transgenic plant plants or parts thereof obtained by any of the described applications in the preparation of heat-resistant plant varieties.

[0027] The use of any of the transgenic plant plants or portions thereof obtained through any of the aforementioned applications as models for studying molecular mechanisms of plant heat tolerance or pathways of response to high-temperature stress.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] Based on blueberry heat stress transcriptome data, this invention is the first to discover that only VcDELLA-1 in the DELLA family contains the DLPYAD motif, and the expression level of this gene in the salicylic acid (SA) treatment group was significantly higher than that in the control group, suggesting that it is a key gene for heat tolerance in blueberries. Functional verification was performed by constructing an overexpression vector and genetically transforming Arabidopsis thaliana and blueberries. The results showed that under high temperature and SA treatment conditions, VcDELLA-1 expression was significantly upregulated in transgenic plants, and the degree of leaf curling and wrinkling was significantly less than that in control plants. This confirms that the VcDELLA-1 gene can significantly improve the heat stress tolerance of plants, filling a gap in research on the heat tolerance function of the blueberry DELLA gene.

[0030] This invention further elucidates the physiological and molecular mechanisms by which the VcDELLA-1 gene enhances plant heat tolerance. Physiological indicators showed that under high temperature and SA treatment, transgenic plants exhibited significantly decreased superoxide dismutase activity and significantly increased peroxidase activity, malondialdehyde (MDA), and hydrogen peroxide content, indicating that this gene enhances plant heat tolerance by regulating reactive oxygen species (ROS) balance. Molecular detection results showed that heat stress-related genes such as bZIP-2, bZIP-3, HSP20-1, HSP20-2, HSP20-3, HSP70, and WRKY were highly expressed in VcDELLA-1 transgenic blueberries. The VcDELLA-1 gene showed a consistent expression trend under high temperature and SA treatment, suggesting that it enhances plant heat resistance by regulating the expression of downstream heat stress-related genes. This invention provides an important gene resource for breeding new heat-tolerant plant varieties and can be widely applied to various plants such as blueberries, Arabidopsis thaliana, tomatoes, peppers, and strawberries, demonstrating broad application prospects. Attached Figure Description

[0031] Figure 1 Multiple alignment diagram of VcDELLA, a key heat-resistant gene in blueberries;

[0032] Figure 2 Evolutionary tree diagram of the VcDELLA family of key heat-resistant genes in blueberries;

[0033] Figure 3 The diagram shows the genetic transformation process and PCR amplification results of Arabidopsis thaliana; a: Arabidopsis planting; b: Arabidopsis infection; c: Arabidopsis transformation; d: Arabidopsis screening; e: Arabidopsis transplanting; f: Arabidopsis seed harvesting; g: PCR amplification results.

[0034] Figure 4 This is a diagram showing the growth status of transgenic Arabidopsis plants; A: SPAD value; B: N content;

[0035] Figure 5 The figure shows the results of the expression analysis of heat stress-related genes in transgenic Arabidopsis thaliana.

[0036] Figure 6 Figure 1 shows the changes in phenotype and leaf SPAD and nitrogen content in transgenic Arabidopsis thaliana under high temperature and exogenous SA treatment; A: Phenotypic changes in Arabidopsis thaliana under high temperature treatment; B: Phenotypic changes in Arabidopsis thaliana under exogenous SA treatment; C: SPAD value of leaves in VcDELLA Arabidopsis thaliana; D: N content of leaves in VcDELLA Arabidopsis thaliana.

[0037] Figure 7 Figure 1 shows the expression of the VcDELLA-1 gene in various transgenic Arabidopsis lines after high temperature and exogenous SA treatment; A: High temperature treatment; B: SA treatment;

[0038] Figure 8-1The graph shows the expression levels of heat stress-related genes ATPase, BZIP-2, HSP20-2, HSP20-3, BZIP-3, and HSPF C-1 in positive seedlings of transgenic blueberries.

[0039] Figure 8-2 The graph shows the expression levels of heat stress-related genes HSF A-3, HSP20-1, HSP70, and WRKY in positive seedlings of transgenic blueberries.

[0040] Figure 9 Figure showing phenotypic changes in transgenic blueberries after high temperature and exogenous SA treatment;

[0041] Figure 10 The graph shows the changes in the expression level of the VcDELLA-1 gene in transgenic blueberries after high temperature and exogenous SA treatment; A: High temperature treatment; B: SA treatment. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is 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, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0043] Example 1

[0044] 1. Materials and Methods:

[0045] (1) Plant materials

[0046] Two-year-old healthy and uniformly growing southern highbush blueberry cultivar 'O'Neal' seedlings were selected from the Jiangsu Provincial 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 seedlings were brought back in liquid nitrogen and stored at -80°C.

[0047] (2) Gene cloning

[0048] The open reading frame (ORF) of the VcDELLA-1 (gene-Vadar_030397) gene was obtained using BioXM 2.6 software. Correction was performed using the existing three-generation full-length transcript set. Candidate gene cloning primers were designed using Oligo 6.0 software (Table 1). 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.

[0049] Table 1 Primer information for cloned genes

[0050]

[0051] 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 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 transformed into ampicillin-resistant LB medium, and incubated on a shaker for 8 h (37℃, 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. The 15 μL colony PCR system consisted of: 7.5 μL 2× T5 Super PCR Mix, 0.75 μL front primer, 0.75 μL back primer, 1.5 μL bacterial culture, and 4.5 μL ddH2O. The bacterial culture PCR reaction program was as follows: 98 ℃ for 2 min; 98 ℃ for 10 s, 55 ℃ for 10 s, 72 ℃ for 15 s, for 35 cycles; 72 ℃ for 2 min, followed by incubation at 4 ℃. After sequencing, plasmids were extracted from the correctly sequenced bacterial cultures using a plasmid extraction kit (Beijing Biotech Biotechnology Co., Ltd.) and stored at -20 ℃ for subsequent experiments.

[0052] (3) Expression level analysis

[0053] Based on the FPKM and log2Fold Change values ​​in the VcDELLA gene family transcriptome data, expression trends and pattern heatmaps were calculated and plotted using TB tools software.

[0054] (4) Analysis of the physicochemical properties of proteins

[0055] 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 was analyzed 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 of the protein were predicted using the online tool MEME (http: / / meme-suite.org / ) with default parameters and a motif count set to 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 / ).

[0056] (5) Sequence alignment and phylogenetic tree analysis

[0057] DNAMAN 6.0 software was used to perform comparative analysis on the VcDELLA gene family in blueberry transcriptome data. The NCBI database was used to find the 20 protein sequences with the highest similarity to the target gene (DELLA). The Clustal W function in MEGA 11 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.

[0058] (6) Materials and equipment for tissue culture seedling experiments

[0059] Quantitative samples of different blueberry tissues were selected from healthy 'O'Neill' cultivars cultivated in the greenhouse of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. Tissue culture seedlings were obtained 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'. Arabidopsis thaliana was a laboratory-reserved Columbia wild-type. Agrobacterium GV3101 used for genetic transformation was purchased from Nanjing Qingke Biotechnology Co., Ltd. Instruments (Table 2), reagents and their preparation (Table 3), and culture medium preparation methods (Table 4) are shown in the tables.

[0060] Table 2 Instruments

[0061]

[0062] Table 3 Reagents and their preparation

[0063]

[0064] Table 4. Culture medium preparation method

[0065]

[0066] (7) Construction of overexpression vectors

[0067] Overexpression vectors were constructed using the plant binary expression vector pCAMBIA1303 (resistance kan). Agrobacterium competent cells stored at -80 °C 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 rapidly frozen in liquid nitrogen for 5 min, then incubated in water at 37 °C 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 °C 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 °C. 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 ℃ for subsequent genetic transformation experiments.

[0068] (8) Agrobacterium-mediated transformation of blueberries and Arabidopsis thaliana

[0069] Blueberry tissue culture seedlings were cut into 1 cm segments and inoculated into a pre-culture medium, then cultured in the dark for 2 days. The pre-cultured blueberry stem segments were then removed and placed in Agrobacterium resuspension for 10 min under normal pressure. The inoculated stem segments were then inoculated into a co-culture medium and cultured at 25 °C under light for 4 days. After co-culture, the stem segments were placed in a sterilization solution for 8 min, with occasional shaking to effectively remove Agrobacterium. The sterilized stem segments were then inoculated into a selection medium and cultured at 25 °C in the dark for 10 days. Finally, they were cultured under light for 50 days, and the stem growth was observed and recorded.

[0070] Before infecting Arabidopsis thaliana, select robust Arabidopsis 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, wrap the plants 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 inoculation. Once the Arabidopsis thaliana matures, 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 matures, 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.

[0071] (9) Quantitative detection of transgenic plants

[0072] Total RNA was extracted from blueberry transgenic plants according to the instructions of the Bioteke Geneporation kit (Beijing Bioteke Co., Ltd.; Cat#RP3302). Total RNA was extracted from Arabidopsis transgenic plants according to the instructions of the FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novizan 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 (Table 7), with the blueberry GADPH gene and the Arabidopsis AtUBC (GenBank: AT5g25760) gene as internal controls, to analyze the expression of key genes. -ΔΔCtMethods for calculating gene expression levels. 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. The PCR amplification program was set on a QuantStudio 3 quantitative instrument (ABI, Thermo Fisher Scientific, USA) as shown in Table 5.

[0073] Table 5 PCR amplification reaction procedure

[0074]

[0075] Gene expression levels in transgenic blueberry and Arabidopsis plants were analyzed, and blueberry and Arabidopsis lines with high VcDELLA-1 expression were screened. Literature review on Arabidopsis responses to heat stress revealed that McMYB52, CHIP, SIL, HSF-16, ERECTA, ZmCIPH, and 13MBF-1L play important roles in Arabidopsis heat stress response. The expression of these heat stress-related genes in Arabidopsis lines with high VcDELLA-1 expression was analyzed (primer sequences are shown in Table 6).

[0076] Based on blueberry transcriptome data, 10 genes that were significantly upregulated and associated with heat stress response were selected, including ATPase, bZIP-2, bZIP-3, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, and WRKY. RT-qPCR was performed using TB Green Premix Taq II (Tli RNASEH Plus) (TaKaRa, Dalian, China) to analyze the expression of these heat stress-related genes in the high-expression VcDELLA-1 blueberry line (primer sequences are shown in Table 6).

[0077] Table 6. Primers for gene quantification

[0078]

[0079] (10) High temperature test, phenotypic observation and physiological index measurement

[0080] Based on the phenotypic changes and leaf condition of plants after short-term high-temperature treatment (24 h), physiological indicators of leaves were measured in the SA treatment group and the CK group. SPAD values ​​and nitrogen content of 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 nitrocyanate-blue tetrazolium method; and hydrogen peroxide (H2O2) content and catalase (CAT) and peroxidase (POD) activities were determined using kits from Nanjing Jiancheng Biotechnology Co., Ltd.

[0081] 2. Results:

[0082] (1) Cloning and sequence analysis of key genes

[0083] Based on transcriptome data annotation and differential analysis, gene-Vadar_030397 was identified as a DELLA protein, suggesting it might be a key gene regulating heat resistance in blueberries. The ORF sequence of the target gene was obtained by PCR amplification and verified by Sanger sequencing. First-generation sequencing confirmed that the assembled fragment matched the expected target gene sequence. The ORF of VcDELLA-1 was 1626 bp (SEQ ID NO.1), encoding a 541-amino acid polypeptide (SEQ ID NO.2) with the stop codon TAA. Furthermore, to further investigate whether similar mechanisms of action exist among its related family members, three VcDELLA family gene members were screened from blueberry heat-resistant leaf transcriptome data (Table 7). The minimum free energy (MFE) and centroid secondary structure of the VcDELLA family gene mRNAs were analyzed using RNAfold software. The results showed that VcDELLA-2 had the highest total free energy (-634.42), while VcDELLA-1 had the lowest (-655.14).

[0084] Table 7. Statistical analysis of expression patterns of key heat-resistant genes in blueberries and their family members.

[0085]

[0086] (2) Analysis of protein physicochemical properties and structure

[0087] The physicochemical properties of the proteins translated by each gene were analyzed using ProtParam software (Table 8). The results showed that the relative molecular weights of the VcDELLA family genes were 59.2, 62.3, and 63.7 kDa, with theoretical isoelectric points ranging from 4.99 to 5.7 and instability coefficients from 44.91 to 52.51. Subcellular localization indicated that all VcDELLA family genes were located in the cell nucleus. The gene structure motifs of VcDELLA family members in the transcriptome data were analyzed using the online analysis website MEME. The results showed that the DLPYAD motif was only present in VcDELLA-1, suggesting it may play a key role in the regulation of heat stress in blueberries. The conserved domains of VcDELLA family genes were analyzed using the NCBI CD searc online tool. The results showed that all three genes in the VcDELLA family contain both DELLA and GRAS domains. The secondary structure of the proteins translated by each gene was analyzed using the SOPMA online tool (Table 9). The results showed that the proteins translated from VcDELLA family genes consisted of α-helices (39.57-42.51%), β-sheets (7.76-8.43%), and random coils (49.05-52.58%).

[0088] Table 8. Analysis of the physicochemical properties of key heat-resistant genes in blueberries and their family members.

[0089]

[0090] Table 9. Secondary structure of proteins encoded by key heat-resistant genes in blueberries and their family members.

[0091]

[0092] (3) Analysis of protein phosphorylation and glycosylation sites

[0093] The NetPhos online tool was used to predict potential phosphorylation sites in proteins, and the NetNGlyc-1.0 online tool was used to predict protein glycosylation sites. The results showed that VcDELLA-1 had 50 phosphorylation sites and 2 glycosylation sites with probabilities of 0.5852 and 0.5138, respectively; VcDELLA-2 had 45 phosphorylation sites and 2 glycosylation sites with probabilities of 0.5673 and 0.4488, respectively; and VcDELLA-3 had 54 phosphorylation sites and 6 glycosylation sites with probabilities of 0.6747, 0.7341, 0.6281, 0.5936, 0.6447, and 0.5451, respectively.

[0094] (4) Phylogenetic tree analysis and expression level analysis

[0095] 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. Figure 1 The study found that VcDELLA-2 and VcDELLA-3 are most closely related to DELLA protein GAI1-like (quinoa), but less closely related to DELLA protein SLR1-like (camellia sinensis). Figure 2 Furthermore, VcDELLA-1 expression was highest in the second-stage fruit. VcDELLA-1 expression was highest in blueberry leaves in July, followed by August. The expression trend of VcDELLA-1 in blueberry stem segments consistently showed an increase in expression level with increasing lignification of the stem segment.

[0096] (5) Genetic transformation of Arabidopsis thaliana and growth observation

[0097] The plant overexpression vector pCAMBIA1303 was constructed for genetic transformation of Arabidopsis thaliana, a model plant. The plant conditions at each stage of the genetic transformation process of the VcDELLA-1 gene in Arabidopsis thaliana were recorded, including proliferation, infection, screening, transplanting, and seed harvesting. Figure 3 af), and PCR amplification and detection of potential positive lines ( Figure 3 g) Individual Arabidopsis thaliana plants that tested positive were harvested and sown, becoming the T2 generation. The T2 generation seeds underwent repeated screening, transplanting, RNA extraction, PCR testing, and individual harvesting of positive seedlings, successfully obtaining T3 generation transgenic Arabidopsis thaliana seeds.

[0098] T3 Arabidopsis thaliana seeds were sown, their growth was recorded, and the SPAD value and N content of the leaves were measured. Figure 4Analysis of SPAD values ​​and N content in different Arabidopsis thaliana strains revealed that the SPAD values ​​of VcDELLA-1 transgenic Arabidopsis thaliana plants were all higher than those of the wild type, with the della-1 strain having the highest value at 37.97% and the della-6 strain having the lowest value at 31.54%. The N content in the leaves of the della-1 and della-2 strains was higher than that of the wild type, at 11.9% and 10.8%, respectively; while the N content in the leaves of the della-3, della-4, della-5, and della-6 strains was lower than that of the wild type, at 10.0%, 9.8%, 9.8%, and 9.8%, respectively. Furthermore, at the same growth stage, compared with wild-type plants, the della-1 transgenic Arabidopsis thaliana plants had approximately 114% more leaves, the della-2 line had approximately 42% more leaves, and the della-3, della-4, della-5, and della-6 lines had approximately 21% fewer leaves than the wild-type. Therefore, in terms of leaf number, leaf SPAD value, and N content, the VcDELLA-1 transgenic Arabidopsis thaliana > WT Arabidopsis thaliana.

[0099] (6) Analysis of expression levels of key genes in Arabidopsis thaliana positive seedlings

[0100] Quantitative detection of the VcDELLA-1 gene in transgenic Arabidopsis thaliana and analysis of gene expression were performed. Figure 5 Based on the gene expression results of different Arabidopsis thaliana lines with different genotypes, three lines (della-2, della-5, and della-6) with high VcDELLA-1 expression were selected from high to low. Among them, della-5 had the highest expression level (181.8), and a total of six high-expressing Arabidopsis thaliana lines were selected for subsequent 45 ℃ high-temperature experiments. In addition, when analyzing and summarizing the expression of heat stress-related genes in transgenic Arabidopsis thaliana, it was found that ( Figure 5 Compared to WT Arabidopsis, heat stress-related genes were significantly upregulated in VcDELLA-1 transgenic Arabidopsis. Among them, McMYB52, CHIP, ERECTA, 13MBF-1L, and ZmCIPH were expressed at the highest levels in the della-2 line, with ERECTA gene expression being about 4 times that in WT. SIL was expressed at the highest level in the della-5 line, about 11 times that in WT.

[0101] (7) Phenotypic and physiological changes in Arabidopsis transgenic plants after high temperature and SA treatment

[0102] Further experiments were conducted on Arabidopsis thaliana lines (della-2, della-5, and della-6) that highly expressed VcDELLA-1, using a 45℃ high-temperature test and 200 μmol / L exogenous SA treatment. The plant phenotypes at 0 h, 6 h, and 24 h after high-temperature and exogenous SA treatment were observed and recorded. Figure 6 A and B) were used to analyze the changes in SPAD value and N content of plant leaves after high-temperature treatment for 0 h and 24 h. Figure 6 (C, D). The results showed that, compared with WT Arabidopsis, della-2, della-5, and della-6 plants did not show yellowing of leaves and had relatively milder water deficiency symptoms after 6 h of high-temperature treatment. After 24 h of high-temperature treatment, both VcDELLA-1 transgenic plants and WT plants showed varying degrees of leaf yellowing, wilting, and water deficiency. However, compared with WT plants, VcDELLA-1 transgenic plants showed less leaf curling and wrinkling, and in terms of the severity of leaf yellowing, wilting, and water deficiency, WT > della-2, della-5, and della-6. Under SA treatment, there were no significant changes in any Arabidopsis lines. The results of changes in SPAD value and N content of leaves of different Arabidopsis lines showed that after 24 h of high-temperature treatment, the SPAD value and N content of leaves of both VcDELLA-1 transgenic plants and WT plants decreased. Among them, the SPAD value of leaves of WT plants decreased by 24.8%, and the N content decreased by 23.3%; the SPAD value of leaves of VcDELLA-1 transgenic Arabidopsis decreased by 33.1%, 23.1%, and 27.1%, respectively, and the N content decreased by 28.9%, 22.2%, and 25.5%, respectively.

[0103] Enzyme activities and MDA and H2O2 contents in leaves of Arabidopsis thaliana under high-temperature treatment and SA treatment were measured and analyzed (Table 10). The results showed that SOD activity decreased in both WT and DELLA Arabidopsis leaves after high-temperature treatment, while SOD activity increased after exogenous SA treatment. The change in CAT activity was the opposite of SOD; CAT activity increased in WT and DELLA Arabidopsis after high-temperature treatment, while it decreased after exogenous SA treatment. POD activity, MDA content, and H2O2 content all increased in WT and DELLA Arabidopsis after both high-temperature and exogenous SA treatments. POD activity and MDA content were significantly higher in high-temperature treatment than in exogenous SA treatment, while H2O2 content was significantly lower in high-temperature treatment than in exogenous SA treatment.

[0104] Table 10. Changes in antioxidant physiological parameters of transgenic Arabidopsis thaliana after high temperature and exogenous SA treatment.

[0105]

[0106] Note: The lowercase letters are the results of the significance test with p < 0.05, and the uppercase letters are the results of the significance test with p < 0.01.

[0107] (8)Gene expression in transgenic Arabidopsis plants after high - temperature and SA treatment

[0108] Gene quantitative analysis was performed on leaf samples of each Arabidopsis line treated with high - temperature, exogenous SA for 0, 6, 12, and 24 h ( Figure 7 ). The results showed that the expression level of the VcDELLA - 1 gene in WT increased with the extension of high - temperature and SA treatment time. The change trend of the VcDELLA - 1 gene expression level was the same among different VcDELLA - 1 transgenic Arabidopsis lines. The expression level of the VcDELLA - 1 gene reached the maximum at 12 h of high - temperature and SA treatment in della - 2, della - 5, and della - 6 transgenic lines, and then decreased. Among them, the expression level of the VcDELLA - 1 gene in della - 6 was as high as 3193.4 after 12 h of high - temperature treatment, which was 228.5 times the expression level in WT at the same time; the expression level of the VcDELLA - 1 gene in della - 5 was as high as 554.5 after 12 h of SA treatment, which was 15.4 times the expression level in WT at the same time. Comprehensive analysis showed that the expression pattern of the VcDELLA - 1 gene under high - temperature and SA treatment was similar, that is, the expression level of the VcDELLA - 1 gene in WT plants under high - temperature and SA treatment continued to increase; the expression level of the VcDELLA - 1 gene reached the maximum at 12 h and then decreased.

[0109] (9)Identification of transgenic blueberry positive seedlings and expression analysis of heat - stress - related genes

[0110] Taking the blueberry 'Tianhou' of the same period with empty vector / without genetic transformation as the control group, the stem segments in the pre - culture, co - culture, and screening culture stages during the genetic transformation process of blueberry stem segments were observed and recorded, and the final 9 VcDELLA - 1 genotype blueberry plants were detected for positive seedlings. According to the results of fluorescence quantitative PCR analysis, among 8 transgenic blueberry lines with relatively high VcDELLA - 1 expression (DELLA - 1, DELLA - 2, DELLA - 3, DELLA - 4, DELLA - 6, DELLA - 7, DELLA - 8, DELLA - 9), the expression level of the VcDELLA - 1 gene in the DELLA - 9 line was the highest, which was 932.44. According to the gene expression levels of different transgenic lines of blueberry in each genotype from high to low, 3 VcDELLA - 1 high - expression blueberry lines (DELLA - 6, DELLA - 7, DELLA - 9) were selected for subsequent 45°C high - temperature experiments. When analyzing and sorting out the expression of heat - stress - related genes in transgenic blueberries, it was found that ( Figure 8-1 、 8-2The expression levels of bZIP-2, bZIP-3, HSP20-1, HSP20-2, HSP20-3, HSP70, and WRKY in VcDELLA-1 transgenic blueberries were all significantly higher than those in CK. For example, the expression level of the WRKY gene in the DELLA-9 strain was about 193 times that in CK.

[0111] (10) Phenotypic and physiological and biochemical changes in transgenic blueberry plants after high temperature and SA treatment

[0112] Further, VcDELLA-1 high-expression transgenic blueberry lines were subjected to a 45℃ high-temperature test and 200 μmol / L exogenous SA treatment for 24 h. The phenotypic changes of the plants were observed and recorded. Figure 9 The results showed that after 24 h of exogenous SA treatment, there were no significant changes in the phenotype of blueberry plants. However, after high-temperature treatment, VcDELLA-1 and VcbZIP-1 transgenic plants and CK plants all showed varying degrees of yellowing, withering, and water shortage in their leaves. However, compared with CK plants, 10-20% of the leaves of VcDELLA-1 transgenic plants were still in good condition.

[0113] The activities of antioxidant enzymes and the contents of MDA and H2O2 in the leaves of blueberries under high-temperature and SA treatments were measured and analyzed (Table 11). The results showed that SOD activity in both CK and VcDELLA-1 blueberries was significantly reduced after high-temperature and SA treatments, and there was no significant difference in SOD activity between the high-temperature and SA treatment groups for both CK and VcDELLA-1 blueberries. CAT activity in CK was significantly reduced after high-temperature and SA treatments, while CAT activity in VcDELLA-1 blueberries was significantly increased. Furthermore, in VcDELLA-1 blueberries, CAT activity in the SA treatment group was significantly reduced compared to the high-temperature treatment group. POD activity in both CK and VcDELLA-1 blueberries was significantly increased after high-temperature and SA treatments, and except for VcFDH-1 blueberries, POD activity in the SA treatment group was significantly increased compared to its high-temperature treatment group. After high temperature and SA treatment, the MDA content in both CK and VcDELLA-1 blueberries significantly increased. In VcDELLA-1 blueberries, there was no significant difference in MDA content between the high-temperature and SA treatment groups. However, in VcFDH-1 blueberries, the MDA content in the SA treatment group was significantly lower than that in the high-temperature treatment group. After high temperature and SA treatment, the H2O2 content in both CK and VcDELLA-1 blueberries significantly increased. Compared to the high-temperature treatment group, the H2O2 content in the SA treatment group was significantly higher. 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.

[0114] Table 11 Changes in antioxidant physiological indicators of transgenic blueberries after high temperature and exogenous SA treatment

[0115]

[0116] Note: Lowercase letters indicate significance test results of p < 0.05, and uppercase letters indicate significance test results of p < 0.01.

[0117] (11) Changes in gene expression in transgenic blueberries after high temperature and SA treatment

[0118] Gene quantification analysis was performed on leaf samples of transgenic blueberries from various lines treated with high temperature and exogenous SA for 0, 6, 12, and 24 h. Figure 10 The results showed that under high-temperature treatment, the expression level of the VcDELLA-1 gene in the control group (CK) increased with the extension of treatment time; under SA treatment, the expression level of the VcDELLA-1 gene in the CK first increased, reached its maximum at 12 h, and then decreased. The expression trend of the VcDELLA-1 gene was consistent among different VcDELLA-1 transgenic blueberry lines. After high-temperature and SA treatments, the expression level of the DELLA-6, DELLA-7, and DELLA-9 lines first decreased and then increased, reaching its maximum at 12 h, and then decreased again. Specifically, after 12 h of high-temperature treatment, the VcDELLA-1 gene expression level in DELLA-9 reached as high as 1689.1, which was 351.8 times that in the control group (WT) at the same time point; after 12 h of SA treatment, the VcDELLA-1 gene expression level in DELLA-9 reached as high as 1769.3, which was 10.6 times that in the control group (WT) at the same time point. Comprehensive analysis revealed that the expression level of the VcDELLA-1 gene in transgenic blueberries showed a consistent trend under high temperature and SA treatment.

[0119] 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. The application of the blueberry VcDELLA-1 gene in improving the plant's tolerance to high-temperature stress, characterized by, include: The blueberry VcDELLA-1 gene was overexpressed in plants, and the nucleotide sequence of the blueberry VcDELLA-1 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application includes the following steps: S1: Construct a plant expression vector for the VcDELLA-1 gene; S2: Transform the plant expression vector of the VcDELLA-1 gene into plant tissues or cells; S3: Cultivate and screen transgenic plants with improved tolerance to high-temperature stress; The plant in question is either blueberry or Arabidopsis thaliana.

3. The application according to claim 2, characterized in that, The method for constructing the plant expression vector of the VcDELLA-1 gene is as follows: the expression cassette of the blueberry VcDELLA-1 gene is inserted into the vector backbone, and the vector backbone contains plant selective marker genes and / or terminator sequences.

4. The application according to claim 3, characterized in that, The method for constructing the expression cassette of the blueberry VcDELLA-1 gene is as follows: the blueberry VcDELLA-1 gene is linked to a plant functional promoter to form an expression cassette.

5. The application according to claim 4, characterized in that, The plant functional promoter is the cauliflower mosaic virus 35S promoter.

6. The application according to claim 2, characterized in that, The transformation was carried out using an Agrobacterium-mediated genetic transformation method.

7. The application according to claim 1, characterized in that, The improved plant tolerance to high-temperature stress is manifested in the following ways: under high-temperature stress conditions, the transgenic plants, compared with wild-type plants, possess at least one characteristic selected from the group consisting of: (a) The degree of leaf damage is reduced; (b) Increased superoxide dismutase activity; (c) Increased catalase activity; (d) Increased peroxidase activity; (e) Malondialdehyde content decreased; (f) The expression levels of heat stress-related genes are upregulated.

8. The application according to claim 7, characterized in that, The heat stress-related genes are selected from at least one of the following genes: McMYB52, CHIP, SIL, HSF16, ERECTA, ZmCIPH, bZIP-2, bZIP-3, HSF A-3, HSP20-1, HSP20-2, HSP20-3, HSF C-1, HSP70, WRKY.

9. The use of the transgenic plant plants or parts thereof obtained by any one of claims 1-8 in the preparation of heat-resistant plant varieties.

10. The use of transgenic plant plants or portions thereof obtained by any one of claims 1-8 as models for studying molecular mechanisms of plant heat tolerance or pathways of response to high-temperature stress.

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