Eggplant heat-resistance-related gene SmLOX5 and its application

Abstract

The present invention belongs to the field of plant molecular biology technology and specifically discloses an eggplant heat-tolerance-related gene SmLOX5 and its application. The present invention provides the application of the eggplant heat-tolerance gene SmLOX5 or its expression product in regulating plant heat tolerance. The nucleotide sequence of the heat-tolerance gene SmLOX5 is shown in SEQ ID NO.1, and the amino acid sequence of the expression product of the gene SmLOX5 is shown in SEQ ID NO.2. Subcellular localization, VIGS experiments, and overexpression experiments in Arabidopsis thaliana showed that SmLOX5 is localized to chloroplasts and can affect the heat tolerance of eggplant under high temperature stress. The SmLOX5 gene is significantly induced and upregulated under high temperature stress conditions. When the SmLOX5 gene is silenced in eggplant, the heat tolerance of the silenced plants is reduced. Overexpression of the SmLOX5 gene in Arabidopsis thaliana can improve the heat tolerance of plants under high temperature stress conditions. Changes in JA content demonstrate that the SmLOX5 gene-silenced plants regulate the plant's heat tolerance by affecting JA synthesis in eggplant under high temperature stress.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to an eggplant heat-resistance-related gene SmLOX5 and an application thereof. Background Art

[0002] Eggplant (Solanum melongena L.) is an economically important vegetable crop of the Solanaceae family, cultivated worldwide, with China being the world's largest producer and consumer of eggplant. However, during the production process, eggplant is subject to various biotic and abiotic stresses, of which high temperature stress is one of the most important abiotic stresses. High temperature stress can inhibit the growth and development of eggplant, manifesting as a shortened flowering period, weak pollen viability, incomplete floral development, abnormal pollination and fertilization, low fruit set rate, increased flower and fruit drop, and increased fruit deformity and corking, ultimately leading to a decrease in eggplant quality and yield. Therefore, improving eggplant's tolerance to high temperature stress is an urgent issue to be addressed in this field.

[0003] Recent studies have shown that lipoxygenases and their derivatives, such as jasmonic acid, divinyl ether, and volatile aldehydes, play a crucial role in enhancing plant tolerance to environmental stresses. Lipoxygenases (LOXs) are involved in the synthesis of these substances. Lipoxygenases (EC 1.13.11.12) are non-heme iron-containing, sulfur-free fatty acid dioxygenases that are both regiospecific and stereospecific and are ubiquitous in plants and animals. To date, at least 40 LOX genes have been isolated and identified in plants. Andre and Hou first discovered and reported LOXs in soybeans. Lipoxygenases are a highly conserved class of proteins, primarily composed of two domains: a C-terminal lipoxygenase domain and an N-terminal PLAT domain. Current research suggests that plant lipoxygenases all contain a representative 38-amino acid motif (His-(X)4-His-(X)4-His-(X)17-His-(X)8-His). This motif plays a crucial role in the stability and activity of lipoxygenases and also provides binding sites for non-heme iron-containing dioxygenases. Plant lipoxygenases are widely present in various plant organs, including roots, stems, and leaves. Their genes have diverse subcellular localization patterns, primarily in plastids, vacuoles, and the cytoplasm. Studies have shown that the LOX genes in Arabidopsis thaliana are located in chloroplasts, and the LOX genes in potato and tomato (Solanaceae) are also primarily located in chloroplasts, microsomes, and the cytoplasm. In higher plants, lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids to form hydroperoxides (HPOs), with linoleic acid (LA) and linolenic acid (LEA) being the primary substrates. Based on this catalytic reaction characteristic of lipoxygenases, plant lipoxygenases that catalyze the oxygenation of LA at positions C9 and C13 are divided into two types: 9-LOX and 13-LOX. The catalytic products are 9-HPOs and 13-HPOs, respectively. On this basis, subsequent researchers have made a more accurate classification of plant lipoxygenases based on their primary structures. Based on the similarity of their amino acid sequences and whether they have chloroplast transit peptides, they are divided into two subfamilies: Type I-LOX and Type II-LOX. Type I-LOX has high sequence similarity but lacks a chloroplast transit peptide, while Type II-LOX has low sequence similarity among its members but has a chloroplast transit peptide, and all Type II-LOX are 13-LOX.

[0004] Research has shown that the lipoxygenase gene family plays an important role in plant stress responses and growth and development. For example, AtLOX6 in Arabidopsis thaliana has been found to help improve the resistance of young leaves to the sea moth (Spodopteralittoralis); TomloxD in tomato is associated with heat tolerance, and overexpression can improve its high-temperature tolerance; and DkLOX3 positively regulates persimmon's tolerance to abiotic stress. In pepper and maize, CalLOX1 and ZmLOX6, respectively, have been reported to be associated with drought tolerance. Furthermore, the LOX gene family is involved in growth and development processes such as fruit ripening, fertility, and aroma compound synthesis. For example, in Arabidopsis thaliana, AtLOX3 and AtLOX4 are associated with male flower fertility; mutations in both genes result in male sterility, which can be restored by exogenous JA application. AtLOX5 is required for lateral root development; and in apple, MdLOX23 is hypothesized to be involved in preharvest fruit ripening. In summary, the LOX gene family has been extensively studied in plant responses to abiotic stress. High temperature stress severely affects eggplant growth and development, but the relationship between SmLOX genes and high temperature stress is currently limited and requires further investigation. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention provides a heat-tolerance-related gene, SmLOX5, in eggplant and its applications. The relative expression level of the SmLOX5 gene increases significantly under high-temperature stress conditions, while plants with the SmLOX5 gene silenced exhibit reduced heat tolerance. Therefore, overexpressing the SmLOX5 gene in Arabidopsis thaliana can improve plant tolerance under high-temperature stress conditions. Therefore, the SmLOX5 gene can be used as a candidate heat-shock response gene for breeding heat-tolerant plant varieties.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide an eggplant heat-resistant gene SmLOX5 or its expression product for use in regulating plant heat tolerance. The nucleotide sequence of the eggplant heat-resistant gene SmLOX5 is shown in SEQ ID NO.1, and the amino acid sequence of the gene SmLOX5 expression product is shown in SEQ ID NO.2.

[0008] Preferably, the application includes improving the heat resistance of plants under high temperature stress conditions and cultivating plant varieties with enhanced heat resistance under high temperature stress conditions.

[0009] Preferably, the plants include eggplant and Arabidopsis thaliana.

[0010] To identify the relationship between SmLOX5 and heat tolerance in eggplant, the present invention overexpressed the SmLOX5 gene in Arabidopsis thaliana and evaluated the heat tolerance of the identified OE-SmLOX5 transgenic Arabidopsis plants. The results showed that heterologous overexpression of SmLOX5 improved Arabidopsis's tolerance to high temperatures. The present invention further analyzed the correlation between the improved heat tolerance of the overexpressed plants and the plant hormone JA, demonstrating that SmLOX5 expression is positively correlated with JA content, indicating that overexpression of SmLOX5 can increase the expression of genes in the JA signaling pathway in Arabidopsis plants.

[0011] The second object of the present invention is to provide a reagent for specifically detecting the gene SmLOX5 or its expression product for use in preparing a product for detecting the heat resistance of eggplant under high temperature stress, wherein the product uses a real-time fluorescence quantitative PCR method to detect the SmLOX5 gene or its expression product.

[0012] Preferably, the product includes a real-time fluorescence quantitative PCR kit, which contains: a primer pair for specifically detecting the SmLOX5 gene, whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] The present invention further verified the expression pattern of the SmLOX gene under high temperature stress through qRT-PCR experiments. After 6 hours of high temperature treatment, the relative expression level of SmLOX5 increased most significantly, with the highest increase of nearly 10 times. Therefore, the SmLOX5 gene or its expression product can be used as a molecular marker to identify the heat tolerance of plants.

[0014] The third object of the present invention is to provide a reagent for silencing SmLOX5 gene expression for use in constructing an eggplant model with reduced heat resistance under high temperature stress, wherein the reagent comprises: primers for amplifying a fragment silencing SmLOX5, whose sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0015] Preferably, the constructed eggplant model reduces the level of plant hormone JA by silencing the SmLOX5 gene, thereby reducing the heat resistance of eggplant under high temperature stress conditions.

[0016] We analyzed the SmLOX5 gene through subcellular localization experiments and proved that SmLOX5 is located in the chloroplast. We also conducted VIGS silencing experiments in eggplant itself. The results showed that silencing the SmLOX5 gene reduced the tolerance of eggplant to high temperature stress conditions.

[0017] The present invention also provides the use of a recombinant vector, expression cassette, transgenic cell line, or transgenic recombinant bacteria containing the heat-resistant gene SmLOX5 in cultivating transgenic heat-resistant plants. Overexpressing the SmLOX5 gene in plants can improve the heat tolerance of the plants. Plants with improved heat tolerance are obtained by transforming the vector, expression cassette, transgenic cell line, or transgenic recombinant bacteria that overexpress the SmLOX5 gene into plant cells, tissues, or organs, and then cultivating the transformed plant cells, tissues, or organs.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This study identifies SmLOX5, a gene from the eggplant LOX gene family whose expression is most significantly induced under high-temperature stress conditions. The study also investigates SmLOX5's heat tolerance, the molecular mechanisms involved in heat stress, and its potential applications. Based on SmLOX5's involvement in eggplant's response to heat stress, it is expected that overexpressing SmLOX5 in eggplant or genetically modifying it in other plants could improve their heat tolerance, thereby increasing plant quality and yield, with significant social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Analysis of the expression pattern of the LOX gene family in eggplant; A. High-temperature transcriptome data analysis of the SmLOX gene family; B. Relative expression levels of the SmLOX gene family at different stages of high-temperature treatment (42°C); Data are expressed as mean ± standard deviation, and different letters (ac) indicate significant differences (p < 0.05).

[0021] Figure 2 Subcellular localization of SmLOX5; A. Prediction result of online tool SUBA5; B. Subcellular localization of SmLOX5 in eggplant protoplasts; GFP represents green fluorescent protein, and ChloroohyⅡ represents chloroplast autofluorescence.

[0022] Figure 3 Effects of silencing SmLOX5 on heat tolerance of eggplant, including: A. expression levels of SmLOX5 in control plants and SmLOX5-silenced plants; B. phenotypes of control plants and SmLOX5-silenced plants under high temperature stress (42°C, 7 d); C. relative electrical conductivity of leaves of control plants and SmLOX5-silenced plants under high temperature stress (42°C, 7 d); D. MDA content in leaves of control plants and SmLOX5-silenced plants under high temperature stress (42°C, 7 d); Data are expressed as mean ± SD (n = 3) and significance was analyzed using Student's t test compared with the control (**, p < 0.01).

[0023] Figure 4Analysis of changes in JA content and JA signaling pathway gene expression in silenced and control plants under high temperature stress (**, p<0.01).

[0024] Figure 5 Effects of heterologous overexpression of SmLOX5 on heat tolerance in Arabidopsis thaliana; A. Phenotypes of wild-type plants and OE-SmLOX5 transgenic plants under normal growth conditions and high temperature stress (43°C, 2 days), scale bar = 2 cm; B: Chlorophyll fluorescence of leaves of wild-type plants and OE-SmLOX5 transgenic plants under normal growth conditions and high temperature stress (43°C, 2 days), scale bar = 2 cm; C: MDA content and Fv / Fm value in leaves of wild-type plants and OE-SmLOX5 transgenic plants under high temperature stress (43°C, 2 days); Data are expressed as mean ± standard error (n = 27), and significance was analyzed using Student's test compared with the control (*, P < 0.05, **, P < 0.01).

[0025] Figure 6 Figure 2 Changes in JA content and expression of JA signaling pathway genes in overexpression plants and control plants under high temperature stress (*, P<0.05, **, P<0.01). DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention and comparative examples. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0028] We identified 12 LOX gene family members in the eggplant genome. Based on their chromosomal location order, the SmLOX genes were named SmLOX1-SmLOX12. The nucleotide sequence of the SmLOX5 gene is shown in SEQ ID NO.1, and the amino acid sequence of its expression product is shown in SEQ ID NO.2.

[0029] Example 1: Analysis of the expression pattern of the LOX gene family in eggplant under high temperature stress conditions

[0030] 1.1 Materials

[0031] Eggplant material "E390" was used as the test material.

[0032] 1.2 Experimental Procedure

[0033] Using the Heat Map function in TBtools software, we created a gene expression heat map based on the existing high-temperature transcripts. Using cDNA from leaves of eggplant accession "E390" at different stages of high-temperature stress (42°C) as templates, we analyzed the relative expression levels of the LOX gene family at different stages of high temperature using qRT-PCR.

[0034] 1.3 Test Method

[0035] 1) Analysis of the expression pattern of the eggplant LOX gene family

[0036] Based on the existing high-temperature transcriptome data of eggplant (Liu et al., 2023), the transcriptome data of the LOX gene family at different stages after high-temperature treatment were retrieved, and a gene expression heat map was drawn using TBtools software.

[0037] 2) Real-time quantitative PCR (qRT-PCR)

[0038] Quantitative primers were designed using Primer Premier 5 software. The primers are shown in Table 1. SmCyclophilin is an eggplant internal reference gene. qRT-PCR was performed using the HiScript III RT SuperMix for qPCR Reagent Kit with gDNAWiper (Vazyme, Nanjing) on ​​a Bio-Rad CFX 384 Touch Fluorescence Quantitative PCR Instrument (Bio-Rad, USA). Three technical replicates were performed for each sample, using 2 -ΔΔCt Quantitative data analysis was performed.

[0039] 1.4 Results Analysis

[0040] The role of SmLOX genes in eggplant's resistance to high temperature stress was analyzed based on the research team's published eggplant high temperature transcription data and qRT-PCR experiments. The high temperature transcriptome data showed that among all SmLOX genes, SmLOX5 and SmLOX9 had high expression levels, and their expression levels increased significantly after 6 hours of high temperature treatment; while the expression levels of other SmLOX genes were relatively low ( Figure 1-A). qRT-PCR experiments were further used to verify the expression pattern of SmLOX genes under high temperature stress. The results showed that the relative expression levels of SmLOX1, SmLOX3, SmLOX8, SmLOX10, and SmLOX11 showed an overall downward trend, while the expression levels of SmLOX2, SmLOX4, SmLOX5, and SmLOX6 increased significantly after 6 hours of high temperature treatment. Among them, the relative expression level of SmLOX5 increased most significantly, with the highest increase of nearly 10 times, while no expression of SmLOX7 and SmLOX9 was detected ( Figure 1 -B). Except for SmLOX9, the qRT-PCR test results were basically consistent with the transcriptome data.

[0041] Table 1 Primers used

[0042]

[0043]

[0044] Note: The underlined sites are enzyme cutting sites.

[0045] Example 2 Effect of Silencing SmLOX5 on Heat Tolerance of Eggplant

[0046] 2.1. Experimental materials: Eggplant material “19009” was used as the experimental material.

[0047] 2.2 Experimental Procedure

[0048] The specific fragment of SmLOX5 was obtained through the Sol Genomics Network online website (https: / / solgenomics.net / ). Homologous recombination primers were designed using CE Design V1 software based on the specific fragment sequence and the restriction site of the pTRV2 vector. The primers are shown in Table 1. Using the leaf cDNA of eggplant material "19009" as a template, PrimeSTAR high-fidelity enzyme (Takara, Beijing) was used to amplify the full-length specific fragment and connect it to the pTRV2 vector through homologous recombination. The plasmid was extracted and sequenced. The correctly sequenced plasmid was transformed into Agrobacterium competent GV3101 (Weidi Biotechnology, Shanghai) using the heat shock method. The centrifuged bacteria were resuspended in infection solution (200μmol / L AS, 10μmol / L MgCl2, 10μmol / L MES, pH 5.7, prepared immediately before use) and the OD was adjusted. 600The bacterial strains were activated in the dark for 1 hour using a pTRV2 blank vector as a control. The pTRV2 empty vector and the constructed pTRV2-LOX5 were mixed with pTRV1 at a 1:1 volume ratio. The bacterial solution was injected into eggplants through the lower epidermis of the cotyledons using a needleless syringe during the flattening stage. After two weeks of normal culture, the silenced plants were evaluated for heat tolerance.

[0049] 2.3 Test Method

[0050] 1) SmLOX5 subcellular localization prediction was performed using the SUBA5 online tool (https: / / suba.live / ). Eggplant subcellular localization assay: The SmLOX5 CDS sequence, with the stop codon removed, was constructed and inserted into the pGreen-GFP vector according to the qRT-PCR method in Example 1. Eggplant protoplasts were prepared and transformed according to the method of Yoo et al. (2007). After 18-24 hours of incubation under low light, protoplasts were observed and photographed using a BX53 biological microscope (OLYMPUS, Japan). A blank vector was used as a control.

[0051] 2) Determine the relative conductivity (REL) of the cell membrane according to the method of Yu Bingwei et al. (2017): Use a 1-cm diameter hole punch to punch 10 small dots on the leaf blade and place them in a 50-mL centrifuge tube containing 15 mL of ultrapure water. Blank ultrapure water is used as a control. After rotating on a shaker at 40 rpm for 90 min at room temperature, the conductivity is measured using a conductivity meter (DDS-307) and recorded as C1. The control is recorded as CK1. The centrifuge tube is placed in a 100°C water bath for 10 min. After cooling naturally to room temperature, the conductivity is measured again and recorded as C2. The control is recorded as CK2. Three technical replicates are performed for each sample.

[0052] Calculation formula: REL = (C1-CK1) / (C2-CK2)*100%.

[0053] 3) Malondialdehyde (MDA) content was determined using the thiobarbituric acid colorimetric method. Weigh 0.5 g of leaves, add 10 mL of 10% TCA solution (added twice), grind on ice to form a homogenate, centrifuge at 8000 rpm at 4°C for 10 minutes, take the supernatant, and place on ice for testing. Take 2 mL of the test solution and 2 mL of 0.5% TBA, shake well, and react the mixture in a boiling water bath for 10 minutes. After rapid cooling, centrifuge at 3000 rpm for 10 minutes. Measure the absorbance of the supernatant at 450 nm, 532 nm, and 600 nm, respectively. Perform three technical replicates for each sample.

[0054] Calculation formula: MDA content (mmol / g FW) = [6.452*(A 532 -A 600)-0.559*A 450 ]*Vt / (Vs*FW)

[0055] Where Vt is the total volume of the extract (mL), Vs is the total volume of the extract used for determination (mL), and FW is the fresh weight of the sample (g).

[0056] 2.4 Results Analysis

[0057] Based on the results of Example 1, we speculated that SmLOX5 may be related to the high temperature stress response of eggplant. The SmLOX5 gene was further analyzed by subcellular localization experiments and VIGS experiments. The subcellular localization results showed that the pGreen-GFP empty vector stimulated green fluorescence in both the nucleus and cell membrane of eggplant leaf cells, while the pGreen-GFP-LOX5 fusion expression vector only observed green fluorescence in the chloroplasts, indicating that SmLOX5 is localized in the chloroplasts ( Figure 2 -B), which is consistent with the results predicted by SUBA5 ( Figure 2 -A). To further investigate the role of SmLOX5 in eggplant's resistance to high temperature stress, a VIGS silencing experiment was conducted on eggplant. The results showed that compared with the control plants (pTRV2 empty vector), the gene expression of SmLOX5 in the VIGS-SmLOX5 silenced plants decreased by 68% ( Figure 3 -A). After 7 days of high temperature treatment at 42℃, the leaves of the control plants showed mild symptoms of water loss and wilting, while the leaves of the silent plants showed severe water loss and wilting, especially the first true leaf ( Figure 3 -B). In addition, the relative electrical conductivity (REL) and malondialdehyde (MDA) content of the cell membrane in the leaves of the silenced plants were significantly higher than those in the control plants ( Figure 3 -C and Figure 3 The above results indicate that SmLOX5 is located in chloroplasts and can improve the tolerance of eggplant to high temperature stress.

[0058] Example 3 Analysis of the correlation between SmLOX5 silenced plants and JA

[0059] 3.1 Data Acquisition

[0060] The sequence information of eggplant JA signaling pathway genes SmMYC2a, SmMYC2b, and SmCOI1 were obtained through the eggplant genome database (http: / / www.eggplant-hq.cn / Eggplant / home / index).

[0061] 3.2 Experimental Procedure

[0062] 1) Analysis of gene expression patterns in the eggplant JA signaling pathway

[0063] The expression patterns of JA signaling pathway genes in eggplant were analyzed by qRT-PCR using cDNA from leaves of control plants and pTRV2-SmLOX5 silenced plants before and after high temperature stress as templates.

[0064] 2) JA content determination

[0065] The JA content in leaves was determined using a plant JA enzyme-linked immunosorbent assay kit (YJ83529S, Mlbio, Shanghai).

[0066] 3.3 Test Method

[0067] 1) Real-time quantitative PCR (qRT-PCR)

[0068] Quantitative primers were designed using Primer Premier 5 software. The primers are shown in Table 1. SmCyclophilin is an eggplant internal reference gene. qRT-PCR was performed using the HiScript III RT SuperMix for qPCR Reagent Kit with gDNAWiper (Vazyme, Nanjing) on ​​a Bio-Rad CFX 384 Touch Fluorescence Quantitative PCR Instrument (Bio-Rad, USA). Three technical replicates were performed for each sample, using 2 -ΔΔCt Quantitative data analysis was performed.

[0069] 2) JA content determination

[0070] The JA content of plants was determined according to the instructions of the plant jasmonic acid (JA) enzyme-linked immunosorbent assay kit (Mlbio, Shanghai). The specific experimental steps are as follows:

[0071] A: Extraction of plant jasmonic acid: For specific methods, refer to Li Jinke et al. (2010).

[0072] B: Determination of jasmonic acid in plants:

[0073] (1) Prepare a standard curve: Use the standard sample in the kit according to the instructions to prepare a standard curve.

[0074] (2) Sample addition: Dilute the sample to be tested 5 times with sample diluent and add the sample to the bottom of the ELISA plate well.

[0075] (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min.

[0076] (4) Washing: Wash the plate 5 times with detergent and pat dry.

[0077] (5) Add enzyme: Add 50 μL enzyme-labeled reagent to each well.

[0078] (6) Incubation: Same as (3).

[0079] (7) Washing: Same as (4).

[0080] (8) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, mix well, and develop color at 37°C in the dark for 10 min.

[0081] (9) Termination: Add 50 μL of stop solution to each well to terminate the reaction.

[0082] (10) Determination: After 15 min of reaction, measure the absorbance at 450 nm and calculate the corresponding content based on the standard curve.

[0083] 3.4 Results Analysis

[0084] To analyze whether the reduction in heat tolerance in silenced plants is related to the plant hormone JA, we tested the JA content in pTRV2-SmLOX5 silenced plants and control plants before and after high temperature stress. The results showed that after 6 hours of high temperature stress, except for a few lines, the JA content in pTRV2-SmLOX5 silenced plants was significantly lower than that in control plants ( Figure 4 ). The expression patterns of JA signaling pathway genes SmMYC2a, SmMYC2b, and SmCOI1 in the control plants and pTRV2-SmLOX5 silenced plants were further analyzed. The results showed that before high temperature stress, the relative expression levels of JA signaling pathway genes SmMYC2a, SmMYC2b, and SmCOI1 in the pTRV2-SmLOX5 silenced plants were not significantly different from those in the control plants. After high temperature stress, the relative expression levels of SmMYC2a, SmMYC2b, and SmCOI1 showed an up-regulated expression trend in both the control plants and the pTRV2-SmLOX5 silenced plants, but the relative expression levels of SmMYC2b and SmCOI1 were significantly lower than those in the control plants, while the relative expression level of SmMYC2a in the pTRV2-SmLOX5 silenced plants was lower than that in the control plants, but there was no significant difference between the two. Taken together, these results indicate that the reduction in heat tolerance in silenced plants may be related to the JA signaling pathway.

[0085] Example 4 Overexpression of SmLOX5 in Arabidopsis improves plant heat tolerance

[0086] To verify the role of the SmLOX5 gene in plant resistance to high temperature stress, SmLOX5 overexpressing Arabidopsis plants were constructed.

[0087] 4.1 The specific construction method is as follows:

[0088] A: Vector construction: The SmLOX5 CDS sequence with the stop codon removed was constructed into the pBI121-GFP vector.

[0089] B: Transformed Agrobacterium competent cells.

[0090] C: PCR detection of bacterial liquid.

[0091] D: Shake the bacterial solution and resuspend and activate the bacteria: The infection solution formula is shown in Table 2.

[0092] Table 2 Infection solution formula

[0093]

[0094]

[0095] E: Infecting Arabidopsis:

[0096] (1) Preparation of Arabidopsis thaliana: Cut off the fruit pods of Arabidopsis thaliana during the flowering period.

[0097] (2) Soak the Arabidopsis inflorescence in the infection solution for 1 min.

[0098] (3) Lay the infected Arabidopsis plants flat on a tray, keep them moist, and culture them in the dark at 22°C for 1 day.

[0099] (4) Wash away the residual infection solution on the Arabidopsis inflorescence with clean water and culture normally until harvest.

[0100] F: Identification of positive seedlings:

[0101] (1) Pour T0 generation Arabidopsis seeds into a 2ml centrifuge tube, wash them 3 times with sterilized water, and then sterilize them with 2.5% sodium hypochlorite (prepared immediately before use) for 10 minutes, shaking them up and down during the process to sterilize them as completely as possible. Then wash them 5 times with sterilized water, and use sterilized filter paper to absorb all the excess water on the surface of the seeds. Then spread the seeds on a 1 / 2 MS culture medium plate (containing the corresponding resistance), vernalize them at 4℃ for 3 days, and culture them in a greenhouse under 50% light. After screening for about 7 days, transplant the still green plants into culture soil and culture them normally until harvest. The whole screening process is repeated once again for a second screening. The T2 generation seeds collected later can meet the experimental requirements.

[0102] (2) PCR identification: Genomic DNA was extracted from leaves of T2 plants and PCR amplified using universal primers for the pBI121-GFP vector (Table 3). Plants with detectable target bands were kept and cultured normally until harvest.

[0103] (3) Determination of relative expression of genes in positive seedlings: RNA was extracted from leaves of T2 generation plants and reverse transcribed into cDNA. The expression of genes in positive seedling transformation was detected. The quantitative primers are shown in Table 3.

[0104] Table 3

[0105]

[0106]

[0107] 4.2 Test Method

[0108] Chlorophyll fluorescence measurement: A chlorophyll fluorescence meter was used to detect the chlorophyll fluorescence system of plants according to the method of Liang Yonggui (2023), and PlantExplorer™ software was used for data analysis.

[0109] The JA content was determined using the same method as in Example 3.

[0110] 4.3 Results Analysis

[0111] The present invention used wild-type Arabidopsis and OE-SmLOX5 transgenic Arabidopsis grown for two weeks under normal soil conditions as materials. After being subjected to a high temperature treatment at 43°C for two days, their phenotypes, MDA content, chlorophyll fluorescence, and photosynthetic system parameters were observed and measured. The results showed that after high temperature treatment, wild-type Arabidopsis showed severe stress damage, with severe leaf dehydration and wilting, and the lower part of the leaves completely yellowed and withered, approaching death. However, OE-SmLOX5 transgenic Arabidopsis showed fewer stress symptoms, with leaves showing slight curling ( Figure 5 A). Determination of MDA content in leaves confirmed this phenotype. Under high temperature stress, the MDA accumulated in the leaves of OE-SmLOX5 transgenic Arabidopsis was significantly lower than that of wild-type Arabidopsis. Among them, the OE-SmLOX5-5 line accumulated the least MDA after high temperature treatment and had the least damage to its membrane system ( Figure 5 C). In addition, high temperature stress can damage the photosynthetic system of plants. The present invention further tested the chlorophyll fluorescence and photosynthetic system parameters of the plants. The results showed that under normal conditions, the photosynthetic system activities of wild-type plants and OE-SmLOX5 transgenic plants were similar, and both showed stable photosynthetic systems. However, after 2 days of high temperature treatment, the photosynthetic systems of all strains became unstable, especially in wild-type Arabidopsis, where the photosynthetic system became extremely unstable ( Figure 5 B). This difference was further quantified by measuring the Fv / Fm value. After 2 days of high temperature treatment, the Fv / Fm values ​​of all strains showed a downward trend. The Fv / Fm value of wild-type Arabidopsis decreased the most and was significantly lower than that of OE-SmLOX5 transgenic Arabidopsis ( Figure 5 C) These results indicate that heterologous overexpression of SmLOX5 improves the tolerance of Arabidopsis to high temperature.

[0112] In addition, to further analyze whether the improved heat tolerance of overexpression plants is related to the plant hormone JA, the JA content of OE-SmLOX5 overexpression plants and control plants was tested before and after high temperature treatment. The results showed that after 6 hours of high temperature treatment, the JA content of both control plants and overexpression plants increased. In addition, regardless of whether high temperature treatment was performed or not, the JA level of OE-SmLOX5 overexpression plants was significantly higher than that of wild-type plants ( Figure 6 ). At the same time, the expression of JA signaling pathway genes AtMYC2 and AtCOI1 in OE-SmLOX5 overexpressing plants and control plants was analyzed. The results showed that the relative expression levels of JA signaling pathway genes AtMYC2 and AtCOI1 in OE-SmLOX5 overexpressing plants were higher than those in wild-type plants after high temperature treatment (6). The above results indicate that the expression level of SmLOX5 is positively correlated with JA content, and overexpression of SmLOX5 can increase the expression of JA signaling pathway genes in Arabidopsis plants.

[0113] The above embodiments of the present invention are merely examples for the purpose of clearly illustrating the technical solutions of the present invention and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Overexpression of heat-resistant genes in eggplant SmLOX5 or the use of its expression product in regulating plant heat tolerance, characterized in that, The eggplant heat-resistant gene SmLOX5 The nucleotide sequence of the eggplant heat-resistant gene is shown in SEQ ID NO.

1. SmLOX5 The amino acid sequence of the expression product is shown in SEQ ID NO. 2; the application refers to improving the heat resistance of plants under high temperature stress conditions and cultivating plant varieties with enhanced heat resistance under high temperature stress conditions; the plant is eggplant or Arabidopsis thaliana.

2. A specific method for detecting heat-resistant genes in eggplant SmLOX5 or a reagent containing an expression product thereof in the preparation of a product for detecting the heat resistance of eggplant under high temperature stress, characterized in that: The eggplant heat-resistant gene SmLOX5 The nucleotide sequence of the eggplant heat-resistant gene is shown in SEQ ID NO.

1. SmLOX5 The amino acid sequence of the expression product is shown in SEQ ID NO. 2; The product uses real-time fluorescence quantitative PCR method to detect eggplant heat-resistant genes SmLOX5 or its expression product, eggplant was treated at 42℃ for 6h, when the heat-resistant gene of eggplant was detected SmLOX5 If the relative expression level of Cyclophilin as the internal reference gene increases, the eggplant has heat resistance under high temperature stress.

3. The use according to claim 2, characterized in that The product includes a real-time fluorescence quantitative PCR kit containing a specific detection of eggplant heat-resistant genes SmLOX5 The primer pair, whose sequences are shown in SEQ ID NO. 3 and SEQ ID NO.

4.

4. Contains eggplant heat-resistant genes SmLOX5 The use of the recombinant vector, expression cassette, and transgenic cell line in cultivating transgenic heat-resistant plants is characterized in that: The eggplant heat-resistant gene SmLOX5 The nucleotide sequence of the eggplant heat-resistant gene is shown in SEQ ID NO.

1. SmLOX5 The overexpression vector, expression cassette, and transgenic cell line are transformed into plant cells, tissues, or organs, and the transformed plant cells, tissues, or organs are then cultivated to obtain plants with improved heat resistance; the plants are eggplant or Arabidopsis.

5. Contains eggplant heat-resistant gene SmLOX5 The use of a transgenic recombinant bacterium in cultivating transgenic heat-resistant plants is characterized in that: The eggplant heat-resistant gene SmLOX5 The nucleotide sequence of the eggplant heat-resistant gene is shown in SEQ ID NO.

1. SmLOX5 The overexpressed transgenic recombinant bacteria are transformed into plant cells, tissues or organs, and the transformed plant cells, tissues or organs are then cultivated to obtain plants with improved heat resistance; the plants are eggplant or Arabidopsis.

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