Use of cucumber cshsp17.8a gene in regulating heat resistance and moisture and heat resistance

Abstract

Disclosed in the present application is the use of a CsHSP17.8A gene in regulating heat resistance and moisture and heat resistance, belonging to the technical field of genetic engineering. The nucleotide sequence of the CsHSP17.8A gene is shown as SEQ ID No. 1. Overexpressing the CsHSP17.8A gene improves the tolerance of yeast cells to high temperatures, and silencing the CsHSP17.8A gene reduces the tolerance of cucumbers to moisture and heat stress.

Description

Application of cucumber CsHSP17.8A gene in regulating heat and damp heat resistance

[0001] This application claims priority to Chinese Patent Application No. 2025101891374, filed on February 20, 2025, entitled "Application of Cucumber CsHSP17.8A Gene in Regulating Heat and Humid Heat Resistance", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of genetic engineering technology, and in particular to the application of the cucumber CsHSP17.8A gene in regulating heat resistance and damp heat resistance. Background Technology

[0003] Cucumber is an important economic crop, mainly cultivated in greenhouses. It prefers warm temperatures but is intolerant of heat, and it prefers moist conditions but is intolerant of waterlogging. Temperatures inside greenhouses are generally high in spring and autumn, and poor ventilation further exacerbates the hot and humid environment. Currently, research on high-temperature stress in cucumbers is relatively common, but studies on combined high-temperature and high-humidity environments are relatively scarce. Therefore, investigating the molecular mechanisms by which cucumbers respond to hot and humid stress is of great significance. Summary of the Invention

[0004] The purpose of this application is to provide the application of the cucumber CsHSP17.8A gene in regulating damp heat tolerance and heat tolerance. The CsHSP17.8A gene positively regulates the tolerance of cucumber to damp heat stress and positively regulates the tolerance of yeast cells to high temperature.

[0005] To achieve the above objectives, this application provides the following technical solution:

[0006] The application of the CsHSP17.8A gene in regulating the heat resistance of yeast cells and the damp heat resistance of cucumber, the nucleotide sequence of the CsHSP17.8A gene is shown in SEQ ID No. 1.

[0007] Application of the protein encoded by the CsHSP17.8A gene in regulating the heat resistance of yeast cells and the damp heat resistance of cucumber.

[0008] Application of recombinant vectors containing the CsHSP17.8A gene in regulating the heat resistance of yeast cells and the damp heat resistance of cucumbers.

[0009] Application of host bacteria containing the recombinant vector in the heat resistance of yeast cells and the resistance to damp heat in cucumbers.

[0010] A method for regulating the heat resistance of yeast cells involves using genetic engineering techniques to increase the expression of the CsHSP17.8A gene in yeast cells. Attached Figure Description

[0011] Figure 1 shows the vector spectra. In the figure, A is the pGADT7-Rec vector spectrum and B is the pTRV2 vector spectrum.

[0012] Figure 2 shows the expression level of cucumber CsHSP17.8A in response to hot and humid stress.

[0013] Figure 3 shows the agarose gel electrophoresis results of pGADT7-CsHSP17.8A. In the figure, A is the gel electrophoresis image of a correctly cloned CsHSP17.8A, and B is the gel electrophoresis image of a culture containing pGADT7-CsHSP17.8A.

[0014] Figure 4 shows the growth of single yeast colonies transformed with pGADT7 and pGADT7-CsHSP17.8A under high temperature stress.

[0015] Figure 5 is a schematic diagram of the silencing site of CsHSP17.8A.

[0016] Figure 6 shows agarose gel electrophoresis images. In the figure, A is a gel electrophoresis image of the silent fragment clone of CsHSP17.8A, and B is a gel electrophoresis image of Agrobacterium colony PCR of pTRV2-CsHSP17.8A plasmid.

[0017] Figure 7 shows the electrophoresis diagrams for identifying pTRV1 and pTRV2 vectors.

[0018] Figure 8 shows the silencing efficiency of pTRV2-CsHSP17.8A positive seedlings.

[0019] Figure 9 shows the effects of moist heat treatment on pTRV2 and pTRV2-CsHSP17.8A-silenced plants. In Figure 9, A represents the growth status of pTRV2 and pTRV2-CsHSP17.8A-silenced plants under moist heat treatment and control conditions; B represents the relative expression of CsHSP17.8A in pTRV2 and pTRV2-CsHSP17.8A-silenced plants after moist heat treatment. Detailed Implementation

[0020] This application provides the application of the CsHSP17.8A gene in regulating the heat resistance of yeast cells and the damp heat resistance of cucumbers, wherein the nucleotide sequence of the CsHSP17.8A gene is shown in SEQ ID No. 1. This application describes the use of overexpressing the CsHSP17.8A gene in cucumbers to improve the heat resistance of the yeast cells. This application describes the use of silencing the CsHSP17.8A gene in cucumbers to reduce the damp heat resistance of the cucumbers.

[0021] This application also provides the application of the protein encoded by the CsHSP17.8A gene in regulating the heat resistance of yeast cells and the damp heat resistance of cucumbers. The amino acid sequence of the protein encoded by the CsHSP17.8A gene of this invention is shown in SEQ ID No. 16, specifically: MSLIPSFFGGRRTNVFDPFSLDAWDPFQGFSFSNSLSNLPSSAFANTRIDWKETPQAHIFTADLPGINKQEVKVEVQEGRVLQISGERSKEQEEKNDKWHRIERSSGQFVRRFRLPENAKVDEVKASMENGVLTVTVPKVEEKKPEIIKSIEITDH.

[0022] This application also provides the application of recombinant vectors containing the CsHSP17.8A gene or host bacteria containing the recombinant vectors in regulating the heat resistance of yeast cells and the damp heat resistance of cucumbers.

[0023] This application also provides a method for regulating the heat resistance of yeast cells, utilizing genetic engineering techniques to increase the expression of the CsHSP17.8A gene in yeast cells. The genetic engineering techniques described in this application include ligating the CsHSP17.8A gene into a yeast expression vector, and then transforming the resulting overexpression vector into yeast cells. The yeast expression vector described in this application includes pGADT7. The yeast cells described in this application include AH109 yeast cells.

[0024] This application has the following beneficial effects:

[0025] This application employed gene family analysis to identify candidate genes associated with resistance to abiotic stress, discovering that the CsHSP17.8A gene plays a crucial role in cucumber abiotic stress. To further clarify the regulatory mechanism of the CsHSP17.8A gene under abiotic stress, the target gene was cloned, and a VIGS silencing vector was constructed to study its abiotic function. Results showed that after wet heat treatment, the growth point of control plants remained normal, but the leaf margins of the two true leaves became lighter in color. In contrast, the growth point of pTRV2-CsHSP17.8A-silenced plants was curled, shrunken, and damaged, with extensive damage to the two true leaves and curled leaf margins. Furthermore, after wet heat treatment, the expression level of the CsHSP17.8A gene in silenced plants was significantly lower than that in control plants. Additionally, experiments showed that overexpression of CsHSP17.8A enhanced the heat tolerance of yeast cells under high-temperature stress. These findings confirm that the CsHSP17.8A gene positively regulates cucumber tolerance to wet heat stress and positively regulates yeast cell tolerance to high temperatures.

[0026] The above technical solution will be further described below with reference to the accompanying drawings and embodiments.

[0027] Unless otherwise specified, the methods used in the following examples are conventional methods. Unless otherwise specified, the reagents, culture media, or carriers used in the examples are all commercially available products.

[0028] The test materials and reagents used in the following examples are as follows:

[0029] (1) Test materials

[0030] The material used in this experiment was the Jinchun No.4 cucumber variety, whose seeds were purchased from Tianjin Kerun Cucumber Research Institute.

[0031] Strains and vectors: GV3101 Agrobacterium competent cells, DH5α Escherichia coli competent cells, and AH109 yeast competent cells were all purchased from Shanghai Weidi Biotechnology Co., Ltd.; the yeast eukaryotic expression vector pGADT7-Rec and the gene-silencing VIGS vector system (TRV1 and TRV2) were purchased from NovoPro. The pGADT7-Rec vector map and the pTRV2 vector map are shown in Figure 1 (A, B).

[0032] (2) The test reagents are shown in Table 1.

[0033] Table 1. Reagents used in the experiment

[0034] a. The culture medium formulation is shown in Table 2.

[0035] Table 2 Culture medium formulation

[0036] Note: Adjust the pH to 7.0 with 1 mol / L NaOH, then sterilize at 121℃ for 20 min.

[0037] b. Preparation of 100mL 50×TAE buffer: 24.2g TRIS + 3.72g Na2EDTA·2H2O + 5.71mL glacial acetic acid. Electrophoresis buffer and gel preparation require a 1×TAE buffer: dissolve 10mL of 50×TAE in ddH2O in a 500mL volumetric flask.

[0038] c. Antibiotic Preparation: All antibiotics should first be prepared into a stock solution concentration suitable for storage, and then diluted according to the working concentration before use. The specific formulas are shown in Table 3. The prepared antibiotics should be filtered into sterilized 2mL centrifuge tubes in a laminar flow hood using a 0.22μm filter membrane (Jinteng, Tianjin) and a 5mL syringe, and then stored at -20℃ protected from light.

[0039] Table 3. Preparation formula of antibiotic stock solution (10 mL)

[0040] d. Preparation of activation buffer

[0041] 1M KOH: Weigh 5.6g of KOH and dilute to 100mL of distilled water;

[0042] 1M MES-KOH: Weigh 10.6625g of MES and dilute to 50mL of distilled water. Then adjust the pH to 5.6 with 1M KOH, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0043] 1MAS: Weigh 0.196g of AS and make up to 1mL of DMSO. Sterilize the filter membrane, aliquot into 200μL PCR tubes, and store at -20℃ in the dark.

[0044] 1M MgCl2: Dissolve 9.521g MgCl2 in 100mL ddH2O, filter through a 0.22μm filter membrane for sterilization, and store at 4℃;

[0045] The formulation of the activation buffer is shown in Table 4.

[0046] Table 4 Activation Buffer Formulation (100 mL)

[0047] Example 1: Cucumber CsHSP17.8A response to damp heat stress

[0048] Using the cucumber variety "Jinchun No.4" as the experimental material, cucumber seeds were first soaked in 55℃ hot water for 15 minutes for sterilization before sowing, then soaked at room temperature for 4-6 hours to ensure full water absorption, and then placed in a 28℃ constant temperature incubator for 24 hours for germination. After germination, the seeds were sown in 50-cell trays, and the incubator environment was controlled at 25 / 18℃ (14h / 10h), relative humidity 80%, and daytime photosynthetically active radiation 200 μmol·s⁻¹. -1 ·m -2 When seedlings reached the 2-leaf, 1-heart stage, they underwent moist heat treatment (T, 42℃ + 95% RH), with 25℃ + 80% RH as the control (CK). Samples were taken 6 hours after treatment. RNA was extracted from the leaves of Jinchun No. 4 cucumber seedlings using a plant RNA extraction kit, and cDNA templates were obtained using an EasyScript one-step gDNA removal and cDNA synthesis kit. Specific primers for CsHSP17.8A (SEQ ID No. 2 and SEQ ID No. 3) and actin primer sequences (SEQ ID No. 4 and SEQ ID No. 5) were designed. qPCR detection was performed using a 2×Q3 SYBR qPCR MasterMix real-time kit, with three technical replicates.-ΔΔCt The relative expression levels of the gene were analyzed. The results showed that the expression level of CsHSP17.8A in cucumber was significantly increased after cucumber was subjected to humid and heat stress (Figure 2), indicating that cucumber CsHSP17.8A can respond to humid and heat stress.

[0049] Example 2: Eukaryotic expression of cucumber CsHSP17.8A in yeast

[0050] Primers with homologous arms for amplifying the target fragment were designed as SEQ ID No. 6 and SEQ ID No. 7. The amplification was performed using the obtained cDNA template and a premixed high-fidelity enzyme. The CDS sequence of CsHSP17.8A was cloned using HS (Premix) to obtain the CDS sequence of the CsHSP17.8A gene with the pGADT7 homologous arm sequence (Figure 3A). The gel recovery product with the correct sequencing was selected and ligated into the yeast expression vector pGADT7. The restriction enzyme sites were BamHI and EcoRI to obtain the recombinant plasmid.

[0051] The correctly sequenced recombinant plasmid and the pGADT7 empty vector were used as controls and transformed into competent AH109 yeast cells, respectively. The cells were plated on SD / -Leu solid medium containing Amp resistance. After 3 days, positive single clones were picked and dissolved in 10 μL ddH2O. 2 μL of the bacterial culture was then incubated at 95°C for 10 min for yeast colony PCR detection (Figure 3B). Colony PCR was performed using 2×TaqPCR premixed reagent II. The primer sequences for colony PCR were SEQ ID No. 8 and SEQ ID No. 9.

[0052] Select bacterial suspensions with the correct band size and add them to SD / -Leu liquid deficient medium containing Amp for expansion culture. Incubate at 28℃ and 250 rpm for 12-14 hours until OD is reached. 600 The OD value is approximately 0.8. Colonies are collected by centrifugation, the supernatant is discarded, and the solution is diluted with 1×PBS to achieve an OD value of approximately 0.8. 600 The concentration was set at 0.3, and the culture was then incubated at 28℃, 37℃, and 42℃ at 250 rpm for 12 h. 5 μL of each bacterial culture was spotted onto SD / -Leu plates containing Amp resistance, incubated at 28℃ for 3 days, and then photographed using a stereomicroscope.

[0053] As shown in Figure 4, at 28℃, yeast colonies transformed with pGADT7 and pGADT7-CsHSP17.8A grew normally, with no significant difference in growth status. However, at 37℃ and 42℃, single colonies transformed with pGADT7-CsHSP17.8A showed significantly stronger growth than those transformed with pGADT7, indicating that yeast cells transformed with pGADT7-CsHSP17.8A have stronger heat resistance than those transformed with pGADT7. This also proves that overexpression of heterologous pGADT7-CsHSP17.8A can effectively improve the heat resistance of AH109 yeast cells.

[0054] Example 3: Effect of gene silencing of cucumber CsHSP17.8A on the heat and humidity tolerance of cucumber

[0055] The SGN VIGS Tool from the Sol Genomics Network (https: / / vigs.solgenomics.net / #opennewwindow), a website dedicated to VIGS primer design, was used to design the silencing site for CsHSP17.8A. The silencing fragment was set to 300 bp to ensure that the silenced sequence was unique in the genome. The silencing location is shown in Figure 5. A premixed high-fidelity enzyme was used. The silenced fragment was cloned using HS (Premix), and the cloning primer sequences are shown in SEQ ID No. 10 and SEQ ID No. 11. Agarose gel electrophoresis confirmed that the gene fragment was the same size as the target fragment (Figure 6A), proving that the target fragment was successfully cloned.

[0056] The silenced fragment was ligated into the pTRV2 vector, with BamHI and XhoI restriction enzyme sites. The correctly sequenced target plasmid, pTRV1 plasmid, and pTRV2 plasmid were transformed into GV3101 Agrobacterium competent cells, respectively. The correctly sequenced positive clones (Figure 6B) were selected and transferred into LB liquid medium containing Rif, Kan, and Gent for expansion culture for transformation.

[0057] Each individual plant was numbered, and the cotyledons of the seedlings were injected. When the injected plants grew to 2 leaves and 1 heart, DNA was extracted from the top true leaf of the cucumber seedling for identification of positive seedlings. Primers for pTRV1 (SEQ ID No. 12 and SEQ ID No. 13) and pTRV2 (SEQ ID No. 14 and SEQ ID No. 15) vectors were used for detection, and the detection results were obtained (Figure 7).

[0058] RNA was extracted from positive seedlings showing bright bands on both pTRV1 and pTRV2. Then, qPCR detection of CsHSP17.8A was performed using primers SEQ ID No. 2 and SEQ ID No. 3. Seedlings infected with the pTRV2 empty vector (pTRV1+pTRV2) were used as controls to detect the expression level of CsHSP17.8A. A total of 6 positive seedlings were obtained, with a silencing efficiency of 24%-56% (Figure 8), demonstrating that silencing CsHSP17.8A using the VIGS technique is successful and effective.

[0059] To investigate the response of cucumber seedlings to heat and humidity stress after CsHSP17.8A silencing, cucumber seedlings with two leaves and one bud (pTRV2 and pTRV2-CsHSP17.8A) were subjected to heat and humidity treatment at 42℃ and 95% relative humidity (RH), with a control environment of 25℃ and 80% relative humidity (RH). The results showed that after heat and humidity treatment, the growth point of the control plants remained normal, but the leaf margins of the two true leaves became lighter in color. In contrast, the growth point of the pTRV2-CsHSP17.8A-silenced plants was curled, withered, and damaged, and the two true leaves showed extensive damage and curled leaf margins (Figure 9A). Furthermore, after heat and humidity treatment, the CsHSP17.8A gene expression level in the silenced plants was significantly lower than that in the control plants (Figure 9B). These results indicate that silencing CsHSP17.8A significantly reduces the heat and humidity tolerance of cucumber seedlings.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the CsHSP17.8A gene in regulating the heat tolerance of yeast cells and the damp heat tolerance of cucumber, characterized in that, The nucleotide sequence of the CsHSP17.8A gene is shown in SEQ ID No.

1.

2. The application of the protein encoded by the CsHSP17.8A gene as described in claim 1 in regulating the heat resistance of yeast cells and the damp heat resistance of cucumbers.

3. The application of the recombinant vector containing the CsHSP17.8A gene as described in claim 1 in regulating the heat resistance of yeast cells and the damp heat resistance of cucumber.

4. The use of a host bacterium containing the recombinant vector as described in claim 3 in the heat resistance of yeast cells and the resistance to damp heat in cucumbers.

5. The application as described in any one of claims 1-4, characterized in that, Silencing the CsHSP17.8A gene in the cucumber reduces the cucumber's resistance to damp heat.

6. The application as described in any one of claims 1-4, characterized in that, Overexpression of the CsHSP17.8A gene in the yeast cells improves the heat resistance of the yeast cells.

7. A method for regulating the heat resistance of yeast cells, characterized in that, Using genetic engineering techniques, the expression of the CsHSP17.8A gene described in claim 1 was increased in yeast cells.

8. The method according to claim 7, characterized in that, The genetic engineering method includes ligating the CsHSP17.8A gene into a yeast expression vector and then transferring the resulting overexpression vector into yeast cells.

9. The method as described in claim 8, characterized in that, The yeast expression vector includes pGADT7.

10. The method as described in claim 8, characterized in that, The yeast cells include AH109 yeast cells.

Images