Application of muskmelon CmPP2C4 gene in regulation and control of cold resistance of muskmelon

Abstract

The invention belongs to the technical field of biology, and particularly discloses application of a muskmelon CmPP2C4 gene in regulation and control of cold resistance of muskmelons, the nucleotide sequence of the muskmelon CmPP2C4 gene is shown as SEQ ID NO.1, a muskmelon CmPP2C4 gene silencing vector is constructed through virus-induced instantaneous silencing, the muskmelon CmPP2C4 gene is silenced in muskmelon seedling bodies, and the muskmelon CmPP2C4 gene can be used for regulating and controlling cold resistance of the muskmelons. The muskmelon CmPP2C4 silent plant capable of improving the tolerance to the cold injury stress is obtained. Compared with the prior art, the silent plant is subjected to 4 DEG C cold injury stress treatment, the cold resistance of the silent plant is further improved, the electrolyte permeability and the malondialdehyde content are remarkably lower than those of a control group, and the proline content and the soluble sugar content are remarkably higher than those of the control group; the determination of the contents of antioxidant enzymes (SOD, CAT and APX) and hydrogen peroxide (H2O2) shows that the antioxidant enzyme activity of a CmPP2C4 silent plant is more strongly induced, and the in-vivo H2O2 accumulation amount is obviously lower than that of a control group.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, particularly to melons. CmPP2C4 Application of genes in regulating the cold resistance of melons. Background Technology

[0002] Melons are loved by consumers for their fragrant flavor and have become one of the world's most popular fruits. China is a major producer and consumer of melons, with per capita consumption increasing year by year and market demand gradually expanding. The planting area covers 31 provinces and municipalities, with the north being the main melon producing area (Yang Nian et al., 2019). In recent years, the melon cultivation and production model has gradually shifted from open-field production to facility cultivation. Because facility cultivation has advantages such as (1) the ability to control the environment to a large extent, (2) the regulation of growth and development, and (3) the ability to achieve early market entry and increase economic benefits, its application has been relatively rapid. However, my country's protected horticulture is still in its early stages of development, with generally poor insulation and cold protection capabilities (Li Tianlai et al., 2019). The cold winter and spring climate in northern China easily causes chilling stress on melon production, significantly reducing yield (-10%) (Korkmaz and Dufault, 2003, 2004) and quality (soluble solids in overwintering crops are generally 8-10%) (data measured by our research group), greatly impacting the melon industry and its economic benefits. Unfortunately, there are currently no cold-resistant, high-quality melon varieties on the market. Therefore, cultivating cold-resistant melon varieties is the best way to help the melon industry achieve low input and high output, and it is also a key issue that urgently needs to be addressed. Summary of the Invention

[0003] To solve the above-mentioned technical problems, the present invention provides a melon. CmPP2C4 Application of genes in regulating the cold resistance of melons.

[0004] To achieve the above objectives, the present invention is implemented according to the following technical solution: melon CmPP2C4 Application of genes in regulating the cold resistance of melons, the melons CmPP2C4 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0005] Specifically, melons were constructed through virus-induced transient silencing. CmPP2C4 Gene silencing vector, and the melon seedlings were used to transmit the melon gene silencing vector. CmPP2C4 Gene silencing produces melons with increased tolerance to cold stress. CmPP2C4 Silent plant.

[0006] Compared with existing technologies, this invention subjected silent plants to 4°C cold stress treatment and found that the cold tolerance of silent plants was further improved, as evidenced by significantly lower electrolyte permeability and malondialdehyde content compared to the control, and significantly higher proline and soluble sugar content compared to the control. Measurements of antioxidant enzymes (SOD, CAT, APX) and hydrogen peroxide (H2O2) content revealed… CmPP2C4 The antioxidant enzyme activity of the silent plants was more strongly induced, and the amount of H2O2 accumulated in the plants was significantly lower than that of the control. Attached Figure Description

[0007] Figure 1 To identify 59 PP2C family members from the melon genome, 59 melons were... CmPP2C Phylogenetic analysis of family members and Arabidopsis PP2C members revealed... CmPP2C4 Belonging to subgroup A indicates CmPP2C4 It may have the function of negatively regulating the ABA metabolic pathway in the A subgroup.

[0008] Figure 2 for CmPP2C4 Expression level analysis at different time points after cold stress treatment.

[0009] Figure 3 To silence CmPP2C4 in No. 330 using VIGS technology, and then... CmPP2C4 Growth status of gene-silenced plant seedlings after being subjected to 4℃ cold stress.

[0010] Figure 4 for CmPP2C4 After cold stress treatment, the electrolyte permeability (A), malondialdehyde content (B), proline content (C), and soluble sugar content (D) of the leaves of gene-silenced plants were measured.

[0011] Figure 5 for CmPP2C4 After being subjected to cold stress, the activities of superoxide dismutase (SOD) (A), catalase (CAT) (B), ascorbate peroxidase (APX) (C), and hydrogen peroxide (H2O2) content (D) in the leaves of silent plants were measured. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0013] Example 1

[0014] Fifty-nine PP2C family members were retrieved and identified from the melon genome, and bioinformatics analysis was performed. The specific analysis process is as follows: (1) PP2C family protein sequences from Arabidopsis thaliana and rice were used, with 80 and 78 sequences respectively, totaling 158 sequences. Then, a hidden Markov model was constructed using HMMER 3.0 software on the obtained known PP2C protein family sequences. This model was used to search for all coding protein sequences of Cucumis melo and to find all potential PP2C family sequences in the Cucumis melo protein sequence.

[0015] (2) Use blastp (version: ncbi-blast-v2.10.1+) to align the sequences of all Cucumis melo proteins to obtain the PP2C family reference sequence, set the e-value to 1e-20, and use the aligned sequence as all potential PP2C family sequences.

[0016] All potential sequences from steps 1 and 2 above were merged as candidate PP2C family protein sequences. The obtained candidate sequences were annotated using the software pfamscan (version: v1.6) and the Pfam A (version: v33.1) database. Sequences containing the PF00481 domain were identified as the final PP2C sequences, totaling 59 sequences. The structural information of these 59 PP2C family members was analyzed, and their physicochemical properties were then analyzed using the website http: / / web.expasy.org / protparam / .

[0017] Next, ML trees were constructed using the identified PP2C protein family sequences from *Cucumis melo* and *Arabidopsis thaliana*. The construction method involved multiple sequence alignment using MAFFT (version: v7.427), followed by ML tree construction using MEGA (MEGA10) software. The parameters were set as follows: Jones-Taylor-Thornton (JTT) model, partial deletion for missing data, cutoff of 50%, and bootstrap value of 1000. The bootstrap value was used to verify the reliability of the calculated phylogenetic tree branches. Phylogenetic tree annotation was performed using iTOL v6 (https: / / itol.embl.de / ). The results are as follows. Figure 1 As shown.

[0018] CmPP2C24-like was named CmPP2C4 The coding region is 1407 bp long, the amino acid length is 468 aa, the molecular weight is 51.63 kDa, and the isoelectric point is 8.44. CmPP2C4 belongs to subfamily A.

[0019] The cold-resistant test material No. 330 melon leaf samples were analyzed at different time points after cold stress treatment using real-time quantitative PCR. CmPP2C4 The changes in expression levels are analyzed in detail below: Total RNA was extracted from melon leaves using the Kangwei Biotechnology Ultrapure RNA Extraction Kit (Catalog No.: CW0581M), and digested with DNase I (Catalog No.: M6101, Promega) according to the manufacturer's instructions. cDNA synthesis was performed according to the GoScript reverse transcriptase (Catalog No.: A2790, Promega) manufacturer's instructions. Real-time quantitative PCR (RT-qPCR) experiments were conducted using an ABI 7500 Real-time PCR instrument (Applied Biosystems) paired with a SYBR Green PCR Real MasterMix (Catalog No.: FP217, Tiangen). The actin gene (LOC103499652) was used as an internal control gene, and a 2... -ΔΔCt Method calculation CmPP2C4 Changes in the relative expression levels of genes. Results Figure 2 As shown, CmPP2C4 The expression of this substance was suppressed by cold stress, and the expression level gradually decreased with the extension of cold stress duration, indicating that... CmPP2C4 Responding to cold damage stress.

[0020] Example 2

[0021] Using virus-induced transient silencing (VIGS) technology, the virus was injected into the "Jinquan No. 1" seedlings. CmPP2C4 Gene silencing, the specific procedures are as follows: according to CmPP2C4 Based on the gene sequence, specific primers were designed (Table 1). Approximately 1 μg of total RNA was taken and reverse transcribed into cDNA using the GoScript™ Reverse Transcription Mix, Oligo (DT) (Cat#A2790, Promega, USA) kit. The reaction system consisted of 20 µL of: Nuclease-free water 4 µL GoScriptTM Reaction Buffer, Oligo (DT) 4 µL GoScript™ Enzyme Mix 2 µL RNA 1 µg Nuclease-free water was added to 20 µL. The reaction conditions are as follows: 25 ℃ 5 min 1 cycle 42 ℃ 60 min 1 cycle 70 ℃ 15 min 1 cycle 4 ℃ ∞.

[0022] Table 1. Specific cloning primers

[0023] Using cDNA samples and the high-fidelity enzyme Prime Star A 350 bp specific sequence was cloned using HS DNA Polymerase (Cat#R010Q, TaKaRa, Tokyo, Japan). Fragment amplification was performed using a 20 μL reaction system as follows: pTRV2-gene primer-F 1 μL pTRV2-gene primer-R 1 μL cDNA template 1 μL PrimeSTAR HS DNA Polymerase 0.5 μL PrimeSTAR Buffer 10 μL ddH2O 6.5 μL.

[0024] The PCR reaction procedure is as follows:

[0025] The cloned product was subjected to gel electrophoresis to verify fragment length. Simultaneously, the pTRV2 plasmid was digested using EcoRI and XhoI restriction endonucleases, and a 350 bp specific sequence was ligated into the pTRV2 silencing vector using ligase to construct the pTRV2- CmPP2C4 Silent vector. The ligated plasmid was transferred into TOP10 *E. coli* competent cells using a freeze-thaw method and cultured on solid LB medium containing antibiotic resistance. After obtaining positive plaques, the cells were propagated using liquid LB medium containing antibiotic resistance. The plasmid was extracted from the propagated bacterial culture using a Tiangen plasmid mini-extraction kit, and then transferred into *Agrobacterium tumefaciens* GV3101 competent cells using a freeze-thaw method. Positive plaques were then obtained through antibiotic resistance selection culture on solid LB medium. The resulting plaques containing pTRV2- CmPP2C4Positive bacterial plaques were subjected to resistance propagation with bacterial suspensions containing pTRV1 plasmid and pTRV2-0 (empty) plasmid, respectively. When the OD600 of the bacterial suspension reached approximately 1.5, the bacterial cells were collected by centrifugation. The bacterial cakes were then resuspended in an osmotic suspension (10 mmol / L MES, 100 mmol / L AS, and 10 mmol / L MgCl2·6H2O) to adjust the OD600 to 0.8, and incubated in the dark at room temperature for 3-4 h. A 1:1 mixture of bacterial suspensions containing pTRV1 and pTRV2-0 was used as a negative control. A 1:1 mixture of bacterial suspensions containing pTRV1 and pTRV2-0 was used as a negative control. CmPP2C4 The bacterial suspension was mixed at a 1:1 ratio as the infection solution for silencing the target gene. A small incision was made on the underside of the cotyledon near the main vein using a 1 ml syringe needle. The mixed bacterial suspension was then injected through the incision using a syringe without a needle until the entire cotyledon appeared water-soaked. Infected seedlings were first cultured in the dark for 24 h, then in low light for 24 h, and finally under normal light conditions, with the temperature controlled below 25℃ and humidity between 60% and 70%. The silencing efficiency was assessed when the seedlings reached the three-leaf stage. RNA was extracted from the leaves, reverse transcribed into cDNA, and then analyzed using specific quantitative primers (Table 2). qRT-PCR was performed using the Tiangen SuperReal PreMix Plus (SYBR Green) (Cat#FP205, Tiangen, Beijing, China) real-time fluorescence quantitative PCR kit. The reaction volume was 20 μL as follows: SuperReal PreMix Plus 10 μL gene primer-F 0.6 μL gene primer-R 0.6 μL cDNA template 1 μL RNase-free ddH2O 7.8 μL.

[0026] The reaction procedure is as follows:

[0027] Table 2 Real-time quantitative primers

[0028] Plants with a silencing efficiency of over 60% were used for cold stress treatment and related testing and analysis. Figure 3 It can be seen that after 24 hours of 4℃ cold stress treatment on silent plants, CmPP2C4 Silent plants exhibited a milder stress injury phenotype, while the control group exhibited a more severe stress phenotype.

[0029] Then, the electrolyte permeability, malondialdehyde content, proline content, and soluble sugar content of the leaves were analyzed.

[0030] Electrolyte permeability determination: Take 0.2 g of fresh leaf sample, soak it in distilled water for 2 h, then mix thoroughly by inversion, and measure the conductivity value 1. Subsequently, boil the sample for 20 min, cool it to 25℃, and measure the conductivity value 2. Electrolyte permeability is calculated according to the formula (value 1 / value 2 × 100).

[0031] Determination of malondialdehyde (MDA) content: Take 0.5 g of fresh leaf sample, add 5 mL of 10% trichloroacetic acid solution and grind into a homogenate, centrifuge at 1800 g for 10 min. Take 2 mL of supernatant and mix with 2 mL of 0.6% thiobarbituric acid solution, boil for 15 min. After rapid cooling, centrifuge at 1800 g for 5 min, and take 2 mL of supernatant to measure the absorbance at wavelengths of 532, 600 and 450 nm. Calculate the MDA content according to the formula: MDA content (μmol g-1) = [6.45 × (OD532)] OD600) [0.56×OD450]×V / W, where V is the total volume of the trichloroacetic acid extract (mL) and W is the fresh weight of the sample (g).

[0032] Proline content determination: The proline content was determined using the sulfosalicylic acid colorimetric method (Bates et al., 1973). The specific steps were as follows: 0.5 g of leaf sample was immersed in 5 mL of 3% sulfosalicylic acid solution and boiled for 10 min. After cooling to room temperature and filtering, 2 mL of the filtrate was mixed with 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin solution and boiled for 30 min. After cooling, 4 mL of toluene was added, and the mixture was shaken for 30 seconds and centrifuged at 1000 g for 5 min. The absorbance of the red supernatant was measured at 520 nm, and the proline content (mg / g fresh weight) was calculated according to the standard curve.

[0033] Determination of soluble sugar content: 0.2 g of fresh sample was homogenized with 80% (v / v) ethanol solution, extracted in an 80°C water bath for 30 min, and then centrifuged at 10,000 g for 10 min. The supernatant was transferred to a new centrifuge tube. The residue was extracted twice more using the above steps, and the supernatants from the three extractions were combined. Finally, the volume was adjusted to 10 mL with 80% ethanol solution. The absorbance of the extract was measured at a wavelength of 620 nm, and the soluble sugar content (mg / g fresh weight) was calculated according to the standard curve.

[0034] The results are shown in Figure 4. Figure 4 It can be seen that, CmPP2C4 The electrolyte permeability and malondialdehyde content of gene-silenced plants were significantly lower than those of the control group, while the proline and soluble sugar contents were significantly higher than those of the control group.

[0035] Finally, CmPP2C4 After gene-silenced plants were treated with cold stress, the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and hydrogen peroxide (H2O2) content in the leaves were analyzed.

[0036] Enzyme extraction was performed according to the method of Liu et al. (2020). Superoxide dismutase (SOD) activity was determined using the method of Gianopolitis and Ries (1977), with one unit of activity (U) defined as the amount of enzyme required to catalyze the reduction of 50% nitroblue tetrazolium (NBT) at 560 nm. Catalase (CAT) and ascorbate peroxidase (APX) activities were determined according to the method of Noctor et al. (2016): the CAT activity assay consisted of 0.1 M phosphate buffer (pH 7.5), 40 mM H2O2, and 50 μL of enzyme extract, and was calculated using the H2O2 decomposition rate over 2 min at 240 nm; the APX activity assay consisted of 10 mM ascorbic acid, 0.1 M phosphate buffer (pH 7.5), 20 mM H2O2, and 50 μL of enzyme extract, and was calculated using the ascorbic acid consumption rate over 2 min at 290 nm.

[0037] hydrogen peroxide ( Content determination: Using 0.1 g of fresh leaf sample, according to the method of Bio-Tech Co., Ltd. Follow the instructions for the assay kit (catalog number: AKAO009M) to measure the absorbance at a wavelength of 415 nm and calculate the absorbance based on the standard curve. content.

[0038] Depend on Figure 5 It can be seen that, CmPP2C4 The antioxidant enzyme activity of gene-silenced plants was more strongly induced, and the amount of H2O2 accumulated in the plants was significantly lower than that of the control.

[0039] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Melon CmPP2C4 The application of genes in regulating the cold resistance of melons is characterized by, The melon CmPP2C4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The melon according to claim 1 CmPP2C4 The application of genes in regulating the cold resistance of melons is characterized by, Constructing melons through virus-induced transient silencing CmPP2C4 Gene silencing vector, and the melon seedlings were used to transmit the melon gene silencing vector. CmPP2C4 Gene silencing produces melons with increased tolerance to cold stress. CmPP2C4 Silent plant.

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