Application of melon autophagy gene CmATG8f2 in plant stress resistance and disease resistance

Abstract

The application provides application of a melon autophagy gene CmATG8f2 in plant stress resistance and disease resistance, a nucleotide sequence of the melon autophagy gene CmATG8f2 is shown as SEQ ID NO. 1, and an amino acid sequence is shown as SEQ ID NO. 2; and the melon plant stress resistance and disease resistance are improved by overexpression of the CmATG8f2 gene. The application of the gene in improving salt tolerance and root rot resistance of melon provides a new theory for breeding melon germplasm with salt tolerance and root rot resistance, and has great application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to an application of a melon autophagy gene CmATG8f2 in plant stress resistance and disease resistance. Background Art

[0002] melon( Cucumis melo Melons (L.) are an important economic crop, widely cultivated in many countries around the world. Melons are susceptible to multiple adversities, including root rot and salt stress, when cultivated in arid and semi-arid regions and in greenhouse environments. Excessive soil salinity can impair water absorption, leading to plant shrinkage, growth stagnation, and even death. Root rot, caused by various pathogens, causes root rot and death in the melons. This disease is particularly exacerbated in environments with poor drainage, continuous cropping problems, and severe salinization, severely impacting melon yield and quality.

[0003] The melon's defense mechanisms against salt stress and root rot involve multiple factors. For example, in response to salt stress, melon promotes the synthesis of osmotic regulators (such as proline) to alleviate water imbalances inside and outside cells and maintain normal cellular function. Furthermore, plants activate antioxidant systems (such as superoxide dismutase and catalase) to reduce oxidative damage caused by stress. To combat root rot, melons often prevent the infiltration and spread of pathogens by producing antimicrobial substances, regulating hormone signaling, activating defense-related genes, and strengthening physical barriers.

[0004] Autophagy is an evolutionarily conserved intracellular degradation pathway. Plants form autophagosomes to transport damaged organelles, proteins, and other cellular components to lysosomes or vacuoles for degradation and recycling, thereby helping plants maintain cellular homeostasis under stressful conditions. Previous studies have shown that autophagy plays a key role in plant resistance to various environmental stresses (such as salt, drought, and high temperature) and disease resistance. For example, under salt stress, plants enhance salt tolerance by degrading damaged organelles, maintaining energy and metabolic balance, and clearing excess reactive oxygen species through autophagy. Furthermore, autophagy can regulate immune responses, enhance plant resistance to pathogens, and inhibit the occurrence and progression of diseases. The formation and development of autophagosomes depend on the coordination of autophagy-related proteins, with ATG8 playing a central role in autophagosome formation, elongation, and fusion.

[0005] While the importance of autophagy in stress responses in various plants is widely recognized (e.g., maize, Arabidopsis, and apple), research on autophagy in melon is still in its infancy. In particular, the functions and regulatory mechanisms of key autophagy genes in melon's defense against salt stress and root rot remain largely unexplored. Summary of the Invention

[0006] In view of this, the application finds that CmATG8f2 is significantly up-regulated under salt stress by detecting the expression level of the melon autophagy gene family after the melon is subjected to salt stress, suggesting that it may be involved in the salt tolerance mechanism of melon, and further studies the performance of the gene in disease resistance, and finally finds that it can improve the resistance of melon to root rot.

[0007] To achieve the above object, the application adopts the following technical solutions:

[0008] The application provides application of a melon autophagy gene CmATG8f2 in plant stress resistance and disease resistance, wherein the nucleotide sequence of the melon autophagy gene CmATG8f2 is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

[0009] Further, the stress resistance and disease resistance of melon plants are realized by overexpression of the CmATG8f2 gene.

[0010] In some embodiments, preferably, the primers used for constructing the CmATG8f2 overexpression vector are as follows:

[0011] The forward primer sequence is CGAACGATACTCGAGTAATCTAGAATGTCTACGAGCTCGTTCAAGCA.

[0012] The reverse primer sequence is ACGAAAGCTCTGAGCTTCATCCAAATGTGTTTTCTCCACTGTATGT.

[0013] In some embodiments, preferably, the vector used for constructing the CmATG8f2 overexpression vector is a pBSE403R vector.

[0014] Compared with the prior art, the application has the following beneficial effects:

[0015] The application provides a melon autophagy gene CmATG8f2 and a protein sequence coded by the melon autophagy gene CmATG8f2, and the gene can significantly improve the salt tolerance and root rot resistance of melon. By overexpressing the gene, the melon plants still grow well in Hoagland nutrient solution containing 150 mM NaCl for 7 days, the leaves remain green, the root system is healthy, and the plants have more dry weight and higher photosynthetic efficiency; the root system of the overexpressed melon plants is soaked in a root rot fungus spore suspension for 20 min, and then planted, the disease index of the overexpressed plants is 1.00, the root rot resistance is high, and the plants show significantly enhanced root rot resistance. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The expression amount of 20 autophagy genes of melon under salt stress.

[0017] Figure 2 Figure for autophagy identification results of CmATG8f2 overexpression and gene editing plants of melon.

[0018] Figure 3 Figure for phenotype of CmATG8f2 overexpression and gene editing materials under salt stress for 7 days of melon.

[0019] Figure 4 Figure for physiological index results of CmATG8f2 overexpression and gene editing materials under salt stress for 7 days of melon.

[0020] Figure 5 Figure for results of pathogenic strain isolation and molecular identification of melon.

[0021] Figure 6 Figure for results of CmATG8f2 function identification of melon against root rot disease. DETAILED DESCRIPTION

[0022] The present application will be further described below in conjunction with specific examples, so that those skilled in the art can more clearly understand the present application.

[0023] Example 1

[0024] This example provides the process of screening autophagy genes in response to salt stress in melon, and the specific steps are as follows:

[0025] 1.1 Plant material culture and salt treatment

[0026] The melon variety used is Xizhoumi No. 25, and a modified Hoagland nutrient solution formula (Table 1) is used. The grown melon plants are placed in a nutrient solution containing a concentration of 150 mM NaCl to simulate salt stress. The control group does not add NaCl. Salt treatment lasts for 7 days, and the nutrient solution is replaced regularly to ensure stable salt concentration.

[0027] Table 1 Modified Hoagland nutrient solution formula

[0028]

[0029] 1.2 RT-qPCR detection of autophagy gene expression

[0030] After salt stress treatment, total RNA from melon roots was extracted using TRIZOL reagent (purchased from TransGen Biotech). Total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Vazyme). Quantitative primers for 20 autophagy genes in melon were designed (synthesized by Tsingke) on the primer design website (https: / / www.primer3plus.com / ). The primer sequences are shown in Table 2. RT-qPCR detection was performed using the ChamQ SYBR qPCR Master Mix quantitative PCR detection kit (purchased from Vazyme) with the CmTUB gene as an internal reference to detect the expression level of the autophagy gene family. The results are shown in Table 2. Figure 1 As shown in the results, CmATG8f2 was significantly upregulated under salt stress conditions, suggesting that it may be involved in the salt tolerance mechanism of melon.

[0031] Table 2 qRT-PCR primer sequences for autophagy genes and internal reference genes

[0032]

[0033] The gene ID of melon CmATG8f2 in the Cucurbitaceae database (http: / / cucurbitgenomics.org / ) is MELO3C021148. The nucleic acid sequence encoded by it is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2:

[0034] SEQ ID NO.1: ATGTCTACGAGTCGTTCAAGCAAGAGCATGATTTTGAAAAGAGACATGCTGAGGCAGCCCGGATTAGAGAAAAATACCCCGACAGAATTCCGGTAATTGTGGAGAAGGCAGAGAGAAGTGATATTCCCAACATTGACAAGAAAAAGTATCTAGTGCCTGCTGACCTGACTGTT GGCCAATTTGTTTATGTTTATCCGGAAAAGAATAAAATTGAGTGCAGAAAAGGCAATTTTCATATTCGTGGACAATGTGTTACCGCCAACAGGATCACTGATGTCTGCAATCTATGATGAGAGGAAGGATGAGGATGGGTTTCTCTATGTTACATACAGTGGAGAAAACACATTTGGATGA

[0035] SEQ ID NO. 2: MSTSSFKQEHDFEKRHAEAARIREKYPDRIPVIVEKAERSDIPNIDKKYLV PADLTVGQFVYVIRKRIKLSAEKAIFIFVDNVLPPTGSLMSAIYDERKDEDGFLYVTYSGENTFG*.

[0036] Example 2

[0037] This example provides the construction and identification of melon CmATG8f2 gene overexpression and gene editing plants, the specific steps are as follows:

[0038] 2.1 Overexpression vector construction

[0039] The melon CDNA in 1.2 was used as a template, and the high-fidelity PCR kit (purchased from Vazyme company) and the primers in Table 3 (ATG8f2-403R-F / ATG8f2-403R-R) were used for PCR amplification. The amplification product was separated by 1% agarose gel electrophoresis, and the correct gene band was cut off, and the DNA gel recovery kit (purchased from Axygen company) was used to recover and purify the PCR product, that is, the CmATG8f2 with vector adapter was obtained.

[0040] First, the pBSE403R vector (CN116675749B paragraph 0067) was digested with XbaI and SacI two endonucleases (purchased from NEB company), and the enzyme digestion conditions were 37°C for 30 min, and then the enzyme was inactivated at 65°C for 20 min. The enzyme digestion product was purified using the PCR clean kit (purchased from Axygen company), and then the recombinant cloning kit (purchased from Vazyme company) was used to homologously recombine the CmATG8f2 with the vector adapter into the pBSE403R vector. Finally, the recombinant vector was transformed into competent Escherichia coli DH5α (purchased from Tsingke company), and the identification primers in Table 3 (ATG8f2-JD-F / ATG8f2-JD-R) were used for colony PCR amplification, and then sequencing identification was performed to verify whether CmATG8f2 was successfully inserted into the constructed overexpression vector. The over vector contains a 35S promoter to ensure high-level expression of CmATG8f2 in transgenic melon plants.

[0041] Table 3 Primers for overexpression vector construction

[0042]

[0043] 2.2 Gene editing vector construction

[0044] Based on the CRISPR / Cas9 technology, the target sequence was selected as much as possible in the CDS conservative functional region and the upstream region of the gene by using the CRISPR target search function of the Geneious software. The designed gene editing primers are shown in Table 4. After the gene editing primers atg8f2sg-F / atg8f2sg-R (synthesized by Tsingke Company) were diluted to 100 μM, the two complementary single-stranded primers were reacted in the following system to anneal the primers and form double-stranded DNA.

[0045] Reaction system (50 μl): 1.5 µL forward primer, 1.5 µL reverse primer, 5 µL 10 x NEB buffer 3.1, 42 µL ddH2O, placed in 1L boiling water and naturally cooled to room temperature.

[0046] After the pBSE403R vector was linearized with BsaI endonuclease, the double-stranded primer was connected with the vector using T4 ligase (purchased from NEB company), that is, the gene knockout vector was constructed. Finally, the vector was transformed into E. coli, and the identification primers (U626-JD-F / U626-JD-R) in Table 4 were used for colony PCR amplification and sequencing identification to verify whether CmATG8f2 was successfully inserted into the constructed gene editing vector.

[0047] Table 4 Primers used for gene editing vector construction

[0048]

[0049] 2.3 Obtain super-expression and gene editing melon plants by using Agrobacterium rhizogenes

[0050] The super-expression and gene editing plants of CmATG8f2 were obtained by melon root transformation system. The specific steps are as follows:

[0051] Firstly, the super-expression vector, gene editing vector and empty vector constructed in 2.2 were transformed into Agrobacterium rhizogenes K599 (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Then the Agrobacterium was cultured in LB medium containing 50 mg / L streptomycin and 50 mg / L kanamycin at 28°C 200 r / min overnight (about 14-18 h) in a shaking bed. After the culture was finished, the bacteria were centrifuged at 6000 r / min. Resuspend to OD 600 =0.8-1.0 with MS liquid medium containing 0.1% sucrose and 200 mmol / L acetosyringone. Take 1 mL of the above Agrobacterium solution into a 1.5 mL centrifuge tube for standby.

[0052] When the cotyledons of melon seedlings are flattened, make an oblique cut at the hypocotyl to create a wound. Immerse the cut surface in a centrifuge tube containing Agrobacterium and infect for 30 minutes. Then, add the MS liquid medium from 2.3 to sterilized vermiculite and stir to moisten it. Insert the infected melon seedlings into the moistened vermiculite and incubate at 23°C for 4 days.

[0053] After co-cultivation, mix Hoagland's nutrient solution with a sterilized vermiculite:perlite (3:1) substrate and place in seedling trays. Finally, transfer the melon seedlings to the moistened substrate and cover to seal and retain moisture. After 2-3 weeks, CmATG8f2 overexpressing and gene knockout melon plants will be obtained.

[0054] 2.4 Identification of Autophagy in Overexpression and Gene-Edited Plants

[0055] To test the gene editing efficiency and type of the CmATG8f2 gene-edited melon plants, Hi-TOM sequencing technology was used (sequencing sample management system: http: / / 121.40.237.174 / Hi-TOM). Conventional PCR primers were first designed within 100 bp of the target site. After adding a specific bridging sequence, Hi-TOM sequencing primers were obtained. The specific sequences are as follows:

[0056] atg8f2-hitomF: GGAGTGAGTACGGTGTGCATGTGGTGGGTGCAGAGTGC;

[0057] atg8f2-hitomR:GAGTTGGATGCTGGATGGACTTTTTCTTGTCAATGTTGGGA.

[0058] The sequencing results are as follows Figure 2 As shown in Figure A, the random mutations generated included point mutations and deletions, with a gene editing efficiency of 78.5%. After gene editing, the CmATG8f2 protein produced two amino acid changes (double underlined). The protein sequence is shown in SEQ ID NO. 3.

[0059] SEQ ID NO.3:MSTSSFKQEHDFEKRHAEAARIREKYPDRIPV LM EKAERSDIPNIDKKKYLVPADLTVGQFVYVIRKRIKLSAEKAIFIFVDNVLPPTGSLMSAIYDERKDEDGFLYVTYSGENTFG*.

[0060] Then, the autophagy levels of the root of the three different transgenic plants of empty vector, overexpression and gene knockout were detected by RT-qPCR, Western blot and MDC staining to verify the availability of the transgenic plants.

[0061] The RT-qPCR detection method and quantitative primer were consistent with the method 1.2, and were not repeated.

[0062] Western Blot analysis of the accumulation level of autophagy protein:

[0063] 1. Mix the plant RIPA lysis buffer with PMSF (both purchased from Beyotime Company), and add 0.5 mL lysis buffer per 0.1 g sample.

[0064] 2. After grinding in liquid nitrogen, place on ice for 10 min, centrifuge at 10000 r / min, 4℃ for 5 min, and collect the supernatant.

[0065] 3. Add 2x SDS loading buffer and react at 95℃ for 20 min.

[0066] 4. Use PAGE gel reagent kit (purchased from Yazhen Company) to prepare protein electrophoresis gel, and after loading, perform electrophoresis, and then use standard wet transfer device (purchased from Bio-Rad Company) to transfer the protein band to nitrocellulose membrane.

[0067] 5. After the transfer is completed, block in TBS-T buffer containing 5% skimmed milk powder overnight.

[0068] 6. Use Autophagy ELISA Kit (LC-II Quantitation) cell analysis kit (purchased from Aikangjie Technology Co., Ltd.) for primary antibody and secondary antibody incubation, and chemiluminescence kit (purchased from Beyotime Company) for development.

[0069] 7. The same method was used for electrophoresis, transfer and blocking of the same sample, and Actin antibody (purchased from Beyotime Company) was used for primary antibody incubation for 1 h, goat anti-mouse IgG (H+L) (purchased from Beyotime Company) was used for secondary antibody incubation for 1 h, and finally chemiluminescence kit was used to detect Actin internal reference protein.

[0070] MDC staining to observe autophagosomes:

[0071] 1. Use DMSO to prepare 100x stock solution of MDC (30432, Sigma), and dilute with PBS Buffer (purchased from Beyotime Company) with pH 7.3 to 100 μM for standby use.

[0072] 2. Wash the melon roots and place them in MDC staining solution, vacuum them for 10 minutes using a vacuum pump, and incubate them in a shaker for 20 minutes.

[0073] 3. After staining is completed, aspirate the staining solution and wash the sample with PBS 2-3 times. Finally, select the root tip with smaller thickness for sample preparation.

[0074] 4. Observe using a confocal microscope with an excitation wavelength of 405 nm and an emission wavelength of 510-560 nm.

[0075] The results of RT-qPCR, Western Blot and autophagy staining are as follows Figure 2 The results showed that the expression level of CmATG8f2 gene and the content of autophagy protein in melon overexpression plants were significantly higher than those in empty-load plants ( Figure 2 In contrast, gene expression levels of CmATG8f2 and autophagy protein content were significantly reduced in gene-edited plants. MDC staining revealed a significant increase in the number of autophagosomes in the roots of CmATG8f2-overexpressing melon plants, while autophagosome formation was blocked in gene-edited melon plants, with a significant decrease in their number. These results suggest that autophagy is significantly promoted or inhibited in the CmATG8f2-overexpressing and gene-edited melon plants, respectively.

[0076] Example 3

[0077] This example provides salt tolerance identification of melon CmATG8f2 gene overexpression and gene editing plants, and the specific steps are as follows:

[0078] Overexpression, knockout and empty vector plants were cultured to two leaves and one heart, and then transferred to Hoagland nutrient solution containing 150 mM NaCl for culture. After 7 days of salt treatment, the phenotypes were recorded, and the growth and photosynthetic indices such as dry weight, photosynthetic efficiency, and SPAD were measured. At the same time, Fv / Fm, relative conductivity, MDA, H2O2, · O2 - 、Na + Salt stress related indicators such as content.

[0079] After 7 days of salt treatment, the growth status of melon is as follows Figure 3 As shown in the results, CmATG8f2 overexpressing plants grew well under salt stress, with green leaves and strong root systems. In contrast, the leaves of gene knockout plants withered and yellowed, and their roots were weak and browned.

[0080] The results of physiological index measurement ( Figure 4 ) also proved this conclusion. Under non-salt treatment, overexpression or knockout of CmATG8f2 did not affect the photosynthetic efficiency, relative conductivity and MDA content of melon ( Figure 4B, C, D, E, F), i.e. the level of autophagy does not cause damage to the melon under non-salt stress.

[0081] Under salt stress, melon plants overexpressing CmATG8f2 exhibit significantly enhanced salt tolerance. Their damage indicators, active oxygen content, and Na + content are significantly lower than the control, while they have higher dry weight and higher photosynthetic efficiency. While melon plants edited for the CmATG8f2 gene have significantly lower dry weight and photosynthetic efficiency than the control, their damage indicators, active oxygen content, and Na + content are significantly higher than the control, showing obvious salt sensitivity.

[0082] The above results show that CmATG8f2 plays an important role in the salt tolerance mechanism of melon. Overexpression of CmATG8f2 can significantly improve the salt tolerance of melon plants, while knocking out CmATG8f2 makes melon plants sensitive to salt stress. These findings also show that CmATG8f2 has a significant effect on improving the salt tolerance of melon plants.

[0083] Example 4

[0084] This example provides the identification of the resistance of melon CmATG8f2 gene overexpression and gene editing plants to root rot, and the specific steps are as follows:

[0085] 4.1 Isolation and identification of melon root rot fungus

[0086] Cut the diseased melon root rot tissue and place it on PDA medium (purchased from Solarbio) to culture the pathogenic fungus at 28°C. Observe the colony growth and the morphological characteristics of spores and hyphae under a microscope, and find that the hyphae on the PDA medium are white with pink to blue-purple pigment (A). Small conidia are oval and large conidia are spindle-shaped (B). Figure 5 Figure 5

[0087] After picking the colonies, universal primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) are used to perform PCR gene amplification and sequencing of the pathogenic fungus. The sequencing results are subjected to BLAST homology comparison with the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and the MEGA software is used to construct a phylogenetic tree (C). The results show that the pathogenic fungus strain has the highest homology with Fusarium oxysporum, with a homology of more than 99.9% (D). Figure 5 Figure 5

[0088] ​​​​Through morphological characteristics and molecular biology identification, the pathogenic bacteria is finally identified as Fusarium oxysporum.

[0089] 4.2 CmATG8f2 gene improves the resistance of melon to root rot

[0090] After the strain is activated, the spores of the culture medium are washed and collected with sterile water, and a spore suspension with a spore concentration of 1×10 6 / ml is prepared for standby. After the roots of the overexpression, gene knockout and control melon plants are washed, they are soaked in the spore suspension of the root rot fungus for 20 min, and then planted in the seedling plug tray, and the disease progress is monitored regularly.

[0091] The disease classification refers to the standard of Nagao (1994) as follows: 0 level, healthy and no infection; 1 level, slight discoloration on the root, and healthy hypocotyl; 2 level, 10% root discoloration, and healthy hypocotyl; 3 level, 30% root discoloration, and slightly infected hypocotyl; 4 level, 70% root discoloration, and infected hypocotyl; 5 level, whole root discoloration, and severely infected hypocotyl, or plant death. The disease index is divided into three levels: high resistance (0~2), medium resistance (2~4), and susceptible (≥4)

[0092] The results show that after inoculation of the pathogenic bacteria, the growth and root state of the CmATG8f2 overexpression plant are obviously better than those of the control; and the root rot of the gene knockout plant is more serious, and the resistance is weaker Figure 6 ). The resistance identification results are shown in Table 4: the disease index of the control melon plant is 2.38, and the resistance to root rot is medium; the disease index of the CmATG8f2 overexpression plant is 1.00, and the resistance to root rot is high, which shows that the resistance to root rot is significantly enhanced; the disease index of the gene editing plant is 4.08, and the resistance to root rot is susceptible, and the resistance to root rot is significantly reduced.

[0093] Table 4 Resistance levels of different melon plant materials to root rot

[0094]

[0095] The above results show that CmATG8f2 has an important function in the resistance of melon to root rot, and the overexpression of CmATG8f2 can significantly improve the resistance of melon plants to root rot, and the knockout of CmATG8f2 can significantly reduce the resistance of melon plants to root rot. These results show that CmATG8f2 has a significant effect on improving the resistance of melon plants to root rot.

[0096] The specific raw materials in the application are all existing substances, which can be directly purchased from the market.

[0097] The above merely preferred embodiments of the present application and are not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of the melon autophagy gene CmATG8f2 in plant stress and disease resistance, characterized in that: The nucleotide sequence of the melon autophagy gene CmATG8f2 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; The stress resistance is to overexpress the CmATG8f2 gene to make the melon plants show significantly enhanced salt tolerance; The disease resistance is achieved by overexpressing the CmATG8f2 gene to significantly enhance the resistance of melon plants to root rot; Wherein, the root rot is caused by Fusarium oxysporum ( Fusarium oxysporum )cause.

2. The use according to claim 1, characterized in that The primers used to construct the CmATG8f2 overexpression vector are as follows: Forward primer sequence: CGAACGATACTCGAGTAATCTAGAATGTCTACGAGCTCGTTCAAGCA; Reverse primer sequence: ACGAAAGCTCTGAGCTTCATCCAAATGTGTTTTCTCCACTGTATGT.

3. The use according to claim 1, characterized in that The vector used to construct the CmATG8f2 overexpression vector is specifically the pBSE403R vector.

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