Application of muskmelon autophagy gene CmATG8f2 in stress resistance and disease resistance of plants

Abstract

The invention provides application of a muskmelon autophagy gene CmATG8f2 in stress resistance and disease resistance of plants. The nucleotide sequence of the muskmelon autophagy gene CmATG8f2 is as shown in SEQ ID NO. 1, and the amino acid sequence of the muskmelon autophagy gene CmATG8f2 is as shown in SEQ ID NO. 2; the improvement of the stress resistance and disease resistance of melon plants is realized through the overexpression of the CmATG8f2 gene. By exploring the application of the gene in improving the salt tolerance and the root rot resistance of the muskmelon, a new theory is provided for cultivating salt-tolerant and root-rot-resistant muskmelon germplasm, and the gene has huge application potential.

Description

Technical Field

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

[0002] melon( Cucumis melo L.) is an important economic crop and is widely planted in many countries around the world. When cultivated in arid and semi-arid areas and facility environments, melons are susceptible to multiple adversities such as root rot and salt stress. Excessive soil salinity will affect the water absorption of melons, causing plant shrinkage, growth stagnation and even death. Root rot is caused by a variety of pathogens, which will cause the roots of melons to rot and die. In particular, poor drainage, continuous cropping obstacles and severe salinization environments can easily aggravate the disease, seriously affecting the yield and quality of melons.

[0003] The defense mechanism of melon against salt stress and root rot involves many aspects. For example, in the face of salt stress, melon promotes the synthesis of osmotic regulating substances (such as proline) to alleviate the water imbalance inside and outside the cell and maintain the normal physiological function of the cell; in addition, plants will activate antioxidant systems (such as superoxide dismutase, catalase, etc.) to reduce oxidative damage caused by stress. For root rot, melon often prevents the infection and spread of pathogens by producing antibacterial substances, regulating hormone signals, activating defense-related genes, and strengthening physical barriers.

[0004] Autophagy is an evolutionarily highly conserved intracellular degradation pathway. Plants form autophagosomes to transport damaged organelles, proteins and other cellular components to lysosomes or vacuoles, which are then recycled after degradation, thereby helping plants maintain cellular homeostasis under stress conditions. Studies have shown that autophagy plays a key role in plants' resistance to various environmental stresses (such as salt, drought, and high temperature) and disease resistance. For example, when faced with salt stress, plants enhance their salt tolerance by degrading damaged organelles, maintaining energy and metabolic balance, and removing excessively accumulated reactive oxygen species through autophagy. In addition, autophagy can also improve plant resistance to pathogens and inhibit the occurrence and development of diseases by regulating immune responses. The formation and development of autophagosomes depend on the coordination of autophagy-related proteins, among which ATG8 plays a core role in the formation, extension, and fusion of autophagosomes.

[0005] Although the importance of autophagy in stress responses in many plants has been widely recognized (such as corn, Arabidopsis, apple, etc.), research on autophagy in melon is still in its infancy. In particular, the functions and regulatory mechanisms of key autophagy genes in melon's resistance to salt stress and root rot have not yet been explored. Summary of the invention

[0006] In view of this, the present invention detected the expression level of the melon autophagy gene family after subjecting the melon to salt stress, and found that CmATG8f2 was significantly upregulated under salt stress conditions, suggesting that it may be involved in the salt tolerance mechanism of melon. The performance of this gene in disease resistance was further studied, and it was finally found that it can improve the resistance of melon to root rot.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides an application of a melon autophagy gene CmATG8f2 in plant stress resistance and disease resistance. 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.

[0008] Furthermore, improving the stress and disease resistance of melon plants is achieved by overexpressing the CmATG8f2 gene.

[0009] In some specific embodiments, preferably, the primers used to construct the CmATG8f2 overexpression vector are as follows: Forward primer sequence: CGAACGATACTCGAGTAATCTAGAATGTCTACGAGCTCGTTCAAGCA; Reverse primer sequence: ACGAAAGCTCTGAGCTTCATCCAAATGTGTTTTCTCCACTGTATGT.

[0010] In some specific embodiments, preferably, the vector used to construct the CmATG8f2 overexpression vector is specifically the pBSE403R vector.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a melon autophagy gene CmATG8f2 and a protein sequence encoded therein, and the gene can significantly improve the salt tolerance and root rot resistance of melon. By overexpressing the gene, melon plants still grow well after being cultured in a Hoagland nutrient solution containing 150mM NaCl for 7 days, with leaves remaining green and roots strong, and having more dry weight and higher photosynthetic efficiency; the roots of the overexpressed melon plants are immersed in a root rot fungus spore suspension for 20 minutes and then planted, and the disease index of the overexpressed plants is 1.00, and the root rot resistance is high, showing significantly enhanced resistance to root rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The expression levels of 20 autophagy genes in melon under salt stress.

[0013] Figure 2This is the result of autophagy identification in melon CmATG8f2 overexpression and gene-edited plants.

[0014] Figure 3 These are actual photos of the phenotypes of melon CmATG8f2 overexpression and gene-edited materials under salt stress for 7 days.

[0015] Figure 4 This is the result of physiological indicators of melon CmATG8f2 overexpression and gene-edited materials under salt stress for 7 days.

[0016] Figure 5 This is the result of isolation and molecular identification of pathogenic strains of melon.

[0017] Figure 6 This is the result of functional identification of melon CmATG8f2 for resistance to root rot. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0019] Example 1 This example provides a process for screening autophagy genes in response to salt stress in melon, and the specific steps are as follows: 1.1 Plant material cultivation and salt treatment The melon variety used was Xizhou Mi No. 25. The modified Hoagland nutrient solution formula (Table 1) was used to place the cultivated melon plants in a nutrient solution containing 150mM NaCl to simulate salt stress. No NaCl was added to the control group. The salt treatment lasted for 7 days, and the nutrient solution was replaced regularly to ensure a stable salt concentration.

[0020] Table 1 Modified Hoagland nutrient solution formula

[0021] 1.2 RT-qPCR detection of autophagy gene expression After salt stress treatment, TRIZOL reagent (purchased from TransGen Biotech) was used to extract total RNA from melon roots. A reverse transcription kit (purchased from Vazyme) was used to reverse transcribe total RNA into cDNA. Quantitative primers for 20 autophagy genes in melon were designed on the primer design website (https: / / www.primer3plus.com / ) (synthesized by Tsingke). The primer sequences are shown in Table 2. ChamQ SYBR qPCR Master Mix quantitative PCR detection kit (purchased from Vazyme) was used to perform RT-qPCR detection with CmTUB gene as internal reference to detect the expression level of the autophagy gene family. The results are shown in Figure 1 As shown, CmATG8f2 was significantly upregulated under salt stress conditions, suggesting that it may be involved in the salt tolerance mechanism of melon.

[0022] Table 2 qRT-PCR primer sequences of autophagy genes and internal reference genes

[0023] 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 protein amino acid sequence is shown in SEQ ID NO.2: SEQ ID NO.1: ATGTCTACGAGCTCGTTCAAGCAAGAGCATGATTTTGAAAAGAGACATGCTGAGGCAGCCCGGATTAGAGAAAAATACCCCGACAGAATTCCGGTAATTGTGGAGAAGGCAGAGAGAAGTGATATTCCCAACATTGACAAGAAAAAGTATCTAGTGCCTGCTGACCTGACTGTT GGCCAATTTGTTTATGTTATCCGGAAAAGAATAAAATTGAGTGCAGAAAAGGCAATTTTCATATTCGTGGACAATGTGTTACCGCCAACAGGATCACTGATGTCTGCAATCTATGATGAGAGGAAGGATGAGGATGGGTTTCTCTATGTTACATACAGTGGAGAAAACACATTTGGATGA SEQ ID NO. 2: MSTSSFKQEHDFEKRHAEAARIREKYPDRIPVIVEKAERSDIPNIDKKKYLVPADLTVGQFVYVIRKRIKLSAEKAIFIFVDNVLPPTGSLMSAIYDERKDEDGFLYVTYSGENTFG*.

[0024] Example 2 This example provides the construction and identification of melon CmATG8f2 gene overexpression and gene editing plants, and the specific steps are as follows: 2.1 Construction of overexpression vector Using the melon CDNA in 1.2 as a template, PCR amplification was performed using a high-fidelity PCR kit (purchased from Vazyme) and the primers (ATG8f2-403R-F / ATG8f2-403R-R) in Table 3. The amplified products were separated by 1% agarose gel electrophoresis, and the correct gene band was cut out. The PCR product was recovered and purified using a DNA gel recovery kit (purchased from Axygen) to obtain CmATG8f2 with a vector adapter.

[0025] First, the pBSE403R vector (CN116675749B, paragraph 0067) was digested with two endonucleases, XbaI and SacI (purchased from NEB), at 37°C for 30 min, and then at 65°C for 20 min to inactivate the enzymes. The digestion product was purified using a PCR cleaning kit (purchased from Axygen), and then the CmATG8f2 with vector adapter was homologously recombined into the pBSE403R vector using a recombination cloning kit (purchased from Vazyme). Finally, the recombinant vector was transformed into competent Escherichia coli DH5α (purchased from Tsingke), and colony PCR amplification was performed using the identification primers in Table 3 (ATG8f2-JD-F / ATG8f2-JD-R), followed by sequencing to verify whether CmATG8f2 was successfully inserted into the constructed overexpression vector. The supervector contains the 35S promoter to ensure high-level expression of CmATG8f2 in transgenic melon plants.

[0026] Table 3 Primers used for overexpression vector construction

[0027] 2.2 Construction of gene editing vector Based on CRISPR / Cas9 technology, using the CRISPR target search function of Geneious software, the target sequence was selected as far as possible in the CDS conserved functional region and the upstream region of the gene. The designed gene editing primers are shown in Table 4. The gene editing primers atg8f2sg-F / atg8f2sg-R (synthesized by Tsingke) were diluted to 100 μM and reacted in the following system to anneal the two complementary single-stranded primers to form double-stranded DNA.

[0028] Reaction system (50μl): 1.5μL forward primer, 1.5μL reverse primer, 5μL 10×NEB buffer 3.1, 42μL ddH2O, placed in 1L boiled water and cooled naturally to room temperature.

[0029] After linearizing the pBSE403R vector with BsaI endonuclease, the double-stranded primer was connected to the vector using T4 ligase (purchased from NEB) to construct a gene knockout vector. Finally, the vector was transformed into Escherichia 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.

[0030] Table 4 Primers used for gene editing vector construction

[0031] 2.3 Obtaining overexpression and gene-edited melon plants using Agrobacterium rhizogenes Overexpression and gene-edited plants of CmATG8f2 were obtained through the melon root transformation system. The specific steps are as follows: First, the overexpression 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, Agrobacterium was cultured in LB medium containing 50 mg / L streptomycin and 50 mg / L kanamycin, and cultured overnight in a shaker at 28°C 200 r / min (about 14-18 h). After the culture was completed, the bacteria were harvested by centrifugation at 6000 r / min. The cells were resuspended in MS liquid medium containing 0.1% sucrose and 200 mmol / L acetosyringone to an OD of 0. 600 = 0.8~1.0. Pipette 1 mL of the above Agrobacterium bacterial solution into a 1.5 mL centrifuge tube for later use.

[0032] When the cotyledons of the melon seedlings are flattened, cut obliquely at the hypocotyl to form a wound, and immerse the cut part in a centrifuge tube containing Agrobacterium for infection for 30 minutes. Then, add the MS liquid medium in 2.3 to the sterilized vermiculite and stir to moisten it. Insert the infected melon seedlings into the moist vermiculite and co-cultivate at 23°C for 4 days.

[0033] After the co-cultivation, mix the Hoagland nutrient solution with the sterilized vermiculite:perlite (3:1) matrix and put it into the seedling tray. Finally, the melon seedlings are transferred into the moist matrix and covered to seal and keep moisture. After 2-3 weeks, melon plants with CmATG8f2 overexpression and gene knockout can be obtained.

[0034] 2.4 Identification of autophagy in overexpression and gene-edited plants To detect the gene editing efficiency and type of melon CmATG8f2 gene-edited plants, Hi-TOM sequencing technology was used (sequencing sample management system: http: / / 121.40.237.174 / Hi-TOM). First, conventional PCR primers were designed within 100 bp of the target site, and Hi-TOM sequencing primers were obtained after adding specific bridge sequences. The specific sequences are as follows: atg8f2-hitomF: GGAGTGAGTACGGTGTGCATGTGGTGGGTGCAGAGTGC; atg8f2-hitomR:GAGTTGGATGCTGGATGGACTTTTTCTTGTCAATGTTGGGA.

[0035] The sequencing results are as follows Figure 2 As shown in A, the random mutations generated include point mutations and deletions, and the gene editing efficiency is 78.5%. After gene editing, the CmATG8f2 protein produced two amino acid changes (double underlined), and the protein sequence is shown in SEQ ID NO.3.

[0036] SEQ ID NO.3:MSTSSFKQEHDFEKRHAEAARIREKYPDRIPV LM EKAERSDIPNIDKKKYLVPADLTVGQFVYVIRKRIKLSAEKAIFIFVDNVLPPTGSLMSAIYDERKDEDGFLYVTYSGENTFG*.

[0037] Next, RT-qPCR, Western blot and MDC staining were used to detect the autophagy levels in the roots of three different melon transgenic plants: empty load, overexpression and gene knockout, to verify the availability of transgenic plants.

[0038] The RT-qPCR detection method and quantitative primers are consistent with those in 1.2 and will not be repeated here; Western Blot analysis of the accumulation level of autophagy proteins: 1. Mix plant RIPA lysis buffer and PMSF (both purchased from Beyotime) and add 0.5 mL of lysis buffer for every 0.1 g of sample.

[0039] 2. After grinding with liquid nitrogen, place on ice for 10 minutes, centrifuge at 10,000 r / min, 4°C for 5 minutes, and collect the supernatant.

[0040] 3. Add 2×SDS loading buffer and react at 95℃ for 20 min.

[0041] 4. Prepare protein electrophoresis gel using PAGE gel kit (purchased from Yazyme), perform electrophoresis after loading the sample, and then transfer the protein bands to nitrocellulose membrane using standard wet transfer device (purchased from Bio-Rad).

[0042] 5. After transfer, block overnight in TBS-T buffer containing 5% skim milk powder.

[0043] 6. Use Autophagy ELISA Kit (LC-II Quantitation) cell analysis kit (purchased from Aimi Technology Co., Ltd.) for primary and secondary antibody incubation and chemiluminescence kit (purchased from Beyotime) for development.

[0044] 7. The same method was used to perform electrophoresis, transfer, and blocking on the same samples. Actin antibody (purchased from Beyotime) was used for primary antibody incubation for 1 hour, and goat anti-mouse IgG (H+L) (purchased from Beyotime) was used for secondary antibody incubation for 1 hour. Finally, a chemiluminescence kit was used to detect the Actin internal reference protein.

[0045] MDC staining to observe autophagosomes: 1. Prepare MDC (30432, Sigma) with DMSO to a 100× stock solution for storage. Dilute to 100 μM with PBS buffer (pH 7.3) (purchased from Beyotime) for use.

[0046] 2. Wash the melon roots and place them in MDC staining solution, evacuate them with a vacuum pump for 10 minutes, and incubate them in a shaker for 20 minutes.

[0047] 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.

[0048] 4. Observe using a confocal microscope with an excitation wavelength of 405 nm and an emission wavelength of 510-560 nm.

[0049] The results of RT-qPCR, Western Blot and autophagy staining are shown in 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 2B and C); while the gene expression of CmATG8f2 and the content of autophagic proteins in gene-edited plants were significantly reduced. MDC staining results showed that the number of autophagosomes in the roots of CmATG8f2 overexpressed melon plants increased significantly, while the formation of autophagosomes in gene-edited melon plants was blocked and the number was significantly reduced. The above results indicate that the autophagic function of the created melon CmATG8f2 overexpressed and gene-edited materials was significantly promoted or inhibited, respectively.

[0050] Example 3 This example provides salt tolerance identification of melon CmATG8f2 gene overexpression and gene editing plants, and the specific steps are as follows: The 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. The phenotypes were recorded after 7 days of salt treatment, 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.

[0051] After 7 days of salt treatment, the growth status of melons is as follows Figure 3 As shown in the figure, CmATG8f2 overexpressing plants grew well under salt stress, with green leaves and strong roots, while the leaves of gene knockout plants withered and yellowed, and the roots were weak and browned.

[0052] 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 4 B, C, D, E, and F), that is, the level of autophagy will not cause harm to melon under non-salt stress.

[0053] Under salt stress, melon plants overexpressing CmATG8f2 showed significantly enhanced salt tolerance. + The content was significantly lower than that of the control, and the plants had higher dry weight and photosynthetic efficiency. The dry weight and photosynthetic efficiency of the melon plants edited with the CmATG8f2 gene were significantly lower than those of the control, and the damage index, active oxygen content and Na + The content was significantly higher than that of the control, showing obvious salt sensitivity.

[0054] 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 gene knockout of CmATG8f2 makes melon plants sensitive to salt stress. These findings also show that CmATG8f2 has a significant effect in improving the salt tolerance of melon plants.

[0055] Example 4 This example provides identification of root rot resistance of melon CmATG8f2 gene overexpression and gene-edited plants, and the specific steps are as follows: 4.1 Isolation and identification of melon root rot pathogens The root rot tissue of the diseased melon was cut and placed on PDA medium (purchased from Solarbio) at 28°C to culture the pathogen. The growth of the colony and the morphological characteristics of the spores and hyphae under the microscope were observed. It was found that the hyphae on the PDA medium were white with pink to blue-purple pigments ( Figure 5 A). Microconidia are oval, macroconidia are spindle-shaped ( Figure 5 B).

[0056] After picking the colonies, the pathogens were amplified and sequenced using universal primers: ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC). The sequencing results were compared with the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) by BLAST homology, and the phylogenetic tree was constructed using MEGA software ( Figure 5 C). The results showed that the pathogenic bacteria strain had the highest homology with Fusarium oxysporum, with a homology of more than 99.9% ( Figure 5 D).

[0057] Through morphological characteristics and molecular biological identification, the pathogen was finally identified as Fusarium oxysporum (F. oxysporum).

[0058] 4.2 CmATG8f2 gene improves melon root rot resistance After the strain was activated, the spores in the culture medium were washed with sterile water and collected to prepare a spore concentration of 1×10 6 / ml spore suspension was prepared for later use. The roots of overexpression, knockout and control melon plants were washed, immersed in the spore suspension of root rot pathogen for 20 minutes, and then planted in seedling trays, and the disease progress was monitored regularly.

[0059] The disease classification is based on the Nagao (1994) standard as follows: 0, healthy and uninfected; 1, slight discoloration on the roots, healthy hypocotyls; 2, 10% root discoloration, healthy hypocotyls; 3, 30% root discoloration, slight infection on hypocotyls; 4, 70% root discoloration, hypocotyl infection; 5, whole root discoloration, severe infection on hypocotyls, or plant death. The disease index is divided into three levels: highly resistant (0~2), moderately resistant (2~4), susceptible (≥4) The results showed that after inoculation with the pathogen, the growth and root status of the CmATG8f2 overexpressing plants were significantly better than those of the control; however, the root rot of the gene knockout plants was aggravated and the resistance was weaker ( Figure 6 ). The results of resistance identification are shown in Table 4: the disease index of the control melon plants was 2.38, and the root rot resistance was moderate; the disease index of the CmATG8f2 overexpression plants was 1.00, and the root rot resistance was high, showing a significantly enhanced resistance to root rot; the disease index of the gene-edited plants was 4.08, and the root rot resistance was susceptible, and the root rot resistance was significantly reduced.

[0060] Table 4 Resistance levels of different melon plant materials to root rot

[0061] The above results show that CmATG8f2 plays an important role in melon's resistance to root rot. Its overexpression can significantly improve the resistance of melon plants to root rot, while gene knockout of CmATG8f2 leads to a significant decrease in the resistance of melon plants to root rot. These results show that CmATG8f2 has a significant effect in improving the resistance of melon plants to root rot.

[0062] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0063] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of melon autophagy gene CmATG8f2 in plant stress resistance 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.

2. The use according to claim 1, characterized in that: Improving the stress and disease resistance of melon plants is achieved by overexpressing the CmATG8f2 gene.

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

4. The use according to claim 2, characterized in that: The vector used to construct the CmATG8f2 overexpression vector is specifically the pBSE403R vector.

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