MiERF109-like or MiERF113 protein and application of coding gene thereof in mango fruit softening regulation

Abstract

The invention discloses an application of a MiERF109-like or MiERF113 protein and a coding gene of the MiERF109-like or MiERF113 protein in regulation and control of softening of mango fruits. The expression mode of MiERF109-like or MiERF113 changing along with time is detected through real-time fluorescent quantitative PCR, and it is found that the expression mode of MiERF109-like or MiERF113 is closely related to the mango fruit softening process. According to the invention, a VIGS technology is further utilized to silence the ethylene response factor of the mango fruit, and results show that the hardness index of the fruit with the silent gene MiERF109-like or MiERF113 of the mango fruit is significantly changed, which indicates that the MiERF109-like or MiERF113 gene is a key gene affecting the softening of the mango fruit, and the Mango fruit with the silent gene miERF109-like or MiERF113 gene is a key gene affecting the softening of the mango fruit. The method can be applied to regulation and control of softening of mango fruits and cultivation of new storage and transportation-resistant mango varieties, and a new idea is provided for preservation of harvested mangoes.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetic technology, and specifically relates to the application of MiERF109-like or MiERF113 protein and its encoding gene in regulating the softening of mango fruits. Background Art

[0002] Mango (Mangifera indica L.) is the second largest tropical fruit in the world, and its production and consumption continue to grow (Liu et al., 2022). As a typical climacteric fruit, mango can only be stored at room temperature for 4 - 10 days after harvest before entering the ripening stage, accompanied by complex physiological changes such as fruit softening, color transformation, and flavor substance accumulation (Ali et al., 2022; Hussain et al., 2024). Its ripening process is triggered by the peak of ethylene release and the jump of respiration rate (Eccher Zerbini et al., 2015). However, the characteristics of short ripening period and rapid postharvest softening of mango result in a significant shortening of the fruit shelf life, exacerbating postharvest losses, severely restricting the long-distance transportation and annual balanced supply of fresh fruits, and posing challenges to the economic benefits of the fruit and vegetable industry in tropical regions.

[0003] The plant cell wall structure is the core factor maintaining fruit firmness and postharvest quality (Yashoda et al., 2006). During mango ripening, the contents of cell wall components such as cellulose, hemicellulose, and protopectin decrease significantly, while the proportion of water-soluble pectin increases (Yashoda et al., 2006), and this process is closely related to the enhanced activities of key enzymes such as polygalacturonase (PG), cellulase (CL), pectin methylesterase (PME), and β-galactosidase (β-GAL) (Akbar Anjum et al., 2022; Lv et al., 2020). The above enzymes directly drive fruit softening by decomposing the cell wall polysaccharide network (Chourasia et al., 2006; Nguyen et al., 2021). In recent years, the ethylene response factor (ERF) family has been confirmed to be the core transcription factor regulating fruit ripening, and it regulates cell wall metabolism by activating the expression of pectin-degrading enzyme genes (such as PL, PG2a, PME) (Peng et al., 2022). For example, MdAP2-like in apples accelerates softening by inducing Mdβ-GAL18 (Wang et al., 2024), PpERF / ABR1 in peaches directly binds to the PpPG promoter to promote enzyme activity (Cheng et al., 2022), and MiERF2 and MiERF8 in mangoes can positively regulate the expression of MiPG (Li et al., 2022). Nevertheless, there are still a large number of unknowns in the molecular mechanism of ERF family members in mango softening, especially the targeted regulatory network of key genes has not been resolved.

[0004] Based on this, the system clarifies the regulatory network of mango fruit softening, excavates core functional genes and analyzes their action mechanisms, which has important application value for developing postharvest preservation technologies, cultivating new varieties resistant to storage and transportation, and optimizing the industrial chain supply mode. Summary of the Invention

[0005] The object of the present invention is as follows: aiming at the above existing problems, the present invention provides the application of MiERF109-like or MiERF113 protein and its encoding gene in the regulation of mango fruit softening. The specific scheme is as follows:

[0006] One object of the present invention is to provide the application of MiERF109-like protein with the amino acid sequence shown in SEQ ID NO.3 and its encoding gene and / or MiERF113 protein with the amino acid sequence shown in SEQ ID NO.4 and its encoding gene in regulating mango fruit softening.

[0007] Another object of the present invention is to provide the application of the silencing vector of MiERF109-like / MiERF113 gene or its specific fragment and the silencing strain of MiERF109-like / MiERF113 gene or its specific fragment in inhibiting mango fruit softening. The nucleotide sequence of the MiERF109-like gene is shown in SEQ ID NO.1, and the nucleotide sequence of the specific fragment of the MiERF109-like gene is shown in SEQ ID NO.5; the nucleotide sequence of the MiERF113 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the specific fragment of the MiERF113 gene is shown in SEQ ID NO.6.

[0008] Further explanation, the silencing vector is obtained by constructing the MiERF109-like gene or MiERF113 gene and its specific fragment into the vector pTRV.

[0009] Further explanation, the strain of the MiERF109-like gene or MiERF113 gene and its specific fragment is obtained by introducing the silencing vector of the gene into Agrobacterium.

[0010] The present invention also provides a method for inhibiting mango fruit softening, which includes inhibiting or down-regulating the expression of MiERF109-like or MiERF113 protein in mango, and can delay fruit softening. The amino acid sequence of the MiERF109-like protein is shown in SEQ ID NO.3, and the amino acid sequence of the MiERF113 protein is shown in SEQ ID NO.4.

[0011] Further explanation: The down-regulation includes knocking out or silencing the MiERF109-like or MiERF113 gene in the genome of mango; or transferring a down-regulator into mango by transgenic technology. The nucleotide sequence of the MiERF109-like gene is shown as SEQ ID NO.1, and the nucleotide sequence of the MiERF113 gene is shown as SEQ ID NO.2.

[0012] Further explanation: The knocking out or silencing is achieved by introducing a specific fragment of the gene into a silencing vector, and the nucleotide sequences of the specific fragments are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0013] The present invention also provides a method for identifying the softening degree of mango fruits obtained by the above method, by observing and / or detecting the fruit hardness to evaluate the influence of the MiERF109-like or MiERF113 gene on the softening of mango fruits.

[0014] In summary, the beneficial effects of the technical solution of the present invention are as follows:

[0015] The present invention uses the VIGS technology to determine that the MiERF109-like or MiERF113 gene plays an important role in the ripening and softening of mango fruits. In view of the above research, the present invention determines that the MiERF109-like or MiERF113 gene may be a key gene related to the softening of mango fruits, providing some ideas and experimental basis for further analyzing the physiological functions and action mechanisms of the ERF gene family in the process of mango fruit ripening and softening, providing a theoretical basis for clarifying the functions of the MiERF109-like or MiERF113 gene and its role in the process of fruit ripening and softening. At the same time, it provides gene resources for cultivating new mango varieties with firm flesh, resistance to storage and transportation, and also provides new ideas and technical means for the post-harvest preservation of mangoes, having great academic value and important production application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the expression pattern of the MiERF109-like and MiERF113 genes of the present invention during three different ripening processes of mango fruits.

[0017] Figure 2 It is a schematic diagram of the influence of the silencing of the MiERF109-like and MiERF113 genes of the present invention on the softening of mango fruits. Among them, A: Fruit phenotype; B: Schematic diagram of the relative expression levels of genes during storage of control (pTRV2-pTRV1) and RNAi (pTRV2-MiERF109-like, pTRV2-MiERF113) fruits. DETAILED DESCRIPTION OF THE INVENTION

[0018] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following will describe the specific embodiments of the present invention in detail with reference to the accompanying drawings. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0019] Example 1: Expression characteristics of MiERF109-like or MiERF113 genes in mango fruits

[0020] (1) Detect the expression pattern of ethylene response factors over time by real-time fluorescence quantitative PCR

[0021] Using "Tainong" mango (Mangifera indica L.) as the material, three different mature processes of mango fruits were obtained with 1-MCP (1-methylcyclopropene), ETH (ethylene), and a blank control. The expression pattern of the ethylene response factor gene over time was detected by real-time fluorescence quantitative PCR.

[0022] Total RNA was extracted using the FastPure Plant RNA Extraction Kit, and cDNA was synthesized using the PrimeScript TM Reverse Transcription Kit. TB Premix was used for real-time fluorescence quantitative PCR (qRT-PCR). Actin was used as an internal reference, and the gene expression level was calculated by the 2-ΔΔCt method.

[0023] The results are shown in Figure 1 , and we used RT-qPCR to verify the expression patterns of MiERF1-MiERF118 in the RNA of mango fruits under three different mature treatments. The ETH treatment strongly induced the expression levels of MiERF109-like and MiERF113, while the 1-MCP treatment inhibited them compared with the control group. These results indicate that ethylene-mediated regulation of mango ripening is related to the expression levels of ERF genes (MiERF109-like, MiERF113).

[0024] (2) Clone the ethylene response factor family genes MiERF109-like and MiERF113 by homologous cloning

[0025] Based on the results of the previous mango transcriptome sequencing data, gene-specific primers (Table 1) were designed using Primer3Puls (Primer3, Maryland, USA) software and synthesized by Beijing Tsingke Biotechnology Co., Ltd. The full-length target fragment was amplified using post-harvest mango pulp cDNA as a template. The amplification reaction system (50 μL): 2×Phanta Flash Master Mix (Dye Plus) 25.0 μL, 10.0 μmol / L forward and reverse primers 2.0 μL each, cDNA template 2.0 μL, supplemented with ddH2O to 50.0 μL. The amplification program: pre-denaturation at 98 °C for 30 s, denaturation at 98 °C for 10 s, annealing at 58 °C for 5 s, extension at 72 °C for 10 s, for a total of 35 cycles. The PCR products were detected by 1.0% agarose gel electrophoresis, gel-cut and recovered, ligated to the pMD19-T vector, transformed into E. coli DH5α competent cells, and positive clone bacterial solutions were screened on LB solid medium containing ampicillin and sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing.

[0026] Table 1 Clone primer information

[0027]

[0028] The amino acid sequence of MiERF109-like is as shown in SEQ ID NO.3:

[0029] MNSAPKRVISGDTIEDDEGATSLFTSTSTTQNEPLDHTTLFLPAVAETCQICKIEGCLGCNFFP

[0030] EQTDEPDDQKKIKYRGVRRRPSDKWAAEIWNPVGAERVWLGTFETAQAAARAYDEAAIKF

[0031] RHLQGLKAKLNFPLSDYDINQIAQRTYDDISL

[0032] The open reading frame (ORF) length of the MiERF109-like gene is 471 bp, encoding 156 amino acids, with a predicted protein molecular weight of 21.42 kDa, a theoretical isoelectric point (pI) of 4.94, a total average hydrophilicity of -0.553, 12 phosphorylation sites (including 4 serine phosphorylations, 7 threonine phosphorylations, and 1 tyrosine phosphorylation), no transmembrane helix structure, and no signal peptide. Protein domain prediction shows that MiERF109-like has an AP2 / ERF domain, which is located at 77 - 143 aa. The subcellular localization prediction of the MiERF109-like protein is in the nucleus.

[0033] The amino acid sequence of MiERF113 is as shown in SEQ ID NO.4:

[0034] MSAMVSALTQVIGTTDDQSNMVQSNPSTVSSDLPQPAQDQGTTRKRHYRGVRQRPWGKW

[0035] AAEIRDPKKAARVWLGTFETAEDAAMAYDRAALKFKGTKAKLNFPERVQGMPEFFYLMG

[0036] GDSSSNTRSFNDQNVRPPMINNPPPPPIHQEVYPDLLRYAQILSSDDASFNYYTSSLFNQEGF

[0037] SPHSSSTLSSSTSSSHRQQQQELMRFSSNFEDIHDDHRKEFDDSNG

[0038] Bioinformatics methods were used to analyze the basic physicochemical characteristics, conserved domains, protein structure, evolutionary relationships, etc. of MiERF113; the results showed that: the open reading frame (ORF) length of the MiERF113 gene was 681 bp, encoding 227 amino acids, the predicted protein molecular weight was 25.61 kDa, the theoretical isoelectric point (pI) was 6.07, the total average hydrophilicity was -0.883, there were 32 phosphorylation sites, no transmembrane helix structure, and no signal peptide. Protein domain prediction showed that the MiERF113 protein contained only 1 conserved AP2 domain, and the 14th and 19th amino acids in this domain were alanine and aspartic acid respectively, which conformed to the typical structural characteristics of the ERF subfamily.

[0039] Example 2: Experiment on silencing the ERF gene in sweet mango fruits by VIGS method and its effect on fruit softening

[0040] 1. Experimental materials and reagents used

[0041] 1.1 Fruit materials

[0042] "Tainong" mangoes (Mangifera indica L.) were harvested at about 90 days after flowering at commercial maturity. Selected fruits were of uniform size (about 200 g to 220 g), and fruits with mechanical damage or pests and diseases were discarded.

[0043] 1.2 Strains and vectors

[0044] Strains: Escherichia coli DH5α competent cells (cloning host for plasmid amplification), Agrobacterium tumefaciens EHA105 competent cells (Agrobacterium strain for VIGS infection), both purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0045] Plasmids: pTRV1, pTRV2 (vectors for VIGS infection, purchased from Changsha Abiway Biotechnology Co., Ltd.)

[0046] 1.3 Reagents and Kits

[0047] RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Plasmid Mini Kit, Ordinary Agarose Gel DNA Recovery Kit, 2×PhantaFlash Master Mix (DyePlus) High-Fidelity Premix Enzyme, Homologous Recombination Kit ClonExpress Ultra One Step Cloning Kit V2, DNA Marker DL2000 (100 - 2000bp) were purchased from Nanjing Novoprotein Biotechnology Co., Ltd.; cDNA Synthesis Kit, Fast qPCR Reagent TB Premix Ex Taq TM II FAST qPCR was purchased from Takara Biotechnology (Beijing) Co., Ltd.; Restriction Endonucleases BamHI and XmaI were purchased from Thermo Fisher Scientific; Acetosyringone (AS), 2-(N-Morpholino)ethanesulfonic acid (MES), 4S Green Plus Non-Toxic Nucleic Acid Dye, LB Liquid Medium and Solid Medium, 50X TAE Electrophoresis Buffer, Kanamycin Sulfate, Ampicillin, Rifampicin Antibiotics, Agarose were purchased from Sangon Biotech (Shanghai) Co., Ltd.; YEP Liquid Medium was purchased from Qingdao Hope Bio-Technology Co., Ltd.

[0048] 1.4 Reagent Preparation

[0049] 1mol / L MES stock solution: Weigh 21.325g of MES and dissolve it in sterile water. First, weigh MES into a beaker and add 80mL of sterile water, then heat and stir in a 50°C water bath. After dissolution, adjust the pH to 5.6 with 1mL of 10mol / L NaOH, and make up the volume to 100mL with a 100mL volumetric flask. Filter through a 0.22mm water-based filter membrane and store at 4°C.

[0050] 1mol / L MgCl2·6H2O stock solution: Weigh 20.33g of MgCl2·6H2O and dissolve it in sterile water. Make up the volume to 100mL with a volumetric flask, filter through a 0.22mm water-based filter membrane to sterilize, and store at 4°C.

[0051] 100 mM Acetosyringone: Weigh 0.1962 g, first dissolve it in 5 mL DMSO and then add water to 10 mL. Sterilize it by passing through a 0.22 mm aqueous filter membrane.

[0052] 50 mg / mL Rif antibiotic: Weigh 0.5 g and dissolve it in 10 mL DMSO. Store it at -20 °C. When using, add 50 μL of 50 mg / mL Rif antibiotic to 100 mL of LB medium, and the working concentration is 25 μg / mL.

[0053] Kanamycin sulfate (Kanamycin, Kana) solution: Weigh 2.5 g of kanamycin sulfate with an electronic balance, dissolve it with deionized water and make up the volume to 50 mL to prepare a working solution with a concentration of 50 mg / ml. Sterilize it by passing through a 0.22 mm aqueous filter membrane and then aliquot it into sterilized 1.5 mL centrifuge tubes. Store it at -20 °C.

[0054] Ampicillin (Ampicillin, Amp) solution: Weigh 5.0 g of ampicillin with an electronic balance, dissolve it with deionized water and make up the volume to 50 mL to prepare a solution with a concentration of 100 mg / mL. Sterilize it by passing through a 0.22 mm aqueous filter membrane and then aliquot it into 1.5 mL sterilized centrifuge tubes. Store it at -20 °C.

[0055] 1X TAE electrophoresis buffer (1 L): Measure 20 mL of TAE stock solution (50X) and make up the volume to 1 L.

[0056] 1.5 Primer list

[0057] The primers for detecting expression levels were designed by Primer3Puls (Primer3, Maryland, USA). The primers for constructing different gene VIGS silencing vectors were first used to select appropriate VIGS silencing fragments by SGN-VIGS and then designed by the primer design website of Novogene's homologous group. The designed primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The primers for this experiment are shown in Table 2 and Table 3.

[0058] Specific fragment for VIGS silencing of MiERF109-like (SEQ ID NO.5):

[0059] TGAACTCCGCACCGAAGCGCGTAATCTCAGGCGATACTATTGAAGATGATGAAGGGGCTACGTCCTTGTTTACTTCGACCAGTACTACACAGAATGAGCCATTAGATCACACAACGTTATTTCTGCCTGCAGTTGCCGAAACATGTCAGATATGCAAAATCGAGGGCTGTCTTGGCTGCAACTTTTTCCCCGAACAAACAGATGAACCGGATGACCAAAAGAAGATCAAGTACAGGGGAGTTAGGCGGAGACCATCAGATAAATGGGCAGCCGAGATTTGGAACCCGGTCGGGGCAGAA

[0060] Specific fragment for VIGS silencing of MiERF113 (SEQ ID NO.5):

[0061] TCAAGTCATTGGCACCACCGATGACCAGTCAAACATGGTGCAATCCAACCCTTCAACAGTTTCTTCAGATTTGCCCCAACCAGCTCAAGATCAAGGAACAACAAGGAAGAGACATTACAGAGGGGTTAGACAAAGACCTTGGGGCAAATGGGCGGCTGAAATTCGAGATCCTAAAAAGGCAGCTCGAGTGTGGCTCGGCACATTCGAGACTGCTGAGGATGCAGCCATGGCATATGATAGAGCAGCCCTCAAATTTAAAGGAACCAAGGCTAAACTTAATTTCCCTGAAAGAGTTCAAGG

[0062] Table 2 RT-qPCR primer information

[0063]

[0064]

[0065] Table 3 Cloning primer sequences

[0066]

[0067] 2. Gene acquisition

[0068] 2.1 Gene information

[0069] Based on the analysis of the mango transcriptome data by the previous research group, the differentially expressed genes MiERF109-like and MiERF113 were selected, and the gene sequences were obtained from the known mango gene database (CATAS_Mindica_2.1). The nucleotide sequence of the MiERF109-like gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the MiERF113 gene is as shown in SEQ ID NO.2.

[0070] (1)SEQ ID NO.1:

[0071] ATGAACTCCGCACCGAAGCGCGTAATCTCAGGCGATACTATTGAAGATGATGAAGGGGCTACGTCCTTGTTTACTTCGACCAGTACTACACAGAATGAGCCATTAGATCACACAACGTTATTTCTGCCTGCAGTTGCCGAAACATGTCAGATATGCAAAATCGAGGGCTGTCTTGGCTGCAACTTTTTCCCCGAACAAACAGATGAACCGGATGACCAAAAGAAGATCAAGTACAGGGGAGTTAGGCGGAGACCATCAGATAAATGGGCAGCCGAGATTTGGAACCCGGTCGGGGCAGAACGTGTGTGGCTTGGCACTTTCGAGACTGCGCAGGCTGCTGCCAGAGCTTATGATGAGGCCGCCATCAAGTTCCGCCACTTACAGGGACTTAAAGCTAAGCTTAATTTTCCATTGTCAGACTATGATATAAATCAAATTGCACAACGCACATATGATGACATCTCTTTATAG

[0072] (2)SEQ ID NO.2:

[0073] ATGTCAGCCATGGTTTCTGCTCTAACTCAAGTCATTGGCACCACCGATGACCAGTCAAACATGGTGCAATCCAACCCTTCAACAGTTTCTTCAGATTTGCCCCAACCAGCTCAAGATCAAGGAACAACAAGGAAGAGACATTACAGAGGGGTTAGACAAAGACCTTGGGGCAAATGGGCGGCTGAAATTCGAGATCCTAAAAAGGCAGCTCGAGTGTGGCTCGGCACATTCGAGACTGCTGAGGATGCAGCCATGGCATATGATAGAGCAGCCCTCAAATTTAAAGGAACCAAGGCTAAACTTAATTTCCCTGAAAGAGTTCAAGGAATGCCTGAGTTTTTCTATTTAATGGGTGGAGATTCTTCTTCAAACACTAGGTCTTTTAATGACCAAAATGTAAGGCCACCAATGATTAATAATCCTCCTCCTCCTCCAATTCACCAAGAAGTTTATCCCGACTTGCTTCGATATGCTCAAATTCTTTCAAGCGATGATGCTAGTTTTAATTATTACACTTCAAGTCTCTTTAATCAGGAAGGTTTTTCACCTCATTCTTCTTCAACGTTATCTTCTTCCACCAGCTCATCACATCGCCAGCAGCAACAGGAGCTTATGAGATTTTCATCAAATTTTGAAGATATTCATGATGATCATAGGAAGGAATTTGATGACTCAAATGGGTAG

[0074] 2.2 Total RNA extraction from mango

[0075] The method was referred to the operation of the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit.

[0076] The concentration of the extracted RNA was determined by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA), and the integrity of the RNA was verified by 1% agarose gel electrophoresis.

[0077] 2.3 cDNA synthesis

[0078] (1) Removal of genomic DNA: Place the template RNA on ice. Prepare a mixture according to the genomic DNA removal system in the following table (Table 4), mix thoroughly and centrifuge. Incubate at a constant temperature of 42 °C for 2 min on a PCR instrument, and then place on ice.

[0079] Table 4 Reaction system for removing genomic DNA

[0080]

[0081] (2) Reverse transcription reaction: Add 4 μL of 5X RT Premix to the reaction solution in step (1) on ice. Mix gently and immediately perform a reverse transcription reaction at 37 °C for 10 min and 85 °C for 5 s. Then store the product at -20 °C.

[0082] 2.4 Cloning of genes

[0083] Clone the gene fragment for VIGS silencing using the cDNA as a template. The PCR amplification system is shown in Table 5.

[0084] Table 5 PCR amplification system for genes

[0085]

[0086] The reaction program is shown in Table 6.

[0087] Table 6 PCR reaction program

[0088]

[0089] The PCR products are detected by electrophoresis on a 1% agarose gel at a voltage of 100 V for 25 min. Cut the target band of the correct size with a clean scalpel or gel cutter and recover the target fragment according to the instructions of the TIANgelMini PurificationKit ordinary agarose gel DNA recovery kit Gel DNAExtraction Mini Kit for gel recovery to obtain the target fragment, and store it at -20 °C for subsequent homologous recombination.

[0090] 3. Treatment of vectors

[0091] Digest and recover the pTRV2 vector. Add components according to the digestion system in Table 7, mix well and incubate in a water bath at 37 °C for 30 min, and then in a water bath at 80 °C for 5 min. Detect the digested products by electrophoresis on a 1% agarose gel at a voltage of 100 V for 25 min to ensure that the vector is cut. Cut the band with a clean scalpel or gel cutter and recover it according to the instructions of the TIANgelMini PurificationKit ordinary agarose gel DNA recovery kit Refer to the instruction manual of Gel DNA Extraction Mini Kit for gel recovery to recover the linearized vector, and store it at -20°C for subsequent homologous recombination.

[0092] Table 7 Vector digestion system

[0093]

[0094] 4. Homologous recombination and transformation

[0095] 4.1 Recombination reaction

[0096] Operate ClonExpress Ultra One Step Cloning Kit V2 using the Novoprotein homologous recombination kit. Refer to Table 8 for adding components to the recombination reaction system. For single-fragment recombination reaction, incubate at 50°C for 5 min; cool to 4°C or immediately place on ice. The resulting ligation product is used for subsequent transformation into the cloning strain DH5α.

[0097] Table 8 Recombination reaction system

[0098]

[0099] 4.2 Transformation of ligation product into DH5α

[0100] Thaw the competent cells on ice. Take 10 μL of the ligation product and add it to 100 μL of competent cells. Gently flick the tube wall to mix and let it stand on ice for 30 min. Heat in a 42°C water bath for 45 s, then immediately cool on ice for 2 min. Add 900 μL of antibiotic-free LB medium and shake the bacteria at 37°C for 1 h (rotation speed 200 rpm). Preheat the LB solid medium plate with the corresponding antibiotic in a 37°C incubator. Centrifuge at 5000 rpm for 5 min. Discard 900 μL of the supernatant, resuspend the bacteria in the remaining medium, and spread it on a plate containing the corresponding antibiotic (Kana) using a sterile spreader. Incubate the plate upside down in a 37°C incubator for 12 - 16 h.

[0101] 5. Colony PCR to detect positive clones and sequencing

[0102] Pick a single colony into a 1.5 mL EP centrifuge tube containing 600 (containing 50 mg / L Kana) of LB liquid medium. Shake and culture at 37°C with a rotation speed of 200 rpm for 3 - 4 h, then perform colony PCR detection. The reaction system is as shown in Table 9 below, and the reaction program refers to Table 10.

[0103] Table 9 Colony verification PCR reaction system

[0104]

[0105] Table 10 Colony verification PCR reaction program

[0106]

[0107] After the reaction ended, the PCR amplification products were detected by 1% agarose gel electrophoresis. 200 μL of the bacterial solution showing positive bands was taken and sent to Beijing Tsingke Biotechnology Kunming Branch for sequencing.

[0108] 6. Plasmid extraction

[0109] Extract the plasmids of the strains with correct sequencing. The extraction method refers to the operation of the Novoprotein Plasmid Mini Kit (FastPure Plasmid Mini Kit - V21.2) and store them at -20 °C for subsequent transformation into Agrobacterium.

[0110] 7. Transformation into Agrobacterium EHA105

[0111] (1) Take the Agrobacterium competent cells stored at -80 °C to room temperature. Wait for them to partially melt (in an ice - water mixture state) and then insert them into ice.

[0112] (2) Add 1 μL of plasmid DNA (the plasmid extracted in 5., containing pTRV2 - target gene) to every 100 μL of competent cells. Mix by gently flicking the bottom of the tube and then let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and in an ice bath for 5 min in sequence.

[0113] (3) Add 700 μL of antibiotic - free YEB liquid medium and culture it with shaking at 28 °C for 2 - 3 h.

[0114] (4) Centrifuge at 6000 rpm for 1 min to collect the bacteria. Leave about 100 μL of the supernatant, gently pipette to resuspend the bacterial pellet, and spread it on an LB plate containing the corresponding antibiotics (50 μg / mL Kana and 25 μg / mL Rif), and culture it in an incubator at 28 °C for 2 - 3 days.

[0115] 8. Agrobacterium - mediated infection of mango

[0116] (1) Pick a single colony containing pTRV2 - target gene into a 1.5 mL EP centrifuge tube containing 600 (containing 50 mg / L Kana and 25 μg / mL Rif) of YEB liquid medium and culture it overnight.

[0117] (2) Inoculate Agrobacterium (EHA105) carrying pTRV2 - target gene, pTRV2, and pTRV1 into 20 mL of YEB medium (containing 50 mg / L Kana and 25 μg / mL Rif) at a ratio of 1:100, and culture it overnight at 28 °C and 220 rpm.

[0118] (2) Enrich the bacterial solution by centrifuging at 5000 rpm for 10 min, then discard the culture medium and suck out the excess culture medium of the bacterial mass with a pipette tip until it is clean.

[0119] (3) Wash the bacterial mass 2 - 3 times with sterile water to remove antibiotics.

[0120] (4) Wash the bacterial mass with the infection solution (final concentration: pH 5.6, 10 mM MES, 10 mM MgCl₂, 200 μM acetosyringone) once. After adding the penetration solution, pipette and blow to mix evenly, centrifuge at 5000 rpm for 10 min, and discard the penetration solution; 1 mL MES (1 M) + 1 mL MgCl 2* 6H₂O (1 M) + 200 μL acetosyringone (100 mM), make up the volume to 100 ml with Wahaha water.

[0121] (6) Finally, adjust the OD₆₀₀ of the bacterial solution to about 0.8 with the infection solution. Then mix pTRV2 - target gene and pTRV2 with the pTRV1 infection solution at a ratio of 1:1 respectively. After mixing well, recover in the shaker at 50 rpm in the dark for 3 h, and confirm that the OD₆₀₀ does not exceed 1.0 before injection.

[0122] (7) Inject at three positions in the center of the vertical axis of the mango with a 1 mL sterile syringe, injecting 0.5 mL at each position.

[0123] (8) Place the injected fruits in the dark at 24 °C for 24 h. Then take pictures and observe every two days, measure the hardness of the injection part of the fruits, and store the tissue of the injection part at -80 °C for subsequent qPCR detection.

[0124] (9) Measure the fruit hardness using a GY - 4 hardness meter with a probe diameter of 5 mm, and the measurement result is expressed in Newtons (N).

[0125] 9. Detect the relative expression level of the silenced gene by qPCR after infection

[0126] Extract the RNA from the injection part and synthesize cDNA according to the operations in 2.2 and 2.3. Use TB Premix ExTaq TM II FAST qPCR for fluorescence quantitative detection, and select the MiAtin gene as the internal reference gene for relative quantification. The real - time fluorescence quantitative PCR reaction system is shown in Table 11.

[0127] Table 11 Real - time fluorescence quantitative PCR reaction system

[0128]

[0129] 10. Results

[0130] From Figure 2 On the 4th day after Agrobacterium infection, the injected part of the empty vector (EV, EV: pTRV1-pTRV2) turned yellow normally, while the infected parts of pTRV2-MiERF109-like / 113 fruits showed green to varying degrees.

[0131] To clarify the mechanism of ERFs silencing on mango fruit softening, the expression of ERFs and softening-related genes in control and pTRV2-ERFs infiltrated fruits was monitored by RT-qPCR. As Figure 2 shown in

[0132] B, the level of MiERFs in pTRV2-MiERFs fruits was significantly down-regulated 4 days after injection.

[0133] Table 12 Firmness of mango fruits after silencing MiERF109-like and MiERF113

[0134]

[0135] Different lowercase letters indicate significant differences between treatments (P<0.05).

[0136] As can be seen from the above table, 4 days after injection, the firmness of fruits infected with pTRV2-MiERF109-like and pTRV2-MiERF113 was significantly higher than that of the EV (pTRV1-pTRV2) control.

[0137] In summary, the experimental results show that: MiERF109-like and MiERF113 genes are closely related to mango fruit softening.

[0138] The above-described embodiments merely represent several embodiments of the present invention. Their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Use of the MiERF109-like protein with the amino acid sequence shown in SEQ ID NO.3 and its coding gene and / or the MiERF113 protein with the amino acid sequence shown in SEQ ID NO.4 and its coding gene in regulating mango fruit softening.

2. Application of a silencing vector of the MiERF109-like / MiERF113 gene or its specific fragment, and a silencing strain of the MiERF109-like / MiERF113 gene or its specific fragment in inhibiting the softening of mango fruits, characterized in that, The nucleotide sequence of the MiERF109-like gene is shown in SEQ ID NO.1, and the nucleotide sequence of the specific fragment of the MiERF109-like gene is shown in SEQ ID NO.5; the nucleotide sequence of the MiERF113 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the specific fragment of the MiERF113 gene is shown in SEQ ID NO.

6.

3. The application according to claim 2, characterized in that, The silencing vector is obtained by constructing the MiERF109-like gene or MiERF113 gene and its specific fragment into the vector pTRV2.

4. The application according to claim 2, wherein The strain of the MiERF109-like gene or MiERF113 gene and its specific fragment is obtained by introducing the silencing vector of the gene into Agrobacterium.

5. A method for inhibiting the softening of mango fruits, characterized in that, The method includes inhibiting or down-regulating the expression of MiERF109-like or MiERF113 protein in mango, which can delay fruit softening. The amino acid sequence of the MiERF109-like protein is shown in SEQ ID NO.3, and the amino acid sequence of the MiERF113 protein is shown in SEQ ID NO.

4.

6. The method according to claim 5, characterized in that, The down-regulation includes: knocking out or silencing the MiERF109-like or MiERF113 gene in the mango genome; or transferring a down-regulator into mango by transgenic technology. The nucleotide sequence of the MiERF109-like gene is shown in SEQ ID NO.1, and the nucleotide sequence of the MiERF113 gene is shown in SEQ ID NO.

2.

7. The method according to claim 6, wherein The knocking out or silencing is achieved by introducing the specific fragment of the gene into the silencing vector, and the nucleotide sequences of the specific fragments are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively.

8. A method for identifying the softening degree of mango fruits obtained by the said method, characterized in that, By observing and / or detecting the fruit firmness to evaluate the effect of the MiERF109-like or MiERF113 gene on mango fruit softening.

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