Application of ERF118 protein and coding gene thereof in mango fruit softening regulation and control

Abstract

According to the application of the ERF118 protein and the coding gene thereof in the mango fruit softening regulation, the expression mode of the ERF118 along with the change of time is detected through real-time fluorescent quantitative PCR (Polymerase Chain Reaction), and the expression mode of the ERF118 gene is found to be closely associated with the mango fruit softening process. According to the present invention, the VIGS technology is adopted to construct the silent vector, the agrobacterium-mediated genetic transformation system is further adopted to transform the gene into the mango fruit, and the result shows that the ERF118 gene expression is negatively correlated with the softening, such that the ERF118 gene has the function of regulating the fruit softening process; the ERF118 gene is silenced in mango fruits, so that the color changing time of the fruits is later than that of an empty load (EV) group, the hardness of the fruits is lower than that of the EV group, and the research shows that excellent gene resources are provided for subsequent cultivation of crops with controllable maturing time.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetic technology, and specifically relates to the application of ERF118 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 (Liu et al., 2022). As a climacteric fruit, mango gradually ripens and its quality deteriorates after 4 - 10 days of storage at room temperature (Ali et al., 2022; Hussain et al., 2024), and its ripening process is triggered by ethylene release and an increase in respiration rate (Eccher Zerbini et al., 2015). Mango ripening is accompanied by complex reactions such as color, texture, and nutritional metabolism, among which softening is the core feature (Li et al., 2023).

[0003] The plant cell wall is the key to maintaining fruit firmness and quality (Yashoda et al., 2006). The cell wall consists of a complex network of cellulose, hemicellulose, pectin, and proteins (Peng et al., 2022). Changes in cell wall components and metabolism (such as changes in the activities of pectinase, cellulase, etc.) significantly affect softening (Chourasia et al., 2006; Liu et al., 2020; Nguyen et al., 2021). During mango ripening, the contents of Na2CO3 - soluble pectin, protopectin, chelate - soluble pectin, hemicellulose, and cellulose decrease, while the content of water - soluble pectin increases (Yashoda et al., 2006). The increased activities of enzymes such as polygalacturonase (PG), cellulase (CL), pectin methylesterase (PME), and β - galactosidase (β - GAL) lead to cell wall decomposition and promote softening (Akbar Anjum et al., 2022; Lv et al., 2020).

[0004] Ethylene response factor (ERF) is a key transcription factor family that regulates fruit ripening, and affects the cell wall structure by regulating the expression of pectin - degrading enzyme genes (such as PL, PG2a, PME) (Peng et al., 2022). In apples, MdAP2 - like promotes softening by activating _Mdβ - GAL18_ (Wang et al., 2024); in peaches, PpERF / ABR1 binds to the _PpPG_ promoter to accelerate softening (Cheng et al., 2022); in mangoes, MiERF2 and MiERF8 regulate the expression of _MiPG_ (Li et al., 2022). However, the specific mechanism of ERF in mango softening is still unclear. This patent reveals the relationship between ERF118 and mango fruit softening. 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 ERF118 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 the ERF118 protein with the amino acid sequence shown in SEQ ID NO.2 and its encoding gene in regulating mango fruit softening.

[0007] The present invention also provides the application of the silencing vector of the ERF118 gene or its specific fragment and the silencing strain of the ERF118 gene or its specific fragment in inhibiting mango fruit softening. The nucleotide sequence of the ERF118 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the specific fragment of the ERF118 gene is shown in SEQ ID NO.3.

[0008] Further explanation, the silencing vector is obtained by constructing the ERF118 gene and its specific fragment into the vector pTRV2.

[0009] Further explanation, the strain of the ERF118 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 promoting mango fruit softening. The method includes inhibiting or down-regulating the expression of ERF118 protein in mango, which can promote fruit softening. The amino acid sequence of the ERF118 protein is shown in SEQ ID NO.2.

[0011] Further explanation, the down-regulation includes: knocking out or silencing the ERF118 gene in the genome of mango; or transferring a down-regulator into mango by transgenic technology. The nucleotide sequence of the ERF118 gene is shown in SEQ ID NO.1.

[0012] Further explanation, the knocking out or silencing is achieved by introducing the specific fragment of the gene into the silencing vector. The nucleotide sequences of the specific fragments are shown in SEQ ID NO.3 respectively.

[0013] The present invention also provides a method for identifying the softening degree of the mango fruit obtained by the above method. By observing and / or detecting the fruit hardness, the influence of the ERF118 gene on mango fruit softening is evaluated.

[0014] In summary, the beneficial effects of the technical scheme of the present invention are:

[0015] The present invention constructs a silencing vector using virus-induced gene silencing (VIGS) technology, and further transforms this gene into mango fruits using an Agrobacterium-mediated genetic transformation system. The results show that the expression of the ERF118 gene is negatively correlated with softening, indicating its function in regulating the fruit softening process. In view of the above research, the present invention determines that the ERF118 gene may be a key gene related to mango fruit softening, providing a theoretical basis for clarifying the function of the ERF118 gene and its role in the fruit softening process. Silencing the ERF118 gene in mango fruits results in a later fruit color change time and a lower hardness than the empty vector (EV) group. This research finding provides excellent gene resources for subsequent cultivation of crops with controllable ripening times. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 FIG. is a comparison diagram of the effect of silencing the MiERF118 gene of the present invention on mango fruit softening. Among them, A: fruit phenotype; B: schematic diagram of the relative expression levels of genes during storage of control (pTRV2-pTRV1) and RNAi (pTRV2-MiERF118) fruits. DETAILED DESCRIPTION OF THE INVENTION

[0017] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following detailed description of the specific embodiments of the present invention will be given in conjunction with the accompanying drawings. Many specific details are set forth in the following description to fully understand 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.

[0018] Example 1: Expression Characteristics of the ERF118 Gene in Mango Fruits

[0019] (1) Detect the expression pattern of MiERF118 over time by real-time fluorescence quantitative PCR

[0020] Using 'Tainong' mango (Mangifera indica L.) as the material, three different mature mango fruits were obtained with 1-MCP (1-methylcyclopropene), ETH (ethylene), and blank control. The expression pattern of the ethylene response factor gene over time was detected by real-time fluorescence quantitative PCR (see Table 1).

[0021] Extract total RNA using the FastPure Plant RNA Extraction Kit, and synthesize cDNA using the PrimeScript TM Reverse Transcription Kit. Perform real-time fluorescence quantitative PCR (qRT-PCR) using TB Premix, with Actin as the internal reference, and calculate the gene expression level by the 2 -ΔΔCt method.

[0022] Relative expression levels of MiERF118 during 13 different ripening processes

[0023]

[0024] As shown in the above table, we used RT-qPCR to verify the expression pattern of MiERF118 in mango fruit RNA under three different ripening treatments. The 1-MCP treatment strongly induced the expression level of MiERF118, while the ETH treatment inhibited them compared with the control group. The visible results indicate that ethylene-mediated regulation of mango ripening is related to the expression level of the ERF gene (MiERF118).

[0025] (2) Clone the ethylene response factor family ERF118 gene by homologous cloning

[0026] According to the results of previous mango transcriptome sequencing data, gene-specific primers (Table 2) were designed using Primer3Puls (Primer3, Maryland, USA) software and synthesized by Tsingke Biotechnology Co., Ltd. in Beijing. The full-length target fragment was amplified using post-harvest mango pulp cDNA as a template. 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. 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 Tsingke Biotechnology Co., Ltd. in Beijing for sequencing.

[0027] Table 2 Information of cloning primers

[0028]

[0029] The amino acid sequence of MiERF118 is as shown in SEQ ID NO.2

[0030] MPEFRKQNFNQNKKRFCGESQITRKVRVICDDPYATDSSSSEDESERNDSIVQKRKRFVREIHIPLGPALVTPEAESSLDSNNFVKDPSKKRKSPVSTIPSSSSEKPKGVRQRKWGKWAAEIRDPFKKGRIWLGTYNTKEEAAKAYELKRIEFETRAAAAVANEQSNVSSKSDMSSSSLVASNSHTENNPDVVSSEDFESVLFHNSPASVLDLDTSASNLTTDCNGDLFKEEFDTNFEDLEIPLCLMEQPFEEQLGNFFNDDFEQLFGDFCVNGVEGNESSELPDCDFELDFGNLDFASLEEQAFPLNIACL

[0031] The open reading frame (ORF) of the MiERF118 gene is 939 bp in length, encoding 312 amino acids. The predicted molecular weight of the protein is 35.56 kDa, and the theoretical isoelectric point (pI) is 4.63. The total average hydrophilicity is -0.712. There are 38 phosphorylation sites (including 30 serine phosphorylations, 7 threonine phosphorylations, and 1 tyrosine phosphorylation). There is no transmembrane helix structure and one signal peptide (located at 37 - 50 aa). Protein domain prediction shows that MiERF118 has an AP2 / ERF domain, which is located at 106 - 171 aa. The subcellular localization of the MiERF118 protein is predicted to be in the nucleus.

[0032] Example 2: Experiment on silencing the MiERF118 gene in sweet mango fruits by VIGS method and its effect on fruit softening 1. Experimental materials and reagents used

[0033] 1.1 Fruit materials

[0034] "Tainong" mangoes (Mangifera indica L.) were harvested at about 90 days after flowering at commercial maturity. The selected fruits were of uniform size (about 200 g to 220 g), and fruits with mechanical damage or pests and diseases were discarded.

[0035] 1.2 Strains and vectors

[0036] 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.

[0037] Plasmids: pTRV1, pTRV2 (VIGS infection vectors, purchased from Changsha Abiway Biotechnology Co., Ltd.)

[0038] 1.3 Reagents and reagent kits

[0039] The RNAprep Pure Polysaccharide and Polyphenol Total RNA Extraction Kit for Plants was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the Plasmid Mini Kit, the Common Agarose Gel DNA Recovery Kit, the 2×PhantaFlash Master Mix (DyePlus) High-Fidelity Premix Enzyme, the Homologous Recombination Kit ClonExpress Ultra One Step Cloning Kit V2, and the DNA Marker DL2000 (100 - 2000bp) were purchased from Nanjing Novoprotein Biotechnology Co., Ltd.; the cDNA Synthesis Kit and the Fast qPCR Reagent TB Premix Ex Taq TM II FAST qPCR was purchased from Takara Biotechnology (Beijing) Co., Ltd.; the 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, and agarose were purchased from Sangon Biotech (Shanghai) Co., Ltd.; the YEP liquid medium was purchased from Qingdao Hope Bio-Technology Co., Ltd.

[0040] 1.4 Reagent preparation

[0041] 1 mol / L MES stock solution: Weigh 21.325 g of MES and dissolve it in sterile water. First, weigh MES into a beaker and add 80 mL of sterile water, then heat and stir it in a water bath at 50 °C. After dissolution, adjust the pH to 5.6 with 1 mL of 10 mol / L NaOH, make up the volume to 100 mL with a 100 mL volumetric flask, filter it through a 0.22 mm water-based filter membrane, and store it at 4 °C.

[0042] 1 mol / L MgCl₂·6H₂O stock solution: Weigh 20.33 g of MgCl₂·6H₂O and dissolve it in sterile water. Make up the volume to 100 mL with a volumetric flask, filter it through a 0.22 mm water-based filter membrane to sterilize, and store it at 4 °C.

[0043] 100 mM Acetosyringone: Weigh 0.1962 g and first dissolve it in 5 mL of DMSO, then add water to make up the volume to 10 mL, and filter it through a 0.22 mm water-based filter membrane to sterilize.

[0044] 50 mg / mL Rif Antibiotic: Weigh 0.5 g and dissolve it in 10 mL of DMSO, store it at -20 °C. When in use, add 50 μL of 50 mg / mL Rif antibiotic to 100 mL of LB medium, and the working concentration is 25 μg / mL.

[0045] Kanamycin (Kana) sulfate 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. After sterilization by passing through a 0.22 mm water-based filter membrane, it is dispensed into sterilized 1.5 mL centrifuge tubes and stored at -20 °C.

[0046] 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. After sterilization by passing through a 0.22 mm water-based filter membrane, it is dispensed into 1.5 mL sterilized centrifuge tubes and stored at -20 °C.

[0047] 1X TAE electrophoresis buffer (1 L): Measure 20 mL of TAE stock solution (50X) and make up the volume to 1 L.

[0048] 1.5 Primer list

[0049] The primers for detecting expression levels were designed by Primer3Puls (Primer3, Maryland, USA). For the primers used to construct different gene VIGS silencing vectors, first select appropriate VIGS silencing fragments on the SGN-VIGS website, and then design the primers on the Novogene homologous group primer design website. The designed primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The primers for this experiment are shown in Tables 3 and 4.

[0050] Specific fragment for VIGS silencing of MiERF118 (SEQ ID NO.3):

[0051] AATTACCAGGAAAGTCCGTGTAATTTGTGATGATCCGTACGCTACCGATTCATCTTCAAGCGAAGATGAATCTGAGAGAAATGACTCCATAGTCCAAAAAAGGAAGCGGTTCGTTCGTGAGATTCATATACCGCTTGGCCCAGCTTTGGTCACGCCGGAGGCTGAGAGTTCTCTTGACAGTAACAACTTTGTCAAAGACCCCTCAAAGAAGAGGAAATCGCCCGTATCAACAATACCGTCTTCGTCATCGGAGAAGCCAAAAGGCGTTAGACAACGAAAGTGGGGCAAATGGGCCGCTGA

[0052] Table 3 RT-qPCR primer information

[0053]

[0054]

[0055] Table 4 Cloning primer sequences

[0056]

[0057] 2. Gene acquisition

[0058] 2.1 Gene information

[0059] Using the previous research group's mango transcriptome data analysis, the differentially expressed gene MiERF118 was selected, and the gene sequence was obtained from the known mango gene database (CATAS_Mindica_2.1). The nucleotide sequence of the MiERF118 gene is as shown in SEQ ID NO.1.

[0060] (1) SEQ ID NO.1:

[0061] ATGCCGGAGTTTCGAAAGCAGAATTTTAATCAAAACAAGAAAAGATTTTGCGGAGAGTCTCAAATTACCAGGAAAGTCCGTGTAATTTGTGATGATCCGTACGCTACCGATTCATCTTCAAGCGAAGATGAATCTGAGAGAAATGACTCCATAGTCCAAAAAAGGAAGCGGTTCGTTCGTGAGATTCATATACCGCTTGGCCCAGCTTTGGTCACGCCGGAGGCTGAGAGTTCTCTTGACAGTAACAACTTTGTCAAAGACCCCTCAAAGAAGAGGAAATCGCCCGTATCAACAATACCGTCTTCGTCATCGGAGAAGCCAAAAGGCGTTAGACAACGAAAGTGGGGCAAATGGGCCGCTGAGATTCGGGACCCATTTAAGAAAGGCCGGATTTGGTTGGGTACTTACAACACTAAAGAGGAAGCAGCCAAAGCTTATGAGCTCAAAAGAATTGAATTCGAAACTAGAGCTGCTGCGGCCGTCGCTAATGAGCAGAGCAACGTCTCCTCTAAAAGTGACATGTCATCATCTTCACTCGTGGCATCTAATTCCCACACCGAAAACAATCCTGATGTTGTTTCCTCTGAAGACTTTGAAAGTGTTTTGTTTCATAATTCACCTGCTTCAGTTCTTGACCTGGATACTTCTGCTTCAAATCTCACTACCGATTGCAATGGTGATTTATTTAAAGAAGAATTTGATACCAATTTTGAAGACCTTGAAATACCCTTGTGTTTAATGGAGCAGCCGTTTGAAGAACAGTTGGGCAATTTCTTCAATGACGATTTTGAGCAGCTATTTGGTGATTTTTGTGTTAATGGGGTTGAAGGTAACGAATCTAGCGAGCTTCCTGATTGTGATTTCGAGTTGGATTTTGGCAATTTGGACTTTGCTTCCTTAGAGGAGCAAGCATTCCCCCTCAATATAGCTTGCCTTTAA

[0062] 2.2 Total RNA extraction from mango

[0063] The method was referred to the operation of the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit.

[0064] The concentration of the extracted RNA was measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA), and the integrity of the RNA was verified by 1% agarose gel electrophoresis.

[0065] 2.3 cDNA Synthesis

[0066] (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 5), mix thoroughly and centrifuge, incubate at a constant temperature of 42 °C for 2 min on a PCR instrument, and then place on ice.

[0067] Table 5 Reaction System for Removing Genomic DNA

[0068]

[0069] (2) Reverse transcription reaction: Add 4 μL of 5X RT Premix to the reaction solution in (1) on ice, gently mix well and immediately carry out a reverse transcription reaction at 37 °C for 10 min and 85 °C for 5 s. Then store the product at -20 °C.

[0070] 2.4 Cloning of Genes

[0071] The gene fragment for VIGS silencing was cloned using the cDNA as a template, and the PCR amplification system is shown in Table 6.

[0072] Table 6 PCR Amplification System for Genes

[0073]

[0074] The reaction procedure is shown in Table 7.

[0075] Table 7 PCR Reaction Procedure

[0076]

[0077] The PCR products were detected by electrophoresis on a 1% agarose gel at a voltage of 100 V for 25 min. The target band of the correct size was cut off with a clean scalpel or gel cutter and recovered according to the instructions of the TIANgelMini PurificationKit Ordinary Agarose Gel DNA Recovery Kit Gel DNAExtraction Mini Kit to recover the target fragment, and store it at -20 °C for subsequent homologous recombination.

[0078] 3. Treatment of Vectors

[0079] Digest the pTRV2 vector and recover it. Add components to the digestion system according to Table 8, mix well, incubate in a water bath at 37 °C for 30 min, and then incubate in a water bath at 80 °C for 5 min. Detect whether the vector is cut by subjecting the digestion product to electrophoresis on a 1% agarose gel at a voltage of 100 V for 25 min. Cut the band with a clean scalpel or gel cutter and perform gel recovery according to the instruction manual of the TIANgel Mini Purification Kit (ordinary agarose gel DNA recovery kit) Gel DNA Extraction Mini Kit to recover the linearized vector, and store it at -20 °C for subsequent homologous recombination.

[0080] Table 8 Vector digestion system

[0081]

[0082] 4. Homologous recombination and transformation

[0083] 4.1 Recombination reaction

[0084] Use the Novoprotein homologous recombination kit to operate the ClonExpress Ultra One Step Cloning Kit V2. Add components to the recombination reaction system according to Table 9. 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.

[0085] Table 9 Recombination reaction system

[0086]

[0087] 4.2 Transform the ligation product into DH5α

[0088] 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 well and let it stand on ice for 30 min. Heat in a water bath at 42 °C for 45 s, and 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 bacterial pellet with the remaining medium, and spread it on a plate containing the corresponding antibiotic (Kana) with a sterile spreading rod. Incubate the plate upside down in a 37 °C incubator for 12 - 16 h.

[0089] 5. Detect positive clones by colony PCR and sequencing

[0090] Pick a single colony and transfer it into a 1.5 mL EP centrifuge tube containing 600 μL of LB liquid medium (containing 50 mg / L Kana). Incubate it in a shaker at 37 °C with an oscillation speed of 200 rpm for 3 - 4 h, and then perform colony PCR detection. The reaction system is shown in Table 10 below, and the reaction program refers to Table 11.

[0091] Table 10 Colony PCR Reaction System for Verification

[0092]

[0093] Table 11 Colony PCR Reaction Program for Verification

[0094]

[0095] After the reaction, use 1% agarose gel electrophoresis to detect the PCR amplification products. Pipette 200 μL of the bacterial solution showing positive bands and send it to Beijing Tsingke Biotechnology Kunming Branch for sequencing.

[0096] 6. Plasmid Extraction

[0097] Extract the plasmids of the strains with correct sequencing. The extraction method refers to the operation of Novoprotein Plasmid Mini Kit (FastPure Plasmid Mini Kit - V21.2), and store them at -20 °C for subsequent transformation into Agrobacterium.

[0098] 7. Transformation into Agrobacterium EHA105

[0099] (1) Take the Agrobacterium competent cells stored at -80 °C to room temperature. Wait until they are partially melted (in an ice - water mixture state), and then place them on ice.

[0100] (2) Add 1 μL of plasmid DNA (the plasmid extracted in step 5, containing pTRV2 - target gene) to every 100 μL of competent cells. Mix well by flicking the bottom of the tube, and then incubate on ice for 5 min, in liquid nitrogen for 5 min, in a water bath at 37 °C for 5 min, and on ice for 5 min successively.

[0101] (3) Add 700 μL of antibiotic - free YEB liquid medium and incubate at 28 °C with shaking for 2 - 3 h.

[0102] (4) Centrifuge at 6000 rpm for 1 min to collect the bacteria. Keep about 100 μL of the supernatant, gently resuspend the bacterial pellet by pipetting, 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.

[0103] 8. Agrobacterium - mediated Infection of Mango

[0104] (1) Pick a single colony containing pTRV2 - target gene into a 1.5 mL EP centrifuge tube with 600 μL of YEB liquid medium (containing 50 mg / L Kana and 25 μg / mL Rif), and culture overnight.

[0105] (2) Inoculate Agrobacterium tumefaciens (EHA105) of 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 overnight at 28 °C and 220 rpm.

[0106] (2) Centrifuge the bacterial liquid at 5000 rpm for 10 min for enrichment, then discard the medium, and use a pipette tip to suck out the excess medium from the bacterial pellet.

[0107] (3) Wash the bacterial pellet with sterile water 2 - 3 times to remove antibiotics.

[0108] (4) Wash the bacterial pellet with the infection solution (final concentration: 10 mM MES, pH 5.6, 10 mM MgCl₂, 200 μM acetosyringone), wash once, add the infiltration solution and pipette - blow to mix evenly, centrifuge at 5000 rpm for 10 min, and discard the infiltration solution; 1 mL MES (1 M) + 1 mL MgCl 2* 6H₂O (1 M) + 200 μL acetosyringone (100 mM), make up to 100 mL with Wahaha water.

[0109] (6) Finally, adjust the OD₆₀₀ of the bacterial liquid to about 0.8 with the infection solution. Then mix pTRV2 - target gene, pTRV2, and pTRV1 infection solutions 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.

[0110] (7) Use a 1 mL sterile syringe to inject at three positions in the center of the longitudinal axis of the mango, and inject 0.5 mL at each position.

[0111] (8) The injected fruits are placed 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 fruit, and store the tissue of the injection part at - 80 °C for subsequent qPCR detection.

[0112] (9) The fruit hardness is measured using a GY - 4 hardness meter with a probe diameter of 5 mm, and the measurement result is expressed in Newtons (N).

[0113] 9. Detection of the relative expression level of the silenced gene by qPCR after infection

[0114] RNA extraction and cDNA synthesis of the injected part were carried out according to the operations in 2.2 and 2.3. Use TB Premix ExTaq TM For fluorescence quantitative detection by II FAST qPCR, the MiAtin gene was selected as the internal reference gene for relative quantification. The real-time fluorescence quantitative PCR reaction system is shown in Table 12.

[0115] Table 12 Real-time fluorescence quantitative PCR reaction system

[0116]

[0117] 10. Results

[0118] From Figure 1 In A, on the 4th day after Agrobacterium infection, the injected part of the empty vector (EV, EV: pTRV1 - pTRV2) turned yellow normally, while the infected part of the pTRV2 - MiERF118 fruit showed green.

[0119] To clarify the mechanism of ERFs silencing on mango fruit softening, the expression of MiERF118 in control and pTRV2 - ERF118 - infiltrated fruits was monitored by RT - qPCR. As Figure 1 shown in B, the level of MiERF118 in pTRV2 - MiERF118 fruits was significantly down - regulated 4 days after injection.

[0120] The fruit firmness is shown in Table 13.

[0121] Table 13 Mango fruit firmness after silencing MiERF118

[0122]

[0123] Different lowercase letters indicate significant differences between treatments (p < 0.05).

[0124] From the above table, it can be seen that 4 days after injection, the firmness of the fruits infected with pTRV2 - MiERF118 was significantly lower than that of the pTRV1 - pTRV2 control.

[0125] In summary, the experimental results show that the MiERF118 gene is closely related to mango fruit softening.

[0126] The above - described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they 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 still 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 ERF118 protein with an amino acid sequence shown in SEQ ID NO.2 and its encoding gene in regulating the softening of mango fruits.

2. Application of a silencing vector of the ERF118 gene or its specific fragment, and a silencing strain of the ERF118 gene or its specific fragment in inhibiting the softening of mango fruits, characterized in that, The nucleotide sequence of the ERF118 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the specific fragment of the ERF118 gene is shown in SEQ ID NO.

3.

3. The application according to claim 2, characterized in that, The silencing vector is obtained by constructing the ERF118 gene and its specific fragment into the vector pTRV2.

4. The application according to claim 2, wherein The strain of the ERF118 gene and its specific fragment is obtained by introducing the silencing vector of the gene into Agrobacterium.

5. A method for promoting the softening of mango fruits, characterized in that, The method includes inhibiting or down-regulating the expression of ERF118 protein in mango, which can promote fruit softening, and the amino acid sequence of the ERF118 protein is shown in SEQ ID NO.

2.

6. The method according to claim 5, wherein The down-regulation includes: knocking out or silencing the ERF118 gene in the mango genome; or transferring a down-regulator into mango by transgenic technology, and the nucleotide sequence of the ERF118 gene is shown in SEQ ID NO.

1.

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.3 respectively.

8. A method for identifying the softening degree of mango fruits obtained by the method, characterized in that, By observing and / or detecting the fruit firmness to evaluate the effect of the ERF118 gene on the softening of mango fruits.

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