Application of ERF118 protein and its encoding gene in mango fruit softening regulation

Abstract

The application provides application of ERF118 protein and a coding gene thereof in mango fruit softening regulation, and finds that the expression mode of the ERF118 gene is closely related to the process of mango fruit softening through real-time fluorescent quantitative PCR detection of the expression mode of ERF118 changing with time. The research constructs a silencing vector by using a VIGS technology, and further transforms the gene into mango fruits by using an agrobacterium-mediated genetic transformation system. The results show that the expression of the ERF118 gene is negatively correlated with softening, which indicates that the ERF118 gene has the function of regulating the fruit softening process. Silencing the ERF118 gene in mango fruits makes the fruit turning color later than the empty vector (EV) group, and the hardness is lower than the EV group. The research finding provides an excellent gene resource for cultivating crops with controllable ripening time.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics technology, specifically relating to the application of the ERF118 protein and its encoding gene in the regulation of mango fruit softening. Background Technology

[0002] Mango (Mangifera indica L.) is the world's second most popular tropical fruit (Liu et al., 2022). As a climacteric fruit, mangoes gradually ripen and deteriorate in quality after being stored at room temperature for 4-10 days (Ali et al., 2022; Hussain et al., 2024). The ripening process is triggered by ethylene release and an increase in respiration rate (Eccher Zerbini et al., 2015). Mango ripening involves complex reactions related to color, texture, and nutrient metabolism, with softening being the core characteristic (Li et al., 2023).

[0003] Plant cell walls are crucial for maintaining fruit firmness and quality (Yashoda et al., 2006). Cell walls are complex networks composed of cellulose, hemicellulose, pectin, and proteins (Peng et al., 2022). Changes in cell wall composition and metabolism (such as changes in the activity of pectinase and cellulase) significantly affect softening (Chourasia et al., 2006; Liu et al., 2020; Nguyen et al., 2021). During mango ripening, Na₂CO₃-soluble pectin, protopectin, chelated soluble pectin, hemicellulose, and cellulose decrease, while water-soluble pectin increases (Yashoda et al., 2006). Increased activity of enzymes such as polygalacturonase (PG), cellulase (CL), pectin methylesterase (PME), and β-galactosidase (β-GAL) leads to cell wall breakdown, promoting softening (Akbar Anjum et al., 2022; Lv et al., 2020).

[0004] Ethylene response factors (ERFs) are a family of key transcription factors regulating fruit ripening, influencing cell wall structure by regulating the expression of pectin-degrading enzyme genes (such as PL, PG2a, and PME) (Peng et al., 2022). In apples, MdAP2-like molecules promote softening by activating Mdβ-GAL18 (Wang et al., 2024); in peaches, PpERF / ABR1 binding to the PpPG promoter accelerates softening (Cheng et al., 2022); and in mangoes, MiERF2 and MiERF8 regulate MiPG expression (Li et al., 2022). However, the specific mechanisms by which ERFs play a role in mango softening remain unclear. This patent reveals the relationship between ERF118 and mango fruit softening. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems by providing the application of the ERF118 protein and its encoding gene in the regulation of mango fruit softening, as detailed below:

[0006] One of the objectives of this 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 the softening of mango fruit.

[0007] The present invention also provides a silencing vector of the ERF118 gene or a specific fragment thereof, and an ERF118 gene or a specific fragment thereof silencing strain for use 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] To further explain, the silencing vector is obtained by constructing the ERF118 gene and its specific fragment into the vector pTRV2.

[0009] To further clarify, the strain containing the ERF118 gene and its specific fragments was obtained by introducing a gene silencing vector into Agrobacterium.

[0010] The present invention also provides a method for promoting the softening of mango fruit, the method comprising inhibiting or downregulating the expression of ERF118 protein in mango, which can promote fruit softening, the amino acid sequence of the ERF118 protein being shown in SEQ ID NO.2.

[0011] To further explain, the downregulation includes: knocking out or silencing the ERF118 gene in the mango genome; or transferring the downregulator into the mango through transgenic technology, wherein the nucleotide sequence of the ERF118 gene is shown in SEQ ID NO.1.

[0012] To further explain, the knockout or silencing is achieved by introducing a specific fragment of the gene into a silencing vector, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0013] The present invention also provides a method for identifying the degree of softening of mango fruit obtained by the method, by observing and / or detecting fruit firmness to assess the effect of the ERF118 gene on mango fruit softening.

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

[0015] This invention utilizes virus-induced gene silencing (VIGS) technology to construct a silencing vector, and further employs an Agrobacterium-mediated genetic transformation system to transform this gene into mango fruit. Results showed that ERF118 gene expression was negatively correlated with softening, indicating its function in regulating the fruit softening process. Based on the above research, this invention identifies the ERF118 gene as a potentially 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 fruit resulted in a later color change time and lower firmness compared to the empty vector (EV) group. This finding provides excellent genetic resources for the subsequent cultivation of crops with controllable ripening time. Attached Figure Description

[0016] Figure 1 This is a comparative diagram showing the effect of MiERF118 gene silencing on mango fruit softening. A: Fruit phenotype; B: Schematic diagram of the relative gene expression levels during storage of control (pTRV2-pTRV1) and RNAi (pTRV2-MiERF118) fruits. Detailed Implementation

[0017] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0018] Example 1: Expression characteristics of the ERF118 gene in mango fruit

[0019] (1) The expression pattern of MiERF118 over time was detected by real-time quantitative PCR.

[0020] Using the "Tainong" mango (Mangifera indica L.) as material, three different ripening stages of mango fruit were obtained using 1-MCP (1-methylcyclopropene), ETH (ethylene), and a blank control. The expression patterns of ethylene response factor genes over time were detected by real-time quantitative PCR (see Table 1).

[0021] FastPure Plant RNA Extraction Kit extracts total RNA; PrimeScript TM cDNA synthesized using a reverse transcription kit, TB Premix was used for real-time quantitative PCR (qRT-PCR), with Actin as an internal control. -ΔΔCt Methods for calculating gene expression levels.

[0022] Table 1. Relative expression levels of MiERF118 in 13 different maturation processes.

[0023]

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

[0025] (2) The ERF118 gene of the ethylene response factor family was cloned using homologous cloning.

[0026] Based on previous mango transcriptome sequencing data, gene-specific primers were designed using Primer3Puls (Primer3, Maryland, USA) software (Table 2), and synthesized by Beijing Qingke Biotechnology Co., Ltd. The full-length target fragment was amplified using postharvest mango pulp cDNA as a template. The amplification reaction system (50 μL) consisted of: 25.0 μL of 2×Phanta Flash Master Mix (Dye Plus), 2.0 μL each of 10.0 μmol / L forward and reverse primers, 2.0 μL of cDNA template, and ddH2O to a final volume of 50.0 μL. The amplification program was: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 58℃ annealing for 5 s, and 72℃ extension for 10 s, for a total of 35 cycles. The PCR products were detected by 1.0% agarose gel electrophoresis, excised from the gel, ligated into the pMD19-T vector, transformed into E. coli DH5α competent cells, and positive clones were screened on LB solid medium containing ampicillin and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0027] Table 2 Cloning Primer Information

[0028]

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

[0030] MPEFRKQNFNQNKKRFCGESQITRKVRVICDDPYATDSSSSEDESERNDSIVQKRKRFVREIHIPLGPALVTPEAESSLDSNNFVKDPSKKRKSPVSTIPSSSSEKPKGVRQRKWGKWAAEIRDPFKKGRIWLGTYNTKEEAAKAYELKRIEFETR AAAAVANEQSNVSSKSDMSSSSLVASNSHTENNPDVVSSEDFESVLFHNSPASVLDLDTSASNLTTDCNGDLFKEEFDTNFEDLEIPLCLMEQPFEEQLGNFFNDDFEQLFGDFCVNGVEGNESSELPDCDFELDFGNLDFASLEEQAFPLNIACL

[0031] The open reading frame (ORF) of the MiERF118 gene is 939 bp in length, encoding 312 amino acids. The predicted molecular weight is 35.56 kDa, the theoretical isoelectric point (pI) is 4.63, the overall average hydrophilicity is -0.712, and it has 38 phosphorylation sites (30 serine, 7 threonine, and 1 tyrosine). It lacks a transmembrane helix structure and contains a signal peptide (located at 37-50 amino acids). The protein domains predicted for MiERF118 include an AP2 / ERF domain located at 106-171 amino acids. Subcellular localization of the MiERF118 protein is predicted to be in the cell nucleus.

[0032] Example 2: VIGS Method for Silencing the MiERF118 Gene in Sweet Mango Fruit and Its Effect on Fruit Softening 1. Experimental Materials and Reagents

[0033] 1.1 Fruit Materials

[0034] The "Tainong" mango (Mangifera indica L.) is harvested at commercial maturity approximately 90 days after flowering. Selected fruits are uniform in size (approximately 200 to 220 grams) and free from mechanical damage; fruits affected by diseases or pests are discarded.

[0035] 1.2 Strains and Vectors

[0036] Strains: Escherichia coli DH5α competent cells (cloning host, used for plasmid amplification) and 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 Aibiwei Biotechnology Co., Ltd.)

[0038] 1.3 Reagents and Kits

[0039] RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; Plasmid Mini-Prep Kit, Ordinary Agarose Gel DNA Recovery Kit, 2×PhantaFlash Master Mix (DyePlus) High-Fidelity Premixed Enzyme, Homologous Recombination Kit ClonExpress Ultra One Step Cloning Kit V2, and DNA Marker DL2000 (100-2000bp) were purchased from Nanjing Novizan Biotechnology Co., Ltd.; cDNA Synthesis Kit and Rapid qPCR Reagent TB Premix Ex Taq TM II. FAST qPCR was purchased from Baori Biotechnology (Beijing) Co., Ltd.; restriction endonucleases BamHI and XmaI were purchased from Thermo Fisher Scientific Co., Ltd.; acetylsylsyringone (AS), 2-(N-morpholino)ethanesulfonic acid (MES), 4S Green Plus non-toxic nucleic acid dye, LB liquid and solid culture media, 50X TAE electrophoresis buffer, kanamycin sulfate, ampicillin, rifampicin antibiotic, and agarose were purchased from Sangon Biotech (Shanghai) Co., Ltd.; YEP liquid culture medium was purchased from Qingdao Haibo Biotechnology 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 the MES into a beaker and add 80 mL of sterile water. Then, heat and stir in a water bath at 50 °C until dissolved. Adjust the pH to 5.6 with 1 mL of 10 mol / L NaOH. Make up to 100 mL in a 100 mL volumetric flask, filter through a 0.22 mm aqueous filter membrane, and store at 4 °C.

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

[0043] 100mM acetylsyl syringone: Weigh 0.1962g, dissolve in 5mL DMSO, add water to 10mL, and filter through a 0.22mm aqueous filter membrane for sterilization.

[0044] 50 mg / mLRif antibiotic: Weigh 0.5 g and dissolve in 10 mL DMSO. Store at -20 °C. When using, add 50 μL of 50 mg / mLRif antibiotic to 100 mL LB medium to obtain a working concentration of 25 μg / mL.

[0045] Kanamycin sulfate (Kana) solution: Weigh 2.5g of kanamycin sulfate with an electronic balance, dissolve it in deionized water and bring the volume to 50mL to prepare a working solution with a concentration of 50mg / ml. After sterilization by filtering through a 0.22mm aqueous filter membrane, dispense the solution into sterilized 1.5mL centrifuge tubes and store at -20℃.

[0046] Ampicillin (Amp) solution: Weigh 5.0g of ampicillin using an electronic balance, dissolve it in deionized water, and bring the volume to 50mL to prepare a 100mg / mL solution. After sterilization by filtering through a 0.22mm aqueous filter membrane, dispense the solution into 1.5mL sterile centrifuge tubes and store at -20℃.

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

[0048] 1.5 Primer List

[0049] Primers used to detect expression levels were designed by Primer3Puls (Primer3, Maryland, USA). Primers used to construct VIGS silencing vectors for different genes were first selected using the SGN-VIGS website for appropriate VIGS silencing fragments, and then primers were designed using the Novus Thomson Matrix Primer Design website. The designed primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The primers used in this experiment are shown in Tables 3 and 4.

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

[0051] AATTACCAGGAAAGTCCGTGTAATTTGTGATGATCCGTACGCTACCGATTCATCTTCAAGCGAAGATGAATCTGAGAGAAATGACTCCATAGTCCAAAAAAGGAAGCGGTTCGTTCGTGAGATTCATATACCGCTTGGCCCAGCTTTGGT CACGCCGGAGGCTGAGAGTTCTCTTGACAGTAACAACTTTGTCAAAGACCCCTCAAAGAAGAGGAAATCGCCCGTATCAACAACATCCGTCTTCGTCATCGGAGAAGCCAAAAGGCGTTAGACAACGAAAGTGGGGCAAATGGGCCGCTGA

[0052] Table 3. Primer information for RT-qPCR

[0053]

[0054]

[0055] Table 4 Cloning primer sequences

[0056]

[0057] 2. Gene Acquisition

[0058] 2.1 Genetic Information

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

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

[0061] ATGCCGGAGTTTCGAAAGCAGAATTTTAATCAAAACAAGAAAAGATTTTGCGGAGAGTCTCAAATTACCAGGAAAGTCCGTGTAATTTGTGATGATCCGTACGCTACCGATTCATCTTCAAGCGAAGATGAATCTGAGAGAAATGACTCCATAGTCCAAAAAAGGAAGCGGTTCGTTCGTGAGATTCATATACCGCTTGGCCCAGCTTTGGTCACGCCGGAGGCTGAGAGTTCTCTTGACAGTAACAACTTTGTCAAAGACCCCTCAAAGAAGAGGAAATCGCCCGTATCAACAATACCGTCTTCGTCATCGGAGAAGCCAAAAGGCGTTAGACAACGAAAGTGGGGCAAATGGGCCGCTGAGATTCGGGACCCATTTAAGAAAGGCCGGATTTGGTTGGGTACTTACAACACTAAAGAGGAAGCAGCCAAAGCTTATGAGCTCAAAAGAATTGAATTCGAAACTAGAGCTGCTGCGGCCGTCGCTAATGAGCAGAGCAACGTCTCCTCTAAAAGTGACATGTCATCATCTTCACTCGTGGCATCTAATTCCCACACCGAAAACAATCCTGATGTTGTTTCCTCTGAAGACTTTGAAAGTGTTTTGTTTCATAATTCACCTGCTTCAGTTCTTGACCTGGATACTTCTGCTTCAAATCTCACTACCGATTGCAATGGTGATTTATTTAAAGAAGAATTTGATACCAATTTTGAAGACCTTGAAATACCCTTGTGTTTAATGGAGCAGCCGTTTGAAGAACAGTTGGGCAATTTCTTCAATGACGATTTTGAGCAGCTATTTGGTGATTTTTGTGTTAATGGGGTTGAAGGTAACGAATCTAGCGAGCTTCCTGATTGTGATTTCGAGTTGGATTTTGGCAATTTGGACTTTGCTTCCTTAGAGGAGCAAGCATTCCCCCTCAATATAGCTTGCCTTTAA

[0062] 2.2 Total RNA extraction from mango

[0063] The procedure was performed according to the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit.

[0064] The concentration of extracted RNA was determined using 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 the mixture according to the genomic DNA removal system in Table 5, mix thoroughly and centrifuge, incubate at 42℃ for 2 min on a PCR instrument, and then place on ice.

[0067] Table 5. Genomic DNA Removal Reaction System

[0068]

[0069] (2) Reverse transcription reaction: Add 4 μL of 5X RT Premix to the reaction solution on ice, mix gently, and immediately carry out reverse transcription reaction at 37℃ for 10 min and 85℃ for 5 s. The product is then stored at -20℃.

[0070] 2.4 Cloning genes

[0071] The gene fragment used for VIGS silencing was cloned using 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] PCR products were detected by electrophoresis on a 1% agarose gel at 100V for 25 min. The target band of the correct size was cut off using a clean scalpel or gel cutter and processed according to the TIANgel Mini Purification Kit for DNA Recovery on agarose gel. The instructions for the Gel DNA Extraction Mini Kit are to perform gel extraction to recover the target fragment, which is then stored at -20°C for subsequent homologous recombination.

[0078] 3. Processing carrier

[0079] The pTRV2 vector was digested and recovered. The enzyme digestion system was prepared according to Table 8, and the mixture was incubated at 37°C for 30 min, followed by incubation at 80°C for 5 min. The digestion products were detected by electrophoresis on a 1% agarose gel at 100V for 25 min to ensure vector cleavage. The bands were cut using a clean scalpel or gel cutter and extracted according to the TIANgel Mini Purification Kit standard agarose gel DNA recovery kit. The instructions for the Gel DNA Extraction Mini Kit specify gel extraction to recover the linearized vector, which should be stored at -20°C for subsequent homologous recombination.

[0080] Table 8. Vector Enzyme Digestion System

[0081]

[0082] 4. Homologous recombination and transformation

[0083] 4.1 Recombination reaction

[0084] Using the Novavito single-source recombination kit, operate the ClonExpress UltraOne Step Cloning Kit V2. Add the recombination reaction system according to Table 9. Perform a single-fragment recombination reaction at 50℃ for 5 min. Cool to 4℃ or immediately place on ice to obtain the ligation product, which will be used for subsequent transformation into the cloned strain DH5α.

[0085] Table 9 Recombination Reaction System

[0086]

[0087] 4.2 Conversion of the ligation product to DH5α

[0088] Thaw competent cells on ice. Add 10 μL of ligation product to 100 μL of competent cells, gently tap the tube wall to mix, and incubate 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, incubate at 37°C for 1 h (200 rpm), and preheat LB agar plates containing the appropriate antibiotic at 37°C. Centrifuge at 5000 rpm for 5 min. Discard 900 μL of supernatant, resuspend the cells in the remaining medium, and spread using a sterile spreader onto plates containing the appropriate antibiotic (Kana). Incubate upside down at 37°C for 12–16 h.

[0089] 5. Detection of positive clones by bacterial culture PCR and sequencing

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

[0091] Table 10 PCR reaction system for bacterial culture verification

[0092]

[0093] Table 11 PCR reaction procedure for bacterial culture validation

[0094]

[0095] After the reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis. 200 μL of the bacterial solution with positive bands was taken and sent to Beijing Qingke Biotechnology Kunming Branch for sequencing.

[0096] 6. Extract plasmids

[0097] Extract plasmids from the correctly sequenced strains using the same extraction method as the Novizan plasmid mini kit (FastPurePlasmid Mini Kit-V21.2), and store at -20℃ for subsequent transformation into Agrobacterium.

[0098] 7. Transformed into Agrobacterium EHA105

[0099] (1) Take the competent Agrobacterium cells stored at -80℃ and place them at room temperature until they partially melt (in a mixture of ice and water), then insert them into ice.

[0100] (2) Add 1 μL of plasmid DNA (5. Extracted plasmid containing pTRV2-target gene) to every 100 μL of competent cells, mix by hand, and incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min.

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

[0102] (4) Centrifuge at 6000 rpm for 1 min to collect the bacteria, keep about 100 μL of supernatant, gently pipette to resuspend the bacterial block, spread it on LB plates containing the corresponding antibiotics (50 μg / mL Kana and 25 μg / mL Rif), and incubate at 28℃ for 2-3 days.

[0103] 8. Agrobacterium infection in mangoes

[0104] (1) Pick a single colony containing the 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 incubate overnight.

[0105] (2) Inoculate pTRV2-target gene, pTRV2, and pTRV1 Agrobacterium (EHA105) into 20mLYEB medium (containing 50mg / L Kana and 25μg / mL Rif) at a ratio of 1:100 and incubate overnight at 28℃ and 220rpm.

[0106] (2) Centrifuge the bacterial solution at 5000 rpm for 10 min to enrich it, then discard the culture medium and remove the excess culture medium from the bacterial block with a pipette tip.

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

[0108] (4) Wash the bacterial blocks once with the inoculation solution (final concentration pH 5.6, 10 mM MES, 10 mM MgCl2, 200 μM acetylsuccinone). After washing, add the permeate and mix well by pipetting. Centrifuge at 5000 rpm for 10 min and discard the permeate. Add 1 mL MES (1 M) + 1 mL MgCl2. 2* Add 6H2O (1M) and 200μL acetylsuccinone (100mM), then dilute with Wahaha water to 100ml.

[0109] (6) Finally, adjust the OD600 of the bacterial culture to about 0.8 with the infection solution. Then mix pTRV2-target gene and pTRV2 with pTRV1 infection solution in a 1:1 ratio. After mixing, incubate in the dark at 50 rpm for 3 hours. Before injection, confirm that the OD600 does not exceed 1.0.

[0110] (7) Use a 1mL sterile syringe to inject 0.5mL into three locations at the center of the vertical axis of the mango.

[0111] (8) The injected fruit was placed in the dark at 24℃ for 24 hours. After that, the fruit was photographed and observed every two days, the hardness of the injection site was measured, and tissue samples from the injection site were frozen at -80℃ for subsequent qPCR detection.

[0112] (9) Fruit hardness was measured using a GY-4 hardness tester with a probe diameter of 5 mm. The measurement result is expressed as Newtons (N).

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

[0114] RNA extraction and cDNA synthesis for the injection site were performed according to procedures 2.2 and 2.3. TB was used. Premix ExTaq TM II. Real-time quantitative PCR was performed using FAST qPCR, with the MiAtin gene selected as the internal reference gene for relative quantification. The real-time quantitative PCR reaction system is shown in Table 12.

[0115] Table 12 Real-time Quantitative PCR Reaction System

[0116]

[0117] 10. Results

[0118] from Figure 1 On day 4 after Agrobacterium infection, the injected portion of the empty vector (EV, EV: pTRV1-pTRV2) turned yellow normally, while the infected portion of the pTRV2-MiERF118 fruit turned green.

[0119] To elucidate the mechanism by which ERF silencing affects mango fruit softening, the expression of MiERF118 in control and pTRV2-ERF118-infiltrated fruits was monitored by RT-qPCR. Figure 1 As shown in Figure B, the level of MiERF118 in pTRV2-MiERF118 fruit was significantly downregulated 4 days after injection.

[0120] 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] As shown in the table above, 4 days after injection, the firmness of mangoes infected with pTRV2-MiERF118 was significantly lower than that of the pTRV1-pTRV2 control.

[0125] The experimental results in summary indicate that the MiERF118 gene is closely related to the softening of mango fruit.

[0126] The embodiments described above are merely examples of several implementations of the present invention, and while their descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. ERF118 Gene silencing vectors, or ERF118 The application of gene-silencing strains in promoting mango fruit softening is characterized by, The ERF118 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The silence carrier includes the... ERF118 The gene-specific fragment was constructed into the vector pTRV2; ERF118 The nucleotide sequence of the gene-specific fragment is shown in SEQ ID NO.

3.

3. The application according to claim 1, characterized in that, The ERF118 The gene-silencing strain is obtained by introducing the gene-silencing vector into Agrobacterium.

4. A method for promoting the softening of mango fruit, characterized in that, The method includes inhibiting or downregulating the expression of ERF118 protein in mangoes to promote fruit softening, the amino acid sequence of which is shown in SEQ ID NO.

2.

5. The method according to claim 4, characterized in that, The downregulation includes: knocking out or silencing in the mango genome. ERF118 Genes; the stated ERF118 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, The knockout or silencing is achieved by introducing a specific fragment of the gene into a silencing vector, the nucleotide sequence of which is shown in SEQ ID NO.3.

Images