MaHDAC3 gene and application thereof in regulating fruit ripening

Abstract

The application provides a gene and application thereof in regulating fruit ripening MaHDAC3 The gene and application thereof in regulating fruit ripening belong to the technical field of genetic engineering. MaHDAC3 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the application uses VIGS technology to silence banana fruit MaHDAC3 The gene, and it is found that the fruit of the banana MaHDAC3 Gene, the hardness, soluble sugar and ROS content and other physiological indexes related to fruit ripening and softening of the fruit change significantly, indicating that MaHDAC3 The gene is a key gene affecting the ripening and softening of sweet banana fruit, and can be applied to regulating the ripening or softening of banana fruit and cultivating new banana varieties.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the MaHDAC3 gene and its application in regulating fruit ripening. Background Technology

[0002] As a vital global economic crop and food source, the maintenance of banana (Musaspp.) postharvest quality directly impacts the efficiency of the industry chain and the consumer experience. Banana fruits exhibit typical climacteric characteristics, resulting in rapid and difficult-to-control postharvest ripening, which easily leads to fruit softening, flavor deterioration, increased susceptibility to diseases, and a significantly shortened shelf life. Therefore, in-depth research into the molecular regulatory mechanisms of banana postharvest ripening, particularly identifying key regulatory factors, is urgently needed for developing targeted, efficient, and environmentally friendly postharvest preservation technologies to reduce postharvest losses and ensure the sustainable development of the banana industry.

[0003] Histone deacetylases (HDACs) are a class of highly conserved epigenetic regulators that precisely regulate gene transcription activity by catalyzing the deacetylation of histone lysine residues, altering chromatin structure. Studies have shown that HDACs play a crucial role in plants, widely participating in the regulation of growth and development (such as flowering, seed germination, and organogenesis), stress responses (biotic and abiotic stresses), and various metabolic pathways. Their powerful function lies in their ability to influence the expression networks of numerous downstream genes by reshaping the epigenetic landscape. However, while the central role of HDACs in plant life activities is widely recognized, their specific functions and mechanisms of action in regulating the critical physiological process of postharvest fruit ripening in bananas remain poorly understood. Systematic research into the functions of banana MaHDAC family members in fruit ripening will not only fill research gaps in this field but also provide new breakthroughs in understanding the epigenetic aspects of fruit ripening regulation. Summary of the Invention

[0004] In view of this, the present invention provides the MaHDAC3 gene and its application in regulating fruit ripening to solve the above problems.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a gene, MaHDAC3, that regulates banana fruit ripening. The nucleotide sequence of MaHDAC3 is shown in SEQ ID NO. 1.

[0007] The present invention also provides a protein encoded by the gene MaHDAC3 that regulates banana fruit ripening, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] This invention also provides the application of silencing the gene MaHDAC3 in promoting banana fruit ripening and / or peel softening.

[0009] The present invention also provides a method for promoting banana ripening, comprising the following steps:

[0010] (1) Design primers to amplify the specific sequence of the silenced gene MaHDAC3;

[0011] (2) The specific sequence of the MaHDAC3 gene was ligated to the TRV2 plasmid vector to obtain the TRV2-MaHDAC3 recombinant plasmid;

[0012] (3) The helper plasmids TRV1 and TRV2-MaHDAC3 recombinant plasmids were introduced into Agrobacterium GV3101, and the recombinant Agrobacterium was cultured until the bacterial culture reached OD. 600 =0.8~1.0, centrifuge and discard the supernatant;

[0013] (4) Resuspend the bacterial cells in infection buffer to OD. 600 =1.2, mix equal volumes of Agrobacterium bacterial suspension containing TRV1 and TRV2-MaHDAC3 recombinant plasmids, and incubate for 4-6 hours to obtain infection bacterial suspension;

[0014] (5) Inject the banana fruit with the infecting bacterial solution and culture it in the dark at 21~23℃ for 8~10h, and then culture it at 24~26℃ under the conditions of 16h light / 8h dark.

[0015] Preferably, the sequences of the primers are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0016] Preferably, the specific sequence of the silenced gene MaHDAC3 is shown in SEQ ID NO.3.

[0017] Preferably, the culture conditions for recombinant Agrobacterium in step (4) are: 27~29℃, 180~200rpm.

[0018] Preferably, the infection buffer contains 10 mM MgCl2, 10 mM MES and 200 μM acetosyringone.

[0019] Preferably, the injection volume of the infecting bacterial solution is 1~10mL.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention uses VIGS technology to silence the MaHDAC3 gene in banana fruit. The results show that the fruits with the MaHDAC3 gene silenced have significant changes in physiological indicators related to fruit ripening and softening, such as firmness, soluble sugar and ROS content. This indicates that the MaHDAC3 gene is a key gene affecting the ripening and softening of sweet banana fruit and can be applied to regulate the ripening or softening of banana fruit and the breeding of new banana varieties. Attached Figure Description

[0021] Figure 1 Chromosomal bitmap analysis of members of the banana SBP gene family.

[0022] Figure 2 Evolutionary relationship analysis of members of the banana MaHDACs gene family.

[0023] Figure 3 Analysis of conserved domains for members of the banana MaHDACs gene family.

[0024] Figure 4 Analysis of cis-regulatory elements in the promoter region of the banana MaHDACs gene family.

[0025] Figure 5 Expression heatmap analysis of members of the banana MaHDACs gene family.

[0026] Figure 6 To analyze the expression patterns of members of the banana MaHDACs gene family using qPCR technology.

[0027] Figure 7 The results of VIGS silencing RT-PCR for the MaHDAC3 gene.

[0028] Figure 8 Functional analysis and physiological and biochemical index analysis of the banana MaHDAC3 gene. Detailed Implementation

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1. Genome-wide identification and basic characteristics of the banana MaHDACs gene family

[0031] To further elucidate the banana histone deacetylase (HDAC) family, a combined strategy of HMMER3.3 (HMMprofilePF00850) and BLASTP was used to identify 13 non-redundant HDAC genes in the banana (Musaacuminata, DH-Pahangv4) genome, named MaHDAC-1 to MaHDAC-13 (Table 1). These genes are distributed across 8 chromosomes, with the highest enrichment at chr08 (3 genes), followed by chr01, chr02, and chr06 (2 genes each), and chr03, chr04, chr05, and chr10 (1 gene each). No HDAC members were found on chr07, chr09, or chr11, indicating an uneven distribution of this family at the chromosomal level. Gene structure analysis showed that the open reading frames (ORFs) of MaHDAC ranged from 1089 to 2478 bp in length, encoding 362 to 825 amino acids, with an average length of 500 amino acids. The number of exons ranges from 3 to 16, with MaHDAC-11 having the most (16) and MaHDAC-9 the fewest (3). Notably, five members—MaHDAC-4, MaHDAC-6, MaHDAC-8, MaHDAC-11, and MaHDAC-13—have ≥13 exons, suggesting they may generate multiple transcripts through complex alternative splicing, thereby regulating different biological processes. Physicochemical property predictions indicate that the molecular weight (MW) of MaHDAC proteins ranges from 41.05 to 92.37 kDa, with isoelectric points (pI) ranging from 5.16 to 8.57, and they are generally acidic (12 / 13 members have pI < 7.0). Among them, MaHDAC-4 (92.37 kDa) and MaHDAC-6 (76.80 kDa) have significantly higher molecular weights than other members, and their pI values ​​are 5.90 and 5.16, respectively, suggesting that they may function in the negatively charged environment of the cell nucleus. MaHDAC-13 (pI=8.57) is the only basic member, which may have different subcellular localization or mechanisms of action. In summary, the banana MaHDAC gene family exhibits significant diversity in gene number, chromosome distribution, exon-intron structure, and protein physicochemical properties, laying an important foundation for a deeper understanding of its epigenetic regulatory functions in banana growth, development, and stress response.

[0032] Table 1. Genome-wide identification and basic characteristics of the banana MaHDACs gene family

[0033]

[0034] Example 2. Chromosomal distribution characteristics of the banana MaHDACs gene family

[0035] To elucidate the chromosomal distribution of MaHDAC genes in the banana genome, this invention, based on banana reference genome information, plotted the physical locations of 13 MaHDAC gene members on 11 chromosomes. Figure 1 The results showed that the MaHDAC gene family exhibited a discrete distribution, with no obvious clustering observed. However, significant differences in gene density were observed across different chromosomes, suggesting that its expansion process may primarily involve dispersed replication. MaHDAC genes were distributed across eight chromosomes: chr01, chr02, chr03, chr04, chr05, chr06, chr08, and chr10. No members were detected on chr07, chr09, and chr11. Chr08 contained three genes (MaHDAC-10 / -11 / -12), representing a high-density distribution region. Chr01, chr02, and chr06 each carried two members; while chr03, chr04, chr05, and chr10 each contained only one member.

[0036] Example 3. Evolutionary relationship analysis of members of the banana MaHDACs gene family

[0037] To further elucidate the evolutionary origin and taxonomic classification of the banana MaHDACs gene family, this invention constructs a maximum likelihood (mL) phylogenetic tree using 13 full-length MaHDACs proteins as the core, combined with previously reported HDAC members from Arabidopsis and rice. The results show ( Figure 2 Based on the characteristics of typical HDAC domains and interspecies topological relationships, all sequences can be divided into five subfamilies: RPD3 / HDA1-subgroup I, RPD3 / HDA1-subgroup II, RPD3 / HDA1-subgroup III, HD2, and SIR2. Each subfamily exhibits a clear pattern of cross-species orthologous homology and phylogenetic-specific expansion. RPD3 / HDA1-subgroup I contains nine MaHDACs (MaHDAC-1 / 3 / 4 / 5 / 7 / 8 / 10 / 12 / 13), RPD3 / HDA1-subgroup II consists of three MaHDACs (MaHDAC-2 / 6 / 11), and RPD3 / HDA1-subgroup III consists of one MaHDAC (MaHDAC-9). However, no homologous banana MaHDACs were found in HD2 and SIR2, suggesting that bananas may have selectively lost these two subgroups during evolution.

[0038] Example 4. Conserved motifs and gene structure analysis of banana MaHDACs gene family members

[0039] To further elucidate the structural diversity and potential functional differentiation of MaHDACs family members, conserved motif analysis of 13 MaHDACs protein sequences was performed using the MEME tool, and the exon and intron structures of the corresponding genes were visualized. The results showed that ( Figure 3 Ten significantly enriched conserved motifs (conserved motifs 1-10) were identified. All MaHDACs members contained conserved motif 3, while the distribution differences of the remaining motifs showed clear clade specificity, indicating that conserved motif 3 may be a typical DNA domain in the MaHDAC structure. Based on the three major evolutionary subgroups, subgroup I contained more than eight conserved motifs (except MaHDAC13), suggesting the specificity and diversity of gene functions in this subgroup. Subgroups II and III consisted of four to five conserved motifs. Exon-intron structure analysis showed that members of subgroup I had 4-14 exons, indicating significant variation in gene structure. Members of subgroup II generally contained more than eight exons, suggesting that functional diversification may be achieved through alternative splicing or mRNA stability regulation. Members of subgroup III contained nine exons. In summary, the motif and structural diversity of the MaHDAC family reveals its functional diversity, suggesting its important role in banana growth and development and external stress.

[0040] Example 5. Analysis of cis-acting elements in the promoter region of the banana MaHDACs gene family

[0041] To elucidate the potential molecular mechanisms of MaHDACs genes in the regulation of banana response to abiotic stress and development, PlantCARE was used to predict cis-acting elements in 13 representative MaHDACs promoters (2.0 kb upstream of the ATG). The results showed that ( Figure 4 A total of 17 functional elements were annotated. Among them, the light-response, jasmonic acid (JA)-response, and abscisic acid (ABA)-response elements had the highest frequency of occurrence in the promoter region of the entire family, reaching 53%, 13%, and 9%, respectively (total element occurrences 277). Specifically, the three most frequent elements in subgroup I were light-response, JA-response, and ABA-response; in subgroup II, they were light-response, JA-response, and low-temperature response; and in subgroup III, they were light-response, drought-induced, and ABA-response. This indicates that the MaHDACs family may play a key role in circadian rhythms and photomorphogenesis, methyl jasmonic acid and ABA hormone signaling, and temperature response.

[0042] Example 6. Analysis of cis-acting elements in the promoter region of the banana MaHDACs gene family

[0043] To elucidate the role of the MaHDACs gene family in fruit ripening regulation, this invention utilized the TCOD (https: / / ngdc.cncb.ac.cn / tcod / home) online transcriptome database to perform expression heatmap analysis of MaHDACs gene family members under ethylene and ethylene inhibitor treatments. The results showed that ( Figure 5 Under ethylene inhibitor treatment, four MaHDAC genes (MaHDAC-6 / 7 / 10 / 12) were upregulated in both the peel and pulp, indicating that these genes may play a negative regulatory role in banana fruit ripening. Under ethylene treatment, three MaHDAC genes (MaHDAC-3 / 4 / 5) were upregulated in both the peel and pulp, suggesting that these genes may play a positive regulatory role in banana fruit ripening.

[0044] Example 7. Analysis of expression patterns of banana MaHDACs gene family members

[0045] To investigate the role of the MaHDAC gene family in fruit ripening regulation, this invention selected six representative MaHDAC genes (MaHDAC-3 / 4 / 8 / 9 / 10 / 12) and analyzed their expression patterns in banana fruits at 25, 45, 65, and 85 days post-budding using qPCR. The results showed that ( Figure 6 Using 25 days after bud break as a reference, the expression levels of MaHDAC-4 / 8 / 10 / 12 genes all showed an initial increase followed by a decrease at different developmental stages of banana, while MaHDAC-3 / 9 showed an increasing trend. Furthermore, overall, MaHDAC-3 showed an initial decrease followed by an increase in the later stages of development, while MaHDAC-9 showed a decreasing trend in the later stages of development. In conclusion, the diverse expression patterns of the MaHDAC genes during banana fruit development indicate the diversity of their functions in regulating banana fruit ripening.

[0046] Example 8. The MaHDAC3 gene negatively regulates banana fruit ripening by affecting cell wall and ROS content.

[0047] To further explore the specific functions of the MaHDACs gene family in regulating fruit ripening, this invention utilizes VIGS transient silencing technology to transiently silence the MaHDAC3 gene in the "Xiangfen No. 3" banana fruit.

[0048] The CDS sequence of the MaHDAC3 gene is 1515 bp, encoding 504 amino acids, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0049] SEQ ID NO.1:

[0050]

[0051] SEQ ID NO.2:

[0052] MWFYLPVSFDLFSLLFSRHPNPIEPKASPRPARVQRPHWYDDSVVIGGGGCWRRRRGGGVAGVGVVRGRGKAAGELLLRADHRGLLLRAGAPDEAPPYPHGPQPCRPLRPPPPHGGLPPFPASAAD IRRFHSDDYVDFLASVSPATAALASAQAQPLSDPASASASSRQLKRFNVGEDCPVFDGLFEFCQASAGGSIGAAVKINRGDADIAINWAGGLHHAKKCEASGFCYVNDIVLGILELLKYRRVLYVD IDIHHGDGVEEAFFTTDRVMTVSFHKYGDFFPGTGHIKDVGFGQGKYYALNVPLNDGMDDESFRGLFPIIQKVMAVYQPDAVVLQCGADSLAGDRLGCFNLSVKGHADCLRYLRSFNVPMLVLGGG GYTMRNVARCWCYETAVAIGVEPDNQLPYNEYYEYFGPDYDLHIRPRSSMENKNSSKELEDIRNMLMDYLSKIEHAPSVQFQSRPPDTEAPEEEDEDMEHRMQPKLWSGEYYDSDSEECPKDPESVP.

[0053] The steps to silence the carrier are as follows:

[0054] First, the SGN VIGS Tool online software (https: / / vigs.solgenomics.net / ) was used to design a VIGS (virus-induced gene silencing) gene silencing fragment. The designed VIGS silencing fragment sequence is shown in SEQ ID NO.3:

[0055] GCTACTTCTACGAGCCGACCATCGGGGACTACTACGGGCAGGGGCACCCGATGAAGCCCCACCGTATCCGCATGGCCCACAACCTTGTCGTCCACTACGCCCTCCACCGCCTCATGGAGGTCTCCCGCCCTTCCCCGCCTCCGCCGCCGACATCCGCCGCTTCCATTCCGACGACTACGTCGACTTCCTCGCCTCCGTGTCCCCGCCACCGCCGCCCTCGCCTCCGCCCAGGCCCAGCCCTTGT CCGACCCCGCCTCTGCCTCCGCCTCCTCCCGCCAACTCAAGCGCTTCAACGTCGGAGAGGACTGCCCCGTCTTCGATGGCCTCTTCGAGTTCTGTCAGGCCTCCGCCGGCGGCTCCATCGGGGCCGCCGTCAAGATCAACCGCGGCGACGCCGACATCGCCATCAATTGGGCCGGCGGCCTCCACCATGCCAAGAAGTGCGAGGCTTCTGGCTTCTGCTACGTCAACGACATCGTCCTCGGCATCCT.

[0056] Then, the siFi21 software was used to perform off-target specificity detection on the above-mentioned VIGS (virus-induced gene silencing) gene silencing fragment to ensure that the fragment is specific to the target gene.

[0057] (1) VIGS silencing fragment amplification

[0058] Using F / R as forward and reverse primers, and the full-length CDS sequence of the HDAC3 gene cloned by TA as a template, PCR amplification was performed. The amplification primers and amplification programs are shown in Tables 2 and 3, respectively.

[0059] F: AGGTTACCGAATTCTTCTAGAGCTACTTCTACGAGCCGACCA (SEQ ID NO. 4).

[0060] R: GAGACGCGTGAGCTCGGTACCAGGATGCCGAGGACGATGTCG (SEQ ID NO. 5).

[0061] Table 2 PCR amplification system (50 μL)

[0062] 10×mix 5 μL High-fidelity enzymes 1 μL dNTP 1 μL Forward primer (10 μM) 1 μL Reverse primer (10 μM) 1 μL Nucleic acid template 1 μL <![CDATA[ddH2O]]> 40 μL

[0063] Table 3 PCR reaction procedure

[0064]

[0065] (2) Enzyme digestion and ligation

[0066] The amplified product was recovered from the gel and ligated into the TRV2 vector. The 50 μL digestion system was as follows: 2 μg vector, 5 μL 10× buffer, 1.5 μL enzyme, and ddH2O to a final volume of 50 μL. Digestion was performed at 37℃ for 1.5 h, with temperature controlled by the PCR instrument. After digestion, the digestion mixture was recovered, and the amplified fragment was then ligated into the vector.

[0067] The 5 μL ligation system consisted of 1 μL of linearized enzyme digestion vector, 1.5 μL of target fragment, and 2.5 μL of homologous recombinase. Ligation was performed at 25℃ for 30 min, with temperature controlled by a PCR instrument, followed by transformation into E. coli.

[0068] (3) Escherichia coli transformation

[0069] Remove competent E. coli cells (DH5α) from the -80°C freezer, place them on ice, and after they have completely thawed, add the above ligation product, gently tap them with your fingertips to mix, and let them stand on ice for 30 minutes.

[0070] Heat shock at 42°C for 50 seconds, then immediately place on ice and let stand for 2 minutes.

[0071] Add 500 μL of empty LB liquid culture medium to the clean bench and incubate at 37°C and 200 rpm for 1.5 h.

[0072] Take 200 μL of the activated bacterial culture and spread it on an LB agar plate containing the corresponding antibiotic. After the liquid on the surface of the plate has dried, seal the plate and incubate it overnight at 37°C with the plate inverted.

[0073] (4) Colony PCR

[0074] Pick 5-10 single colonies from the above plates in a clean bench and place them in 2.0 centrifuge tubes containing 800 μL of the corresponding antibiotic in LB liquid medium. Incubate at 37°C and 200 rpm for 3 hours.

[0075] In a clean bench, 2 μL of the above bacterial solution was taken and colony PCR was performed according to the PCR reaction system below.

[0076] The 20 μL colony PCR amplification system included: 10 μL of 2×mix, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 2 μL of DNA template, and 6 μL of ddH2O; the PCR procedure was the same as in Table 3. The PCR products were detected by gel electrophoresis to determine the presence of the target band. If present, the products were sent to BGI Genomics for sequencing analysis. The sequencing results were compared with the target fragment nucleic acid sequence. If the sequencing results were completely consistent with the target fragment nucleic acid sequence, the vector construction was successful.

[0077] (5) Agrobacterium strain transformation

[0078] Single colonies of Agrobacterium GV3101 that had been successfully activated and transformed were taken from freshly cultured Agrobacterium with helper plasmids TRV1 and TRV2-MaHDAC3 recombinant plasmids, respectively, and cultured in LB medium (with 100 μg / mL kanamycin) at 28°C and 200 rpm until the culture reached OD. 600 The value is 0.8. Transfer more than 1% of the culture to a 50 mL centrifuge tube containing 30 mL of LB liquid medium (containing 200 μM acetylsalicylic acid and 10 mM Mes buffer), and incubate overnight at 28°C and 180 rpm until OD reaches 0.8. 600 The value should be between 0.8 and 1.0. Transfer 30 mL of the cultured bacterial solution to a 50 mL sterile centrifuge tube, centrifuge at 4000 rpm for 8 min, and discard the supernatant.

[0079] Resuspend the bacterial cells to OD using an appropriate volume of infection buffer working solution (10 mM MgCl2, 10 mM MES, and 200 μM Macetosyringone). 600 =1.2, mix equal volumes of Agrobacterium tumefaciens bacterial suspension containing TRV1 and TRV2-MaHDAC3 recombinant plasmids, and incubate at 28°C, 100 rpm in the dark with shaking for 4-6 h, and then infect banana fruits.

[0080] (6) Infection by recombinant Agrobacterium

[0081] Select bananas that are 50% ripe. Starting from the other end of the banana stem, use a 10mL needle tip to repeatedly clear the channel along the central axis. Then, use a 1mL syringe to inject the inoculation solution. Inject 1-2mL of the inoculation solution according to the size of the "Xiangfen No. 3" banana.

[0082] Each treatment was inoculated with 4 samples in 3 replicates. After inoculation, the banana fruits were cultured overnight in the dark at 22°C, and then transferred to a cycle of 16 hours of light at 25°C and 8 hours of darkness at 25°C. The phenotypic changes of the inoculated banana fruits were observed continuously once a day. The phenotype was observed after about 2-3 weeks, and the TRV2 empty vector was used as a control.

[0083] (7) RT-PCR detection of the MaHDAC3 gene in banana fruit silenced by VIGS

[0084] The silencing efficiency of the MaHDAC3 gene in banana fruits was detected using semi-quantitative RT-PCR. Total RNA was extracted from banana fruits 13 days after inoculation and analyzed by RT-PCR.

[0085] The quantitative primers are shown in SEQ ID NO.6 and SEQ ID NO.7:

[0086] HDAC3-qPCR-F: AGGCATTCTGGCTCTAACGA (SEQ ID NO. 6);

[0087] HDAC3-qPCR-R: GCAGATGAGTTGACCAAGCA (SEQ ID NO. 7).

[0088] The test results showed that the expression level of the MaHDAC3 gene was significantly reduced in infected banana fruits. Figure 7 This indicates that the MaHDAC3 gene in banana fruit is effectively silenced.

[0089] (8) Effect of MaHDAC3 gene silencing on banana fruit softening ( Figure 8 )

[0090] ① Compared with the TRV2 empty vector control, banana fruits exhibited early ripening after 13 days of silencing the MaHDAC3 gene, and the fruit firmness was significantly lower than that of the control, indicating that the degree of degradation of the fruit cell wall was significantly greater than that of the control.

[0091] ② After silencing the MaHDAC3 gene, the chlorophyll content in the pulp and peel was significantly lower than that in the control, which is consistent with the color of the peel.

[0092] ③ After silencing the MaHDAC3 gene, the content of soluble sugar and ROS in the pulp and peel was significantly higher than that in the control. This is consistent with the role of ROS signaling in promoting fruit ripening, and also indicates that the sweetness of the fruit begins to accumulate and the edibility of the fruit increases.

[0093] In summary, MaHDAC3 negatively regulates banana fruit ripening by affecting processes such as banana fruit cell wall degradation, soluble sugar accumulation, and ROS.

[0094] As can be seen from the above embodiments, the present invention provides the MaHDAC3 gene and its application in regulating fruit ripening. Silencing the MaHDAC3 gene can promote banana fruit ripening.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Genes regulating banana fruit ripening MaHDAC3 Its characteristics are, Gene MaHDAC3 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The gene for regulating banana fruit ripening as described in claim 1 MaHDAC3 The encoded protein is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Silencing the gene as described in claim 1 MaHDAC3 Application in promoting banana fruit ripening and / or peel softening.

4. A method for promoting banana ripening, characterized in that, Includes the following steps: (1) Design primers to amplify silent genes MaHDAC3 Specific sequences; (2) Silent genes MaHDAC3 The specific sequence was ligated into the TRV2 plasmid vector to obtain TRV2- MaHDAC3 Recombinant plasmids; (3) Add the helper plasmids TRV1 and TRV2 respectively. MaHDAC3 Recombinant plasmid was introduced into GV3101 Agrobacterium, and the recombinant Agrobacterium was cultured until the bacterial culture reached OD. 600 =0.8 ~ 1.0, centrifuge and discard the supernatant; (4) Resuspend the bacterial cells in the infection buffer until OD. 600 =1.2, containing TRV1 and TRV2- MaHDAC3 Mix equal volumes of Agrobacterium tumefaciens bacterial suspension containing recombinant plasmids and incubate for 4-6 hours to obtain infected bacterial suspension; (5) Inject the banana fruit with the infecting bacterial solution and culture it in the dark at 21~23℃ for 8~10h, and then culture it at 24~26℃ under the conditions of 16h light / 8h dark. Gene MaHDAC3 The nucleotide sequence is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that, The sequences of the primers are shown in SEQ ID NO.4 and SEQ ID NO.

5.

6. The method according to claim 4, characterized in that, The silent gene MaHDAC3 The specific sequence is shown in SEQ ID NO.

3.

7. The method according to claim 4, characterized in that, The culture conditions for recombinant Agrobacterium in step (4) are: 27~29℃, 180~200rpm.

8. The method according to claim 4, characterized in that, The infection buffer contains 10 mM MgCl2, 10 mM MMES, and 200 μM acetosyringone.

9. The method according to claim 4, characterized in that, The injection volume of the infecting bacterial solution is 1 to 10 mL.

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