MiERF5 gene and application of protein of MiERF5 gene in regulation and control of maturing and coloring of mangoes

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to an application of a MiERF5 gene and a protein thereof in regulating and controlling maturing and coloring of mangoes. The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the gene is a mango ethylene response gene and can regulate and control mango coloring. Experiments prove that the gene is a positive regulation factor responding to an ethylene signal and has a positive regulation effect on a mango coloring key gene MiPAL1; the MiERF5 gene is over-expressed in mango fruits, along with the increase of the expression quantity of the MiERF5 gene, the expression of MiPAL1 is also up-regulated, the enzyme activity of PAL is improved, the content of anthocyanin on fruit peels is also increased, and the color of the fruit peels is deepened, so that the regulation and control of the MiERF5 gene on the coloring of mangoes are realized. The invention lays a theoretical foundation for researching a mango coloring mechanism, also provides a gene resource for cultivating a new mango variety, and has a relatively good potential application value.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to the application of MiERF5 gene and protein thereof in regulating the ripening and color change of mango. Background Art

[0002] Mango (Mangifera indica L.), a globally important tropical fruit, is widely cultivated in South my country (including Hainan, Guangdong, and Guangxi), making my country the world's second-largest mango producer. However, mango is a typical respiratory climacteric fruit, rapidly softening and deteriorating during postharvest ripening, resulting in a short storage period and limited shelf life. To meet the needs of long-distance transportation, mangoes are usually harvested immature (green-ripe) at this stage. At this time, the fruit has a hard texture, a green color, a distinct astringency, and a low commercial value. Therefore, postharvest artificial ripening has become a key link in the mango industry chain. By precisely controlling the ripening process, it can promote fruit color change, softening, and the synthesis of flavor compounds, thereby enhancing the consumer experience and market value. Currently, exogenous ethylene treatment such as ethephon is the mainstream ripening method, but its mechanism of action is still unclear and can easily lead to uneven ripening or quality fluctuations. In-depth analysis of the molecular regulatory network of ripening is urgently needed.

[0003] In the fruit ripening regulatory mechanism, transcription factors, especially the ERF (Ethylene Response Factor) family, have been proven to be the core hub of the ethylene signaling pathway. ERF proteins are widely involved in ethylene-mediated physiological processes, including stress response, organ development, fruit ripening and pigment metabolism, by binding to the GCC-box elements of target gene promoters. Studies in model fruits such as tomatoes and bananas have shown that specific ERF members can positively or negatively regulate ripening-related pathways such as carotenoid synthesis and cell wall degradation enzyme expression. However, current research on the molecular mechanism of mango ripening is mostly focused on the physiological and biochemical levels (such as pigment content and changes in enzyme activity), and there is a serious lack of understanding of the transcriptional regulatory network that forms key qualities such as color change, especially the lack of systematic verification of the functions of key transcription factors such as ERF.

[0004] While the release of mango genome data has provided a foundation for gene function research, the core genes regulating fruit coloration and their mechanisms of action remain largely unknown. Existing technologies struggle to precisely control the color change process of mangoes, resulting in unstable postharvest ripening results and poor product consistency. Therefore, analyzing the molecular pathways by which mango ERF transcription factors regulate pigment synthesis and identifying key target genes is not only scientifically significant for clarifying fruit ripening theory but also provides new strategies for developing gene editing, molecular marker-assisted breeding, or targeted ripening technologies, ultimately breaking through industry bottlenecks. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the application of MiERF5 gene and protein thereof in regulating the ripening and color change of mango.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A first aspect of the present invention is to provide a gene for regulating the color change of mango ripening. The gene is the MiERF5 gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] The second aspect of the present invention is to provide a protein encoded by the gene for regulating mango ripening and color change according to the first aspect of the present invention, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The third aspect of the present invention is to provide specific primers for amplifying the MiERF5 gene, including a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4.

[0010] The fourth aspect of the present invention is to provide a kit comprising the specific primers described in the third aspect of the present invention.

[0011] The fifth aspect of the present invention provides the use of the MiERF5 gene described in the first aspect of the present invention, the specific primer described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in regulating the ripening and color change of mangoes.

[0012] As a preferred technical solution, the MiERF5 gene affects the coloring of mango peel by regulating the coloring gene MiPAL1.

[0013] As a preferred technical solution, the MiERF5 gene is overexpressed, the expression of the coloring gene MiPAL1 is upregulated, the PAL enzyme activity is increased, and the color of the mango peel is deepened.

[0014] The present invention has the following advantages: It provides a mango ethylene-responsive gene, MiERF5, which can regulate mango coloration. By treating commercially mature mangoes with ethylene and analyzing the expression of MiERF5, it was confirmed that the gene is a positive regulator of ethylene signals. Yeast one-hybrid and dual-luciferase assays further confirmed that MiERF5 positively regulates MiPAL1, a key gene for mango coloration. Finally, overexpressing the MiERF5 gene in mango fruit revealed that increased MiERF5 expression also upregulated MiPAL1 expression, increasing PAL enzyme activity and anthocyanin content in the peel, leading to a darker peel color. This demonstrates the regulation of mango coloration by the MiERF5 gene. The MiERF5 gene obtained in this invention lays a theoretical foundation for studying the mechanism of mango color change and provides a genetic resource for breeding new mango varieties, demonstrating its potential application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the transcriptional expression analysis diagram of MiERF5 gene after ethylene treatment.

[0016] Figure 2 This is a diagram of the analysis of cis-acting elements of the MiERF5 gene.

[0017] Figure 3 Figure 2 is the plasmid map of the pGADT7 vector and the pHis2 vector, wherein Figure a is the plasmid map of the pGADT7 vector and Figure b is the plasmid map of the pHis2 vector.

[0018] Figure 4 This is the yeast one-hybrid verification result.

[0019] Figure 5 Figure 2 is the plasmid map of pGreen II 62-SK vector and p Green II 0800 vector, wherein Figure a is the plasmid map of pGreen II 62-SK vector and Figure b is the plasmid map of p Green II 0800 vector.

[0020] Figure 6 The results were verified by dual luciferase assay.

[0021] Figure 7 The expression of MiERF5 and MiPAL1 genes in the peel of MiERF5 gene-overexpressing mangoes and control mangoes.

[0022] Figure 8 This is a diagram showing the color changes in mango peel between MiERF5 gene-overexpressing mangoes and the control.

[0023] Figure 9 These are the results of anthocyanin content and PAL activity determination in the peel of mangoes with MiERF5 gene overexpression and the control mangoes. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments. The protection scope of the present invention is not limited to the following:

[0025] The experimental materials and reagents used in the present invention, unless otherwise specified, are all consumables and reagents that can be obtained from conventional commercial channels. The mango materials used were all provided by the Mango Planting Industrial Park in Yazhou District, Sanya City, Hainan Province.

[0026] Example 1: Detection of MiERF5 gene transcription expression in mango after ethylene treatment

[0027] The mango MiERF5 gene was discovered by the inventors through transcriptome analysis of postharvest mango fruit during ripening. Transcriptome sequencing was performed by Wuhan Maiwei Biotechnology Co., Ltd. The treatment used was 500 mg·L⁻¹ ethephon and clean water (CK) for 24 hours. The transcript expression levels of the mango MiERF5 gene after ethylene treatment were as follows: Figure 1 shown.

[0028] from Figure 1 It was found that after ethylene treatment, the transcriptional expression of the mango MiERF5 gene gradually increased in mangoes, reaching a peak on the 18th day. After 6 days of treatment, the expression level of the MiERF5 gene in the ethylene-treated group was significantly higher than that in the control group, especially on the 18th day, when it was five times that of the control group. These results indicate that the mango MiERF5 gene is a potential ethylene-responsive regulatory gene.

[0029] The full-length CDS sequence of the MiERF5 gene is 783 bp in length, and the specific nucleotide sequence is: 5’-ATGGCGTATCAAGACGAAGCTTTTACCTTAGAGTATATCAGCCAGTACCTTCTCAATGA TTGTAATGCTTCCATGGAATCCTTCATCAAAAATTTTAATTTTCAACCCCAACAGCAACTTAAACAAGTGAATTCTCAGACACATCAGACACAGAGTCATCCAACTCTTGTAAAATCTTCGAGTACTTTGAGCCAAAGAAAACCATCTATAAACGTGGCGATTCCTCCTGCGGCAGCCTTTACTTCGACACCCAACAATTACCCAGTTGTGAAAAAAGAAGCAGAACCGGAAGCGGAGAAGGAAAAGCACTACAGAGGAGTGAGGAGAAGGCCATGGGGAAAATACGCGGCGGAAATCCGTGACCCGAATAGAAAAGGGACTCGGGTCTGGCTAGGAACTTTTGACACCGCCATTGAAGCTGCAAAAGCTTATGATAACGCAGCGTTTAGGCTACGTGGAAGCAAAGCCATCTTGAACTTTCCTCTTGAGATTGGGAATTCAAGTTCGAGCCCCGTGGAATCCAAATTACAAGTGAATTTAGGGAAAAAGAGGAAGAACGAAGAGACAGAAGAGGGTCCTATGGAGAGAAAAGTTGTGAAGAAGGAAGAAGCGTACGAAGAATCGACGCCGGCTAACATAATCACAGGGGATCATCCTTTGACGCCGTCAAGTTGGACGGGGTTCTGGGATATTGGTAACGGTAACGGGGTTTTCAGTGTGCCGCCGTTATCTCCGTTATCTCCTCATCCAAGTTTGGGATACCCTCAGCTCCCGGTTATGTGA-3’SEQ ID NO.1 The amino acid sequence of the encoded protein is:

[0030] MESFIKNFNFQPQQQLKQVNSQTHQTQSHPTLVKSSSTLSQRKPSINVAIPPAAAFTSTPNNYPVVKKEAEPEAEKEKHYRGVRRRPWGKYAAEIRDPNRKGTRVWLGTFDTAIEAAKA YDNAAFLRGSKAILNFPLEIGNSSSSPVESKLQVNLGKKRKNEETEEGPMERKVVKKEEAYEESTPANIITGDHPLTPSSWTGFWDIGNGNGVFSVPPLSPLSPHPSLGYPQLPVMSEQ ID NO.2

[0031] Example 2: Analysis of cis-acting elements

[0032] The cis-acting elements of MiERF5 gene were analyzed using the online tool PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). Figure 2 As shown. Figure 2 It can be found that the MiERF5 promoter region contains three ethylene signal response elements (EREs).

[0033] Example 3: Construction and functional analysis of yeast single hybrid vector

[0034] The specific steps and results are as follows:

[0035] (1) The full-length CDS sequence of the target gene MiERF5 was constructed into the pGADT7 vector (the vector was obtained from Nanjing Novozymes Biotechnology Co., Ltd.), with EcoRI and BamHI restriction sites. The promoter region sequence of the gene MiPAL1 was constructed into the pHis2 vector, with EcoRI and XhoI restriction sites. The vector map and insertion site are as follows: Figure 3 shown.

[0036] The promoter sequence of the MiPAL1 gene used is:

[0037]

[0038] (2) The vector plasmid pHis2-MiPAL1pro was transferred into yeast Y187 and cultured in LB solid medium at 28°C for 2 days.

[0039] (3) Single colonies of pHis2-MiPAL1pro were picked and dissolved in 40 μL of water, and then inoculated onto LB solid medium containing different concentrations of 3AT. The culture was carried out at 28°C for 2 days, and the appropriate 3AT concentration for inhibiting promoter self-activation was screened out, which was 10 mM.

[0040] (4) pGADT7-53+pHis-53 (positive control), pGADT7-53+pHis-2 (negative control), and pGADT7-MiERF5+pHis2-MiPAL1pro were co-transfected into yeast Y187, inoculated into two LB solid media containing the corresponding 3AT and without 3AT, cultured at 28°C, and observed and photographed after 3 days. The results are as follows: Figure 4 shown.

[0041] Depend on Figure 4 The results showed that the negative control pGADT7-53+pHis-2 could not grow normally under 10Mm 3AT conditions, while the positive control and pGADT7-MiERF5+pHis2-MiPAL1pro could grow normally, indicating that ERF5 can directly bind to the promoter of the MiPAL1 gene.

[0042] Example 4: Dual luciferase vector construction and functional analysis

[0043] The specific steps and results are as follows:

[0044] (1) The full-length CDS sequence of the target gene MiERF5 was constructed into the pGreen-II 62-SK vector (the vector was obtained from Nanjing Novozymes Biotechnology Co., Ltd.), with the restriction sites of BamHI and EcoRI. The promoter region sequence of the gene MiPAL1 was constructed into the pGreen0800-LUC vector, with the restriction sites of XhoI and BamHI. The vector map and insertion site are as follows Figure 5 shown.

[0045] (2) Transformation of positive clone plasmid into GV3101 Agrobacterium strain: thaw 20 μL of competent Agrobacterium in an ice bath, add 3-5 μL of constructed expression vector plasmid, mix gently and place on ice for 30 min, freeze in liquid nitrogen for 5 min, take out, place in a 37°C water bath for 5 min, then place on ice for 5 min, add 200 μL of antibiotic-free LB liquid culture medium, shake at 28°C, 200 rpm for 4 h, evenly spread 100 μL of bacterial solution on LB solid culture medium containing kanamycin and rifampicin, culture inverted at 28°C for 2-5 days, pick a single colony for bacterial P verification, and then transfer to LB liquid culture medium containing kanamycin and rifampicin for expansion and culture as mother solution.

[0046] (3) Agrobacterium infection: Take 1 ml of the mother liquid and add it to 40 ml of LB liquid medium containing kanamycin and rifampicin, shake for 3-6 hours, measure the OD600 value, and stop when it reaches about 0.7; centrifuge at 5000 rpm for 5 minutes, pour off the supernatant, add 2 / 3 volume of the suspension, measure the OD600 value, and adjust it to 0.8-1 with the suspension, add 200 mM acetosyringone, and let it stand at room temperature in the dark for 3 hours before mixing the promoter and transcription factor bacterial solution in a ratio of 1:9 for infection;

[0047] (4) Use a syringe needle to poke a hole on each side of the main vein of the test tobacco leaf. Use a 1mL syringe to draw an equal amount of the bacterial solution after it has been allowed to stand, and inject the bacterial solution into the hole on the back of the leaf. After 24 hours, according to the Dual- The Reporter Assay System kit uses a single-tube luminescence detector (GLOMAX) to detect firefly luciferase LUC and Renilla luciferase REN. Reporter expression activity = LUC / REN. The results are as follows: Figure 6 shown.

[0048] Figure 6 The results showed that the LUC / REN ratio of the MiERF5+MiPAL1 pro group was twice that of the SK+MiPAL1 pro group, indicating that MiERF5 positively regulates the MiPAL1 gene.

[0049] Example 5: Functional verification of MiERF5 gene overexpression

[0050] The specific steps and results are as follows:

[0051] (1) The plasmid used for the MiERF5 gene overexpression vector was consistent with the expression vector pGreen-II62-SK-MiERF5 in the dual luciferase experiment. The constructed gene overexpression vector was transformed into Agrobacterium GV3101, and the positive clones were picked and added to a 10 mL LB liquid culture medium containing kanamycin and rifampicin, and cultured at 28°C, 200 rpm, and shaken for 48 h.

[0052] (2) Take the bacterial solution and transfer it to LB medium containing kanamycin and rifampicin at a volume ratio of 1:100. Incubate at 28°C and 200 rpm until the OD600 value is approximately 1.0.

[0053] (3) Centrifuge at 5000 rpm for 5 min at room temperature. Discard the supernatant and resuspend the cells in a suspension solution containing a final concentration of 10 mM MES buffer and 10 mM MgCl2. Mix thoroughly and adjust the OD600 value to approximately 0.6-0.7. Then, add acetosyringone to a final concentration of 200 mM and let stand at room temperature for 3 h.

[0054] (4) During the static period, several mango peels were taken out with a 0.5 cm hole puncher, and then immersed in the infection solution for half an hour. The peel discs were taken out and the excess bacterial solution was absorbed with absorbent paper. The peel discs were placed on 1 / 2 MS culture medium and cultured in the dark at 28°C for 6 days. The phenotypic changes of the peel before and after treatment were recorded by taking pictures, and samples were collected for analysis of physiological indicators and related gene expression.

[0055] (5) Gene expression analysis was performed as follows: RNA was extracted using the Tiangen RNA extraction kit (Tiangen, Beijing, China). Reverse transcription was then performed using a cDNA synthesis kit (Thermo Fisher Scientific, USA). qRT-PCR primers were designed using Primer 5 software. The primers for the MiERF5 gene were:

[0056] F:5'-CGACGCCGGCTAACATAATC-3', SEQ ID NO.3

[0057] R:5'-CACATAACCGGGAGCTGAGG-3'; SEQ ID NO.4

[0058] The primers for the MiPAL1 gene are:

[0059] F:5'-TGGCCAAGAAACTCCTGACA-3', SEQ ID NO.5

[0060] R:5'-CTCCTTTCACCATTGGCCA-3'; SEQ ID NO.6

[0061] The ChamQ Universal SYBR qPCR Master Mix (Vaz-yme, China) kit was used according to the manufacturer's instructions. The PCR reaction system included 1 μl of cDNA template, 5 μl of SYBR Mix, 0.4 μl of forward and reverse primers, and 3.25 μl of ddH2O. The amplification program was an initial denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 5 s, for a total of 40 cycles. MiActin was used as an internal control, and each sample was biologically replicated three times. The Ct value was read using 2 -ΔΔCt Method to analyze data.

[0062] The physiological index determination methods are as follows: anthocyanin content was determined by spectrophotometry, and PAL activity was determined by L-phenylalanine method. The above index determinations were repeated 3 times.

[0063] Genetic testing results such as Figure 7 As shown in Figure 3, the expression level of the MiERF5 gene in the peel of overexpressing mango plants was significantly higher than that in the control plants, indicating that the MiERF5 gene overexpression system was successfully established. Compared with the control group, the expression of the pigmentation gene MiPAL1 was activated, indicating that the MiERF5 transcription factor can affect mango peel coloration by regulating MiPAL1.

[0064] Phenotypic observation and physiological and biochemical index determination results are as follows Figure 8 and Figure 9 Compared with the control group, mangoes overexpressing the MiERF5 gene showed darker peel color, increased PAL activity, and increased anthocyanin accumulation.

[0065] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and they are all covered by the scope of protection of the present invention.

Claims

1. A gene for regulating mango ripening and color change, characterized in that: The gene is MiERF5 gene, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the gene for regulating mango ripening and color change according to claim 1, wherein the amino acid sequence is shown in SEQ ID NO.

2.

3. The specific primer for amplifying the MiERF5 gene according to claim 1, characterized in that: It includes a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.

4.

4. A kit, characterized in that The method comprises the specific primer according to claim 3.

5. Use of the MiERF5 gene according to claim 1, the specific primer according to claim 3, or the kit according to claim 4 in regulating the ripening and color change of mangoes.

6. The use according to claim 5, characterized in that The MiERF5 gene affects the coloration of mango peel by regulating the coloration gene MiPAL1.

7. The use according to claim 6, characterized in that Overexpression of the MiERF5 gene upregulated the expression of the coloring gene MiPAL1, increased PAL enzyme activity, and deepened the color of the mango peel.

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