CN116004656B - Banana maturation associated gene MabHLH130 and application thereof - Google Patents

Abstract

The invention discloses a banana ripening related gene MabHLH130 and its application. The nucleotide sequence of the CDS of the gene MabHLH130 is shown in SEQ ID NO: 1; the protein MabHLH130 encoded by the gene MabHLH130 has an amino acid sequence shown in SEQ ID NO: 2. Show. The present invention screened out a functional gene MabHLH130 that plays an important role in regulating post-harvest ripening of pink banana fruits through real-time quantitative PCR technology; the present invention constructed a plant expression vector pCAMBIA1304_MabHLH130, transferred it into Agrobacterium GV3101, and used Agrobacterium infection technology to MabHLH130 from Pink Banana was integrated into the tomato genome. In the independent transgenic lines obtained, experiments have shown that MabHLH130 participates in the biosynthesis of ethylene by regulating the expression of key enzyme genes for ethylene biosynthesis, thereby regulating fruit ripening and promoting the growth of tomato fruits. Postharvest ripening provides genetic resources for genetic improvement of bananas.

Description

Banana ripening related gene MabHLH130 and its application

Technical field

The invention relates to the field of biotechnology, specifically to a banana ripening-related gene MabHLH130 and its application.

Background technique

Banana is an important tropical fruit and plays an important role in world economy and trade. Among them, pink banana (Musa ABBPisang Awak, FJ) belongs to the Musa banana hybrid variety. Compared with Musa acuminate L.AAA groupcv.Cavendish (BX), the flavor and taste are significantly different. It is rich in nutrients, sweet and sour, and its ripening process Faster than Brazilian banana, it is widely welcomed in the international and domestic markets and has broad application prospects.

Therefore, in-depth research on the molecular regulatory mechanism of postharvest ripening of pink banana fruits, excavating the key genes that regulate the rapid ripening of pink banana fruits, and genetic improvement of bananas through biotechnology can further increase the commodity value of the pink banana industry and expand the sustainability of the banana industry. Development is one of the national strategic needs for developing efficient agriculture with tropical characteristics.

At present, no genes related to postharvest ripening of bananas have been found in the banana genome. Therefore, it is urgent to find a meaningful gene that regulates banana fruit ripening, thereby promoting postharvest ripening of bananas and achieving genetic improvement of bananas.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a banana ripening related gene MabHLH130 and its application. The present invention contributes to the excavation of genes related to banana ripening, and also provides genetic resources for production issues related to the development and application of early ripening genes of fruits.

In order to achieve the above object, the present invention designs a banana ripening related gene MabHLH130. The nucleotide sequence of the CDS of the gene MabHLH130 is shown in SEQ ID NO: 1.

The present invention also provides a protein MabHLH130 encoded by the above-mentioned gene MabHLH130, the amino acid sequence of which is shown in SEQ ID NO: 2.

The present invention also provides a primer pair for obtaining the above-mentioned maturity-related gene MabHLH130, and the primer pair is:

Forward primer F: 5′-ATGTACGGTCTCCTTCGGGACGGC-3′, as shown in SEQ ID NO: 3;

Reverse primer R: 5′-GTAGGGCTTCTGCCTGCCAGCTGAAC-3′, as shown in SEQ ID NO: 4.

The invention also provides a method for obtaining the maturity-related gene MabHLH130 using the above primer pair, which includes the following steps:

Using the cDNA of Brazilian banana fruit as a template and the primer pair of the above-mentioned ripening-related gene MabHLH130, PCR amplification was performed, and the ripening-related gene MabHLH130 was purified.

The present invention also provides a recombinant expression vector, which contains a plant expression vector of the above-mentioned maturity-related gene MabHLH130, wherein the plant expression vector is pCAMBIA-1304.

The above-mentioned expression vector can use commonly used vectors in the field of gene recombination, such as viruses, plasmids, etc.; the present invention is not limited to this.

The invention also provides a method for constructing the above-mentioned recombinant expression vector, which includes the following steps:

Introduce the target fragment into the cloning vector 18T, and then clone the cloning vector MabHLH130-/> containing the target gene. 18T and pCAMBIA-1304 were digested with NcoⅠ and SpeⅠ double enzyme digestion, and then the obtained target fragment was connected to the digested pCAMBIA-1304 vector to obtain the recombinant expression vector pCAMBIA-1304-MabHLH130.

The invention also provides a host cell containing the above recombinant expression vector, and the host cell is Agrobacterium GV3101.

The application of one of the following items in regulating ethylene biosynthesis in fruit ripening, including:

(1) The above-mentioned maturation-related gene MabHLH130;

(2) The above-mentioned recombinant expression vector;

(3) The above-mentioned host cell.

As a preferred version, the plant is tomato or banana.

The application of one of the following items in cultivating new varieties with easy-to-ripe fruits, including:

(1) The above-mentioned maturation-related gene MabHLH130;

(2) The above-mentioned recombinant expression vector;

(3) The above-mentioned host cell.

The above new varieties are tomatoes or bananas;

Take tomato as an example; the Agrobacterium bacterial liquid of the pCAMBIA-1304 vector plasmid (pCAMBIA-1304-MabHLH130) containing the banana ripening related gene MabHLH130 is activated, infects the tomato leaves, and the explants after co-culture are placed in bud induction culture Subculture was carried out in the base, and then rooting culture was carried out to obtain transgenic tomato plants with MabHLH130 gene.

Principle of the invention:

The present invention screened out a functional gene MabHLH130 through real-time quantitative PCR technology, which plays an important role in regulating the post-harvest ripening of pink banana fruits, and belongs to the bHLH protein gene family member.

Basic helix-loop-helix (bHLH) proteins are the most widespread type of transcription factors in eukaryotes and regulate gene expression through interactions with specific motifs in target genes. bHLH transcription factors are not only involved in plant growth and metabolism, photomorphogenesis, light signal transduction and response to drought, salt and cold stress, but also play an important role in the regulation of life processes such as the development of plant reproductive organs and secondary metabolism. effect.

The bHLH transcription factor contains a highly conserved basic/helix-loop-helix domain, which is composed of functionally completely different basic amino acid regions and helix-loop-helix regions. The basic amino acid region binds to DNA cis elements. It can regulate gene transcription, and helix-loop-helix can form homo- or heterodimers and interact with other proteins. The latest research on model plants Arabidopsis, rice and other plants shows that bHLH plays an important role in the regulation of plant reproductive organ development (regulation of flowering, stamens, pollen, seed development) and the biosynthesis of metabolite anthocyanins. effect. These bHLH domain-containing proteins typically regulate the expression of their target genes as homo- or heterodimers.

The MabHLH130 sequence was Blast analyzed in the Arabidopsis genome (https://www.arabidopsis.org/cgi-bin/Blast/TAIRblast.pl) and found that it is consistent with AT1G51140.1 (AKS1, CFLAP1, FBH3) of Arabidopsis thaliana. , AT2G42280.1 (FBH4 and AKS3), AT2G42280.3 (FBH4 and AKS3), AT4G09180.1 (FBH2) have high homology. FLOWERING BHLH1 (FBH1), FBH2, FBH3 and FBH4 were first discovered as transcriptional activators of the CONSTANS (CO) gene. They activate the transcriptional expression of CO by binding to the E-box cis element in the CO promoter, leading to early flowering. SnRK1 kinase can directly phosphorylate FBH4, and Arabidopsis JMJ28 interacts with FBH transcription factors to remove the inhibitory effect of H3K9me2 from the CO locus on CO transcription, thereby activating CO transcription expression. The homologues of Arabidopsis FBH (FLOWERING BHLH) in sugarcane, ScFBH1, ScFBH2 and ScFBH3, bind to the ScACS2 (Sugacane ACS2) promoter to activate its transcription and regulate ethylene biosynthesis.

The present invention verifies that the expression level of gene MabHLH130 gradually increases at different stages of fruit development of transgenic tomato plants with MabHLH130 gene. It was further verified that the expression levels of the key genes for ethylene synthesis SIACS2, SIACS4, SIACO1 and SIACO3 in different lines transfected with the MabHLH130 gene at different stages of fruit development and maturity were significantly increased or even significantly higher compared with the wild-type control. At the same time, it was found that ethylene was The release amount of tomato fruits with MabHLH130 gene at different stages of postharvest ripening was significantly higher than that of non-transgenic (wild-type) tomato fruits. This indicates that MabHLH130 regulates ethylene biosynthesis in tomato fruits by regulating the expression of ethylene biosynthesis-related genes during tomato fruit development.

Beneficial effects of the present invention:

The present invention screened out a functional gene MabHLH130 that plays an important role in regulating post-harvest ripening of pink banana fruits through real-time quantitative PCR technology; the present invention constructed a plant expression vector pCAMBIA1304_MabHLH130, transferred it into Agrobacterium GV3101, and used Agrobacterium infection technology to The 14-3-3 protein gene MabHLH130 of pink banana is integrated into the tomato genome to regulate banana fruit ripening, promote banana post-harvest ripening, and achieve genetic improvement of bananas.

Description of the drawings

Figure 1 shows the results of amplification electrophoresis of gene MabHLH130;

In the figure, M is DL2000 DNA Marker, and lane 1 is the gene MabHLH130 PCR product;

Figure 2 shows the identification map of pCAMBIA-1304-MabHLH130 vector;

In the figure, M is DL2000 DNA Marker, lane 1 is the pCAMBIA-1304-MabHLH130 vector enzyme digestion identification electrophoresis pattern, and lane 2 is the gene MabHLH130 PCR identification pattern;

Figure 3 shows the expression of gene MabHLH130 in pink banana fruits at 0, 2, 3, 4, 5 and 6 days after harvest;

Figure 4 is a diagram of the genetic transformation process of the ornamental tomato variety "Micro-Tom" transformed by the leaf disk method in the present invention.

In the figure, A, B and C from left to right are the induction of callus, the screening of effective callus and the differentiation of callus respectively;

Figure 5 shows the PCR detection results of different strains of tomato plants overexpressing the MabHLH130 gene;

In the figure, 1-11 are the PCR amplification results of leaf DNA of OE1-11 respectively, 12 is the positive control, and 14, 15 and 16 are the negative control, DNA extraction blank control and ddH ₂ O control respectively.

Figure 6 is a graph showing the relative expression levels of exogenous gene MabHLH130 in different lines of MabHLH130 overexpressing plants.

In the figure, OE1, OE3 and OE6 are three independent transgenic lines selected.

Figure 7 shows the relative expression levels of the exogenous gene MabHLH130 in the over-expressing MabHLH130 gene lines OE1, OE3 and OE6. The development and maturity stages are 25 days after flowering, green ripening stage, color change stage and red ripening stage fruits respectively;

Figure 8 shows the relative expression level of the endogenous gene SIACS2 in the overexpressing MabHLH130 gene OE1, OE3, OE6 and WT lines, and the development and maturity stages are 25 days after flowering, green ripening stage, color turning stage and red ripening stage fruits respectively. picture;

Figure 9 shows the relative expression level of the endogenous gene SIACS4 in the overexpressing MabHLH130 gene OE1, OE3, OE6 and WT lines, and the development and maturity stages are 25 days after flowering, green ripening stage, color change stage and red ripening stage fruits respectively. picture;

Figure 10 shows the relative expression level of the endogenous gene SIACO1 in the overexpressing MabHLH130 gene OE1, OE3, OE6 and WT lines, and the developmental maturity is 25 days after flowering, green maturity stage, color change stage and red maturity stage fruits respectively. picture;

Figure 11 shows the relative expression of the endogenous gene SIACO3 in the overexpressing MabHLH130 gene OE1, OE3, OE6 and WT lines, and the developmental maturity is 25 days after flowering, green maturity stage, color change stage and red maturity stage fruits respectively. picture;

Figure 12 shows the ethylene release in green-ripened fruits of the overexpressing MabHLH130 gene OE1, OE3, OE6 and WT lines at different postharvest stages.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments to facilitate understanding by those skilled in the art.

Example 1 Banana ripening related gene MabHLH130

Using pink banana fruit cDNA as a template, use the following primer pairs:

5′-ATGTACGGTCTCCTTCGGGACGGC-3′,

5′-GTAGGGCTTCTGCCTGCCAGCTGAAC-3′ was amplified by PCR to obtain a fragment with a nucleotide sequence of 1278 bp (Figure 1). After sequencing analysis, the nucleotide sequence of the pink banana ripening-related gene MabHLH130 CDS and the protein MabHLH130 amino acid sequence were obtained, respectively. As shown in SEQ ID NO:1 and SEQ ID NO:2:

ATGTACGGGTCTCCTTCGGGACGGCCGTCGGAAGATCTCAACCTTCCTTACCCTCCCGCCGGTCCGTTCAGTGGACAACGAACAGCGGAAGCCGAATCCGATCTCCTCCGCCGCCAGCATCAGCATCAGCAGCAGCAGATGAGCTCCGGCCTCCTCCGGTATCGGTCAGCCCCGAGCTCGCTGCTCGGCGAAGTCTGCGAGGACTTCCTTTCCGTTAGAGCCTCCAGCCCCGAGACCGAGACATGCTCCGCCCGG TTCTTGGCGCCGGATCTCCGACGAGACCCAGGACGGTCCTTCCGGTGGCGCCGCCACCGCTAGCGGCCAGAGCAGCCCTCACTTTCCACCCCCGCAGCTGCCGCCTTCTGCACAGGAGGTCAAAGAACAGCAGAGCAGAGGATTTACCTCTGCCCCGCAGATGATATTCCGTCCTCAGCAGCAGCAGCGGCAGATGCCGAACCATAGCTCATCGGAGAGCCCGTTCCGGGCCGTCATGGGATCATTGACGATGGAGGCGGC ACAACTCAAGCACGGAGACTTCGGCAGTCCCTCTAATCTCATCAGGCACAGTAGCTCTCCTGCTGGCCTGTTTTCTCACTTGAACGTGGACGAAGGTTATGGTATGAGGAGAGGGACGAGTGGCTTCATGATGGATGCAACAGATAGATCAAAGGGTCAGATAAGCTTCTCGCCGAGGCAAAACTCCGTGATGTCGCAGATCTCCGAGATGGAAAGCGACGACATGGATGGGAGTAGCAGCCCCGGGACGGCGGAGGC CGGCCGGAGTTACATACCGGGATTCCCGGTTGGCTCTTGGGACGATTCCTCTCCCTTCAACAACAACAGCTTGTCAGGTCTAAAGGGAAGCAGAGACGGCGAGGAGAAGATGGTCACAGGTCTCAGTCCACTGGAGCTGCCACAGAATGGAGAGGTGAGGAACCATGGGTTGGGTCTGTTTAGCTTGCCGAGGTCTACGTCGGAGATAGCCACGATAGAAAAGTTCCTACAGTTCCATGACGGCAGTCCCCTGCAAGATCAG AGCTAAACGAGGCTGCGCCACTCACCCGCGAAGCATAGCCGAGAGGGTAAGGAGGACTAGAATTAGCGAGAGGATGAAGAAGCTCCAGGAGCTTGTTCCTAACATGGATAAGCAAACCAACACAGCAGACATGCTAGATTTGGCAGTGGATTATATCAAGGATCTCCAGAAACAGGTGAAGGCATTATCGGA

AAGCCGGGCGAGCTGCAGCTGTTCAGCTGGCAGGCAGAAGCCCTAC

MYGSPSGRPSEDLNLPYPPAGPFSGQRTAEAESDLLRRQHQHQQQQMSSGLLRYRSAPSSLLGEVCEDFLSVRASSPETETMLARFLAPDLRDETQDGPSGGAATASGQSSPHFPPPQLPPSAQEVKEQQSRGFTSAPQMIFRPQQQQRQMPNHSSSESPFRAVMGSLTMEAAQLKHGDFGSSSNLIRHSSSPAGLFSHLNVDEGYGMRRGTSGFMMDATDRSK GQISFSPRQNSVMSQISEMESDDMDGSSSPKDGGGGRSYIPGFPVGSWDDSPFNNNSLSGLKGSRDGEEKMVTGLSPLELPQNGEVRNHGLGLFSLPRSTSEIATIEKFLQFHDAVPCKIRAKRGCATHPRSIAERVRRTRISERMKKLQELVPNMDKQTNTADMLDLAVDYIKDLQKQVKALSESRASCSCSAGRQKPY

Example 2 Relative expression analysis of banana ripening-related gene MabHLH130 at different postharvest stages of pink banana fruits

Eight-ripe pink banana fruits (Musa ABB group, cv Pisang Awak, FJ) were collected in bunches from the experimental base, returned to the laboratory and combed, rinsed with tap water, dried, and treated with 0.1% sodium hypochlorite solution at night. Soak for surface disinfection for 10 minutes, then place in a well-ventilated 25°C culture room (200 μmol·m-2·s-1 light conditions, 16h light/8h dark, 70% relative humidity and 25°C) to dry, without treatment, take Fruits on days 0, 2, 3, 4, 5 and 6 were quick-frozen in liquid nitrogen and stored in a -80°C refrigerator for later use.

Total RNA from pink bananas at different postharvest periods was extracted according to the instructions of the RNAprep Pure Plant Kit (DP441, Tiangen, China). The extracted total RNA was tested for band clarity and integrity through 1.2% agarose gel electrophoresis, and Agilent2100Bioanalyzer (Agilent RNA 6000Nano Kit) was used to detect the concentration, RIN value, 28S/18S and fragment size of the total RNA, and the purity of the sample. Use UV spectrophotometer NanoDropTM for detection. cDNA synthesis was performed according to the instructions of Promega's M-MLV Transcriptase reverse transcription kit. qRT-PCR analysis was performed using the first strand of cDNA from normal fruits at 0, 2, 3, 4, 5 and 6 days after harvest as a template. Primer sequence:

MabHLH130-F is 5′-TAGCCGAGAGGGTAAGGAGG-3′, and MabHLH130-R is 5′-CCGGCTTCCGATAATGCCT-3′.

The qRT-PCR reaction system is: SYBR Premix Ex Taq (2×) (Takara) 12.5 μL, Rox referenceDyeⅡ (50×) (TAKARA) 0.5 μL, 10 μmol/L primer 0.75 μL, cDNA template 1 μL, and then make up to 20 μL with water . Each sample was repeated 3 times.

The reaction program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 5 s, annealing at 60°C for 15 s, and extension at 72°C for 20 s, a total of 40 cycles. After the number of cycles, melting curve analysis was performed at 94-56°C.

The 2-ΔΔΔCT relative quantification method was used to analyze the differences in gene expression. The expression level of MabHLH130 gradually increased from 1 to 4 days after banana harvest, reaching the highest level on day 4, and then gradually decreased. This is closely related to the postharvest ethylene release of pink banana fruits. It initially indicates that MabHLH130 may be involved in the ripening regulation process of pink banana fruits. The results are shown in Figure 3.

Example 3 Construction of recombinant expression vector pCAMBIA-1304-MabHLH130

The 1278bp MabHLH130 nucleotide obtained from the above cloning was connected to the pCAMBIA-1304 vector using homologous recombination. The cloning primers used were:

5′-ATGTACGGTCTCCTTCGGGACGGC-3′,

5′-GTAGGGCTTCTGCCTGCCAGCTGAAC-3′,

The correct clone verified by PCR was further verified and confirmed by sequencing (Figure 2), and the recombinant expression vector pCAMBIA-1304-MabHLH130 was obtained.

Example 4 Transformation of tomatoes with recombinant expression vector pCAMBIA-1304-MabHLH130

1. Transformation of Agrobacterium with pCAMBIA-1304-MabHLH130 expression vector

Take 100 μl of GV3101 Agrobacterium competent cells, thaw them on ice, add 100 ng of pCAMBIA-1304-MabHLH130 recombinant plasmid, place on ice for 30 minutes, freeze in liquid nitrogen for 5 minutes, bathe in 37°C water for 5 minutes, add 700 μl of YEB liquid medium, Pre-incubate at 28°C, 180rpm for 2-3 hours, pipet 100μl and spread on YEB solid medium plate containing 50mg/L rifampin and kanamycin, incubate at 28°C for 2-3 days, select single colonies, and verify by PCR The correct strain was used for the next experiment. And stored in -80℃ ultra-low temperature refrigerator for later use. The commonly used YEB medium formula for culturing Agrobacterium is 10 g/L tryptone, 1 g/L yeast extract, 0.4 g/L MgSO ₄ ·7H ₂ O, pH 7.0. After dispensing, autoclave at 121°C for 20 minutes.

2. Agrobacterium-mediated genetic transformation of tomatoes

(1) Take a certain amount of "Micro-Tom" tomato seeds, soak them in 75% ethanol for 30 seconds, and then pour them out quickly. Then soak and disinfect with 2% sodium hypochlorite for 10 minutes. After disinfection, wash with sterile water 8-9 times, washing thoroughly each time to completely remove residual disinfectant. After the seeds are washed, they are sown on 1/2MS medium. Cultivate in the dark for 2-3 days. After the seeds germinate, move to 25°C and cultivate under a photoperiod of 16h/8h (light/dark). The cotyledons will be used for transformation before they grow into true leaves.

(2) Take the sterile cotyledons that have not grown true leaves in the above (1), cut off the leaf tips and petioles, cut the remaining cotyledons into ^10mm2 squares, place them on antibiotic-free MS solid medium, 28°C Pre-culture for 2 days.

(3) Activate and expand the pCANBIA1304-MabHLH130-GV3101 strain stored in a -80°C ultra-low temperature refrigerator in YEB medium. The activated bacterial solution was centrifuged at 5000 rpm for 6 minutes, and the precipitate was diluted with sterile water until the OD600 was about 0.6. 0.1% acetosyringone was added to prepare an Agrobacterium infection solution for infecting tomato explants. Use the prepared Agrobacterium infection solution to infect the tomato cotyledon explants in (2) above for 10 minutes. The infected explants were placed on MS solid medium without antibiotics and cultured in the dark at 28°C for 2 days. The co-culture medium is MS+2mg/LZT+0.2mg/L IAA+10mg/L hygromycin.

(4) Take out the explants after 2 days of co-culture, place them in a bud induction medium, culture them under light for 7 days, and then transfer them to a new medium for subculture. The medium was changed every two weeks until the explants were fully developed. The composition of shoot induction medium is MS+2mg/LZT+0.2mg/L IAA.

(5) After the bud induction period, when the explant sprouts are about 2-3cm long, they are transferred to the bud elongation medium and cultured for 3-4 weeks. The composition of shoot elongation medium is MS+0.5mg/LZT+0.2mg/L IAA+10mg/L hygromycin.

(6) When the bud length is about 4-5cm, cut off the callus and transfer the buds to the rooting medium and culture them for 3-4 weeks. The rooting medium composition is 1/2MS+2mg/LIBA+10mg/L hygromycin.

(7) Transfer the seedlings that have vigorous roots and grown to a certain height into water and culture them for about a week. After new roots grow, put them into soil pots and continue culturing. The conversion process is shown in Figure 4.

2. Screening and purification of MabHLH130 transgenic tomatoes

(1) Extract DNA from tomato leaves that grow normally during the soil culture stage, and use gene-specific primers for PCR identification. The PCR identification results are shown in Figure 5.

The positive plants tested were cultured normally and labeled as T1 generation. The PCR primers used to detect MabHLH130 are:

MabHLH130-F is 5′-TAGCCGAGAGGGTAAGGAGG-3′,

MabHLH130-R is 5′-CCGGCTTCCGATAATGCCT-3′.

(2) To obtain homozygous transgenic lines, harvest T1 generation seeds from a single plant and sow them to obtain T2 generation transgenic seedlings.

(3) Extract T2 generation transgenic tomato leaf DNA, take out the gene-isolating plants, harvest T2 generation seeds, and plant the harvested seeds to obtain T3 generation transgenic tomato seedlings.

(4) PCR detection of the isolation of T3 generation transgenic tomato populations. Lines with no genetic segregation in the T3 generation are homozygous transgenic lines. Detection primer sequence:

MabHLH130-F is 5′-TAGCCGAGAGGGTAAGGAGG-3′,

MabHLH130-R is 5′-CCGGCTTCCGATAATGCCT-3′.

(5) qRT-PCR technology was used to detect the relative expression of the MabHLH130 gene in the transgenic lines. The test results showed that the exogenous gene MabHLH130 was expressed in the leaves of MabHLH130OE1-6, among which three lines: MabHLH130OE1, OE3 and MabHLH130OE6 The expression level in is higher as shown in Figure 6.

The primer sequence of qRT-PCR is:

MabHLH130-FP is 5′-TAGCCGAGAGGGTAAGGAGG-3′,

MabHLH130-RP is 5′-CCGGCTTCCGATAATGCCT-3′.

(6) Collect seeds of homozygous transgenic tomato lines for subsequent experiments.

3. Wild-type and MabHLH130 overexpressing tomato plants were sown at the same time in 4:1 nutrient soil and vermiculite, placed in a culture room at 25°C, with a photoperiod of 16h/8h (light/dark), and watered with Hog once a week. Orchid nutrient solution.

4. Analysis of expression levels of exogenous gene MabHLH130 in different transgenic lines

Select 3-4 fruits each in the green ripening stage, color changing stage and red ripening stage of wild type and MabHLH130 overexpressing tomato plants with consistent growth and development. The expression level of the exogenous gene MabHLH130 was measured at different stages of development of overexpressed tomato plants. The results showed that the development stages of the exogenous gene MabHLH130 in the three lines MabHLH130OE1, OE3 and OE6 were 25 days after flowering, the green maturity stage and the color change stage. The expression level gradually increased in fruits at the and red ripening stages, among which the expression level in the MabHLH130OE1 strain was higher, as shown in Figure 7.

The detection primers for MabHLH130 are:

MabHLH130-FP is 5′-TAGCCGAGAGGGTAAGGAGG-3′,

MabHLH130-RP is 5′-CCGGCTTCCGATAATGCCT-3′.

5. Expression analysis of key enzyme genes for ethylene synthesis in different stages of tomato fruit development in MabHLH130 overexpression tomato lines

In the ethylene biosynthetic pathway, ACC synthase and ACC oxidase are rate-limiting enzymes and key enzymes. The expression of rate-limiting enzymes and key enzyme genes determines the amount of ethylene produced. Select 3-4 fruits each of wild type and MabHLH130 overexpression tomato plants with consistent growth and development 25 days after flowering, green ripening stage, color change stage and red ripening stage. The expression levels of genes related to the ethylene synthesis pathway were measured at different stages of development and maturity of wild-type and MabHLH130-overexpressing tomatoes.

The test results are shown in Figures 8, 9, 10 and 11. The expression levels of SIACS2, SIACS4, SIACO1 and SIACO3 at different stages of fruit development of the transgenic tomato lines were significantly or significantly higher than those in wild-type fruits, especially The expression level of the fruit during the veraison period is significantly higher than that of the wild type. It shows that the MabHLH130 gene affects the expression of ethylene synthase gene during tomato fruit development, thereby affecting the ethylene biosynthesis of tomato.

The primer sequences of qRT-PCR used to detect the expression of SIACO1, SIACO3, SIACS2 and SIACS4 genes are:

SlACS2-F: 5-TGTTAGCGTATGTATTGACAACTGG-3;

SlACS2-R: 5-TCATAACATAACTTCACTTTTGCATTC-3;

SlACS4-F: 5-CTCCTCAAATGGGGAGTACG-3;

SlACS4-R: 5-TTTTGTTTGCTCGCACTACG-3;

SlACO3-F: 5-CTCCCATGCGCCACTCTATT-3;

SlACO3-R: 5-AGATCACCGCGTCATTTCCT-3;

SlACO1-F: 5-GCCAAAGAGCCAAGATTTGA-3;

SlACO1-R: 5-TTTTTAATTGAATTGGGATCTAAGC-3;

6. Analysis of ethylene release in different postharvest periods of different lines of MabHLH130 overexpressing tomato plants and wild-type controls

The ethylene content of transgenic and wild-type tomato fruits overexpressing MabHLH130 were measured at different postharvest periods using gas chromatography direct injection method: Tomato fruits of the same strain were grouped, and the fruits in the green ripening stage were picked at the same time. Weighing and measuring volumes. The selection criteria for each component are: 25-30 fruits; 90-100g total fruit volume; 100-110ml total fruit volume. After harvest, tomato fruits are placed in an artificial culture room with ventilation at 25°C; 16 hours of light/8 hours of darkness; and a relative humidity of 70%. Ethylene release was measured at different periods including 0d, 2d, 4d, 6d, 8d, 12d and 14d after harvest. Before ethylene measurement, fruits from different groups were placed in 350 ml stuffy bottles, and the lid outlet was closed with a rubber hose for three hours. A gas chromatograph (Agilent, USA) was used to measure ethylene release. Before collecting gas samples , shake the sealed tank gently, push the sampling air-tight needle (Agilent 1mL air-tight needle) to the bottom, push and pull the needle bar 4 times repeatedly, insert the needle into the rubber hose, and take samples deep into the tank. The chromatographic conditions were Agilent 7890B gas chromatograph. The chromatographic column model is: HP-5 (capillary column) 30.0m×0.32mm×0.25μm. The measurement conditions are as follows: injection port 230.0℃, column temperature 60℃, lasting 5min; hydrogen ion flame detector (FID) temperature 250℃; carrier gas N ₂ , flow rate 1.6mL/min; gas H ₂ , flow rate 30mL/min; air Flow rate 400mL/min; flow rate: 1.6mL/min; split ratio 2:1; makeup gas N2 flow rate 10mL/min; peak time: 3.38min.

The external standard method is used to qualitatively prepare the standard curve based on the peak time of the standard sample and the quantitative peak area. Gas chromatography is used to measure ethylene standard samples of different concentrations. The ratio of the peak area to the injection volume is used to draw the ethylene standard curve. Through the ethylene standard curve and the size of the sample peak area, the ethylene release amount of the fruits at different postharvest periods was calculated. The release amounts of MabHLH130 overexpressed and wild-type control tomato fruits at different postharvest periods are shown in Figure 12.

The results show that in MabHLH130OE1, OE3, OE6 and the wild-type control group, the ethylene release amount of the fruits at different stages after harvest is higher than that of the wild-type control group, especially the ethylene release amount of the fruits of MabHLH130OE1 and MabHLH130OE3 lines at various stages after harvest. Higher or extremely significantly higher than the wild type in the control group. It shows that MabHLH130 can promote fruit ripening by affecting the biosynthesis of ethylene in fruits.

Other parts not described in detail are existing technologies. Although the above embodiments describe the present invention in detail, they are only part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without any inventive step. These embodiments All belong to the protection scope of the present invention.

Claims (7)

1. A banana maturation associated gene MabHLH130, characterized in that: the nucleotide sequence of the gene MabHLH130CDS is shown as SEQ ID NO. 1.

2. A protein MabHLH130 encoded by the gene MabHLH130 as claimed in claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.

3. A recombinant expression vector, characterized in that: the recombinant expression vector contains the plant expression vector of the maturation associated gene MabHLH130 of claim 1, wherein the plant expression vector is pCAMBIA-1304.

4. A method of constructing the recombinant expression vector of claim 3, wherein: the method comprises the following steps:

introduction of the fragment of interest into cloning vectorThen, cloning vector containing target gene is carried outAnd pCAMBIA-1304 is digested with NcoI and SpeI, and the obtained target fragment is connected to the digested pCAMBIA-1304 vector to obtain recombinant expression vector pCAMBIA-1304-MabHLH130.

5. A host cell comprising the recombinant expression vector of claim 4, wherein: the host cell is Agrobacterium GV3101.

6. The use of one of the following in regulating the biosynthesis of mature ethylene in tomato fruits, characterized by: it comprises the following steps:

(1) The maturation associated gene MabHLH130 of claim 1;

(2) The recombinant expression vector of claim 3;

(3) The host cell of claim 5.

7. The application of one of the following in cultivating new variety of tomato fruits easy to mature is characterized in that: it comprises the following steps:

(1) The maturation associated gene MabHLH130 of claim 1;

(2) The recombinant expression vector of claim 3;

(3) The host cell of claim 5.