Application of grape VvJOX1 gene in plant growth and fruit ripening

Abstract

The present invention relates to grapes VvJOX1 The application of genes in plant growth and fruit ripening belongs to the field of genetic engineering technology. The present invention analyzes the gene expression profile and methylation profile of 'Kyoho' grape and its early-maturing bud variant 'Fengzao' during fruit development and finds a JOX The gene expression and methylation levels were significantly upregulated during fruit development, and there were differences between 'Feng Zao' and 'Kyoho', suggesting that it may be involved in the development and ripening of grape fruit. VvJOX1 Genes, verified by performance in plant growth or fruit ripening VvJOX1 The role of genes in regulating plant growth and fruit ripening provides genetic resources and molecular pathways for molecular breeding of fruit trees.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to grape VvJOX1 Application of genes in plant growth and fruit ripening. Background Art

[0002] Jasmonic acid (JA) is an important plant hormone that can regulate processes such as seed germination, plant growth, root growth, fruit development, and stress response. JOX The (JASMONATE-INDUCED OXYGENASES) gene encodes jasmonic acid oxidase, which catalyzes the conversion of jasmonic acid into an inactive state and plays an important role in the jasmonic acid signaling pathway. Summary of the Invention

[0003] In view of the above problems, one object of the present invention is to provide a grape VvJOX1 The present invention provides a gene that can regulate plant growth and fruit ripening. VvJOX1 , providing genetic resources and molecular pathways for molecular breeding of fruit trees.

[0004] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0005] Grape VvJOX1 The application of the gene in plant growth and fruit ripening, the coding region of the VvJOX1 gene is 1086 bp in length, the nucleotide sequence is shown in SEQ ID NO: 1; it encodes 361 amino acids, the amino acid sequence is shown in SEQ ID NO: 2.

[0006] A method for regulating plant growth and fruit ripening by regulating VvJOX1 Genes are overexpressed or suppressed in plants to regulate plant growth and fruit ripening;

[0007] described VvJOX1 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2.

[0008] Furthermore, the method is to overexpress VvJOX1 The method further comprises the following steps:

[0009] Step 1: Amplification VvJOX1 Gene: RNA was extracted from leaves of 'Kyoho' grapes, reverse transcribed into cDNA, and amplified using cDNA as a template. VvJOX1 Gene;

[0010] Step 2: Construct the overexpression vector VvJOX1-pHB: VvJOX1 The gene was connected to the pEASY vector, and the ligation product was transformed into Escherichia coli DH5α competent cells. After successful verification, the plasmid was extracted and the extracted plasmid was used as a template for amplification. VvJOX1 The coding region sequence was amplified by homologous recombination VvJOX1 The coding region sequence was connected to the pHB vector, and after successful verification, the overexpression vector VvJOX1-pHB was obtained;

[0011] Step 3: Arabidopsis genetic transformation: The overexpression vector was transformed into Arabidopsis using the floral dipping method and Agrobacterium-mediated genetic transformation method.

[0012] Get VvJOX1 Overexpression transgenic Arabidopsis thaliana T3 generation positive plants, the positive plants VvJOX1 Overexpression of α-glucanase (α-glucanase) slows growth and delays maturation and senescence in Arabidopsis plants.

[0013] Furthermore, the method is to inhibit expression in plants VvJOX1 Genes are used to promote plant growth and fruit ripening. Furthermore, the specific method includes the following steps:

[0014] Step 1: Amplification VvJOX1 Gene: RNA was extracted from leaves of 'Kyoho' grapes, reverse transcribed into cDNA, and amplified using cDNA as a template. VvJOX1 Gene;

[0015] Step 2: Construct the VvJOX1-pIR suppression vector: VvJOX1 The gene was connected to the pEASY vector, and the ligation product was transformed into Escherichia coli DH5α competent cells. After successful verification, the plasmid was extracted and the extracted plasmid was used as a template for amplification. VvJOX1 Coding region sequence, VvJOX1 The coding region sequence was reversely cloned into the pIR vector to obtain the VvJOX1-pIR suppression vector;

[0016] Step 3: Extract the VvJOX1-pIR plasmid. Three weeks before the color change of 'Kyoho' grapes, mix the VvJOX1-pIR plasmid with an equal amount of the IL-60-BS helper plasmid. Use a fine needle to poke a small hole in the stalk of the grape cluster. Use a capillary to absorb the plasmid mixture and insert it into the small hole. Pull out the capillary after the plasmid is completely absorbed by the cluster. Inject once every five days until the fruit changes color.

[0017] Beneficial effects: The present invention analyzes the gene expression and methylation profiles of 'Kyoho' grapes and their early-maturing bud variant 'Fengzao' during fruit development and finds aJOX The gene expression and methylation levels were significantly upregulated during fruit development, and there were differences between 'Fengzao' and 'Jufeng', suggesting that it may be involved in the development and ripening of grape fruit. We cloned this gene from 'Jufeng' grape and named it VvJOX1. Its coding region is 1086 bp long and encodes 361 amino acids. VvJOX1 Overexpression vector was used to transform Arabidopsis thaliana and T3 transgenic plants were obtained. It was found that the transgenic plants grew slower, flowering and seeding were delayed, and leaf senescence was delayed, indicating that overexpression VvJOX1 Genes can delay the maturation of Arabidopsis plants. VvJOX1 The inhibition expression vector was transiently transformed into grape berries and found to inhibit VvJOX1 After the gene is expressed, the fruit matures early. Therefore, the present invention provides a gene that can regulate plant growth and fruit ripening, providing gene resources and molecular approaches for molecular breeding of fruit trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This figure shows the results of differentially expressed and differentially methylated genes screened using transcriptome and methylome analysis. In the figure, F stands for "Feng Zao" and K stands for "Ju Feng." The numbers indicate different developmental stages: "Feng Zao" ranges from F1 to F5, and "Ju Feng" ranges from K1 to K6. "Feng Zao" matures earlier than "Ju Feng" and therefore lacks a developmental stage. The heat map shows gene expression (expressed in FPKM) and gene methylation levels, with red indicating high expression and green indicating low expression.

[0019] Figure 2 yes VvJOX1 Figure 2. Gene cloning results.

[0020] Figure 3 Overexpression VvJOX1 Figure 1 shows the phenotypic results of Arabidopsis thaliana. WT indicates the wild type; VvJOX1-OE indicates the overexpression line; #1 and #2 indicate two lines.

[0021] Figure 4 Inhibition of VvJOX1 The phenotypic results of the gene. In the figure, the control is the pIR empty vector; VvJOX1-RNAi indicates inhibition VvJOX1 Gene. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0023] In the following examples, unless otherwise specified, the materials or reagents used are commercially available, and the methods or operations used are conventional means.

[0024] 1. Materials and Methods

[0025] 1. Materials

[0026] Five-year-old 'Kyoho' and 'Fengzao' grapevines were grown in the vineyards of the Henan University of Science and Technology Experimental Base (Luoyang, Henan). Arabidopsis seeds were stored in the laboratory of the Engineering Research Center for Quality Control of Horticultural Plants at Henan University of Science and Technology and are readily available. All vectors, competent cells, and reagents used were also stored in the laboratory.

[0027] , gene cloning and vector construction

[0028] RNA was extracted from leaves of the 'Kyoho' grape variety using the Plant Polysaccharide and Polyphenol Total RNA Extraction Kit (TIANGEN, Beijing). RNA was then reverse-transcribed into cDNA using the HiScript First Strand cDNA Synthesis Kit (Novozymes, Nanjing). Forward and reverse primers were designed using the gene sequence from the grape reference genome (SEQ ID NO: Vitvi08g00656) (primer sequences are shown in Table 1). Using the cDNA as a template, high-fidelity enzyme cloning was performed. VvJOX1 The gene coding region is full-length, and the reaction system (50 μL) is: 25 μL high-fidelity enzyme, 1 μL cDNA, 2 μL forward primer, 2 μL reverse primer and 20 μL ddH2O. The band size is detected by agarose gel electrophoresis. The target band is recovered with a gel recovery kit (polymerase, Beijing), connected with the pEASY vector, and the ligation product is transformed into Escherichia coli DH5α competent cells. The positive clone bacterial liquid is selected and sent to a sequencing company for sequencing. After successful sequencing, the bacteria are shaken and the plasmid is extracted with a plasmid extraction kit (polymerase, Beijing), and the gene cloning is completed. Primers for connecting to the pHB vector are designed (primer sequences are shown in Table 1), and restriction sites and 15 bp vector sequences are added before the primers. The above method is used to amplify the obtained VvJOX1 The coding region sequence was ligated with the enzyme-digested pHB vector using a commercially available seamless cloning kit using homologous recombination. After ligation, the competent E. coli was transformed according to the above method, sequenced, and the plasmid was extracted. After enzyme digestion, the gel was run to detect whether the bands were correct, which was the constructed overexpression vector VvJOX1-pHB.

[0029] Table 1 Primer sequences

[0030]

[0031] 3. Arabidopsis genetic transformation

[0032] The overexpression vector was transformed into Arabidopsis thaliana using the floral dip method and Agrobacterium-mediated genetic transformation. Wild-type Arabidopsis seedlings were sown in advance until the bolting and flowering stage for transformation. The VvJOX1-pHB plasmid was transformed into competent Agrobacterium tumefaciens GV3101 cells. The bacterial suspension was aspirated and shaken briefly. A 1% agrobacterium fraction was added to 100 mL of antibiotic-containing LB medium and cultured vigorously until the OD600 reached approximately 1.0. The cells were harvested by centrifugation and resuspended to an OD600 of approximately 1.0. Arabidopsis plants were watered thoroughly one day in advance, and all inflorescences were placed upside down in the suspended bacterial suspension for approximately 30 seconds. Seven days later, the transformation was repeated as described above. After 2-3 weeks, when the plants matured, seeds (T0 generation) were collected and placed in paper bags and placed in a desiccator for 7 days. The collected seeds were then cultured and planted as described above. DNA was extracted from leaves to identify transgenic plants. T3 generation seeds from positive plants were harvested for subsequent experiments.

[0033] , instantaneous transformation of grape fruit

[0034] The same method was used to design primers for connecting to the pIR vector (primer sequences are shown in Table 1). VvJOX1 The gene coding region sequence was reverse-cloned into the pIR vector to generate the VvJOX1-pIR inhibitory vector. The VvJOX1-pIR plasmid was extracted using a plasmid extraction kit. Approximately three weeks before veraison of 'Kyoho' grapes, 200 ng of VvJOX1-pIR plasmid (approximately 50 μL) was mixed with an equal amount of the IL-60-BS helper plasmid; the empty vector pIR was used as a control. A small hole was pierced in the stalk of the grape cluster using a fine needle, approximately half the diameter of the stalk. A capillary tube was used to draw up the plasmid mixture and insert it into the small hole until the plasmid was completely absorbed by the cluster, after which the capillary tube was removed. Injections were repeated every five days until the fruit showed a color change phenotype.

[0035] 2. Results and Analysis

[0036] By analyzing transcriptome and methylome data, we found that a gene encoding jasmonate-induced oxygenase1 (JOX) was significantly differentially expressed during berry development between 'Jufeng' and 'Fengzao' grape varieties, with gene expression gradually increasing with berry development ( Figure 1 a), and the methylation level also changed significantly during fruit development ( Figure 1 b), suggesting that it may be involved in the development and ripening of grape berries.

[0037] To further study the function of this gene, we cloned it from 'Kyoho' grape and named it VvJOX1 ( Figure 2 The coding region (CDS) of this gene is 1086 bp long and encodes 361 amino acids. VvJOX1The gene sequence and amino acid sequence are shown below.

[0038] >VvJOX1, CDS sequence (SEQ ID NO: 1)

[0039]

[0040] >VvJOX1, amino acid sequence (SEQ ID NO: 2)

[0041] MKMMNCLQSWPEPIVRVQSLSDSGIHVLPDRYIRHPSDRPSFTPISTQANIPVIDLHSLLAARDARLRQATLDRISGACREWGFFQVVNHGVRPELMKSIRQLWRDFFHLPLEAKQVYANSPATYEGYGSRLGVEKGAKLDWSDYFFLNYLPESARDENKWPTTPESCRELVHEYSKAVVE LCGILMKILSVNLGLEEDHLQNAFGGDDVGACLRVNYYPKCPQPDLTLGISPHSDPGGMTILLPDDDVSGLQVRKGEHWVTVEPIPDALIVNLGDQIQVISNAIYKSVEHRVIVNSIKERVSLAYFYNPKGDLLIEPAKKLVSKDRPASYSAMTFDQYRLFIRQSGLWGKSQVESLKCSQ*

[0042] By building VvJOX1 We successfully obtained the gene overexpression vector and transformed Arabidopsis thaliana. VvJOX1 Overexpression transgenic Arabidopsis T3 generation. By observing its phenotype and wild type, it was found that at 2 weeks old, the transgenic plants were significantly smaller than the wild type ( Figure 3 a). At 4 weeks old, the wild-type plants had already set seeds, while the transgenic plants had only flowered but not set seeds. The plants were shorter than the wild-type plants ( Figure 3 b), fresh weight was significantly lower than that of wild type ( Figure 3 c). At 5 weeks of age, most leaves of wild-type plants have senesced, while leaves of transgenic plants are still green ( Figure 3 d). The above results indicate that overexpression VvJOX1 It slows down the growth of Arabidopsis plants and delays their maturity and senescence.

[0043] To further confirm the function of this gene in grape berries, we used the pIR repression vector to suppress the expression of VvJOX1 The results showed that the expression of genes was inhibited compared with the control (pIR) VvJOX1 The grapes mature significantly earlier after the gene Figure 4 ), further confirming that this gene can regulate grape fruit ripening.

[0044] It should be noted that the above-described embodiments are to be understood as illustrative and not limiting of the scope of protection of the present invention, which is subject to the claims. It will be apparent to those skilled in the art that non-essential improvements and adjustments to the present invention, without departing from the spirit and scope of the present invention, still fall within the scope of protection of the present invention.

Claims

1. Through overexpression VvJOX1 A method for genetically delaying the growth and fruit ripening of Arabidopsis thaliana, characterized by: described VvJOX1 The coding region of the gene is 1086 bp in length, and the nucleotide sequence is shown in SEQ ID NO: 1; it encodes 361 amino acids, and the amino acid sequence is shown in SEQ ID NO:

2.

2. The method according to claim 1, wherein: The specific method includes the following steps: Step 1: Amplification VvJOX1 Gene: RNA was extracted from leaves of 'Kyoho' grapes, reverse transcribed into cDNA, and amplified using cDNA as a template. VvJOX1 Gene; Step 2: Construct the overexpression vector VvJOX1-pHB: VvJOX1 The gene was connected to the pEASY vector, and the ligation product was transformed into Escherichia coli DH5α competent cells. After successful verification, the plasmid was extracted and the extracted plasmid was used as a template for amplification. VvJOX1 The coding region sequence was amplified by homologous recombination VvJOX1 The coding region sequence was connected to the pHB vector, and after successful verification, the overexpression vector VvJOX1-pHB was obtained; Step 3: Arabidopsis genetic transformation: The overexpression vector was transformed into Arabidopsis using the floral dipping method and Agrobacterium-mediated genetic transformation method. Get VvJOX1 Overexpression transgenic Arabidopsis thaliana T3 generation positive plants, the positive plants VvJOX1 Overexpression of α-glucanase (α-glucanase) slows growth and delays maturation and senescence in Arabidopsis plants.

3. By inhibiting expression VvJOX1 A method for genetically promoting grape growth and fruit ripening, characterized by: The coding region of the VvJOX1 gene is 1086 bp in length, and the nucleotide sequence is shown in SEQ ID NO: 1; it encodes 361 amino acids, and the amino acid sequence is shown in SEQ ID NO:

2.

4. The method according to claim 3, wherein: The specific method includes the following steps: Step 1: Amplification VvJOX1 Gene: RNA was extracted from leaves of 'Kyoho' grapes, reverse transcribed into cDNA, and amplified using cDNA as a template. VvJOX1 Gene; Step 2: Construct the VvJOX1-pIR suppression vector: VvJOX1 The gene was connected to the pEASY vector, and the ligation product was transformed into Escherichia coli DH5α competent cells. After successful verification, the plasmid was extracted and the extracted plasmid was used as a template for amplification. VvJOX1 Coding region sequence, VvJOX1 The coding region sequence was reversely cloned into the pIR vector to obtain the VvJOX1-pIR suppression vector; Step 3: Extract the VvJOX1-pIR plasmid. Three weeks before the color change of 'Kyoho' grapes, mix the VvJOX1-pIR plasmid with an equal amount of the IL-60-BS helper plasmid. Use a fine needle to poke a small hole in the stalk of the grape cluster. Use a capillary to absorb the plasmid mixture and insert it into the small hole. Pull out the capillary after the plasmid is completely absorbed by the cluster. Inject once every five days until the fruit changes color.

Images