CmMYC2 gene for regulating and controlling peel color of sweet melon, encoded protein and application of CmMYC2 gene

Abstract

The invention discloses a CmMYC2 gene for regulating and controlling the color of sweet melon peel, an encoded protein and application, and belongs to the technical field of bioengineering breeding. In order to solve the technical problems of long period and high cost in sweet melon peel color identification in the prior art, the invention provides a CmMYC2 gene for regulating and controlling the sweet melon peel color, an encoded protein and application of the CmMYC2 gene in regulating and controlling the sweet melon peel color. The invention identifies and verifies a CmMYC2 gene participating in sweet melon pericarp color regulation, through field phenotypic identification, a BSA-seq technology is utilized to finely locate a gene MELO3C003412.2 for controlling the yellow / green pericarp traits of melons, and the gene MELO3C003412.2 is named as CmMYC2. The invention provides a solid theoretical technology for technicians in the field to screen and identify germplasm resources of different peel colors of muskmelons and molecular marker-assisted selection, and provides a new idea for new variety breeding.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering breeding technology, and specifically relates to a method for regulating the color of melon peel. CmMYC2 Genes, encoded proteins and applications. Background Art

[0002] melon( Cucumis melo L Cantaloupe is an important cash crop of the Cucurbitaceae family (Cucumis genus) and one of the world's top ten fruits. Statistics show that my country is the world's largest producer and consumer of melons, accounting for approximately 50% of the world's total cultivated area and production. It is also the world's largest producer of cantaloupe melons cultivated in greenhouses. As a high-yield cash crop best suited to greenhouse cultivation, cantaloupe plays a vital role in increasing agricultural efficiency. Cultivating high-quality, innovative cantaloupe varieties has far-reaching significance for the development of the melon industry.

[0003] Melon peel color is genetically rich and diverse, and as a key appearance trait, it directly influences consumer choice. However, melon peel color identification requires waiting for the entire fruit development cycle before color trait identification can be performed. This results in a longer single-generation breeding cycle, limiting the number of breeding generations per year and severely delaying the breeding process. Furthermore, the inability to exclude plants with non-target colors during the immature stage results in a continuous consumption of land, water, fertilizer, and management resources for ineffective material, significantly increasing field planting costs and labor input.

[0004] Therefore, those skilled in the art are eager to shorten the breeding cycle, reduce breeding costs, and reduce labor expenditures in melon variety cultivation. Summary of the Invention

[0005] The present invention aims to solve the technical problems of long cycle and high cost in the prior art of identifying the color of melon peel, and provides a method for regulating the color of melon peel. CmMYC2 Genes, encoded proteins and applications.

[0006] One of the objects of the present invention is to provide CmMYC2 Gene or CmMYC2 Application of gene-encoded protein in regulating melon peel color, CmMYC2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CmMYC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0007] In a preferred embodiment of the present invention, the melon peel is yellow or green.

[0008] The second object of the present invention is to provide CmMYC2 Gene or CmMYC2 Application of gene-encoded protein in melon peel color mutation breeding, the CmMYC2The nucleotide sequence of the gene is shown in SEQ ID NO.1; CmMYC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0009] The third object of the present invention is to provide CmMYC2 Gene or CmMYC2 Application of gene-encoded protein in early identification of melon peel color traits, the CmMYC2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CmMYC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0010] The fourth object of the present invention is to provide a method for amplifying CmMYC2 A primer set for a gene, wherein the primer set is: CmMYC2-F-SEQ ID NO.4: 5'-ATCAGCTCCCTTGACCATGT-3'; CmMYC2-R-SEQ ID NO.5: 5'-TTTCCACTCCAAGGCAGTTC-3.

[0011] Beneficial effects of the present invention: This study identifies and validates a gene involved in regulating the color of melon peel. CmMYC2 Gene or CmMYC2 The gene encodes a protein. The present invention uses the yellow-skinned melon inbred line 9316 and the gray-green-skinned melon inbred line 2019-126 as parents to construct an F2 generation segregating population. Through field phenotypic identification, the genetic law of the melon peel color trait is clarified; the BSA-seq technology is used to preliminarily locate the candidate genes that regulate the melon peel traits, and then the molecular marker technology and the screening of recombinant individual plants are used to finely locate the candidate genes. Finally, the gene that controls the yellow / green peel trait of the melon is located as MELO3C003412.2 , named CmMYC2 , which can be specifically expressed as CmMYC2 The gene was expressed at the highest level in the peel of the yellow-skinned melon inbred line 9316, which was significantly higher than the expression level in the peel of the gray-green-skinned melon inbred line 2019-126.

[0012] The present invention provides a method for regulating the color characteristics of melon peel CmMYC2 The discovery of this gene can accurately and quickly identify the melon peel color at the seed or seedling stage; it provides solid theoretical technology for technicians in this field to screen and identify melon germplasm resources with different peel colors, and for molecular marker-assisted selection; it helps to deeply supplement and enrich the "formation mechanism of melon peel color" and provide new ideas for the breeding of new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The phenotypes of the yellow-skinned melon inbred line 9316 and the gray-green-skinned melon inbred line 2019-126 are shown. Figure 2 To control the color of melon peel CmMYC2 Fine positioning map of the gene; A is the Join Map; B is the preliminary positioning map; C is the fine positioning map; Figure 3 To control the color of melon peel CmMYC2 Gene homology comparison analysis diagram; Figure 4 To control the color of melon peel CmMYC2 Statistical graph of gene expression at different stages, with the vertical axis Relative Expression representing the expression level. DETAILED DESCRIPTION

[0014] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0015] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0016] Example 1: Regulating the color of melon peel CmMYC2 Gene mapping and genetic analysis (1) Selection of test materials: The test materials include maternal, paternal, F1 and F2 generation populations; The male parent material is: yellow peel melon inbred line 9316; The female material is: gray-green peel melon inbred line 2019-126; The above-mentioned maternal and paternal materials were provided by Qiqihar Agricultural Technology Extension Center; The F1 generation population is: the yellow-skinned melon inbred line 9316 and the gray-green-skinned melon inbred line 2019-126 are hybridized to obtain the green-skinned F1 generation population; The F2 generation population is: a population of 141 F2 plants obtained by self-pollination of the above-mentioned F1 generation.

[0017] (2) Genetic separation and identification Using the yellow-skinned muskmelon inbred line 9316 and the gray-green-skinned muskmelon inbred line 2019-126 as parents, the green-skinned F1 generation population was obtained by hybridization. The phenotypes of the parents and the F1 generation population are as follows: Figure 1 As shown in the figure, 120 F2 plants were obtained by self-pollination of the F1 generation. The fruit peel color phenotype of the F2 population was identified during the melon ripening period. The identification results showed that in the F2 population, the number of individual plants with yellow peel was 29, and the number of individual plants with green peel was 91. It can be seen that the segregation ratio of fruit peel color was green:yellow = 91:29, which is consistent with the Mendelian genetic segregation ratio of 3:1 (χ 2 >χ 2 0.05=3.841), indicating that the green peel trait is controlled by a dominant single gene.

[0018] (3) By combining whole-genome resequencing of parents with BSA pooling technology of F2 generation population (such as Figure 2 As shown in the figure, the mixed gene pool of extreme individuals in the F2 genetic population was resequenced, and the gene controlling the fruit skin color trait was preliminarily located within a 3.5 Mb segment of chromosome 4 of the melon genome; by developing 12 CAPS (Cleaved Amplified Polymorphic Sequences) markers, 10 dCAPS (Derived Cleaved Amplified Polymorphic Sequences) markers and 1 InDel (Insertion and Deletion) marker within the above 3.5 Mb segment, and using the F2 genetic population, the gene controlling the fruit skin color trait was finely located within a 14.377 Kb segment.

[0019] Based on the melon genome DHL92 v3.6.1, a 14.377 kb region contains a candidate gene, MELO3C003412.2 Therefore, determine MELO3C003412 The gene is a candidate gene with a full length of 2688 bp (the nucleotide sequence is shown in SEQ ID NO.1), only one exon, the exon length is 2688 bp, and the length of the amino acid encoded is 723aa (the amino acid sequence is shown in SEQ ID NO.2).

[0020] In order to further clarify the genes regulating melon peel, the present invention MELO3C003412The candidate gene and its upstream 2000 bp promoter sequence were compared to analyze whether there were no base insertion / deletion or SNP mutation sites on the gene, while there were 4 SNP mutation sites in the upstream 2000 bp promoter sequence. MELO3C003412.2 The full-length gene DNA and upstream promoter sequence were cloned and sequenced for the first generation, wherein the MELO3C003412.2 (CmMYC2) The primer sets for the genes are as follows: CmMYC2-F-SEQ ID NO.4: 5'-ATCAGCTCCCTTGACCATGT-3'; CmMYC2-R-SEQ ID NO.5: 5'-TTTCCACTCCAAGGCAGTTC-3.

[0021] The full-length of 2688 bp was obtained. CmMYC2 gene (nucleotide sequence is shown in SEQ ID NO.1) and 2000bp upstream promoter (nucleotide sequence is shown in SEQ ID NO.3). After comparing the parental sequences, it was found that MELO3C003412 There are indeed no base insertion / deletion or SNP mutation sites in the candidate gene, and there are indeed 4 SNP mutation sites in the upstream promoter.

[0022] Found through sequence comparison of Cucurbitaceae website MELO3C003412.2 The gene is highly homologous to the melon MYC2 transcription factor, e.g. Figure 3 As shown, and according to previous studies, MYC2 transcription factor is found to be involved in regulating signal transduction mediated by plant pigments as a molecular switch. Therefore, the present invention speculates that MELO3C003412.2 The gene is a candidate gene for regulating the color trait of melon peel and is named CmMYC2 .

[0023] Example 2: CmMYC2 Gene or CmMYC2 Application of gene-encoded proteins in regulating melon peel color (1) Extraction of genomic DNA: The peels of the yellow-skinned muskmelon inbred line 9316 and the gray-green-skinned muskmelon inbred line 2019-126 were sampled 15 and 20 days after pollination, respectively. DNA was extracted from watermelon leaves using the modified CTAB method. The specific steps are as follows: The steps for extracting DNA using the modified CTAB method described in the following examples are: S1: Place the sample to be tested in a 2 mL centrifuge tube, bring it back to the laboratory with an ice pack, use a vacuum freeze dryer to dry it, and then grind it into powder using a grinder; S2: Add the ground solution in S1 to the centrifuge tube. 1. Preheat the CTAB solution (add 2% β-mercaptoethanol) in a 65°C water bath for 1 h, shake to ensure full contact between the powder and the liquid, and place in a 65°C water bath for 1 h, mixing up and down 3-4 times during this period; S3: After cooling to room temperature, centrifuge at 4°C and 12,000 r / min for 20 min, transfer the supernatant to a new 2 mL centrifuge tube, and add The mixture (chloroform and isoamyl alcohol were mixed in a volume ratio of 24:1) was shaken thoroughly and centrifuged at 12,000 rpm for 30 min at 4°C. This step was repeated twice. S4: Pipet the supernatant after centrifugation in S3 , placed in a 1.5 mL centrifuge tube, and added Add 4°C pre-cooled anhydrous ethanol, shake thoroughly, and precipitate at 4°C for 1 h; centrifuge at 4°C and 12,000 rpm for 30 min, aspirate the supernatant, and retain the precipitate; S5: Wash the precipitate obtained in S4 2-3 times with 75% ethanol, dry it at room temperature, and add distilled water to make up the volume. , obtain the melon DNA sample to be tested; The DNA quality was checked by 1% agarose gel electrophoresis and its concentration was determined by UV spectrophotometer. .

[0024] (2) Fluorescence quantitative PCR analysis: RNA was extracted from the test samples (peel DNA from yellow-peeled muskmelon inbred line 9316 and gray-green-peeled muskmelon inbred line 2019-126) at 15 and 20 days after pollination using a plant total RNA purification kit (Tiangen, China). cDNA was generated by reverse transcription using a cDNA synthesis kit (Cwbio, China). The nucleotide sequences of the gene-specific primers for the internal reference gene actin are shown in SEQ ID NOs. 6-7. CmMYC2 The nucleotide sequences of the qRT-PCR primers for the genes are shown in SEQ ID NO. 8-9. Real-time quantitative PCR was performed using the StepOnePlus real-time PCR platform (Applied Biosystems, Foster City, CA, USA). Three biological replicates and three technical replicates were set up. The qRT-PCR reaction system was as follows: DNA template was ( )\TB Green Premix Ex Tagll (2x), upstream and downstream primers ( ), ddH2O The total reaction volume is The qRT-PCR amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, and 72°C for 30 s, for 40 cycles; and constant heating from 60°C to 95°C to obtain the melting curve.

[0025] The results are as follows Figure 4 As shown, at different developmental stages (15 and 20 days after pollination) of the yellow-skinned melon inbred line 9316 and the gray-green-skinned melon inbred line 2019-126, CmMYC2 (MELO3C003412.2) The gene expression level was highest in the peel of the yellow-skinned muskmelon inbred line 9316, which was significantly higher than that in the peel of the gray-green-skinned muskmelon inbred line 2019-126. CmMYC2 (MELO3C003412.2) It is a candidate gene controlling melon peel color.

[0026] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. CmMYC2 Gene or CmMYC2 The application of gene-encoded protein in regulating the color of melon peel is characterized in that: described CmMYC2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CmMYC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The melon peel is yellow or green.

3. CmMYC2 Gene or CmMYC2 The application of gene-encoded protein in melon peel color mutation breeding is characterized in that: described CmMYC2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CmMYC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

4. CmMYC2 Gene or CmMYC2 The application of gene-encoded protein in early identification of melon peel color traits is characterized by: described CmMYC2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CmMYC2 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

5. A method for amplification CmMYC2 A primer set for a gene, characterized in that The primer set is: CmMYC2-F-SEQ ID NO.4: 5'-ATCAGCTCCCTTGACCATGT-3'; CmMYC2-R-SEQ ID NO. 5: 5'-TTTCCACTCCAAGGCAGTTC-3.

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