Regulation of balsam pear peel color by mcbai gene
By heterologously overexpressing the McCAB1 gene in white bitter gourd, the color of the bitter gourd peel was significantly regulated, solving the problem of the single color of the bitter gourd peel, realizing diversified peel color breeding, increasing chlorophyll content, and meeting the diversified market demand.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- VEGETABLE RES INST GUANGDONG ACAD OF AGRI SERVICES
- Filing Date
- 2025-10-15
- Publication Date
- 2026-05-12
AI Technical Summary
Current technology has failed to effectively control the color of bitter melon peel, resulting in a limited variety of bitter melons on the market and making it difficult to meet diverse consumer demands.
By heterologously overexpressing the McCAB1 gene derived from green bitter melon B07 in white bitter melon, the pGWB5-McCAB1 vector was constructed, transformed into Agrobacterium, and injected into the pericarp of bitter melon, resulting in a significant greening of the pericarp and an increase in chlorophyll content.
It significantly deepens the color of bitter melon peel, increases chlorophyll content, meets market demand for different peel colors, provides a core target for fruit color regulation, and provides a basis for breeding work.
Smart Images
Figure CN121064306B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant molecular biology and plant genetic engineering technology, specifically involving the regulatory role of the McCAB1 gene on the color of bitter melon peel. Background Technology
[0002] The color of the peel is a prominent agronomical trait of bitter melon (Momordica charantia L.), possessing important quality characteristics and commercial value. Due to differences in peel color, bitter melons exhibit significant differences in taste, nutritional properties, and consumption scenarios, directly leading to a marked differentiation in their commercial value. Currently, green-skinned bitter melons remain the dominant force in the market. Dark green varieties such as Jiangmen Dading and Cui Lü Dading have 30%-40% higher momordicin content than white bitter melons, resulting in a more significant blood sugar-lowering effect, making them suitable for diabetics and health-conscious individuals. Demand is very stable in southern provinces such as Guangdong and Guangxi. Besides market demand advantages, green bitter melons are also widely adaptable to cultivation, possess strong heat resistance, and exhibit stable yields. Currently, its mainstream production areas have established mature and comprehensive supply chain systems, effectively supporting market circulation needs. Furthermore, due to their higher bitterness concentration, green bitter melons offer excellent conditions for extracting momordicin, making them suitable for health food production, thereby generating higher product added value. Meanwhile, white and light green-skinned bitter melons are gradually forming new consumption trends. These types of bitter melons (such as white jade bitter melon and apple bitter melon) have thick flesh, a mild bitterness with a sweet aftertaste, and are suitable for eating raw, in salads, or as a base, perfectly aligning with the healthy and light-eating trend among young people. Furthermore, white and light green bitter melons have higher vitamin C content and are richer in minerals such as potassium and magnesium, further catering to health-conscious consumers. In addition, there is a niche category of yellow / red-skinned bitter melons that, upon ripening, gradually lose their bitterness, and the flesh becomes sweeter, making them suitable for making jams, preserves, or as a natural sweetener. Overall, cultivating bitter melon varieties with different skin colors can diversify and meet the needs of different consumers, and in-depth research into the genes that regulate bitter melon skin color is a crucial foundation for related breeding work. Summary of the Invention
[0003] To overcome the shortcomings of the existing technology, this invention has discovered that the McCAB1 gene, derived from the green bitter gourd variety B07, plays a regulatory role in the color of bitter gourd fruits. In experiments with heterologous overexpression of the McCAB1 gene, the peel color of the bitter gourd was significantly deepened and turned greener, and its chlorophyll content was also significantly increased. This indicates that the McCAB1 gene can be applied to breeding work for improving the appearance quality of bitter gourd fruits and has broad application prospects in related fields.
[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0005] The first aspect of this invention provides the application of the McCAB1 gene in the regulation of bitter gourd peel color, wherein the McCAB1 gene is expressed in green bitter gourd but not expressed at all in white bitter gourd; the protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.3.
[0006] The second aspect of this invention provides the application of the McCAB1 gene in the breeding of bitter gourd fruit appearance quality, wherein the McCAB1 gene is expressed in green bitter gourd but not expressed at all in white bitter gourd; the protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.3.
[0007] The third aspect of this invention provides the application of the McCAB1 gene in the identification of bitter gourd peel color, wherein the McCAB1 gene is expressed in green bitter gourd but not expressed at all in white bitter gourd; the protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.3.
[0008] Preferably, the genomic DNA sequence of the McCAB1 gene is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.2.
[0009] Preferably, the bitter gourd peel color identification is used to assist in identifying the peel color of bitter gourd during the seedling stage.
[0010] The fourth aspect of this invention provides a method for cultivating green bitter gourd varieties, namely, heterologously overexpressing the McCAB1 gene in the bitter gourd variety material to be improved; the protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.3.
[0011] Preferably, the method for cultivating green bitter gourd varieties includes the following steps:
[0012] S1. The full-length CDS sequence of the McCAB1 gene was constructed into the pGWB5 plasmid to obtain the overexpression vector pGWB5-McCAB1. The CDS sequence of the McCAB1 gene is shown in SEQ ID NO.2.
[0013] S2. Transform pGWB5-McCAB1 into Agrobacterium, and then inject the bacterial solution into the pericarp of bitter melon fruit. After the McCAB1 gene is expressed, the pericarp color will turn significantly green.
[0014] More preferably, in S1, the full-length CDS sequence of the McCAB1 gene is inserted into the pGWB5 plasmid between the attR1 and attR2 sites downstream of the 35S promoter, replacing the ccdB gene through gene recombination.
[0015] Preferably, the bitter gourd variety to be improved is a white bitter gourd, or it can be a less common variety such as a yellow / red-skinned bitter gourd.
[0016] Compared with the prior art, the beneficial effects of the present invention are:
[0017] This invention, for the first time, clearly identifies the McCAB1 gene, derived from the green bitter gourd B07, as a key gene regulating bitter gourd fruit color, filling a research gap in the molecular regulation of bitter gourd fruit color and providing a core target for elucidating the mechanism of bitter gourd fruit color formation. Heterologous overexpression experiments verified that this gene can significantly deepen the color of bitter gourd peel and increase its chlorophyll content, fully demonstrating its function in regulating pigment synthesis and providing a reliable basis for related gene function research. Furthermore, the McCAB1 gene can be used for bitter gourd peel color identification or targeted breeding for the appearance quality of bitter gourd fruits, helping to cultivate bitter gourd varieties with excellent color to meet market demands. It also provides a reference for improving the fruit color of cucurbitaceous crops, with broad application prospects. Attached Figure Description
[0018] Figure 1 Transcriptional expression of the McCAB1 gene in white and green bitter gourd materials;
[0019] Figure 2 A map of the overexpression vector pGWB5 plasmid;
[0020] Figure 3 The overexpression and phenotype of the McCAB1 gene in bitter melon pericarp. Detailed Implementation
[0021] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0022] In the following examples, experimental methods without specific conditions were generally performed under standard conditions. Standard conditions can be found in *Molecular Cloning: A Laboratory Manual* by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturers of the reagents or instruments used in the experiments. All commonly used chemical reagents used in the examples were commercially available products. The bitter gourd material used in the examples was provided by the Facility Agriculture New Technology Research Laboratory of our institution.
[0023] Example 1: Transcriptional expression of the McCAB1 gene in white and green bitter gourd materials
[0024] The McCAB1 gene belongs to the chlorophyll a / b binding protein family and was discovered through transcriptome analysis of green bitter gourd (variety B07) and white bitter gourd (variety A06) materials (transcriptome sequencing was commissioned to Shanghai Paisennuo Biotechnology Co., Ltd.). When the bitter gourd fruits reached commercial maturity, data showed that the McCAB1 gene expression level in the green bitter gourd B07 material was 430.08 (measured by rpkm value), while the gene was not expressed at all in the white bitter gourd A06 material. Subsequent qPCR experiments (upstream primer (SEQ ID NO.4): 5'-TCTCGGCAATGGGAGAGTC-3'; downstream primer (SEQ ID NO.5): 5'-TCGAGCTCACGATTCTTGGC-3') confirmed the expression difference, and the results were consistent with the transcriptome analysis. Figure 1 ).
[0025] Genomic DNA sequence of the McCAB1 gene (the bolded sequence is the coding sequence) (SEQ ID NO.1):
[0026]
[0027] The CDS sequence of the McCAB1 gene (SEQ ID NO.2): ATGGCTCTCTCCTCTCCGTCACTCGCCGGAAAGGCGGTGAAGCTCACGCCCTCTTCCCCCGAGCTTCTCGGCAATGGGAGAGTCAGCATGAGAAAATTTGTCTCCAAGTCCGTTTCTTCTGGGAGCCCATGGTACGGACCCGACCGTGTCAAATATTTGGGCCCATTCTCCGGTGAGCCTCCATCCTACCTCACCGGAGAATTCCCTGGAGACTATGGTTGGGATACCGCCGGACTTTCTGCAGATCCCGAGACCTTTGCCAAGAATCGTGAGCTCGAAGTCATACACTCCAGGTGGGCTATGCTTGGGGCCTTGGGCTGCGTGTTCCCAGAGCTTCTATCCCGCAACGGTGTGAAATTCGGGGAAGCGGTATGGTTCAAGGCTGGATCTCAGATCTTCAATGAAGGTGGGCTCGACTACTTGGGCAACCCCAGCTTGATCCACGCACAGAGCATTTTAGCCATTTGGGCTTCCCAAGTAGTTCTAATGGGAGCCGTCGAGGGATACCGTATTGCCGGTGGTCCACTCGGCGAGGTGACCGACCCGATCTACCCGGGTGGGAGCTTTGACCCGTTGGGACTTGCAGATGACCCGGAAGCATTTGCAGAGCTGAAGGTGAAGGAGCTTAAGAATGGAAGGTTGGCGATGTTCTCCATGTTTGGTTTCTTCGTTCAGGCTATTGTCACCGGAAAAGGTCCATTGGAGAACCTTGCTGACCATTTGGCTGACCCAGTCAACAACAATGCTTGGGCTTACGCCACCGACTTTGTTCCTGGAAAATAA。
[0028] The protein sequence encoded by the McCAB1 gene (SEQ ID NO.3): MALSSPSLAGKAVKLTPSSPELLGNGRVSMRKFVSKSVSSGSPWYGPDRVKYLGPFSGEPPSYLTGEFPGDYGWDTAGLSADPETFAKNRELEVIHSRWAMLGALGCVFPELLSRNGVKFGEAVWFK AGSQIFNEGGLDYLGNPSLIHAQSILAIWASQVVLMGAVEGYRIAGGPLGEVTDPIYPGGSFDPLGLADDPEAFAELKVKELKNGRLAMFSMFGFFVQAIVTGKGPLENLADHLADPVNNNAWAYATDFVPGK.
[0029] Example 2: Overexpression of the McCAB1 gene and its application in regulating the color of bitter melon peel.
[0030] 1. Construction of McCAB1 gene overexpression vector
[0031] The full-length CDS sequence of the McCAB1 gene was constructed into the pGWB5 plasmid (plasmid map shown). Figure 2 As shown), the specific insertion location is between the attR1 and attR2 sites downstream of the 35S promoter. During the construction process, through gene recombination exchange (the operation method is referenced in: Saba K, Sameeullah M, Asghar A, et al. Expression of ESAT-6antigen from Mycobacterium tuberculosis in broccoli: An edible plant[J].Biotechnology and Applied Biochemistry, 2020, 67(1): 148-157.), the full-length CDS sequence of the McCAB1 gene was used to replace the original ccdB gene at this site, and finally the overexpression vector of the McCAB1 gene was successfully obtained, which was named pGWB5-McCAB1.
[0032] 2. Construct a gene expression model for transient pGWB5-McCAB1 expression.
[0033] (1) Transform the overexpression vector from Part 1 into Agrobacterium EHA105 and incubate upside down on a medium containing kanamycin (50 ug / mL) for 48 h.
[0034] (2) Pick a single clone and add it to 4 mL of LB medium containing kanamycin (50 ug / mL) and rifampin (50 ug / mL), and shake it at 28℃ and 180 rpm for 24 h.
[0035] (3) Add fresh LB medium containing the corresponding antibiotics and rifampin to the above bacterial culture at a ratio of 1:100 (volume ratio), and continue to shake the culture at 28°C and 180 rpm until OD. 600 The value is approximately 3.0.
[0036] (4) Collect the bacterial cells by centrifugation at 3000 rpm for 5 minutes, resuspend the bacterial cells in a suspension (containing 10 mM MES, 10 mM MgCl2, and water as the solvent), and adjust the OD. 600 Once the value reaches around 0.4, add 200 mM acetylsyl syringone.
[0037] (5) Let the treated bacterial solution stand at room temperature for 3 h.
[0038] (6) Use a 1 mL syringe to draw an equal amount of the bacterial solution after standing, and inject it into the pericarp of the bitter melon fruit. The control group (CK) contains bacterial solution with empty carrier.
[0039] (7) The injected bitter gourd fruits were first cultured in the dark at 28℃ for 24 hours, and then transferred to the conditions of 22℃ in the dark for 12 hours and 28℃ in the light for 12 hours, and continued to be cultured for 3 to 4 days.
[0040] (8) Collect fruit peel and pulp samples from the treatment group and the control group respectively, extract RNA, and use fluorescent PCR to detect the expression level of McCAB1 gene (the detection primers are the same as SEQ ID NO.4 and SEQ ID NO.5).
[0041] Experimental results showed that the McCAB1 gene was highly expressed in the pericarp of bitter melon fruits treated with transient expression, while no expression of the gene was observed in the control (CK) group. Figure 3 A) indicates that the transient expression model of the McCAB1 gene in the pericarp of bitter melon fruit was successfully constructed.
[0042] 3. Phenotypic detection of bitter melon fruit
[0043] Based on the transient expression model of the McCAB1 gene in bitter gourd fruit peel successfully constructed in Part 2, the color changes of the fruit around the injection point were observed and recorded after 3 to 4 days of cultivation. White bitter gourd A06 was used as the injection material, where "T" represents the McCAB1 gene overexpression treatment group and "CK" represents the control group. To ensure the reliability of the experimental results, each bitter gourd fruit was tested in triplicate for both the treatment and control groups, with a total of 5 bitter gourd fruits used in the experiment. The final experimental results showed that after expressing the McCAB1 gene in the peel of white bitter gourd A06, the peel color was significantly greener than the control group (CK), further confirming the regulatory role of the McCAB1 gene on the fruit color of bitter gourd. Figure 3 B).
[0044] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.
Claims
1. The application of the McCAB1 gene in the regulation of bitter melon peel color, characterized in that, Overexpression of the McCAB1 gene in bitter melon can darken the color of the bitter melon peel to green; the protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.
3.
2. The application of the McCAB1 gene in breeding bitter gourd fruit appearance quality, characterized in that, Overexpression of the McCAB1 gene in bitter melon can darken the color of the bitter melon peel to green; the protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.
3.
3. Application of the McCAB1 gene in the identification of bitter melon peel color, characterized in that, The McCAB1 gene is expressed in green bitter melon but not at all in white bitter melon; the protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.
3.
4. The application according to any one of claims 1-3, characterized in that, The genomic DNA sequence of the McCAB1 gene is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.
2.
5. The application according to claim 3, characterized in that, The identification of bitter gourd peel color is used to assist in identifying the peel color of bitter gourd during the seedling stage.
6. A method for cultivating green bitter gourd varietal materials, characterized in that, The McCAB1 gene was heterologously overexpressed in bitter gourd cultivars to be improved. The protein sequence encoded by the McCAB1 gene is shown in SEQ ID NO.
3.
7. The method for cultivating green bitter gourd varieties according to claim 6, characterized in that, Includes the following steps: S1. The full-length CDS sequence of the McCAB1 gene was constructed into the pGWB5 plasmid to obtain the overexpression vector pGWB5-McCAB1. The CDS sequence of the McCAB1 gene is shown in SEQ ID NO.
2. S2. Transform pGWB5-McCAB1 into Agrobacterium, and then inject the bacterial solution into the pericarp of bitter melon fruit. After the McCAB1 gene is expressed, the pericarp color will turn significantly green.
8. The method for cultivating green bitter gourd varieties according to claim 7, characterized in that, In S1, the full-length CDS sequence of the McCAB1 gene is inserted into the pGWB5 plasmid between the attR1 and attR2 sites downstream of the 35S promoter, replacing the ccdB gene through gene recombination.
9. The method for cultivating green bitter gourd varietal materials according to claim 6, characterized in that, The bitter gourd variety to be improved is a white bitter gourd.