Regulation and control effect of McCAB1 gene on bitter gourd peel color

Abstract

The invention belongs to the technical field of plant molecular biology and plant genetic engineering, and particularly relates to a regulating effect of an McCAB1 gene on the color of bitter gourd peel. Research finds that the McCAB1 gene derived from the green balsam pear B07 variety has a regulation effect on the color of balsam pear fruits. After the McCAB1 gene is subjected to heterologous overexpression, the peel color of the balsam pear is obviously deepened and greened, and the chlorophyll content of the balsam pear is also obviously improved. According to the invention, the McCAB1 gene from green balsam pear B07 is determined as a key gene for regulating and controlling the fruit color of the bitter gourd for the first time, the research blank in the field of molecular regulation and control of the fruit color of the bitter gourd is filled, and a core target is provided for analyzing a fruit color forming mechanism of the bitter gourd. Besides, the McCAB1 gene can be used for bitter gourd pericarp color identification or oriented bitter gourd fruit appearance quality breeding, the cultivation of bitter gourd varieties with excellent color is assisted to meet the market demand, reference is provided for fruit color improvement of cucurbitaceae crops, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant molecular biology and plant genetic engineering, and particularly relates to the regulation of McCAB1 gene on the pericarp color of Momordica charantia L. BACKGROUND

[0002] The pericarp color is a prominent agronomic trait of Momordica charantia L., and has important quality characteristics and commercial value. Due to the different pericarp colors, Momordica charantia L. has obvious differences in taste, nutritional characteristics and consumption scenarios, which directly leads to the significant differentiation of its commercial value. At present, the market still relies on green pericarp Momordica charantia L., such as Jiangmen Dadeng and Cui green Dadeng, and the content of momorcharin in the green pericarp Momordica charantia L. is 30%-40% higher than that in white Momordica charantia L., and the effect of reducing blood sugar is more significant, which is suitable for diabetic patients and health crowds, and the demand in southern provinces such as Guangdong and Guangxi is very stable. In addition to the market demand advantage, the green pericarp Momordica charantia L. also has a wide adaptability and strong heat resistance, and the yield performance is stable. At present, the mainstream production area has built a mature and perfect supply chain system, which can effectively support the market circulation demand. Moreover, the green pericarp Momordica charantia L. has high momorcharin content, which can be used for the production of health food, thereby generating high product added value. At the same time, white and light green pericarp Momordica charantia L. is gradually forming a new consumption trend. This kind of Momordica charantia L. (such as white jade Momordica charantia L. and apple Momordica charantia L.) has thick flesh, light bitterness and a sweet aftertaste, which is suitable for eating raw, cold dishes or making salads, and is highly consistent with the health and light food trend pursued by young people. Moreover, the white and light green pericarp Momordica charantia L. has higher content of vitamin C and is richer in minerals such as potassium and magnesium, which further meets the health consumption demand. In addition, there is a kind of mature yellow / red pericarp Momordica charantia L., which belongs to a small category and has a unique flavor. The bitterness of the mature yellow / red pericarp Momordica charantia L. gradually disappears, and the flesh turns sweet, which is suitable for making jam, candied fruit or being used as a natural sweetener. Overall, cultivating Momordica charantia L. varieties with different pericarp colors can meet the needs of different consumers in multiple dimensions and diversities, and further exploring the genes regulating the pericarp color of Momordica charantia L. is an important basis for carrying out related breeding work. SUMMARY

[0003] In order to overcome the shortcomings of the prior art, the present application finds that the McCAB1 gene derived from the B07 variety of green pericarp Momordica charantia L. has a regulation effect on the fruit color of Momordica charantia L.. In the experiment of heterologous overexpression of the McCAB1 gene, the pericarp color of Momordica charantia L. is obviously deepened and green, and the chlorophyll content is also significantly improved. It is shown that the McCAB1 gene can be applied to the breeding work of the appearance quality of Momordica charantia L. fruit, and has a wide application prospect in the related field.

[0004] In order to achieve the above purpose, the technical scheme adopted by the present application is: The application provides the application of the McCAB1 gene in the regulation of the peel color of Momordica charantia, wherein the McCAB1 gene is expressed in green Momordica charantia and is not expressed at all in white Momordica charantia; and the protein sequence coded by the McCAB1 gene is shown in SEQ ID NO. 3.

[0005] The application provides the application of the McCAB1 gene in the breeding of the appearance quality of Momordica charantia fruits, wherein the McCAB1 gene is expressed in green Momordica charantia and is not expressed at all in white Momordica charantia; and the protein sequence coded by the McCAB1 gene is shown in SEQ ID NO. 3.

[0006] The application provides the application of the McCAB1 gene in the identification of the peel color of Momordica charantia, wherein the McCAB1 gene is expressed in green Momordica charantia and is not expressed at all in white Momordica charantia; and the protein sequence coded by the McCAB1 gene is shown in SEQ ID NO. 3.

[0007] 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.

[0008] Preferably, the identification of the peel color of Momordica charantia is the auxiliary identification of the peel color of Momordica charantia at the seedling stage.

[0009] The application provides a method for cultivating green Momordica charantia variety materials, that is, heterologously overexpressing the McCAB1 gene in Momordica charantia variety materials to be improved; and the protein sequence coded by the McCAB1 gene is shown in SEQ ID NO. 3.

[0010] Preferably, the method for cultivating green Momordica charantia variety materials comprises the following steps. S1, constructing the CDS full-length sequence of the McCAB1 gene into a pGWB5 plasmid to obtain an overexpression vector pGWB5-McCAB1, wherein the CDS sequence of the McCAB1 gene is shown in SEQ ID NO. 2; S2, transforming the pGWB5-McCAB1 into Agrobacterium, and then injecting the bacterial liquid into the peel part of the Momordica charantia fruit, so that the peel color is significantly green after the McCAB1 gene is expressed.

[0011] More preferably, in S1, the insertion position of the CDS full-length sequence of the McCAB1 gene on the pGWB5 plasmid is between the attR1 and attR2 sites downstream of the 35S promoter, and the ccdB gene is replaced by gene recombination exchange.

[0012] Preferably, the Momordica charantia variety material to be improved is white Momordica charantia, and can also be yellow / red peel Momordica charantia, which is a small category.

[0013] Compared with the prior art, the present application has the beneficial effects that: The McCAB1 gene derived from B07 of Momordica charantia L. for the first time is a key gene for regulating fruit color of Momordica charantia L., fills the research gap in the field of molecular regulation of fruit color of Momordica charantia L., and provides a core target for analyzing the formation mechanism of fruit color of Momordica charantia L. The gene can significantly deepen the color of the pericarp of Momordica charantia L. and improve the chlorophyll content of Momordica charantia L. through heterologous overexpression experiments, fully confirming its function of regulating pigment synthesis, and providing a reliable basis for related gene function research. In addition, the McCAB1 gene can be used for identification of the pericarp color of Momordica charantia L. or directed for breeding of the appearance quality of Momordica charantia L., helping to cultivate Momordica charantia L. varieties with excellent color to meet market demand, and also providing a reference for improvement of fruit color of Cucurbitaceae crops, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Transcriptional expression of the McCAB1 gene in white and green Momordica charantia L. materials; Figure 2 Map of the overexpression vector pGWB5 plasmid; Figure 3 Overexpression of the McCAB1 gene in the pericarp of Momordica charantia L. and phenotype. DETAILED DESCRIPTION

[0015] The specific embodiments of the present application will be further described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0016] In the following examples, the experimental methods not specified in the specific conditions are usually operated according to the conventional conditions. The conventional conditions can refer to the related conditions recorded in the book “Molecular Cloning: A Laboratory Manual” by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or can be performed according to the recommended conditions provided by the manufacturers of the reagents or instruments used in the experiments. The various common chemical reagents used in the examples are commercially available products. The Momordica charantia L. materials used in the examples are provided by the New Technology Research Office of the Unit Facilities.

[0017] Example 1: Transcriptional expression of the McCAB1 gene in white and green Momordica charantia L. materials The McCAB1 gene belongs to the chlorophyll a / b binding protein family, and is a gene mined through transcriptome analysis (transcriptome sequencing was entrusted to Shanghai Paissen Biotechnology Co., Ltd.) of green balsam pear (variety B07) and white balsam pear (variety A06) materials. When the balsam pear fruit enters the commercial mature stage, the detection data show that the expression amount of the McCAB1 gene in the green balsam pear B07 material is 430.08 (measured by the rpkm value), and the gene is not expressed at all in the white balsam pear A06 material; subsequent verification by qPCR experiment (upstream primer (SEQ ID NO. 4): 5'-TCTCGGCAATGGGAGAGTC-3'; downstream primer (SEQ ID NO. 5): 5'-TCGAGCTCACGATTCTTGGC-3') shows that the results are consistent with the transcriptome analysis results, further confirming the expression difference Figure 1 ).

[0018] The genomic DNA sequence of the McCAB1 gene (the bold sequence is the coding sequence) (SEQ ID NO. 1):

[0019]

[0020] CDS sequence of the McCAB1 gene (SEQ ID NO. 2): ATGGCTCTCTCCTCTCCGTCACTCGCCGGAAAGGCGGTGAAGCTCACGCCCTCTTCCCCCGAGCTTCTCGGCAATGGGAGAGTCAGCATGAGAAAATTTGTCTCCAAGTCCGTTTCTTCTGGGAGCCCATGGTACGGACCCGACCGTGTCAAATATTTGGGCCCATTCTCCGGTGAGCCTCCATCCTACCTCACCGGAGAATTCCCTGGAGACTATGGTTGGGATACCGCCGGACTTTCTGCAGATCCCGAGACCTTTGCCAAGAATCGTGAGCTCGAAGTCATACACTCCAGGTGGGCTATGCTTGGGGCCTTGGGCTGCGTGTTCCCAGAGCTTCTATCCCGCAACGGTGTGAAATTCGGGGAAGCGGTATGGTTCAAGGCTGGATCTCAGATCTTCAATGAAGGTGGGCTCGACTACTTGGGCAACCCCAGCTTGATCCACGCACAGAGCATTTTAGCCATTTGGGCTTCCCAAGTAGTTCTAATGGGAGCCGTCGAGGGATACCGTATTGCCGGTGGTCCACTCGGCGAGGTGACCGACCCGATCTACCCGGGTGGGAGCTTTGACCCGTTGGGACTTGCAGATGACCCGGAAGCATTTGCAGAGCTGAAGGTGAAGGAGCTTAAGAATGGAAGGTTGGCGATGTTCTCCATGTTTGGTTTCTTCGTTCAGGCTATTGTCACCGGAAAAGGTCCATTGGAGAACCTTGCTGACCATTTGGCTGACCCAGTCAACAACAATGCTTGGGCTTACGCCACCGACTTTGTTCCTGGAAAATAA.

[0021] The protein sequence encoded by the McCAB1 gene (SEQ ID NO.3): MALSSPSLAGKAVKLTPSSPELLGNGRVSMRKFVSKSVSSGSPWYGPDRVKYLGPFSGEPPSYLTGEFPGDYGWDTAGLSADPETFAKNRELEVIHSRWAMLGALGCVFPELLSRNGVKFGEAVWFK AGSQIFNEGGLDYLGNPSLIHAQSILAIWASQVVLMGAVEGYRIAGGPLGEVTDPIYPGGSFDPLGLADDPEAFAELKVKELKNGRLAMFSMFGFFVQAIVTGKGPLENLADHLADPVNNNAWAYATDFVPGK.

[0022] Example 2: Overexpression of the McCAB1 gene and its application in regulating the color of bitter melon peel.

[0023] 1. Construction of McCAB1 gene overexpression vector 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.

[0024] 2. Construct a gene expression model for transient pGWB5-McCAB1 expression. (1) Transform the overexpression vector in Part 1 into Agrobacterium EHA105 and incubate upside down on a medium containing kanamycin (50 ug / mL) for 48 h.

[0025] (2) Pick a single clone and add it to 4 mL LB medium containing kanamycin (50 ug / mL) and rifampicin (50 ug / mL), and shake at 28°C, 180 rpm for 24 h.

[0026] (3) Add fresh LB medium containing the corresponding antibiotics and rifampicin to the above bacterial solution at a ratio of 1:100 (volume ratio), and continue to shake at 28°C, 180 rpm until the OD 600 value is about 3.0.

[0027] (4) Centrifuge at 3000 rpm for 5 minutes to collect the bacterial cells, resuspend the bacterial cells with suspension liquid (containing 10 mM MES, 10 mM MgCl2, solvent is water), and adjust the OD 600 value to about 0.4, and then add 200 mM acetosyringone.

[0028] (5) The treated bacterial solution is placed at room temperature for 3 h.

[0029] (6) Use a 1 mL syringe to suck an equal amount of the settled bacterial solution, and inject it into the peel part of the bitter gourd fruit. The control group (CK) contains bacterial solution containing empty vector.

[0030] (7) The injected bitter gourd fruit is first cultured at 28°C in the dark for 24 h, and then transferred to 22°C in the dark for 12 h / 28°C in the light for 12 h, and then cultured for 3 to 4 days.

[0031] (8) Collect the peel and pulp samples of the treatment group and the control group, extract RNA, and use fluorescent PCR to detect the expression level of McCAB1 gene (detection primers are SEQ ID NO. 4 and SEQ ID NO. 5).

[0032] The experimental results show that the McCAB1 gene in the peel of the bitter gourd fruit treated by transient expression of McCAB1 gene shows a high expression level, while the gene in the control (CK) group has no expression (A), indicating that the transient expression model of McCAB1 gene in the peel of the bitter gourd fruit is successfully constructed. Figure 3

[0033] 3, Bitter gourd fruit phenotype detection ​For the successful construction of McCAB1 gene in the fruit pericarp transient expression model in Part 2, the fruit color change around the injection point was observed and recorded after 3 to 4 days of culture. White A06 was selected as the injection material, and "T" represents the treatment group of McCAB1 gene overexpression, and "CK" represents the control group. To ensure the reliability of the experimental results, the treatment group and the control group of each bitter gourd fruit were set up 3 times, and the total number of bitter gourd fruits used in the experiment was 5. The final experimental results show that after expressing McCAB1 gene in the pericarp of white A06, the pericarp color appears a significant green phenomenon compared with the control group (CK), further confirming the regulation of McCAB1 gene on the color of bitter gourd Figure 3 B}.

[0034] The above detailed the embodiments of the present application, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments without departing from the principles and spirits of the present application still fall within the protection scope of the present application.

Claims

1. Use of the McCAB1 gene in the regulation of the color of the pericarp of Momordica charantia, characterized in that, The McCAB1 gene is expressed in green balsam pears, but not expressed in white balsam pears at all; the protein sequence coded by the McCAB1 gene is shown as SEQ ID NO.

3.

2. Use of the McCAB1 gene in breeding of the fruit appearance quality of Momordica charantia, characterized in that, The McCAB1 gene is expressed in green balsam pears, but not expressed in white balsam pears at all; the protein sequence coded by the McCAB1 gene is shown as SEQ ID NO.

3.

3. Use of the McCAB1 gene in the identification of the color of the pericarp of Momordica charantia, characterized in that, The McCAB1 gene is expressed in green balsam pears, but not expressed in white balsam pears at all; the protein sequence coded by the McCAB1 gene is shown as SEQ ID NO.

3.

4. Use according to any one of claims 1 to 3, characterized in that, The genomic DNA sequence of the McCAB1 gene is shown as SEQ ID NO. 1, and the CDS sequence is shown as SEQ ID NO.

2.

5. Use according to claim 3, characterized in that, The balsam pear peel color is identified as auxiliary identification of the peel color of balsam pear at the seedling stage.

6. A method of cultivating green bitter gourd variety material, characterized by, Heterologous overexpression of the McCAB1 gene in balsam pear variety materials to be improved; The protein sequence coded by the McCAB1 gene is shown as SEQ ID NO.

3.

7. The method of claim 6, wherein the green bitter gourd variety material is cultivated by the steps of: The method comprises the following steps: S1, the CDS full-length sequence of the McCAB1 gene is constructed on the pGWB5 plasmid to obtain the overexpression vector pGWB5-McCAB1, and the CDS sequence of the McCAB1 gene is shown as SEQ ID NO. 2; S2, the pGWB5-McCAB1 is transformed into Agrobacterium, and then the bacterial liquid is injected into the peel part of the balsam pear fruit, so that the peel color can be significantly green after the McCAB1 gene is expressed.

8. The method of claim 7, wherein the green bitter gourd variety material is cultivated by the steps of: In S1, the insertion position of the CDS full-length sequence of the McCAB1 gene on the pGWB5 plasmid is between the attR1 and attR2 sites downstream of the 35S promoter, and the ccdB gene is replaced by gene recombination exchange.

9. The method of claim 6, wherein the green bitter gourd variety material is cultivated by the steps of: The balsam pear variety material to be improved is white balsam pear.

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