Cloning and Expression Analysis of CMB1 Gene in Pepper

Abstract

【Objective】CMB1 gene plays an important role in the regulation of inflorescence structure and fruit ripening in plants, but the function and regulatory mechanism of CMB1 gene in pepper have been rarely reported. Therefore, the role of transcription factors related to the regulation of carotenoid synthesis and CMB1 gene in pepper was studied so as to provide a reference for pepper breeding.【Method】The coding sequences of CMB1 were cloned by the homology sequence method, and the expression patterns of CMB1 in fruits of red and yellow peppers with wrinkled skin at different development stages (green ripening stage, color change stage, mature stage) were analyzed by real-time quantitative PCR.【Result】The ORF length of CMB1 gene was 732 bp in both red pepper and yellow pepper, encoding 243 amino acid residues. Sequence alignment showed that the mutation occurred at the 11th site of CMB1 base sequence, G mutated to T, resulting in amino acid sequence mutated from G (Glycine) to V (Valine). Bioinformatics analysis revealed that the CMB1-encoded protein of wrinkled skin red pepper had a molecular mass of 20 923.21, a theoretical isoelectric point of 5.59, an instability coefficient of 57.28, and a total mean hydrophilicity (gravimetric) index of -1.001. The CMB1-encoded protein of wrinkled skin yellow pepper had a molecular mass of 27 958.47, a theoretical isoelectric point of 7.10, an instability coefficient of 53.54, and a total mean hydrophilicity (gravimetric) index of -0.809. Both CMB1 of wrinkled skin red and yellow peppers were unstable hydrophobic proteins, both contained 38 phosphorylation sites, both were devoid of transmembrane structures and signal peptides, and both had typical MADS-box structural domains and K-box structural domains. Evolutionary tree analysis revealed that pepper CMB1 was most closely related to Solanaceae and most distantly related to Leguminosae. The results of fluorescence quantitative PCR showed that the expression of CMB1 increased sequentially in the three stages of green ripening, color change, and maturity of pepper fruits, and the expression of CMB1 in the three stages was significantly higher in wrinkled skin red pepper than in wrinkled skin yellow pepper.【Conclusion】Pepper CMB1 is a member of MADS-box, and its expression in pepper fruit is similar to that of carotenoid in pepper, suggesting that CMB1 is mainly involved in regulating the synthesis of carotenoid like capsanthin.

Keywords

• pepper
• carotenoid
• transcription factor
• gene cloning
• cmb1