Cloning and expression analysis of CsCYP79A1 gene in tea plant

【Objective】The CYP79 enzyme gene CsCYP79A1 of tea plant cytochrome P450(CYP) enzyme system was cloned, and its bioinformatics analysis and expression analysis were performed to provide a theoretical basis for in-depth understanding of the mechanism of CsCYP79A1 gene involved in the defense response of tea plant and its function in the production process of oolong tea.【Method】The CsCYP79A1 gene was cloned using Huangguanyin leaves as materials, and the gene was analyzed by analysis softwares. Real-time fluorescent quantitative PCR(qRT-PCR) was used to detect its expression in different tea varieties, different tenderness leaves and different shaking times to analyze its expression characteristics. Gas chromatography-mass spectrometry(GC-MS) was used to determine the volatilization of phenyl-acetonitrile in leaves of different treatments to analyze its relationship with the expression of CsCYP79A1 gene.【Result】The cloned tea plant CsCYP79A1 gene coding region(CDS) was 1617 bp in length, encoded 538 amino acids, had a relative molecular mass of 61.07 kD and a theoretical isoelectric point(pI) of 7.64, and it was an unstable liposoluble hydrophilic protein. The amino acid sequence of CsCYP79A1 protein had the highest similarity with TXG46886.1(Actinidia chinensis), reaching 87%. The CsCYP79A1 protein had two transmembrane structures, no signal peptide, and had 40 phosphorylation sites. In the secondary structure, α-helix, β turn, extended chain and random coil accounted for 49.07%, 4.28%, 11.71%, and 34.94% of the amino acid sequence, respectively. The similarity between the tertiary structure and the crystal structure of rust alcohol synthase(5ylw. 1. A) was 26.61%. After four times shaking treatments, the expression of CsCYP79A1 gene in the third leaves of Jinxuan and Huangguanyin was significantly higher than that of Zhenong 113, Baiye No. 1 and Fuding Dabaicha, indicating that the expression level of CsCYP79A1 gene was higher in varieties which were suitable for making oolong tea. The expression level in the third leaves was significantly higher than that in the first leaves and terminal bud(P<0.05) and with the increase of the number of shaking, the expression level first increased and then decreased. After 4 shaking treatments, the third leaf released a large amount of phenylacetonitrile, while the first leaf and apical buds released a little or no phenylacetonitrile. In leaves with different shaking times, the volatilization of phenylacetonitrile increased with the increase of shaking times.【Conclusion】The expression level of CsCYP79A1 gene is related to tea variety, leaf tenderness and the degree of shaking, and both the expression level and the release of phenylacetonitrile are the highest in the third leaf. Therefore, it is speculated that CsCYP79A1 gene is one of the key genes in the synthesis of phenylacetonitrile.