Cloning and Expression Patterns of LmC3H1 Gene in Lonicera macranthoides and Its Correlation with Chlorogenic Acid Content / 中国实验方剂学杂志

ABSTRACT

Objective:To clone p-coumaroyl quinate/shikimate 3' -hydroxylase gene from Lonicera macranthoides, and analyze its bioinformatics and expression patterns with chlorogenic acid content, in order to speculate the functions of LmC3H1 gene from L. macranthoides. Method:The full-length cDNA sequence of LmC3H1 gene was cloned by reverse trascription polymerase chain reaction(RT-PCR) and RACE techniques. The bioinformatics analysis of the gene sequence was carried out by using relevant software.Real-time fluorescence quantification PCR(Real-time PCR) and HPLC were used to determine relative expression of LmC3H1 and content of chlorogenic acid in stems, leaves and flowers of different flowering stages. Result:The LmC3H1 (GenBank MN177695) gene was cloned, and the open reading frame (ORF) of it was 1 533 bp in length and encoded 510 amino acids. The molecular formula was C2618H4134N718O727S22, the relative molecular mass was 58 005.32, and the isoelectric point was 8.92.It was a hydrophilic protein located in the chloroplast with a transmembrane region LLLIPAVLFLISLVYPLI, and contained a conserved domain CYTOCHROME_P450(433-422 aa) in cytochrome P450.The results of Real-time PCR showed that LmC3H1 was expressed in different degrees in stems, leaves and different flowering stages of L. macranthoides. In the flower development stage, the relative expression of white bud stage was the highest, followed by flower buds and white flowering stage. The ratio of flower to stem and leaf was the highest, and the relative expression of flower was the highest. The HPLC results showed that the content of chlorogenic acid increased from greenish white to golden yellow in flowering stage and golden yellow flowering stage. Among the different organs, the flower had the highest chlorogenic acid, and the stem showed the lowest. Conclusion:The LmC3H1 gene of L. macranthoides is cloned, suggesting that LmC3H1 might be involved in the biosynthesis of L. macranthoides chlorogenic acid. This study provides a basis for further studying the functions of the gene and exploring the biosynthesis and regulation mechanism of L. macranthoides chlorogenic acid, while laying the foundation for the genetic improvement of L. macranthoides.