Cloning and Expression Analysis of the GeCYP85A1 Gene in Gelsemium elegans
ZHANG Yao1, CHEN Wen-Qiang1, PAN Li-Mei2, MU De-Tian1, ZHOU Yu1, LIN Xiao-Dong1, LIU Zhao-Ying3,*, TANG Qi1,*
1 College of Horticulture/National Research Center of Engineering Technology for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha 410128, China; 2 Guangxi Botanical Garden of Medicinal Plants, Nanning 510023, China; 3 College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China
Abstract：Gelsemium elegans is a valuable traditional Chinese herb in China. The whole plant is highly toxic, but has anti-tumour and analgesic effects, as well as fattening effects on pigs and other livestock, and its main active constituents are terpenoid indole alkaloids (TIAs). Among them, the most representative ingredient is gelsenicine, which has the strongest toxicity and highest activity. In this study, 209 genes related to cytochrome P450 (CYP450) were screened based on the genomic and transcriptomic data of G.elegans, combined with metabolomic data, an important candidate gene GeCYP85A1 (GenBank No. OQ653843) related to the biosynthesis pathway of Oryza japonica was screened by "genome+expression profile+metabolome". The full-length cloning of this gene was performed by PCR, and its bioinformatics analysis was carried out. The expression pattern of the GeCYP85A1 gene in different tissues of G. elegans was analysed by qPCR. The results showed that the GeCYP85A1 gene was 1 392 bp long, encoding 463 amino acids, with a theoretical relative molecular mass of 53.390 06 kD and a theoretical isoelectric point of 9.07. It was a non-secretory protein with a subcellular localization in chloroplasts, a transmembrane region and no signal peptide. It was similar to Coffea arabica, Coffea eugenioides, Erythranthe guttata, Nicotiana tabacum, Olea europaea subsp. europaea, Phtheirospermum juttata, Salvia miltiorrhiza, Sesamum indicum, and Vitis riparia, and the amino acid sequences of the related CYP85A1 of the above 9 species were highly similar and belonged to a subfamily of the CYP450 family. By qPCR, the expression pattern of GeCYP85A1 in different parts of the plant was found to be consistent with the trend of the relative content of gelsenicine in different parts of the plant, which were root>flower>stem>leaf. This study provides a reference for further elucidation of the biosynthetic pathway for the TIAs-like alkaloids in G. elegans.
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