Cloning and Analysis of the UrIO Gene and Its Promoter in Uncaria rhynchophylla
ZHENG Hao-Jie, WANG Xiao-Hong, LI Yong-Quan, LI Xue, SHANGGUAN Li-Yang TIAN Yu-Hang, ZHANG Ming-Sheng*
College of Life Sciences/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China
Abstract Iridoid oxidase (IO) belonging to the CYP76 family A26 subfamily and is the step 4 rate-limiting enzyme of the iridodial pathway in the upstream synthetic pathway of terpenoid indole alkaloids, catalyzing the synthesis of 7-deoxyloganetic acid from iridoid. In this study, UrIO (GenBank No. ON227515) was cloned based from Uncaria rhynchophylla transcriptome data, which encoded 516 amino acids. Phylogenetic relationship showed that the relationship between UrIO of U. rhynchophylla and CcIO of Cinchona calisaya was the closest, followed by CrIO of Catharanthus roseus. Multiple sequence alignment analysis showed that UrIO had 3 typical domains of cytochrome P450 proteins (CYP) family. The expression level of UrIO in stem was the highest, followed by fruit, and the expression of UrIO was significantly increased under methyl jasmonate (MeJA) treatment. However, it was not induced by abscisic acid (ABA). The promoter of UrIO was cloned by fusion primer and nested integrated PCR (FPNI-PCR) method as well, which contained stress response and hormone response elements by plant cis-acting regulatory elements (PlantCARE) analysis. Transient transformation experiments in tobacco (Nicotiana tabacum) showed that the promoter could drive GUS reporter gene expression. Furthermore, yeast one hybrid (Y1H) confirmed the interaction between UrMYC2 and UrIO promoter. This study provides a reference for further analysis of the synthetic pathway of indole alkaloids from U. rhynchophylla.
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