Screening Of Candidate Functional Genes In The Biosynthetic Pathway Of Tetrandrine And Study On The Function Of Noraconitine-6-O-methyltransferase

Fangji the traditional Chinese medicine is the dried root of Stephania tetrandra S.Moore which has a medicinal history of more than 2000 years in China.Modern research shows that the main active ingredient in Fangji is Tetrandrine which has anti-pneumoconiosis anti-hypertension anti-inflammatory anti-tumor and other pharmacological effects.However with the growth of demand and the destructive excavation of wild resources fangji has gradually seen a price increase and a shortage of supply.Tetrandrine belongs to the class of bisbenzylisoquinoline alkaloids.At present the biosynthetic pathways of various benzylisoquinoline alkaloids including morphine have been fully resolved and have been engineered but there is still little information about Biosynthesis of bisbenzylisoquinoline alkaloids such as tetrandrine.In order to achieve the complete analysis of the tetrandrine biosynthesis pathway and the heterogeneous production of tetrandrine this study performed transcriptome sequencing on S.tetrandra and obtained a series of functional genes that may be involved in the biosynthesis of tetrandrine.The obtained St6 OMTs gene was cloned and heterologously expressed and the gene function was verified by in vitro enzymatic reaction.The main research contents are as follows:1.Transcriptome measurement and comparative transcriptomics analysis of S.tetrandra.The relative content of related alkaloids was measured in the four tissue parts of S.tetrandra velamens roots stems and leaves.Then the second-generation combined with the third-generation sequencing technology was used to perform transcriptome sequencing on the above four tissue parts and 38(S)-norcoclaurine synthase(NCS)genes 12norcoclaurine-6-O-methyltransferase(6OMT)genes and 64 coclaurine N-methyltransferase(CNMT)genes were obtained from the transcriptome data.Combined with the accumulation pattern of alkaloids the genes with higher expression levels in the underground parts that may be involved in the downstream pathway of powder tetrandrine biosynthesis were selected including12 O-methyltransferase genes and 23 CYP450 genes.The obtained transcriptome data will provide the premise for functional verification of upstream genes and complete analysis of downstream pathways.In this work the transcriptome of S.tetrandra was sequenced and the genes related to tetrandrine biosynthesis were screened from the transcriptome.This work laid the foundation for the verification of functional genes.2.Full-length cloning and sequence analysis of St6 OMT.Based on the 6OMT gene sequence obtained by transcriptome sequencing primers were designed.Using velamen cDNAas a template four St6 OMTs were cloned and analyzed for bioinformatics and gene expression.Multi-sequence comparison between St6 OMTs and 6OMT with verified functions revealed that all St6 OMTs have 6OMT characteristic conserved sequences.In the evolutionary tree analysis St6OMT1 is more clustered with 6OMT in other plants with known functions.In gene expression analysis the relative expression levels of St6OMT1 and St6OMT2 in the underground part were much higher than those in the above-ground part while the relative expression levels of St6OMT3 and St6OMT4 in each tissue site were lower.In this work St6 OMTs were cloned and sequenced which indicated the direction for the verification of gene function in vitro.3.The expression and function verification of St6 OMTs.The St6 OMTs was constructed into the prokaryotic expression vector pET32 a using seamless splicing technology.The recombinant plasmid was successfully transformed into BL21(DE3)strain and expressed the corresponding soluble protein.using(S)-norcoclaurine as the substrate and S-adenosyl-L-methionine as the methyl donor to perform enzymatic reaction in vitro to verify the function.The results show that St6OMT1 can catalyze(S)-norcoclaurine to produce the expected product(S)-coclaurine.In the St6OMT2 reaction system a new chromatographic peak with an m/z of 300 was detected presuming that it was a product of continuous methylation but further research is needed.St6OMT3 and St6OMT4 showed no catalytic activity for substrate(S)-norcoclaurine.In this work the function of 6OMT in the tetrandrine biosynthesis pathway was identified which laid the foundation for the complete analysis of the tetrandrine biosynthesis pathway and also provided alternative genetic elements for the heterogeneous production of BIAs.