Functional Research On Flavonoid Prenyltransferase Genes From Epimedium Pubescens Maxim.

Objective:Based on the results of genome-wide assembly of Epimedium pubescens Maxim.,this thesis comprehensively investigated and identified the flavonoid prenyltransferase genes in E.pubescens and explored their evolutionary relationships.Candidate genes were cloned and constructed into yeast expression vectors,and the gene functions were verified by yeast incubation with various substrates.The results decipher the key step of biosynthetic pathways of icariin flavonoids and provide a basis for the in vitro synthesis of such compounds and facilitate the breeding cultivar of Epimedium medicinal materials with high content of active ingredients.Methods:In the first section of this thesis,all the predicted protein sequences of E.pubescens were used to construct a local protein database,and the HMM search was combined with the online database of PFAM and NCBI Batch CD-search to comprehensively identify the UbiA PTs.The candidate genes of flavonoid prenyltransferases were screened by analyzing the phylogenetic relationships,chromosomal locations,and gene expression patterns among different tissues.Next,nested PCR technology was used to clone candidate genes using the cDNA of E.pubescens leaves.The sequence characteristics such as gene structure,physicochemical properties,and transmembrane domain were analyzed.The analysis results provide evidences for the functional verification of the candidate genes.In the second section of this thesis,the function of cloned candidate genes was studied.Firstly,the complete protein-coding sequences EpPTs and the truncated gene sequence without transit peptide EpPT?TPs were amplified,and then the entry vectors pENTR/D-TOPO-EpPTs/EpPT?TPs was constructed through the TOPO cloning reaction.The yeast expression vectors pDR196GW-EpPTs/EpPT?TPs were constructed by site-specific recombination with LR Clonase.The constructed yeast expression vectors were transformed into yeast strain DD104.The positive yeast recombinant confirmed by colony PCR were incubated with the presumed 17 types of flavonoid substrates,respectively.Finally,HPLC was used to detect the reaction products.Results:According to the results of genome assembly of E.pubescens,a total of 24 PT sequences containing UbiA conserved domains were identified.Through analysis of phylogeny,expression patterns,and gene replication events,11 possible flavonoid prenyltransferase candidate gene sequences were found(EVM0033754,EVM0036836,EVM0018117,EVM0011267,EVM0010669,EVM0011343,EVM0022490,EVM0029975,EVM0031923,EVM0032839 and EVM0033386).The analysis results suggested that the flavonoid prenyltransferase in Epimedium might evolved from the genes involved in vitamin E biosynthesis.Seven gene sequences were cloned from cDNA of E.pubescens leaves by nested PCR: EpPT1 a and EpPT1b(two different transcripts of EVM0029975),EpPT2(EVM0022490),EpPT3(EVM0010669),EpPT4～EpPT6(no complete genome sequences were found in the genome).Besides,EpPT7(EVM0032839)was not successfully cloned,so the complete protein-coding sequence was synthesized.Through further bioinformatics analysis,all the EpPTs showed typical sequence characteristics of plant flavonoid prenyltransferase.The complete protein-coding sequences EpPTs and the truncated gene sequence without transit peptide EpPT?TPs were amplified by PCR.A total of 10 yeast expression vectors including PDR196GW(empty vector),pDR196GW-EpPT2(positive control),pDR196GW-EpPT2 ? TP82,pDR196GW-EpPT2 ? TP32,pDR196GW-EpPT3,pDR196GW-EpPT3 ? TP51,pDR196GW-EpPT4 ? TP32,pDR196GW-EpPT5,and pDR196GW-EpPT5?TP49 were constructed and transformed into yeast strain DD104.The yeast incubation tests showed that EpPT2 mainly catalyzed the prenylation of kaempferol,and catalyzed quercetin and apigenin to produce a small number of products as well.However,none of the products were detected when reacted with the remaining 14 types of flavonoid substrates.Conclusion:In this paper,based on the results of genome assembly and transcriptome sequencing of E.pubescens,a detailed bioinformatics analysis of UbiA gene family members was carried out to clarify the evolutionary relationship of flavonoid prenyltransferase.Combined with molecular cloning technology and yeast expression technology,a flavonol prenyltransferase gene,EpPT2,was cloned and identified,and the main product was prenylkaempferol.EpPT2 may be the key enzyme in the biosynthesis of icariin active ingredients.This study provides a basis for further analysis of the flavonoid biosynthesis pathway in Epimedium and lays a foundation for continuing to obtain other flavonoid prenyltransferases from Epimedium.