Woad Wooden Fat Students Synthetic Ways Five Key Enzyme Gene Function Research

Objective: IiC3H and IiPLR involved in the lignan metabolic pathway were successfullycloned from Isatis indigotica for the first time.The characteristic and function of this pathwaygenes including IiC3H，IiPLR，Ii4CL，IiCAD and IiUGT were then studied.These studiesprovided qualification and strategy for enhancing desired components content by using metabolicengineering,also provided basic to explore metabolic regulation mechanism of the lignanbiosynthesis.Methods: Molecular cloning of IiC3H and IiPLR from I. indigotica were carried out byRapid amplification of cDNA ends (RACE) method. The related bioinformatics analysis wascarried out by the software. We detected the expression level of five key genes in differenttissues (leaves, roots, stems and flowers) and under different treatments(MeJA and UV) indifferent periods by Real-time PCR (RT-PCR). The recombinant Ii4CL, IiCAD, IiUGT, IiPLRin bacteria were induced by IPTG and the expression of fusion protein were examined by SDS-PAGE. The expression levels of IiC3H and IiPLR genes were examined by RT-PCR andcontent of compounds involved in the lariciresinol metabolic pathway in transgenic hairy rootsby LC-MS-MS.Results: We cloned two key genes from I. indigotica by RACE and designated them asIiC3H (GenBank accession: JF826963) and IiPLR（Genbank accession No. JF264893）. Thefull-length cDNA of IiC3H was1830bp containing an open reading frame (ORF) of1527bp,encoding a polypeptide of509amino acids. Comparative and bioinformatics analysis revealedthat IiC3H showed high homology with other known C3Hs. The386bp and249bp intronswere present in the genomic DNA.The full-length cDNA of IiPLR has a total length of1062bpwith ORF of953bp, and is predicted to encode a protein of317amino acid residues，sharinghigh degree of homology with PLRs from other plants. RT-PCR analysis revealed that fivegenes were expressed in all tissues,Ii4CL, IiUGT and IiPLR were preferentially expressed inroot compared with other tissues,the highest expression of IiC3H is in the Stem, IiCAD inflower.When I. indigotica hairy roots were induced by MeJA and UV, these five genesexpression were also up-regulated. The recombinant Ii4CL, IiCAD, IiUGT and IiPLR inbacteria were induced by IPTG and the expression of fusion protein was revealed by SDS- PAGE. In the IiC3H, IiPLR transgenic affairs, the RT-PCR result showed that the expression ofIiC3H, IiPLR were up-regulated4and8-fold respectively. The accumulation of Lariciresinoland Sinapyl alcohol were dramatically up-regulated by overexpression IiC3H, bothLariciresinol and Sinapyl alcohol content ncreased to4fold and9fold of CK. The contents oflariciresinol in IiPLR transgentic hairy roots increased9-fold over control.Conclusion: We first cloned two key genes from I. indigotica making all genes cloned inthe lariciresinol metabolic pathway, providing basis for further studying metabolic regulationmechanism in this pathway. In the two transgenic affairs, the expression of IiC3H and IiPLRwere up-regulated and contents of secondary metabolites were successfully increased.This studyeffectively boosts the lariciresinol metabolic flux,providing available strategy for plantsecondary engineering breeding.