Identification And Characterization Of A4-coumarate:CoA Ligase Gene Family In Isaits Indigotica Fort

The enzyme4-coumarate:CoA ligase (4CL) activates cinnamic acid and itshydroxylated derivatives by forming the corresponding CoA thioesters. These serve assubstrates for biosynthesis of phenylpropanoid-derived secondary metabolites for themedicinal herbal Isatis indigotica. The4CL genes exist in plants as a family with multiplemembers.To investigate the function of4CL genes in Isatis indigotica, we havecharacterized these genes named Ii4CL2with Genbank accession No. KC430622andIi4CL2with Genbank accession Genbank accession No. GU937875. The full-length cDNAof Ii4CL1was1975bp andcontained a1692bp ORF encoding a564aminoacid protein,meanwhile, the full-length cDNA of Ii4CL2was1932bp andcontained a1692bp ORFencoding a543aminoacid protein.Phylogenetic analysis places the Ii4CL1into a thirdgroup distinct from the common type I and type II4CL,but the Ii4CL2and Ii4CL3into thegroup of type II4CL.Real-time quantitative PCR analysis indicated that all Ii4CLs were expressed in roots,stems, leaves and flowers of I. indigotica, with the highest expression level in roots andflowers. The elicitor treatment experiments using methyl jasmonate (MeJA), and UV-Brevealed that Ii4CL respond to these elicitors in different manners. Yellow fluorescentprotein location studies revealed that the Ii4CLs’ proteins are localized in the cytosl. Thefull-length of ORF was sub-cloned into bacterial expression vector pET32a(+), which wasthen transferred into E.coli BL21(DE3). The recombinant protein had high expression levelin E.coliBL21(DE3) with IPTG induction. Affinity-purified recombinant proteins weretested with several substrates, including cinnamic acid and several of its hydroxy-ormethoxy-derivatives, for their ability to form the corresponding CoA thioesters. Whereasboth4CL2and4CL3catalyzed the formation of CoA esters of caffeic acid and ferulic acidcinnamic acid.Several single-gene over-expression vectors containing4CL1,4CL2,4CL3, andseveral single-gene suppress-expression vectors containing bypass-enzyme4CL1,4CL2,4CL3, respectively, were successfully constructed and transformed to I. indigotica hairyroot. In the Ii4CL1knockout transgenic affairs, results showed that the expressions of4CL2,4CL3, PLR and contents of Lariciresinol nearly no changed. Whereas, in theIi4CL2knockout transgenic affairs, Results showed that the expressions of4CL3,PLR up-regulated2to3and3-fold respectively and contents of lariciresinol increased about5-fold over control. Then, in the Ii4CL3knockout transgenic affairs, results showed that theexpressions of4CL1,4CL2up-regulated2and5-fold respectively but the expressions ofPLR down-regulated to60%of the control. Meanwhile, contents of lariciresinol decreasedto about0.4over control.Therefore,we revealed that4cl3may mainly affects on thelariciresinol synthesis pathway. Profiling analysis of genes and metabolites (intermediates)involved in the synthesis pathway was being performed in details. This study provided anew responsive model system to profile modulations in biosynthesis of important plantsecondary metabolites, and will certainly help us to globally and deeply understandmetabolic flux of the phenylpropanoid-derived secondary metabolites synthesis, both atstressed-elicitation and genetic-regulation levels, also be a great motivation forbioengineering the secondary metabolics in I. indigotica..