Functional Identification Of Bryophyte Steroid Synthase

Terpenoid is a kind of huge and diverse natural organic compound founded in plants,which plays a very important physiological and ecological effects in plant defense,communication and pollination value,it also has important application value in the agricultural,commercial and pharmaceutical industries.In plant,the synthesis of terpenoids is mevalonate(MVA)pathway and 2-methyl-erythritol-4-phosphate(MEP)pathway.Terpene synthases(TPS)play a key role in the formation of various terpenoids through precursors.Bryophytes not only the earliest plants on the land,also the second plant following the angiosperm now.The results of natural compounds in bryophyte,showed that bryophytes had a large proportion of terpenoids,so it was necessary to study the terpenoids and their synthetases in bryophytes.In recent years,the development of high-throughput sequencing technology and the accumulation of bryophytes transcriptional data make it possible to study the terpene synthase-like gene in bryophytes by bioinformatics and experimental methods.In this study,representative bryophytes were used as the research object.First,we identified seven bryophyte TPSL genes(LPSF-TPSL1,WSPM-TPSL1,IGUH-TPSL1,GOWD-TPSL1,GOWD-TPSL3 and VBMM-TPSL3)to study the evolution,sequence conservation and motif analysis of TPSL genes.Second,the protease activity and the function of these genes was verified by prokaryotic expression.Third,we are obtained seven mutations genes that D232K-LNSF-TPSL1,D198K-WSPM-TPSL1,D190K-IGUH-TPSL1,D198K-LNSF-TPSL3,K190D-GOWD-TPSL1,L198D-GOWD-TPSL3 and D206K-VBMM-TPSL3 by site-directed mutagenesis,and analyzed the impact of these mutation genes function.The result show:1.Through the analysis of the evolutionary tree and structure domain,we found that LNSF-TPSL1,LNSF-TPSL3,WSPM-TPSL1,IGUH-TPSL1 and VBMM-TPSL3 genes were located on the TPS-h subfamily branch but not belong to any TPS subfamilies,and their domains are completely different from the GOWD-TPSL1 and GOWD-TPSL3 which belong to the TPS-h subfamily.2.After constructed prokaryotic expression vector,expressed protein and analyzed by GC-MS of Seven bryophytes TPSL genes,we found that trans-geraniol,cis-geraniol and(-)-lavandulol by qualitative analysis of the compounds detected after LNSF-TPSL1,WSPM-TPSL1,IGUH-TPSL1,LNSF-TPS3,GOWD-TPSL1,GOWD-TPSL3 and VBMM-TPSL3 genes expressed protein reacted with GPP.The compounds were only(+)-nerolidol was analyzed by qualitative analysis after GOWD-TPSL3 gene expressed protein reacted with(E,E)-FPP,whereas the compounds of VBMM-TPSL3 gene expressed protein reacted with(E,E)-FPP by qualitative analysis are(Z)-?-farnesene and?-bisabolene.So the seven moss TPSL genes have monoterpene synthase function,and GOWD-TPSL3 and VBMM-TPSL3 genes have sesquiterpene synthase function.3.In order to identify the key amino acid residues in TPSL gene,we used the same prokaryotic expression method to express the seven Moss TPSL genes after mutations,then used the GC-MS analysis the compounds.The protein expressed by D198K-LNSF-TPSL3 and D206K-VBMM-TPSL3 mutant genes also catalyze the function of GPP to form ?-menthone and fennelone,D206K-VBMM-TPSL3 mutant gene expression of the protein has lost the ability to catalyze(E,E)-FPP formation of sesquiterpene compounds;the protein expressed by D198K-WSPM-TPSL1 mutant gene cann't catalyze GPP;the protein expressed by L198D-GOWD-TPSL3 mutant gene can not catalyze(E,E)-FPP.In summary,the aspartic acid which at position 198 in the WSPM-TPSL1 and LNSF-TPSL3 genes was associated with its monoterpene synthase function,the function of 206 aspartic acid in VBMM-TPSL3 gene is related to its function of monoterpene synthase and sesquiterpene synthase and the leucine at position 198 in the GOWD-TPSL3 gene was associated with its sesquiterpene functional activity.The mutations of the amino acid residues in the remaining genes did not affect the monoterpene synthase and the sesquiterpene synthase.The study of TPSL gene function in bryophytes not only enriches the resources of TPS gene,but also contributes to the evolution of TPS gene.Although we have not studied many of the moss TPSL genes,our study helping people to learn more about the TPSL genes in bryophytes.