Cloning?expression And Functional Study Of Laicases From Salvia Miltiorrhiza Bunge(Smlacs)

Dan Shen(Salvia Miltiorrhiza Bunge,S.miltiorrhiza),is a kind of traditional Chinese medicine with long history mainly used to cure cardiovascular disease.Rosmarinic acid(RA)and Lithospermate-B(LAB)are the main water-soluble active ingredients inside the plant.It was found that rosmarinic acid(RA)was an important precursor for the synthesis of LAB,and it was suggested that laccase is a key enzyme that catalyzes the transformation of RA into LAB in plants.The purpose of this study is to validate this hypothesis and to explore the function and function of the laccase gene family in the phenolic acid metabolic pathway.We obtained the laccases from the genomic database of S.miltiorrhiza and obtained the target laccase gene by means of various means to explore its role in the metabolic pathway of salvianolic acid in vitro and in vivoThis paper shows studies including functions of SmLACs in vivo and in vitro.1.Commercial Rhus verniciflua laccases were used to catalyze rosmarinic acid(RA),and obtained signal of lithospermate B(LAB)in the production.This results indicated that plant laccases might be the key enzyme which turns RA into LAB.2.Then we did bioinformatics analysis of genome database to dig out full of the laccases information in S.miltiorrhiza.The results of blast between laccase genes from NCBI and the genome database of S.miltiorrhiza shows that 29 pieces of complete SmLACs exist totally,while the characteristics of the proteins had been computed,such as physical and chemical properties,protein structure and the sequence identity etc.3.Combined the results of induction of MeJA(increase with the drug)and the tissue specific expression(high expression in root),we finally got 3 target genes SmLAC7,SmLAC2 and SmLAC28.These three would be the research object of our study.4.We got these three target genes by PCR reactions and did protein expression by using Pichia pastoris.We have completed the construction of the vectors and imported them into GS115 cell strain.5.We bulit RNA interference system,then obtained the transgene hairy roots showed distinctive morphological characteristics with control ones by RT-PCR and qPCR.The expression of target genes apparently lower than that in control,besides,the diversity of shape pattern among the samples was obvious.The assay of RA and LAB in transgene hairy roots was proceed by LC-MS/MS,we saw that the accumulation of LAB decreas.However,the accumulation of RA increased a little.Actually,laccases in S.miltiorrhiza could influnence LAB synthesis in vivo.6.For further study,we did metabolic engineering study about Smlac7 and Smlac20 by inducing hairy roots with over-expressing these genes.Results of RT-PCR and qPCR showed that the expression of target genes improved in various degrees.The gene over-expressing hairy roots,with wider cambium layer and bigger protoxylem,grew faster and thicker than that of control.At the same time,the accumulation of LAB in transgene group was much higher than that in control,and the accumulation of RA was not decreasing.Over-expressing SmLAC in vivo could be a novel mean to regulate content of RA and LAB in S.miltiorrhiza.In summary,this study screened laccases in S.miltiorrhiza by bioinformatic analyze and got 3 target genes to verified their function.Detected the inside connection between RA and LAB on the pathway of secondary metabolism for the first time.Extend the knowledge of laccases on the regulation of secondary metabolites.This could be a theory base for genetic reorsanization and breeding.At the same time,the experimental results also remind us that there are other genes that may be involved in the phenolic acid metabolic pathway in the luciferine family,and we need to conduct a comprehensive exploration.