Cloning And Functional Verification Of Triterpene Cyclase Gene In Chuanshegan

ObjectiveChuanshegan is a dried rhizome of Iris tectorum Maxim.About 300 species of iris plants have been found all over the world and about 60 species in China.They have ornamental and medicinal value.Chuanshegan is a medicinal material adopted by Chinese Pharmacopoeia.The main effects are clearing heat and detoxification,eliminating phlegm and benefiting the pharynx.Recently,a series of novel new iridal-type triterpenes have been identified from Iris tectorum Maxim,which have good anticancer activity.For example,four monocyclic iridal-type triterpenes(iritectols A,iritecols B,isoiridogermanal,and iridobelamal A)have good cytotoxic activity on human cancer cell.It indicates that iridal-type triterpenes have medicinal prospects in the treatment of cancer.Chuan Shegan contains many types of triterpenes,in addition to the common tetracyclic or pentacyclic triterpenes,there are a series of structurally specific triterpenes such as irisaldehyde triterpenes.However,the research on the triterpene biosynthesis pathway of Chuanshegan is still unclear,and the biosynthesis and molecular mechanism of iridal-type triterpenes have not been reported.Therefore,the cloning and verification of the key enzyme gene(2,3-oxdiosqualene cyclase)in the triterpene biosynthesis pathway of Chuanshegan will provide clues for the study of iridal-type skeleton biosynthesis.It will find new catalytic enzymes and provide a new way for biological transformation and discover anticancer lead compounds.Finally,it has important significance for utiling the plentiful iris plants resource.Methods(1)The rhizomes,leaves and flowers of Chuanshegan were frozen in liquid nitrogen and sent to a company for transcriptome sequencing.The candidate OSC genes were sceened out by local blast procedure from the data of transcriptome.The open reading frame of candidate OSC gene was analyzed to determine whether it was a full-length sequence.(2)For full-length ItOSCs,they were constructed into yeast expression vector by gene cloning and homologous connection.Their biochemical functions were verified in yeast system.For non full-length ItOSCs,the full-length sequence was obtained by RACE technology.Then construct the yeast expression vector and verificate the biochemical functions.Codon optimization of ItOSC4,and construct yeast expression vector and verify it’s biochemical function(3)ItOSC2 and ItOSC4 were constructed into tobacco expression vector.Their biochemical functions were verified in tobacco expression system with infection approach of Agrobacterium tumefaciens.The results between yeast expression system and tobacco expression system were compared.(4)The expression of ItOSCs in rhizome,leaf and flower was analyzed by the technology of Quantitative Real-time PCR(q PCR).The content of triterpenoids in rhizomes and leaf of Chuanshegan was analyzed by GC-MS.Compare the content distribution of triterpenes with the gene expression of ItOSCs.(5)By using the crystal structure of human lanosterol synthase(Hs LSS)as a template,establishing a homologous modeling molecular model of ItOSCs.The QM/MM-MD simulations and sequence comparison with other OSC sequences were used to analyze the protein sequence characteristics of ItOSCs and key catalytic amino acids of ItOSCs.Site-directed mutagenesis experiments were performed to verify the catalysis of these key amino acid residues and the catalytic mechanism of enzymes.(6)A phylogenetic tree was constructed by alignment the protein sequences of known plant OSCs,and the evolutionary relationship of ItOSCs were analysed.Results(1)A total of 6 candidate ItOSCs genes were obtained from the transcriptome data,and they were named ItOSC1,ItOSC2,ItOSC3,ItOSC4,ItOSC5,ItOSC6.ItOSC1,ItOSC2,ItOSC6 have full-length gene sequences,but ItOSC3,ItOSC4,ItOSC5 have not.The full-length sequence of the ItOSC3,ItOSC4,ItOSC5 were obtained by 3’-RACE technology.The gene sequence of ItOSC5 is consistent with ItOSC4,so it is judged that ItOSC5 and ItOSC4 are actually the same gene.Totally,5 candidate ItOSCs genes were cloned from Chanshegan.(2)The biochemical functions of the four genes ItOSC2,ItOSC3,ItOSC4,and ItOSC6 were verified in the yeast expression system.Because ItOSC1 was failed to construct to yeast vector,its biochemical function has not yet been verified.ItOSC2 is a multifunctionalα-amyrin synthase whose products are α-amyrin,β-amyrin,and δ-amyrin(22: 1.7: 1).The product of ItOSC3 is cycloartenol.ItOSC4 was failed to produce a catalytic product in the yeast system at first.After its codons were optimized,the biochemical function of ItOSC4 was β-amyrin synthase.The catalytic product of ItOSC6 was lupeol.(3)The products of ItOSC2 in tobacco expression system and yeast expression system are consistent,and are all α-amyrin,β-amyrin,and δ-amyrin.The product of ItOSC4 in tobacco expression system β-amyrin is the same as the catalytic product of ItOSC4 in yeast expression system after codon optimization.Therefore,the catalytic functions of ItOSC2 and ItOSC4 were verified in the both system.(4)ItOSC1 and ItOSC3 have similar expression distribution.The order of ItOSCs’ expression levels in various parts from high to low is flowers,leaves,and rhizomes.ItOSC2 and ItOSC4 in order expressed in leaves,flowers,and rhizomes.ItOSC6 in order expressed in leaves,flowers,rhizomes.ItOSC4 highest expressed in rhizomes,and ItOSC4 and ItOSC6 have similar expression in flowers.The distrubitions of α-amyrin,β-amyrin,and lupeol in iris plant were analyzed by GC-MS.The lupeol content was 0.624%,0.008%(dry weight ratio)in the leave and rhizomes respectively.The content of α-amyrin in the leaves was 0.091%,and it was not detected in the rhizomes.The content of β-amyrin in leaves was 0.117% and the content in rhizomes was 0.005%.There is a positive correlation between the results of gene expressions and compound contents.(5)ItOSC2 has a unique catalytic motif(FLALAR)and some key amino acid residues by homologous modeling and sequences camparing.12 amino acid residues were mutated by site-directed mutations.They are F255 L,F255M,L256 M,L256W,A257 C,L258H,L258 Y,A259C,Y531 F,Y531L,Y531 W,F725I.It is speculated that ItOSC6 has 4 key amino acid residues,which were mutated in L255 F,M256L,Y531 W,and F549 L,respectively.The special catalytic mechanisms of ItOSC2 and ItOSC6 were explained by molecular dynamics calculations and site-directed mutations.(6)The phylogenetic tree was constructed including ItOSC2,ItOSC3,ItOSC4,ItOSC6 and other OSCs.The results showed CASs were clearly classified between monocots and dicots.ItOSC3 clustered in monocot CAS clusters which is consist with the actual situation.The other OSCs in dicots and monocots are completely separated from CASs,and they also seperated between dicots and monocots.So we determine the ancient CAS gene(important for plants as primary metabolism)firstly evoluted from low algae.With the differentiation of the monocots and dicots plants and genome duplication,the ancient CAS genes have maintained their original functions in the monocots and dicots.Other OSCs were evoluted from the replications of ancient CAS independly in monocots and dicots.The biochemistry functions of OSCs changed from CAS.It is the reasons for the diversity of triterpoids in plants.Although monocots and dicots OSCs can both produce amyrins,they are clustered into different clusters,which indicated that the evolution of OSC occurred after the differentiation of monocots and dicots.Presently,monocots OSCs are rarely identified.ItOSC2,ItOSC4 and ItOSC6 have special motif.The study of ItOSCs in Chuanshegan can better understand the evolutionary history of plant OSCs genes.ConclusionIn this thesis,five ItOSCs candidate genes were excavated and cloned from the Iris tectorum Maxim.Four ItOSCs were biochemically verified through the yeast heterologous expression system and tobacco heterologous expression system.Among them,ItOSC2 catalyzed the production of α-amyrin(87%),β-amyrin,δ-amyrin,is a kind of rare multifunctional α-amyrin synthase in nature.ItOSC3 catalyzes the production of cycloartenol,a conserved monocot CAS enzyme.ItOSC4 is a single functional β-amyrin synthase,ItOSC6 is a single functional lupeol synthase,and the biochemical function of ItOSC1 is on-going.Through homology modeling,site-directed mutation,and phylogenetic tree analysis,it was found that ItOSC2 and ItOSC6 in monocots iris have unique catalytic motif and unique catalytic mechanism.