Identification And Functional Analysis Of PT, TPS And P450 Genes In The Pineal Moss Synthesis Pathway

Terpenoids are a kind of natural product subiquitous in plants,bacterial and fungal,and have important physiological and ecological value.Isoprene(C5)as terpenoids compounds important units,on the basis of unit number terpenoids divided into monoterpenes,sesquiterpenes and diterpenes,etc.Terpenoids biosynthesis is complex with a variety of enzymes participation and coordinating role.Precursors C5 were catalyzed by prenyltransferases to generate intermediums C10,C15 and C20,etc.Terpene synthases(TPSs)are key enzymes in terpenoids biosynthesis.The monoterpene synthases,sesquiterpene synthases,and diterpene synthases catalyzed intermediums to make monoterpenes,sesquiterpenes,and diterpenes.Some terpene can be modified by cytochrome P450 to form terpenoids with specific functions.Pinus taeda,gymnosperm pinus,is the most important fast-growing conifer species in American,which have important economic and ecological value.Due to the pinus genome is extremely big and complex genome sequencing work is a big challenge.Pinus taeda genome sequencing was completed by science team from University of California in 2014,as currently the biggest genome among completed sequencing genome.Enzymes involved in the terpenoids biosynthsis research mostly concentrated in angiosperms,but the research is less in gymnosperm.In order to explore enzymes in the terpenoids biosynthesis pathway in gymnosperm,this study used bioinformatics method genome-wide investigations of the enzyme genes responsible for pinus taeda terpenoid biosynthesis,identification,classification,comprehensively analysis gene structure and conserved motif,expression profile analysis in different tissues and stages,function and evolution analysis.The results showed as follow:1.Identification and analysis of enzymes genes in MVA and MEP pathway.Based on hidden markov prediction model and BLAST analysis,we identified full-length enzymes involved in terpenoids biosynthsis in pinus taeda:MCT,CMK,MDS,HDR and DXR contains only one gene,HDS contains four genes,DXS contain three genes in MEP pathway;With the exception of the MVD and PMK containing only one gene,AACT contains four genes,HMGS contains three genes,HMGR contains 43 gene,IDI contains seven genesinMVA pathway.Compared to these enzymes in arabidopsis thaliana,the number of HMGR in MVA pathway varies greatly.Analysis of Oryza sativa,Sorghum bicolor,Populus trichocarpa,Zea mays,we found that the number of HMGR gene is associated positively with the genome size.2.Identification and function analysis of prenyltransferase.The prenyltransferase,FPS,GPS,GFPPS,GGPS with full-length,has identified in pinus taeda:1 FPS gene,one GPS gene,two GFPPS genes,and 23 GGPS genes.Evolutionary analysis found that the four kinds of prenyltransferases divided into three obvious evolution clades in phylogenetic tree due to the sequence difference and function differentiation.FPS and GPS form independent clade respectively,GFPPS and GGPS forming a clade.Prenyltransferase in pinus taeda and other annotated prenyltransferases were constructed sequence similarity network with 507 nodes and 1692 edges.Based on sequence similarity network prenyltransferase function were annotated.3.Identification and function analysis of TPS.Based on Hidden Markov Model 68 full-length TPSs genes in pinus taeda was identified.Compared with angiosperm pinus taeda TPS genes have amplified in evolution,and forming the independent TPS-d family.Pinus taeda TPS still contain a classic DDXXD motif based on multiple suquence alignment.Gene structure analysis showed pinus taeda TPS genes contain a lot of long intron,the largest intron about 25 kilobases.Pinus taeda TPSs were divided into TPS-d1,TPS-d2,TPS-d3 subfamilies.Some pinus taeda TPSs function were predicted based on phylogenetic tree.Promoter analysis showed 110 cis-acting elements were found in the promoters of pinus taeda TPS genes.TPSs in pinus taeda and other annotated TPSs were constructed sequence similarity network with 465 nodes and 6796 edges.Based on sequence similarity network some TPSs function were annotated.4.Identification and analysis of P450.257 P450 genes were identified in pinus taeda.These P450 genes divided into 35 families,of which the A type P450 has 15 families totally 161 genes and non-A type P450 contains 20 families totally 96 genes.Compared with angiosperms,Member number of CYP73?CYP78 and CYP98 family in pinus taeda,which exist before angiosperms and gymnosperm differentiation,is consistent with angiosperms.5.Function correlation analysis of genes involved in terpenoids biosynthesis.Coexpression network were constructed by genes involved in terpenoids biosynthesis,which contain 446 nodes representing gene and 4992 edges representing correlation among gene expression.Coexpression ntwork analysis found two groups of relating gene in terpenoids biosynthesis:(A)HMGS,HMGR,GGPS,TPS,P450,it may speculated that function of the group relating genes are related to diterpene biosynthesis.(B)HMGS,HMGR,PMK,IDI,FPS,TPS,P450,it may speculated that function of the group relating genes are related to sesquiterpene biosynthesis.Conclusions:this study investigated genome-widely the PT,TPS,P450 genes involved in terpenoid biosynthesis in pinus taeda and deeply analyzed these genes using bioinformatics method.Moreover based on phylogenetic tree and sequence similarity network the function of these enzymes were predicted.Finally based on coexpression network analysis function correlation of genes involved in terpenoids biosynthesis were identified.The research may provide important theoretical basis for function study of gene involved in terpenoids biosynthesis and terpenes application.