Identification And Functional Analysis Of TPS Gene In Perilla Frutescens

Terpenoid volatile oil is an important component of plants,which is involved in the regulation of plant growth and development,response to stress,formation of flavor and taste.Perilla frutescens is an important oil crop rich in volatile oil.The discovery and functional analysis of the key genes of terpenoid volatile oil synthesis regulation can provide an important gene source for the creation of new crop germplasm with high yield,stress resistance and high quality.In this study,based on the identification of chemical types of perilla volatile oil,the key genes of terpenoid volatile oil regulation in perilla were screened and analyzed by transcriptome sequencing technology,and the function of candidate genes was analyzed by genetic transformation of model plant Arabidopsis thaliana.The main research contents and results are as follows:1.Chemical types of P.frutescens volatile oil.GC-MS technology was used to detect the volatile oil from four different varieties of P.frutescens leaves.It was found that the relative contents of volatile oil components in different varieties were quite different.according to this,they were divided into four chemical types:Perillaldehyde type,Perillaketone type,Perillaene type and Menthone type.The typical substances contained were Perillaldehyde 68.01μg·ml^-1,Perillaketone 88.76μg·ml^-1,Perillaene71.65μg·ml^-1 and Menthone 61.20μg·ml^-1。2.Key genes of volatile oil synthesis and regulation.The leaf cDNA libraries of four chemical P.frutescens species were constructed,and the transcriptome was sequenced to analyze the regulatory network of volatile oil formation and explore the key genes.It was found that 834 genes were up-regulated in Perillaldehyde type,373in Perillaketone type,329 in Perillaene type and 542 in Menthone type during the formation of volatile oil.Among them,the genes involved in the regulation of volatile oil in Perillaldehyde type were the most,including the terpenoid synthase gene PfTPS86which was up-regulated in this chemical type and Co-expressed with other genes in isoprene pathway.GO and KEGG enrichment analysis showed that the above differences were mainly concentrated in isoprene pathway,and 78 genes participated in this regulatory pathway.A total of 3195 transcription factors were obtained by genome prediction analysis of P.frutescens,of which 75 were up-regulated and co-expressed with 15 PfTPS genes in isoprene pathway.According to the above results,the candidate key gene PfTPS86 for volatile oil regulation was obtained,and qRT-PCR analysis verified the expression consistency of the gene.3.Characteristics of PfTPS gene family.To study the possible properties and biological functions of PfTPS86 gene,bioinformatics analysis was carried out on PfTPS gene family of P.frutescens.It was found that there were 118 members of PfTPS gene family belonging to 5 subfamilies,among which PfTPS-a subfamily was the largest,with 64 family members,while PfTPS86 belonged to PfTPS-a subfamily,which had promoter elements such as photoresponse and salicylic acid.There are differences in phylogeny,gene structure,expression characteristics,physicochemical properties and cis-acting element composition among members of PfTPS family,which indicates that PfTPS gene family is complex and rich in functions.4.Cloning of PfTPS gene.The gene PfTPS86 was cloned from aldehyde perilla frutescens by homologous cloning technology.Bioinformatics analysis showed that the gene had obvious conserved domain of terpene synthetase,with ORF of 1758 bp,encoding 586 amino acids,molecular weight of 68.18532 KDa,isoelectric point（pI）of5.09,hydrophilicity of-0.347,and the encoded protein was acidic hydrophilic protein.PfTPS86 gene has the highest homology with Perilla citriodora,and phylogenetic tree found that PfTPS86 gene can be divided into one class with wild-type P.citriodora.5.Biological function of PfTPS gene.A plant overexpression vector pCambia2301-35S-PfTPS with 35S as promoter and GUS as reporter gene was constructed and verified by colony PCR.Genetic transformation of PfTPS gene into model plant A.thaliana by flowering method was carried out to study the gene function.Twelve independent transgenic plants of T₀ generation were obtained by GUS histochemistry and genome PCR identification.The growth and development of T₁generation and qRT-PCR detection of leaves showed that transgenic A.thaliana（TP）grew 2～3 days faster than wild type（WT）,and the plants were tall,the seeds were large,and the veins and petioles had color changes.The expression of PfTPS in TP type was higher than that in WT type.The expression of PfTPS gene can promote the growth and development of recipient plants.