Cloning,Expression And Functional Study Of Terpenoid Synthase Genes CpTPS10 And CpTPS14 From Chimonanthus Praecox

Chimonanthus praecox(Linn.)Link is a well-known traditional flower and tree in China.Its fragrant flowers blossom in the winter when everything withers.It has beautiful posture and is widely used in cut flowers,green planting,garden cultivation and industrial spice production.It has high greening and commercial value.In this study,based on the results of previous studies on volatile substances in flowers of Fragrant Plum H29 and Fragrant Plum SW001 at different stages,CpTPS 10 and CpTPS 14 genes of terpene synthase were screened by transcriptome analysis of H29-SW001.The relationship between gene expression and volatile substances was found by real-time quantitative expression analysis and volatile substance analysis.Subsequently,the cDNA,gDNA and promoter sequences of these two genes were cloned,and their sesquiterpene genes were preliminarily identified by biological analysis.By constructing pCAMBIA2300 binary vector and transforming early-blooming tobacco to verify its preliminary function,the main results are as follows:1.Terpene synthase gene(TPS)downstream of terpene metabolic pathway may play an important role in the difference of flower fragrance.The differentially expressed genes CpTPS10 and CpTPS14 of Prunus chinensis H29 and SW001 were identified by analyzing the transcriptome library data.Real-time quantitative expression analysis of CpTPS10 and CpTPS14 in five floral organ samples of Chimonanthus chinensis was carried out.It was found that the expression trends of CpTPS10 and CpTPS14 genes in H29 and SW001 Chimonanthus chinensis were different.2.We cloned the DNA sequences of CpTPS10 and CpTPS14 genes from H29 and SW001 strains respectively,and found no difference.We constructed a molecular phylogenetic tree and clustered the two genes in TPS-a subfamily.3.The gDNA sequences of CpTPS10 and CpTPS14 genes cloned in H29 and SW001 respectively showed that the gDNA sequence of CpTPS10 had 7 exons and 6 introns in H29,while there was no intron in SW001,and the sequence was identical with the sequence of CpTPS10 and CpTPS14.The gDNA sequence of CpTPS14 has 6 exons and 5introns in H29 and SW001.There are 69 differences between them and two deletions of14 bp and 51 bp,respectively.Both deletions are in the intron.4.The promoter sequences of CpTPS10 and CpTPS14 genes cloned from H29 and SW001 respectively showed that there were 26 base differences in CpTPS10 promoter between H29 and SW001.The cis-reaction elements ABRE3 a,ABRE4 and chs-CMA2 a specific to H29 may be related to abscisic acid response and light response.The unique MBS and O2-site in SW001 are cis-acting regulators involved in drought-induced MYB loci and zein metabolism.CpTPS14 promoter has five base differences and one 5bp fragment difference in H29 and SW001.There is no difference in the types of cis-response elements of promoter.SW001 has two TATA-boxes more than H29.5.Tobacco plants overexpressing CpTPS10 and CpTPS14 were obtained by leaf disc method.The aroma components were identified by GC-MS.It was found that the contents of sesquiterpene caryophyllene and aristolocene in CpTPS10 transgenic tobacco were significantly higher than those in the control.The contents of monoterpene linalool,sesquiterpene 4,5-di-epi-aristolene in CpTPS14 transgenic tobacco were significantly higher than those in the control.The content of linalool increased significantly,6.8 times as much as that of the control.