Functional Identification And Promoter Activity Analysis Of CiDXR,a Key Gene For Terpenoid Synthesis In Chrysanthemum Indicum Var.Aromaticum

Chrysanthemum indicum var.aromaticum is a perennial herb of Compositae Chrysanthemum.The plant has a strong aroma,and its main chemical components are terpenoids such as ?-Thujone,?-Thujone,Borneol,etc.It is a precious spice plant.DXR gene is a key rate-limiting enzyme in the MEP pathway of terpenoids synthesis and has an important effect on the synthesis of terpenoids.In order to explore and improve the expression of key enzyme genes in the terpene synthesis pathway of C.indicum var.aromaticum,in this study,the key enzyme genes of the terpenoid synthesis pathway were predicted and analyzed,and the CiDXR gene and its promoter were cloned and analyzed.The results are as follows:In the transcriptome data of C.indicum var.aromaticum treated with methyl jasmonate,a total of 21 genes were identified for up-regulation of terpenoid synthesis pathways,and no genes for down-regulation were found.The GO biological process annotation mainly focuses on the biological processes of redox process,metabolic process,lyase,helicase and terpene synthase activity.A total of 68 differentially expressed gene fragments were found in the transcriptome of C.indicum var.aromaticum after UV-B treatment,36 were up-regulated and 32 were down-regulated.The GO biological process is annotated to the redox,metabolic processes and catalytic activity.Six genes were annotated to the terpene synthase activity process.The target gene was obtained from the cDNA of C.indicum var.aromaticum leaves by PCR amplification of the target fragment.The gene sequence ORF was 1419bp in length,encoding 472 amino acids,and the sequence had 2 DXR functional domains:LPADSEHSAI and NKGLEVIEAHY.The gene was named CiDXR.The protein structure of CiDXR predicts that a typical conserved domain of NADPH binding is embedded in the domain.qRT-PCR analysis of the expression of CiDXR gene showed that the expression level of CiDXR gene was highest in leaves,and the expression level in stems and roots decreased sequentially.The expression level of CiDXR gene treated with 0.5%methyl jasmonate showed an increasing trend and then a decreasing trend with time.The expression level was highest at 24 hours after treatment.The CiDXR promoter was obtained by cloning from the gene sequence,with a sequence length of 833 bp and containing a variety of cis-acting elements,including 18 promoter core elements TATA-box,10 core elements common to the promoter and enhancer CAAT-box,and 5 optical signal response related elements ACE,G-box,TCCC-motif,TCT-motif,2 methyl jasmonate response regulatory elements CCGCA-motif,TGACG-motif,2 abscisic acid response element ABRE,3 stress response elements MYB,MYC in total,2 zein metabolism regulation element O2-site.The CiDXR gene promoter and the 5 'deletion fragment were fused with the GUS reporter gene to construct a transient expression vector.Agrobacterium-mediated transformation of tobacco was performed.qRT-PCR experiments verified that all three promoter fragments had promoter activity.