Isolation And Founctional Preliminary Analysis Of MYC2L1 And MYC2L2 Gene In Ophiorrhiza Pumila

Ophiorrhiza pumila which belongs to Rubiaceae Ophiorrhiza,is a perennial herb,containing the anticancer drug camptothecin(CPT)and its derivatives.Camptothecin derivatives are widely used in the clinical treatment of cancer,but the shortage of drugs has become a huge bottleneck for the development and application of camptothecin compounds.The development of plant secondary metabolism engineering provides a good solution to solve the shortage of drug sources and increases the content of medicinal components.In this study,the related genes involved in the regulation of camptothecin biosynthesis were excavated by transcriptome sequencing and genetic engineering of Ophiorrhiza pumila,and the function of the genes was preliminary studied,and the transgenic material with high yield of camptothecin was obtained.1.According to the annotation of Arabidopsis gene sequence,16 full-length bHLH transcription factors that may be involved in the regulation of secondary metabolism synthesis were selected for subsequent analysis.Through gene cloning,domain and function prediction,tissue expression profiling,etc,the two transcription factors,OpMYC2L1 and OpMYC2L2,were finally selected for functional studies.2.In order to confirm the roles of OpMYC2L1 and OpMYC2L2 in the camptothecin synthesis pathway,the overexpression and inhibition expression vectors of OpMYC2L1 and OpMYC2L2 were constructed,respectively,and functional verification was performed in Ophiorrhiza pumila.In the over-expressed strains,the content of camptothecin was increased by more than 2 times,and Seconloganin was decreased,but the decrease was not significant.3.The yeast one-hybrid system was used to analyze the regulation which between OpMYC2L1,OpMYC2L2 and the TDC,CPR,G10 H and SLS genes in the camptothecin synthesis pathway.The results showed that OpMYC2L1 and OpMYC2L2 can bind to the G-box in the key enzyme SLS promoter.Therefore,we speculate that OpMYC2L1 and OpMYC2L2 may be involved in the regulation of the expression of the key enzyme SLS.4.In order to analyze the roles of OpMYC2L1 and OpMYC2L2 in the methyljasmonic acid pathway,the yeast two-hybrid and BiFc systems were used tostudy the interaction between these two transcription factors and upstream JAZ.The yeast two-hybrid study showed that JAZ1,JAZ2,JAZ3,JAZ4,and JAZ5 interact with OpMYC2L1,and JAZ1,JAZ2 can interact with OpMYC2L2.The BiFc experiment also verified that JAZs can interact with OpMYC2L1 and OpMYC2L2.Therefore,it is speculated that there is an interaction between OpMYC2L1 and OpMYC2L2 and JAZ,which may participate in the response induced by exogenous methyl jasmonate and affect the expression of SLS gene downstream of camptothecin synthesis.In this thesis,the functions of two genes,OpMYC2L1 and OpMYC2L2,were studied,which provided a theoretical basis and technical support for the establishment of the synthesis regulation network of camptothecin.