The Application Of Transcription Factor ORA1 From Camptotheca Acuminata In Camptothecin Biosynthesis

Camptothecin(CPT)is an important secondary metabolite in Camptotheca acuminata.Its derivatives are recognized as anti-tumor drugs in the clinic.Camptothecin is mainly extracted from the leaves and fruits of C.acuminata.Camptothecin is extracted from C.acuminata leaves and fruits.Because of its low content in plants,only a large supply of raw materials can meet the huge market demand.As an important regulator of secondary metabolites in medicinal plants,transcription factors had attracted much attention in the study of biosynthesis and regulation of camptothecin.Studies have shown that both vinblastine and camptothecin belong to the terpenoid indole alkaloids,and there are some common upstream metabolic pathways.In addition,studies have shown that ORCA(Octadecaniod-derivative Responsive Catharanthus AP2-domain protein)in Catharanthus roses had a significant promoting effect on the biosynthesis of vinblastine by acting on key enzyme genes in the upstream pathway.However,ORA transcription factors in C.acuminata have rarely been reported.Therefore,it is important to clone such transcription factors and study their functions in C.acuminata.In this paper,homologous sequence aliagnment was used to find a transcription factor highly homologous to ORCA in the transcriptome database of C.acuminata,and name it ORA1.The ORA1 was cloned from the leaves of C.acuminata by molecular biology.The expression vector p BI121GD-ORA1 and the low temperature-induced expression vector rd29A::ORA1 were constructed.Phylogenetic analysis showed that the protein sequence of ORA1 is highly homologous to the protein sequence of ORCA protein in C.roses and several ERF genes in the Arabidopsis,and their domains are very conserved.The results of primary,secondary and tertiary structure predictions showed that ORA1 has a molecular formula of C₁₂₀₉H₁₉₂₄N₃₄₆O₃₇₄S₄,it has a composition of 249 amino acids and with a size of 27.4KDa;61.04%of the total length of ORA1 protein is random coil,23.29%and 12.05%of the total length is?-helix and extended chain,respectively;the three-dimensional structure prediction result was consistent with the secondary structure prediction.Then,the ORA1 was transiently overexpressed in the leaves from C.acuminata by Agrobacterium-mediated transient transformation.The effect of constitutive overexpression of ORA1 on the biosynthesis of camptothecin in the leaves of C.acuminata was determined by HPLC.The results showed that the accumulation of camptothecin increased 68%.Meantime,real time PCR was used to determine the transcription levels of key enzyme genes in the camptothecin biosynthesis pathway in the constitutive overexpression group and low tempreture-induced expression group(0 h,24 h and 48 h)of ORA1.The results showed that Ca7DLGT,Ca G8O,Ca7DLS,Ca CYC1 and Ca STR were up-regulated by overexpression of ORA1 to 4.25,85.0,261.85,5.51 and 5.00 times of the control group,respectively.The JA response gene Ca JR3 and the JA biosynthetic gene Ca AOC2 were upregulated significantly.In addition,after 24 h of cold induction at 4°C,the expression of upstream key enzyme genes in the camptothecin biosynthesis pathway was up-regulated in the experimental group compared with the control group.For example,Ca G8O,Ca7DLS,Ca7DLGT and Ca STR were increased to 42.50,9.23,5.02 and 5.00 times of induced 0 h,respectively,and Ca AOC2 was increased to 14.57 times,and the transcription level of the above genes were increased with the increase of cold induction time.The above results indicated that overexpression of ORA1 in the leaves of C.acuminata promoted the expression of Ca STR,Ca G8O and Ca7DLS in the upstream pathway of camptothecin biosynthesis,and promoted the biosynthesis of camptothecin.At the same time,ORA1 was associated with JA signal.In this study,the transcription factor ORA1 from C.acuminata was successfully cloned.The accumulation of camptothecin was improved by over-express of ORA1 in C.acuminata leaves.And research shows that ORA1 may promote the expression of Ca STR,Ca G8O and Ca7DLS in the upstream pathway of camptothecin biosynthesis by promoting JA synthesis and its signal transduction,and ultimately promote the biosynthesis of camptothecin.This paper provides data support for the study of the molecular mechanism by transcription factor ORA1 regulats the synthesis of camptothecin,and provides a new research strategy for the final regulation of camptothecin biosynthesis by transcription factors in C.acuminata.