Cloning And Functional Analysis Of Transcriptional Factor CaIAA27 In Camptotheca Acuminata

Camptothecin(CPT)is the second broad-spectrum anticancer drug extracted from plants after Taxus chinensis extract paclitaxel.It is also the only natural plant discovered to date to inhibit cytotoxicity by inhibiting topoisomerase I Active ingredient.The main plant source of CPT is Camptotheca acuminata,but the content in Camptotheca acuminatais extremely low,only a few thousandth of the dry weight,resulting in extremely low total yield.At present,it is difficult to prepare CPT by chemical synthesis,and there is no large-scale production.In addition,Camptotheca acuminata has become a rarely and endangered plant in China,with limited natural resources and the increasing demand for CPT in the market,the resources of CPT are continuously overexploited and utilized.Therefore,the price of CPT is not only expensive but also in short supply situation.It is important to use different methods to increase the content of CPT in Camptotheca acuminata,in which hormones are involved in the biosynthesis of alkaloids as signal molecules.indole-3-acetic acid(IAA)is the earliest discovered and most important class of plant hormones,its main role is to promote growth and promote differentiation,but the impact of exogenous IAA treatment of Camptotheca acuminata on its growth and in accumulation CPT content has not been reported.According to the research of this thesis,the accumulation of CPT increased significantly under the treatment of hormone IAA,and the relative expression of key enzyme genes in CPT synthesis increased.It was determined that hormone IAA would change the accumulation of CPT in camptotheca acuminata.Transcription factors are effective tools for regulating the co-expression of multiple genes.A transcription factor related to CPT synthesis can synergistically control the expression of multiple synthetase genes in the CPT metabolic pathway,thereby effectively promoting the mass of CPT.Synthetic,thus functionally more economical,comprehensive and effective.Based on the known characteristics of enzyme gene expression and accumulation of metabolites in the biosynthesis pathway of CPT,the high-throughput transcriptome database of young leaves and mature leaves of Camptotheca acuminata was used to analyze the similarity of genes.A transcription factor involved in the regulation of CPT synthesis by the hormone IAA signal is obtained,and the candidate gene is cloned and a full-length coding sequence is obtained.The overexpression vector driven by constitutive promoter(35S)and inducible promoter(rd29A)was constructed,and Agrobacterium transformation and identification were carried out.The leaves of Camptotheca acuminata were infested by injection infiltration,and the treated leaves were collected and RNA was extracted.Real-time quantitative PCR(q RT-PCR)analysis of several key enzyme genes on the CPT biosynthesis pathway and HPLC analysis of camptothecin accumulation.The main purpose of this study was to screen and identify a transcription factor related to hormonal signaling,and to reveal the molecular mechanism of hormone signaling to regulate the synthesis of CPT,in order to provide the necessary genetic components for the genetic engineering of transcription factors to regulate camptothecin synthesis.The theoretical basis to meet the growing market demand for CPT.In this study,one of the two candidate transcription factor genes(CaIAA27)with similarity in gene expression characteristics and metabolite accumulation characteristics was cloned and transient overexpression analysis was performed.The results showed that the content of CPT in the overexpressed leaves of p BI121-35S-GD-CaIAA27 increased by 1.72 times,reaching a significant level,and several key enzyme genes CaG8 O,Ca7DLS,CaCYC1,CaSTR,Ca7 DLGT and CaTDC1 increased by 257.83,117.95,83.49,19.03,and 11.40 times,respectively,and CaG8 O and Ca7 DLS reached significant levels.The relative expression levels of key enzyme genes CaG8 O and Ca7 DLS increased by 2.69 and 1.74 times,respectively,after p BI121-rd29A-GD-CaIAA27 overexpression at low temperature for 24 h.After 48 hours of induction,only the relative expression level of CaTDC1,a key enzyme gene,increased by 1.04 times,and the difference was not significant.The samples from different parts of the wild-type Camptotheca acuminata seedlings of different growth time were ground,total RNA was extracted,and the expression level of CaIAA27 gene was analyzed by q RT-PCR.The 30-day CaIAA27 gene was transcribed at different sites compared with 60 days.The average height of leaves,phloem and root reached2.36,4.74 and 10.10 times,respectively,and the difference reached a significant level.The epidermis was 3.22 times,respectively,and the difference was not significant.The results showed that exogenous IAA can promote the synthesis of CPT.The transient expression of CaIAA27 can significantly promote the expression of the enzyme gene in the CPT synthesis pathway,and promote the synthesis of CPT.CaIAA27 is an important hormone signal transduction pathway.The transcription factor,which mediates the hormonal signal regulation of CPT biosynthesis,provides a new way for hormones to regulate plant secondary metabolite production.