Cloning And Functional Analysis Of Transcriptional Factor CaTCP1 And CaZHD6 In Camptotheca Acuminata

Camptothecin(CPT)is a kind of monoterpene indole alkaloids with anti-cancer effect,which has very important application in clinical medicine.At present,CPT is mainly extracted from Camptotheca acuminata,a deciduous tree of Davidiaceae,which is unique to China.However,the content of natural CPT in Camptotheca acuminata is very low,even in the most abundant tender leaves,it is only a few thousandths.In addition,CPT has become a rare and endangered protected plant in China,and its natural resources are limited.As a result,the yield of CPT is far from meeting the growing demand.Market demand,therefore,is very important for how to improve the biosynthesis and accumulation of CPT.Transcription factors are effective tools to regulate the co-expression of multiple genes.A transcription factor related to the synthesis of CPT can coordinately control the expression of multiple synthase genes in the metabolicpathway of CPT,which can effectively promote the synthesis of CPT in large quantities,so it is more economical,comprehensive and effective in function.The main purpose of this study is to screen and identify two transcription factors of Camptotheca acuminata,in order to provide the necessary gene elements and theoretical basis for the regulation of CPT synthesis by gene engineering of transcription factors,in order to meet the growing market demand of CPT.Based on the characteristics of gene expression and metabolite accumulation and distribution of known enzymes in CPT biosynthesis pathway,the similarity analysis of gene co-expression was carried out using the constructed high-throughput transcriptome database of young and mature leaves of Camptotheca acuminata,and candidate transcription factor genes were quickly obtained.The candidate genes were cloned and the full-length coding sequence was obtained.Overexpression vectors driven by constitutive promoter(35S)and inducible promoter(rd29A)were constructed,transformed and identified by Agrobacterium tumefaciens,infected Camptotheca acuminata leaves by injection and osmosis,collected and treated leaves,extracted RNA,analyzed by real-time fluorescence quantitative PCR(qRT-PCR)for relative expression of several key enzymes genes in CPT biosynthesis pathway and CPT.The accumulation was analyzed by HPLC.The results showed that two of the three candidate transcription factor genes(CaTCP1 and CaZHD6)with similar gene expression characteristics and metabolite accumulation characteristics were cloned.The sequencing results showed that the full length of CaTCP1 coding sequence was 951 bp,encoding 316 amino acids,the relative molecular weight was 35.7 k D,and the isoelectricpoint was 9.80.The content of CPT in pBI121GD-CaTCP1 constitutive overexpression leaves increased by20.57%,and the relative expression of several key enzyme genes Ca7 DLS,CaG8 O and CaCYC1 in metabolicpathway increased by 5.1,2.7 and 2.8 times,respectively.The relative expression of the key enzymes CaG8 O,CaCYC1 and Ca7 DLS increased by 3.5,3.2 and 2.4times respectively after the overexpression of pBI121GD-rd29A-CaTCP1 induced by low temperature for 24 hours.The relative expression of key enzyme genes CaG8 O,CaCYC1 and Ca7 DLS increased 17.4,37.1 and 10.2 times respectively after 48 hours of induction,while the expression of CaSTR began to increase 7.1 times after 48 hours.The full length of CaZHD6 coding sequence is 1002 bp,encoding 333 amino acids,with a relative molecular weight of 35.7 k D and an isoelectricpoint of 7.68.The content of CPT in pBI121GD-CaZHD6 overexpressed leaves increased by 43.98%,and the relative expression of several key enzyme genes Ca7 DLS,CaCYC1 and CaSTR in metabolicpathway increased by 3.4,2.8 and 2.4 times,respectively.PBI121GD-rd29A-CaZHD6 overexpression induced by low temperature for 24 hours only increased the relative expression of the key enzyme gene CaTDC1 to 3.5 times.The relative expression of key enzyme genes Ca7 DLGT,CaG8 O,Ca7DLS and CaCYC1 increased 4.8,37.3,7.0 and 13.8 times respectively after 48 hours of induction,while the expression of CaTDC1 and CaSTR increased significantly,reaching 4.3and 12.0 times respectively.The roots of growth tree at 7 weeks and 14 weeks of growth were observed.The expression patterns of CaTCP1 and CaZHD6 genes in different tissues were studied by qRT-PCR in xylem,leaves and bark.The results showed that the relative expression level of CaTCP1 in the parts of Camptotheca acuminata was 2-10 times that of 7 weeks of growth.The bark was 3.1 times,the xylem was 4.7 times,the root was 10.1,and the leaf was 2.4 times.The relative expression of CaZHD6 in the parts of Camptotheca acuminata growing for 14 weeks was 1-94 times that of the 7-week-old tree.The bark was 1.7 times,the xylem was 66.3times,the root was 94.6 times,and the leaf was 11.8 times.It is concluded that CaTCP1 gene can simultaneously regulate the expression of CaG8 O,CaCYC1 and Ca7DLS,thereby promoting the biosynthesis and accumulation of CPT.CaZHD6 gene can simultaneously regulate the expression of CaSTR,CaCYC1 and Ca7 DLS,thereby promoting the biosynthesis and accumulation of CPT.The regulation of the key enzyme gene CaTDC1 needs to induce the accumulation of time.The longer the growth period of the Camptotheca acuminata,the higher the expression levels of the genes CaTCP1 and CaZHD6 in each organ.This provides a possibility for geneticengineering of transcription factors in Camptotheca acuminata,and lays a foundation for promoting the biosynthesis of CPT and increasing the content of CPT in Camptotheca acuminata.