CaERF1 And CaERF2 Mediate Abscisic Acid Regulation Of Camptothecin Biosynthesis

Camptotheca acuminata Decne is one of the unique anticancer medicinal plants in China.Camptothecin,a monomer component isolated from Camptotheca acuminata,as the third largest plant anticancer drug in the world,is widely used in clinical treatment of cancer diseases.However,the accumulation of camptothecin in plants is very limited,and the inefficient synthesis in plants limits the application of camptothecin in medical treatment.Therefore,exploring the regulation mechanism of camptothecin has important theoretical value and significance.The biosynthesis of camptothecin is affected by many factor.It is found that AP2/ERF transcription factors(TF)are involved in regulating the biotic and abiotic stress responses of plants,and regulating the synthesis of plant secondary metabolites.Meanwhile,abscisic acid plays a key role in the growth and development of higher plants,and is an important factor in regulating metabolic pathway.In previous studies,AP2/ERF transcription factors Ca ERF1 and Ca ERF2 were successfully cloned from Camptotheca acuminata by coexpression analysis,and overexpression of these two transcription factors promoted the expression of key enzyme genes in camptothecin synthesis pathway,thus promoting camptothecin accumulation.The expression level of response genes in ABA signaling pathway was up-regulated after transient overexpression of Ca ERF1 and Ca ERF2.Therefore,this paper takes this as a breakthrough point to further explore the molecular mechanism of ABA-regulated camptothecin biosynthesis mediated by transcription factors Ca ERF1 and Ca ERF2,and provide a new strategy for the efficient synthesis of camptothecin.Firstly,Ca ERF1 and Ca ERF2 were silenced by virus-induced gene silencing(VIGS),and the accumulation level of camptothecin in C.acuminata was detected by high performance liquid chromatography(HPLC).Meanwhile,fluorescence quantitative PCR(q RT-PCR)analysis showed that the transcription levels of key enzyme genes in camptothecin synthesis pathway were all down-regulated in different degrees.When Ca ERF1 was silenced,Ca G8 O was down-regulated to 42%,Ca TDC1 was down-regulated to 23%,Ca7 DLGT was down-regulated to 41%,Ca ERF1 was down-regulated to 58%,and Ca ERF2 was down-regulated to 70%.When Ca ERF2 is silenced,Ca G8 O was down-regulated to 40%and Ca7 DLGT was down-regulated to 22%,which indicated that Ca ERF1 and Ca ERF2 could regulate the biosynthesis of camptothecin,and Ca ERF1 and Ca ERF2 had mutual regulation.Secondly,in order to study the regulation mode between transcription factors Ca ERF1 and Ca ERF2 and ABA,wild-type C.acuminata leaves were treated with exogenous hormone ABA,and q RT-PCR results showed that ABA synthesis gene Ca NCED and ABA receptor protein gene Ca P707 A were up-regulated by 22 times and 14 times respectively.Ca PYL8 was up-regulated by 26 times,and the relative expression level of ABA related genes in Camptotheca acuminata leaves was significantly up-regulated.The gene expression levels of Ca ERF1 and Ca ERF2 were also greatly increased,up-regulated by 27 times and 20 times respectively.The transcription level of camptothecin synthesis pathway enzyme gene was up-regulated in different degrees;The above results indicated that ABA responsive genes in Camptotheca acuminata were successfully induced by exogenous ABA,and ABA could regulate the transcription levels of Ca ERF1 and Ca ERF2.Thirdly,exogenous ABA was further used to treat C.acuminata leaves silenced by two transcription factors,Ca ERF1 and Ca ERF2.It was found that the relative expression of Ca ERF1 and Ca ERF2,the key enzyme genes in camptothecin synthesis pathway had different degrees of callback in the silencing of Ca ERF1 and Ca ERF2 after the intervention of exogenous ABA.The results showed that after silencing Ca ERF1,the sensitivity of Ca ERF1 and Ca ERF2 to ABA signal decreased.When Ca ERF2 is silenced,the response intensity of Ca ERF1 and Ca ERF2 to ABA signal decreases,and the transcription levels of Ca ERF1 and Ca ERF2 are up-regulated through the intervention of exogenous ABA signal.To sum up,transcription factors Ca ERF1 and Ca ERF2 can mediate ABA signal to regulate camptothecin biosynthesis.Compared with silencing Ca ERF1,after silencing Ca ERF2,the key enzyme genes of camptothecin synthesis pathway were up-regulated rapidly,which indicated that Ca ERF1 was more critical than Ca ERF2 in mediating ABA signal to regulate camptothecin biosynthesis.At last,the subcellular localization of Ca ERF1 and Ca ERF2 was carried out by confocal microscope.the results showed that both Ca ERF1 and Ca ERF2 were specifically expressed in the nucleus,which conformed to the biological characteristics of their transcription factors.In summary,this paper further analyzes the regulation of Ca ERF1 and Ca ERF2 on camptothecin biosynthesis by silencing the transcription factors Ca ERF1 and Ca ERF2.Secondly,the wild-type C.acuminata leaves and the silenced C.acuminata leaves are treated by exogenous ABA,and the expression of ABA response genes and key enzyme genes are analyzed,thus determining the regulation mode of transcription factors Ca ERF1 and Ca ERF2 regulating camptothecin biosynthesis by mediating ABA.It provides good theoretical support and scientific evidence for further elucidating the molecular mechanism of Ca ERF1 and Ca ERF2 regulating camptothecin biosynthesis,and provides a new strategy for efficient production of camptothecin by using plant hormones and transcription factors.