Investigation Of Bax And Tdc Gene Expression On Camptothecin And 10-hydroxycamptothecin Biosynthesis In C. Acuminata Cells

Apoptosis, which is commonly existed in animals and plants, is also known as gene-regulated "programmed cell death". Further research shows that apoptosis plays important roles in plant normal development and biotic and abiotic stress tolernance. Bax, one members of BcI-2 gene family, is involved in triggering apoptosis. The biosynthetic pathway of camptothecin and its derivatives is a complicated process involving many key enzymes and related genes. C.acuminata tryptophan decarboxylase (TDC) gene encodes a key enzyme in camptothecin and its derivatives biosynthetic pathway. It has been cloned, but its role in C.acuminata secondary metabolism remains unclear. In this study, we transformed apoptosis gene Bax into C.acuminata cells with inducible promotor and overexpressed tdc gene in C. acuminata to study the affects on C.acuminata secondary metabolism. The main results are summarized as follows:1. Influence of Bax on cell apoptosis and the camptothecin and 10-hydroxycamptothecin syntesis of C.acuminata.Bax gene was transformed into C.acuminate. PCR detection, RT-PCR, Q-PCR and western blotting analysis confirmed that Bax have been transformed into C.acuminata successfuly. The chang of cell morphology and TTC activity test results showed that inducing Bax gene expression can trigger C.acuminate cell apoptosis.Established and optimized the transgenic lines induced expression system. Without the Est treatment, the Bax transgenic cells had the same camptothecin and 10-hydroxycamptothecin synthesis as wild-type cells. While after treated with 5mg/L Est for two weeks, the content of camptothecin and 10-hydroxycamptothecin in Bax transgenic cells strongly increased, respectively,4.4 and 2.1-folder than the control's.2. Influence of tdc on the camptothecin and 10-hydroxycamptothecin syntesis of C.acuminata.tdc homologous gene tdcl and tdc2 were cloned from C.acuminata, and we successfully constructed pBIN-35S-tdc-GFP expression vector. The constructed tdc expression vector were transformed into Agrobacterium tumefaciens GV3101.Using the optimized leaf disc method to carry tdcl and tdc2 gene into C.acuminate. PCR analysis indicated that tdcl and tdc2 have transformed into C.acuminata. The camptothecin detection results showed that tdc overexpression can promote the accumulation of camptothecin and 10-hydroxycamptothecin.3. Optimized camptothecin and 10-hydroxycamptothecin extraction conditions. RP-HPLC fluorescence detection method was established to detect the content of camptothecin and 10-hydroxycamptothecin in C.acuminata. By comparing various extraction methods to obtain the simultaneous extraction of camptothecin and 10-hydroxycamptothecin, the optimum technology parameters were found as following:homogenation for 8min with 55% ethanol,60℃water bath for 30min and ultrasonic extraction for 15min finally.