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Investigation of Bax and tdc Gene Expression on Camptothecin and 10-hydroxycamptothecin Biosynthesis in C. Acuminata Cells

Author: ShenShaoHua
Tutor: WangLiLin
School: Hangzhou Normal University
Course: Botany
Keywords: C. acuminata Bax gene tdc gene Genetic transformation Camptothecin 10 - hydroxycamptothecin
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 28
Quote: 0
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Abstract


Apoptosis is commonly found in plants and animals in an active manner of death of cells by gene regulation. Apoptosis is not only the regulation and control of plant essential for normal growth and development, is still an important means of plants against pathogens against. Bax gene is a member of the Bcl-2 gene family, the gene expression induced apoptosis. Camptothecin and its derivatives of the biosynthetic pathway is a multi-enzyme complex catalyzed process involving a number of key enzymes and related genes. Of Camptotheca acuminata tryptophan decarboxylase (tryptophan decarboxylase, TDC) gene is a key enzyme in Camptotheca acuminata camptothecin and its derivatives in the biosynthetic pathway, the gene has been successfully cloned, but about it in of Camptotheca acuminata secondary metabolism 's role is not clear. This research will promote apoptosis in the Bax gene and C. acuminata tdc gene into C. acuminata cells were studied pro-apoptotic gene as well as the impact of the Camptotheca acuminata secondary metabolites synthesis camptothecin synthesis of key gene overexpression . The main results are as follows: the influence of the of 1.Bax gene on of Camptotheca acuminata apoptosis and camptothecin synthetic use of PCR testing proved the pro-apoptotic Bax gene (Est promoter) successfully transformed into C. acuminata cells. By RT-PCR, quantitative PCR and Western blotting experiments confirmed that the normal expression of the Bax gene. By morphological observation and TTC cell activity, found that the induced Bax gene expression promotes Camptotheca acuminata apoptosis. Optimized inducible expression system for transgenic lines. The not added Est induced camptothecin and 10 - HCPT content with non-transgenic control group had no significant difference. Add: 5 mg / L Est induced two weeks of transformed cells, the content of the metabolites significantly improved compared with the control group were 2.2 times and 4.3 times of the control group. The 2. The Camptotheca acuminata tdc homologous genetic transformation Camptotheca acuminata and influence synthesis of camptothecin was cloned Camptotheca acuminata the TDC homologous gene tdcl and the tdC2, constructed plant expression vector pBIN-35S-tdcl-GFP and pBIN-35S-tdc2 -GFP, and introduced into Agrobacterium tumefaciens GV3101. Use of the optimized leaf disk acuminata genetic transformation, transgenic cell lines obtained and confirmed by PCR. The camptothecin test results showed that the tdc overexpression can promote the accumulation of camptothecin and 10 - hydroxycamptothecin content. Camptothecin and 10 - of hydroxycamptothecin extraction method optimization and the establishment of the RP-HPLC fluorescence detection method by comparison of a variety of extraction methods to obtain optimal simultaneous extraction of camptothecin and 10 - hydroxycamptothecin The methods are: 55% ethanol homogenate 16min, 60 ℃ water bath for 30min, ultrasonic extraction 15min. RP-HPLC fluorescence detection camptothecin and 10 - hydroxy Camptotheca acuminata content.

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CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
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