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Influence of Gene Overexpression of EuFPS on the Gutta Cells in Eucommia Ulmoides

Author: ZhaoDan
Tutor: ZhaoDeGang
School: Guizhou University
Course: Biochemistry and Molecular Biology
Keywords: Eucommia ulmoides Olive rubber farnesyl pyrophosphate synthase genetic transformation Microscopic observation
CLC: S567.19
Type: Master's thesis
Year: 2009
Downloads: 71
Quote: 2
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Abstract


In this research, the Duzhong (Eucommia ulmoides Olive) farnesyl pyrophosphate synthase (EuFPS) gene was transformed into E. ulmoides by the Agrobacterium-medidated method and then the gutta cells in leaf of the transfenic plant, where were rubber biosynthesis, were observed by light microscope (LM) and transmission electron microscope (TEM). The main results of the research are as follows;(1) Construction of EuFPS Gene Expression VectorThe expression vector pSH-FPS was constructed by using restriction enzymes EcoRI and Bam HI digested pSH737 and pUC-FPS which contains EuFPS gene, reconvering and ligsing the pSH737 backbone and EuFPS DNA fragments. The EuFPS gene and GUS::NPTⅡfusion gene were droved by CaM35S strong promoter respectively.(2) The Genetic transformation by Agrobacterium-mediated in Eucommia ulmoides OliveBy using the Agrobacterium-medidated method, we transformed the 35S::FPS into the hypocotyls of E. ulmoides. The explants of 15-days-old seedlings were immersed for 8min in the Agrobacterium suspension (OD600=0.3-0.6) adding with 50μmol/L acetosyringone (AS) and were incubated on the MS co-cultivation medium for 2 days in the dark at 28℃. Having eliminated Agrobacterium tumefaciens on MS+6-BA0.8mg/L+NAA 0.1mg/L with 300mg/L cefhalosporin (Cef) for 3 days, the explants teansformed A. tumefaciens were transferred into select medium MS+6-BA0.8mg/L+NAA 0.1mg/L with 50mg/L kanamycin(Km)and 300mg/L Cef. we obtained 17 transgenic plants from 279 regenerating plants resistant to Km through GUS staining and PCR detecting.(3) Microscopic observation of the transgenic plants of Eucommia ulmoides OliveBoth the leaf gutta cells of transgenic and wild type plants were observed by using LM and TEM, Much more rubber particles in gutta cells of transgenic plant leaf were found than in wild type. It suggests that farnesyl pyrophosphate synthase plays a role in the forming of rubber particles of Eucommia ulmoides Olive.

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