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Expression of D-AtCGS in E. Coli and Preparation of Polyclonal Antibody Against D-AtCGS

Author: WangYiPeng
Tutor: XuYanLi
School: Northeast Forestry University
Course: Microbiology
Keywords: Methioine Cystathionine-γ-synthase prokaryotic expression transgenic plant test
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 18
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Abstract


Methionine (Met) has very important physiological functions as an essential amino acid, and is used at multiple levels in cellular metabolism:as a protein constituent, in the initiation of. mRNA translation.the form of S-adenosylmethioine(SAM) is the most important methyl-group donor, meanwhile,it also functions as sulphur and aminopropyl carrier, the derivatives S-methylmethioine (SMM) is sulphur-carrier and related to sulphur metabolism. Met can promote growth of body and fur of animals, and has a function of protecting liver, participating in the form of erythrocyte. Met deficiency will cause loss of appetite, rapid weight loss, fatty liver, lecithin lack, hair degeneration, muscle atrophy, and anemia. Met is not be synthesized in animals and human beingonlying rely on daily dietary intake. Soybean is high-quality protein source, but Met content only accounts for 1.1%-1.6% of total protein. Methionine content of soybean varieties in the existing genetic variation is limited,so it’s difficult to cultivate high Met level soybean by conventional breeding methods, Cystathionine-y-synthase (Cystathionine-y-synthase, CGS) is a key enzyme in the synthesis of Met, it atalyzes O-phospho-L-homoserine (OPH) to form cystathionine. Then cystathionine was catalyzed by cystathionine-β-lyase, methionine synthase and S-adenosylmethionine synthetase and at last Met was synthesized.CGS and TS have similar Km, so the efficient of competiting OPH depends on the concentrations of the two enzymes. A large number of studies show that CGS expression is regulated by feedback inhibition of substrates. Therefore, regulated by means of genetic engineering, the molecular structure of CGS gene needs to be changed to reduce feedback inhibition of substrate. CGS in Arabidopsis research showed that 30 amino acids in N-terminal between 99-128 have nothing to do with catalyst, the feedback inhibition significantly decreased after removing it. Overexpression of this N-terminal deletion CGS gene is more conducive to enhanced methionine content in transgenic plants. In our study,N terminal deletion AtCGS gene (D-AtCGS) was transmormed into soybean and legume model plants Lotus japonicus to research the regulation of Met synthesis and breed high-Met soybean by molecular biological methods, a number of transgenic plants has been successfully obtained.In this study, prokaryotic expression vector were constructed to expressing D-AtCGS, and the antiserum against D-AtCGS was prepared, several transgenic D-AtCGS soybean and Lotus japonicus plants obtained by our lab were tested by Western blot and Elisa using Antiserum obtained and good. Results show that the testing signal of transgenic plant more strong than wild type The immunological methods for D-AtCGS transgenic plants testing were established successfully. An ideal technical support for expression and regulation of CGS research and testing D-AtCGS transgenic soybean or other plants was supplied.

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