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Biological Effect of Rare Earth Element and Genetic Transformation on Arnebia Euchroma(Royle) Johnst.Cell

Author: HuangWenHu
Tutor: GeFeng
School: Kunming University of Science and Technology
Course: Biochemical Engineering
Keywords: Arnebia euchroma rare earth element genetic transformation real time-PCR
CLC: S567.239
Type: Master's thesis
Year: 2011
Downloads: 8
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Abstract


There is a huge market potential of Shikonin and its derivatives in medicine, food, cosmetics, printing and dyeing fields, Arnebia euchroma (Royle) Johnst.of "Chinese Pharmacopoeia" of 2010 versions is the raw plant source of Shikonin and its derivatives. However, at present, the environment for A. euchroma growth has been destroyed and the marketable requirement has been increasing, resulting in the conflict between supply and demand, and the need for application of modern biotechnology means to resolve the problem of shortage of raw medicinal herbs is urgent. Thus, under the premise that Arnebia euchroma cells are the subjects of this study, based on plant cell engineering research methods,the effects of rare earth elements on cell growth and shikonin biosynthesis will be focused on.Because cell growth and shikonin biosynthesis are non-associated, the first step is that the effects of rare earth elements on cell growth will be researched in the solid growth medium; the second step is that the effects of rare earth elements on shikonin biosynthesis will be researched in the liquid or solid synthesis medium; and then from the perspective of gene regulation, the molecular mechanism of the regulation of Cerium on shikonin biosynthesis will be discussed preliminarily, to do some basic research for the future use of metabolism Engineering means to improve the synthesis of the target product. In addition, the substitution effect of rare earth elements on kinetin will be studied in solid medium, to discuss the class hormonal effects of rare earth elements. Finally, the initial establishment of optimization system about Agrobacterium-mediated genetic transformation of Arnebia euchroma cells will be completed, to do some work for genetic transformation of cells. The major findings of present study are as following:1.The class hormonal effects of rare earth elements substitution on kinetin was studied. La, Nd, Gd, Sm, Ce, Pr could not match the effect of KT on cells growth and shikonin biosynthesis, however, compared to the process of training without KT, low concentrations of rare earth elements could promote cells growth or shikonin biosynthesis, and relatively high concentrations of rare earth could reduce cells growth or shikonin biosynthesis. Homesis effect was showed. 2. Homesis effect appeared in the effects of six kinds of rare earth elements in the whole range of concentration used in the experiment in the process of cell growth and shikonin biosynthesis. Shikonin content in solid medium was higher than that in liquid medium.3. The molecular mechanism of regulation of Cerium on the shikonin biosynthesis had been researched by real time quantitative PCR qualitatively. DPI,4CL, HMGS, C4H were shikonin biosynthesis enzyme genes. The results were that appropriate concentrations of Ce could improve the expression level of DPI, basically keep the expression level of C4H and 4CL, reduce the the expression level of HMGS, and ultimately shikonin synthesis increased.4. Optimized the parameters of Agrobacterium-mediated genetic transformation of A. euchroma cells. The result was that the optimal Agrobacterium strain EHA105, infection concentration of OD600 0.7, infection time of 10min, culture time of 48h, culture temperature of 25℃, and 50mg/L acetosyringone to activation medium.

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