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The Research of Agrobacterium Tumefaciens-Mediated Transformation of Candida Glycerinogenes

Author: ZhangJunSheng
Tutor: ZhuGeJian
School: Jiangnan University
Course: Fermentation Engineering
Keywords: Agrobacterium tumefaciens Saccharomyces cerevisiae Glycerol production by Candida Glyceraldehyde 3 - phosphate dehydrogenase gene Transformation
CLC: TQ929
Type: Master's thesis
Year: 2006
Downloads: 149
Quote: 5
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Abstract


Glycerin is an important the light chemical raw materials, has been widely used in cosmetics, toothpaste, tobacco, fragrance, water-based ink, textile printing and dyeing, paint, synthetic resin, leather, paper, pharmaceutical, food and defense and a dozen other areas over 1700 kinds of products. The hypertonic yeast resistance the - production glycerinogenes is the laboratory with independent intellectual property glycerol-producing strain, fermentation production of glycerol has the advantages of high-yield, high conversion rates and high recovery, better industrialization in China has been Application. In recent years, the use of genetically engineered bacteria to produce glycerol growing cause for concern. Means of genetic engineering recombinant bacteria glycerol production by Candida metabolic pathways the purposeful transformation, further to improve production glycerinogenes performance, in order to achieve the purpose of glycerol production and process improvement. Achieve this goal we must first obtain a valid conversion method. In this study, by constructing a plasmid pCAM 3300-URA3 transformation of Agrobacterium tumefaciens. The use of the URA3 gene defects complementary selection marker, try using Agrobacterium tumefaciens-mediated transformation of Saccharomyces cerevisiae, a success. To transform S. cerevisiae successful based on the study by constructing plasmid pCAM 3300-Zeocin its electric shock into Agrobacterium tumefaciens. The the 3300-Zeocin the Agrobacterium tumefaciens and the production of glycerol Candida yeast co-culture by the target plasmid pCAM using Zeocin resistance gene as a selection marker, to achieve the transformation of pCAM 3300-Zeocin production glycerinogenes. In the study based on the growth characteristics of the Agrobacterium tumefaciens transformation conditions were optimized in co-culture for 24 h, the proportion of cells is 1:500-1000 highest conversion rate for transformants of 2/104 yeast cells. The initial establishment of Agrobacterium tumefaciens-mediated transformation of the production of glycerol Candida transformation methods lay the foundation for further study of the production glycerinogenes. PCR amplified from S. cerevisiae full-length production of glycerol key enzyme glyceraldehyde 3 - phosphate dehydrogenase gene (glycerol 3-phosphate dehydro-genase gene, GPD1), constructed plasmid pCAM 3300-Zeocin-GPD1 and translate them into the root tumefaciens. Agrobacterium tumefaciens-mediated transformation system for the first time the Saccharomyces cerevisiae GPD1 full-length gene transferred to the production glycerinogenes, positive transformants were picked in zeocin-resistant plates. Positive transformants obtained were screened to obtain the plant containing of corn syrup 5 g / L Urea 2 g / L, glucose 250 g / L of culture medium in 30 ° C, 80 h fermentation of glycerol under the conditions of 200 r / min of The output can be increased to 12.93% of the transformants, named the Candida glycerinogenes-the GPD1, 10 (Cg-G10). Cg-G10 in the cell growth the equilibrium phase glycerol accumulation rate, a 71.7% increase than Cg Gpd1p enzyme activity increased by 22.33%; accompanied by the of glycerol accumulation rate and Gpd1p enzyme activity compared to the CG-G10 consumption of sugar rate 36.92% , fermentation time by nearly 24 h. This shows that by Agrobacterium tumefaciens-mediated transformation GPD1 full-length gene transferred production glycerinogenes and effective expression. Studies show that the the GPD1 full-length gene insertion did not change the CG-G10 cell growth process and to adapt to the phosphorus source traits. CG-G10 passaged stability study results showed that CG-G10 fermented traits can be genetically stable in non-selective medium.

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