應用基因組改組技術選育普魯蘭高產(chǎn)菌.pdf

1、華中科技大學碩士學位論文應用基因組改組技術選育普魯蘭高產(chǎn)菌姓名：陳習娟申請學位級別：碩士專業(yè)：生態(tài)學指導教師：康建雄20090531II華 中 科 技 大 學 碩 士 學 位 論 文 Abstract Pullulan, chatracterized as a unique substance in physics, chemistry and the biology, is a water-soluble extracelluler

2、 polysaccharide produced aerobically by a yeast like fungus Aureobasidium pullulans. Pullulan have a wide range of commercial and industrial applications in many fields like environment polluted disposal, food science, a

3、griculture, pharmacy and health care. It is precondition to breeding strain with high production for Industrial production and bio-flocculant field. We have used genome shuffling to improve the output of Pullulan by Aur

4、eobasidium pullulans. The optimized conditions for protoplast preparation were as follows: the strain being cultured for 16h; glusulase, lysozym and cellulase concentration of 2 mg/mL; incubation at 30℃ for 90min to allo

5、w cell wall lysis; KCl as osmotic solution. Subsequently, studying the optimized conditions for protoplast inactivation methods show that the ultraviolet irradiation and heat treatments at 50℃ are both 25min. Under the o

6、ptimized conditions for protoplast fusion: 25% PEG 6000 containing 15% dimethyl sulfoxide (DMSO), and 10 mM CaCl2, incubation at 30℃ for 7min with gently shaking, and sucrose as osmotic solution, the fusion rate was 0.71

7、‰. After three rounds of genome shuffling, two high yielding strain, designated as A312、A38, was successfully isolated. Its production reached 20.3 mg/L, which was twofold increase compared to the parent strains DES7 and