野油菜黄单胞菌论文.doc

二零一三年六月 摘 要 野油菜黄单胞菌是黄原胶的一种生产菌种。黄原胶是由黄单胞菌发酵，产生的胞外多塘应用。黄原胶的合成是一个精细复杂的过程，需要酶参与，如转移酶I - V、β-淀粉酶、缩酮丙酮酸转移酶、乙酰化酶、尿苷二磷酸葡萄糖脱氢酶Gum A蛋白等。应用基因工程的方法获得优良的产黄原胶菌株是一个新的研究领域。 由于黄原胶的乙酰化程度与黄原胶的稳定性正相关。因此研究野游菜黄单胞菌的乙酰化酶基因对黄原胶的生产有重要影响。本课题将从基因工程的角度，对野游菜黄单胞菌的乙酰化酶基因进行诱变改良。以野游菜黄单胞菌CICC10258菌株为试验菌株，提取并克隆其乙酰化酶基因片段，然后利用稀土经PCR对其进行体外诱变，并测定诱变后乙酰化酶的基因序列及蛋白质空间构型。进而筛选菌株。 本实验提取野油菜黄单胞菌(Xanthomonas campestris pv. campestris) CICC10258菌株的总基因，设计特异性引物，PCR中扩增乙酰化酶基因把扩增的片段与PMD19-Tvecter重组，导入感受态细胞，进而成功筛选阳性克隆子。后期利用不同浓度的稀土离子Nd3+、La+、Er3+对乙酰化酶基因进行PCR体外诱变，将其诱变后的乙酰化酶基因重新转化连接，导入受体细胞，并筛选出阳性克隆子 。对比原始序列与诱变后的碱基序列，发现较多T碱基突变成C碱基。由此推断出稀土对乙酰化酶产生了重要影响。为后期构建表达载体，筛选优良菌株奠定了基础。 关键词：黄原胶；野游菜黄单胞菌；乙酰化酶；PCR扩增；稀土诱变 Abstract Xanthomonas campestris is a production bacteria of xanthan gum. Xanthan gum was fermented by Xanthomonas campestris, It was a kind of extracellular microbial Polysaccharide. It is widely used in various industries. Synthesis of xanthan gum is a fine and complex process, It requires the participation of a variety of enzymes and protein factors, such as the transfer of the enzyme I - V, and polymerase, β-amylase ketal pyruvate aminotransferase ,acetyltransferase, uridine diphosphate glucose dehydrogenase, and Gum A protein. The application of genetic engineering to get excellent production of xanthan gum strains is a new area. the stability of of xanthan gum has positive correlations with the degree of acetylation. Therefore researching the acetyltransferase gene of Xanthomonas has an important impact on the production of xanthan gum. We begin the subject from the point of geneticengineering, xanthan gum production strains Xanthomonas campestris has mutagenesis improvement.We use the Xanthomonas extracellular bacteria CICC10258 strains as the original strains, extracted and cloned acetyltransferase gene fragment, then use the rare earth in vitro mutagenesis by PCR,then take determination of the mutagenesis acetyltransferase gene sequence and space configuration of protein.Then filter out t