【作者】 周世奇；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2005， 硕士

【摘要】 本论文研究的主要内容是以下内容:通过DNA片段原生质转化法,用带有完整recA基因的DNA片段转化产核黄素菌株B.subtilis 24(recA4突变株)原生质体,恢复了B.subtilis 24的同源重组功能。恢复recA基因功能恢复后,菌株的核黄素产量没有下降(为1.3g/L)是比较理想的受体菌。运用基因工程方法构建了带强启动子SPO2和P43及原启动子P1核黄素操纵子的三个B.subtilis整合型质粒,发现三个带核黄素操纵子质粒都可以在大肠杆菌中能产一定量的核黄素,核黄素产量P43>P1≈SPO2。然后,分别通过整合型质粒在B.subtilis D3染色体上定点(amyE位点)整合和随机整合研究了带不同启动子的核黄素操纵子在B.subtilis中的表达情况。定点(amyE位点)整合的转化子中,核黄素产量P1>P43>SPO2。通过比较发现随机整合的效果要比定点(amyE位点)整合好,通过筛选得到了带有P43强启动子核黄素操纵子的高产核黄素菌株,达3.1g/L。研究了不带启动子的rib结构基因构建的多克隆载体重组子在E.coli(Top10)中产核黄素的原因。利用PCR方法从B.subtilis扩增出只含核黄素操纵子中ribA结构基因的DNA片段,克隆到E.coli中发现该菌株已有一定的核黄素表达量。一方面解释了带rib结构基因的多克隆载体在E.coli(Top10)中产核黄素的原因,另一方面说明了单独增加ribA的基因剂量就能增加核黄素的产量。更多还原

【Abstract】 The main work presented in this study is as follows:Riboflavin-producing strain B.subtilis 24, a recA4 mutant, was transformed with a DNA fragment containing whole recA gene by an approach of protoplast transformation with DNA fragments, then the ability of homologous recombination in B.subtilis 24 was recovered. This process has no negative effect on riboflavin yield of the strain, which is about 1.3g/L. This strain is an idea host for engineering strain. Three integration plasmids of B.subtilis were constructed by genetic engineering.The plasmids have riboflavin operon under the control of the original promoter and two strong promoters SPO2 and P43, respectively. All the rib plasmids can be expressed in E.coli. The yield of riboflavin in E.coli containing riboflavin operon under the control of P43 is higher than the other two promoters, which has similar effect on riboflavin synthesis. Then, the expression level of riboflavin operon in B.subtilis was studied by inserting the integration plasmid in the chromosome of B.subtilis D3 at the amyE locus and a random locus. The riboflavin yield of the strain which had riboflavin operon under the control of the original promoter inserted in by a double-cross event at the amyE locus is higher than the other two promoters, and under the control of P43 is higher than that of SPO2. Comparatively, we have filtrated out a high riboflavin yield strain of P43 which was inserted in by a single-cross event, and the yield is about 3.1g/L. The result indicated the expression level by a random insert is higher.In order to open out the reason why the riboflavin operon of promoteless can accumulate riboflavin when it was cloned in E.coli Top10, we have done this work. A structural gene of the riboflavin operon, ribA, was isolated by PCR from B.subtilis. The gene was cloned in E.coli and made the strain accumulate some riboflavin in the medium. The result made the reason of promoteless rib accumulating riboflavin clear and also indicated that riboflavin yield can be increased by only enhancing ribA gene.更多还原