摘要
根据同源重组的原理,构建了用于敲除冰岛硫化叶菌中反螺旋酶(RG)基因的质粒pRG。重组质粒反复转化冰岛硫化叶菌,得到质粒整合到宿主染色体上的单交换菌株,但没有获得反螺旋酶基因缺失的双交换转化子。由此推测反螺旋酶基因是冰岛硫化叶菌必需的功能基因,敲除反螺旋酶基因可导致细胞死亡,或者转化菌株为生存需要存在某种未知机制而阻止双交换发生。单交换菌株可以再次发生同源重组,通过PCR的方法,证实再次重组得到的皆为回复突变而无缺失突变菌株。
Based on the homologous recombination, the plasmid pRG of reverse gyrase gene knockout was constructed and transformed into the Sulfolobus islandicus and the single crossover mutant was isolated. However, the gene knockout mutant could not be isolated in this experiment. The results indicated that RG gene was essential for the Sulfolobus islandicus. Any damages to this gene would be lethal meaning that there may be some unknown mechanisms interfering double crossovers. Reverse mutation of the single crossover strain occurred. The frequency of reverse mutation was calculated by plating selective/non-selective plates and confirmed by PCR. The results showed that all the mutants were caused by reverse mutation.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2009年第1期39-42,共4页
Journal of Huazhong Agricultural University
基金
浙江省重大科技攻关项目(2004C12010)资助
关键词
反螺旋酶
基因敲除
单交换
重组
reverse gyrase
gene knockout
single crossover
recombination
