利用果蝇转座子作载体构建土垢分枝杆菌变异基因文库的研究

目的　利用果蝇mariner转座子作为载体构建土垢分枝杆菌随机变异基因文库以筛选影响脂肪酸代谢的基因。　方法　将果蝇转座子mos1的末端反向重复序列置于卡那霉素抗性基因两侧 ,并将此种基因插入到M2 72B载体 ,用此载体转化土垢分枝杆菌 ,用Southernblot验证外源基因的插入。将变异的基因克隆在不同碳源的培养基上培养 ,筛选只在软脂酸上生长的菌落。　结果　初筛了 4 6 8个菌落 ,有 5个不能在软脂酸上生长 ,其中 2个在ICL基因上发生变异 ,3个在PCKA基因上发生变异。ICL为乙醛酸旁路所必须 ,PCKA编码糖异生的关键酶。　结论　研究证明该转座子载体用于基因变异有效 ,变异筛选分枝杆菌中与脂肪酸代谢的基因可行。

ESTABLISHMENT OF MYCOBACTERIUM SMEGMATISB MUTAGENESIS LIBRARY WITH DROSOPHILA MAURITIANA MARINER ELEMENT VECTOR

Objective Use the Drosophila mauritiana mariner transposable element as a vector to construct Mycobacterium smegmatis random mutagenesis library in order to screen transposon mutants that affect the metabolism of fatty acids. Methods A transposon mutagenesis vector was constructed, which contains the Tn903 kanamycin resistance (KanR) gene flanked by the 5′ and 3′ inverted repeats of the D. mauritiana transposable element Mos1. M. smegmatis was transformed with the vector. The ability of transposon to integrate within the chromosome was identified using southern strategy. M. smegmatis transposon mutants were pooled and screened using different carbon sources. The clones could not grow on palmitate were selected. Results From initial screen of 468 colonies, we isolated one insertion in the isocitrate lyase gene, required for the glyoxalate shunt, and three independent insertions in the phosphoenolpyruvate carboxykinase gene, required for gluconeogenesis. Conclusion These results demonstrate the utility of this transposon mutagenesis vector, as well as the ability of our mutagenesis screen to identify mutations that affect the metabolism of fatty acids by mycobacterium.