利用酿酒酵母转座子文库筛选MTM1基因缺失表型相关基因

Screen in Saccharomyces cerevisiae for transposon insertion sites able to rescue phenotype of MTM1 deletion mutant using mTn-lacZ/LEU2 transposon library

【目的】MTM1基因对于维持锰超氧化物歧化酶的活性和线粒体正常功能十分重要,MTM1基因的缺失会严重影响锰超氧化物歧化酶活性,并损伤线粒体功能,使酵母在非发酵培养基上不能生长。为加深对MTM1基因功能及其相关基因的研究,尝试利用转座子文库筛选MTM1基因缺失表型相关基因,寻找哪些位置的转座子插入能挽救MTM1基因缺失导致的生长缺陷。【方法】因MTM1基因的缺失造成的损伤不可逆,直接转入文库无法筛选得到MTM1基因缺失表型相关基因,本研究利用外源MTM1基因菌株和mTn-lacZ/LEU2酿酒酵母转座子文库进行筛选,寻找能挽救mtm1突变体生长缺陷的转座子插入位点。【结果】发现转座子插入HSL1和TPS2基因能挽救mtm1突变体的生长缺陷。【结论】我们的结果为深入了解MTM1基因的功能提供了线索。

[ Objective] MTM1 gene is essential for superoxide dismutase 2 activity and normal mitochondrial functions. MTM1 deletion results in decreased superoxide dismutase 2 activity, impaired mitochondrial functions and growth defect on nonfermentable carbon source. To promote understanding of MTM1 gene, we started a genetic screen for transposon insertions which are able to rescue the growth defect resulting from MTMI deletion. [ Methods ] Routine screening strategy didnt work because of the irreversible damage caused by MTM1 deletion. So we adopted the following screening strategy： we transformed a plasmid overexpressing MTM1 into wild type before deleting MTM1 in chromosome and got the resulting strain, designated YES2MTM1. Then we transformed mTn-lacZ/LEU2 transposon library into the YES2MTM1 strain. Transformants lost the plasmid overexpressing MTM1 after 5-Flnoroorotic acid treatment. We picked up the yeast strains able to grow on nonfermentable carbon source with MTM1 deletion and some transposon insertion and identified the insertion sites. E Results] We found transposon insertions in two genes were able to rescue the growth defect resulting from MTM1 deletion on nonfermentable carbon source. [ Conclusion ] Our study will provide reference for thorough understanding of MTM1 gene function.