【摘要】 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酵母锰超氧化物歧化酶活性,并损伤线粒体功能,因此在非发酵培养基上不能生长.利用MTM1基因缺失的突变体在非发酵培养基上的生长缺陷,转入酵母基因组文库筛选MTM1抑制基因,发现MTM1基因缺失造成的损伤一旦形成不可逆转,重新引入MTM1基因也无法挽救,直接筛选无法得到抑制基因.为了避免MTM1缺失造成的不可逆损伤,在野生型酵母中先转入带有MTM1基因的质粒,再敲除染色体上的MTM1基因,随后转入基因组文库,再利用药物5-氟乳清酸(5-FOA)迫使细胞丢失表达MTM1基因的外源质粒,再筛选能在非发酵培养基上生长的转化子,通过这种方法筛选发现,POR2等5个基因的过表达可以挽救MTM1基因缺失造成的非发酵培养基上的生长缺陷,为深入了解MTM1基因的功能提供了线索,对筛选其他造成不可逆损伤的突变基因的抑制基因提供了一条可行的研究思路.更多还原

【Abstract】 MTM1 gene is essential for SOD2 activity and normal mitochondrial function. MTM1 deletion results in decreased SOD2 activity, impaired mitochondrial function and growth defect on nonfermentable carbon source. A yeast genomic library was transformed into mtm1 deletion mutant to screen for suppressor genes of MTM1. The damage caused by MTM1 deletion is irreversible and even overexpression of MTM1 can not rescue the growth defect of mtm1 deletion mutant. Another screening strategy was adopted: a plasmid overexpressing MTM1 was transformed into wild type before the MTM1 gene on chromosome was deleted. The resulting strain, designated YES2MTM1, was transformed with a yeast genomic library. Transformants lost the plasmid overexpressing MTM1 after 5-FOA treatment. Yeast strains able to grow on nonfermentable carbon source with MTM1 deletion and overexpression of some DNA fragments were picked up and candidate suppressor genes were identified. Overexpression of five genes were identified to be able to rescue the growth defect on nonfermentable carbon source. The study will provide reference for MTM1 gene function and screening for suppressor of genes whose deletion result in irreversible damage.更多还原