酵母ATG8与PMT1双基因缺失菌株的构建及鉴定  

The construction and identification of ATG8 and PMT1 double-gene deficiency yeast strain

作　　者：崔红晶 袁源 陈亚芹 邱堃佩 赵炜 CUI Hong-jing(Institute of Aging Research,Guangdong Medical University,Dongguan 523808,Chin)

机构地区：[1]广东医科大学衰老研究所 [2]广东省医学分子诊断重点实验室 [3]东莞市衰老与抗衰老重点实验室,广东东莞523808

出　　处：《牡丹江医学院学报》2018年第4期1-3,共3页Journal of Mudanjiang Medical University

基　　金：国家自然科学基金(31701050);广东省医学科学技术研究基金(A2016257);东莞市医疗卫生科技计划一般项目(2016105101289)

摘　　要：目的构建自噬相关ATG8与蛋白质O-甘露糖转移酶1(PMT1)双基因缺失酵母菌株。方法一步基因置换法构建ATG8基因缺失菌株(BY4741 atag8::LEU2);通过将不同配型的酵母细胞杂交、显微镜下四分体拆分,平板印记、缺陷型培养基筛选等方法,构建ATG8和PMT1双基因缺失菌株(BY4742 agt8::LEU2 PMT1::URA3);PCR鉴定阳性克隆基因组DNA。结果 ATG8基因缺失菌株电泳条带为2759 bp和980 bp,ATG8和PMT1双基因缺失菌株电泳条带分别为2759 bp,980bp,1630 bp和731 bp。四分体拆分实验发现ATG8基因缺失酵母细胞形成较小的单克隆。结论成功构建ATG8基因缺失菌株和ATG8与PMT1双基因缺失菌株;缺失ATG8基因酵母细胞形成较小的单克隆。Objective This study was aimed to construct the autophagy related ATG8 andprotein O - mannosyltransferase 1 （PMT1） double -gene deficiency strain. Methods ATG8 deficiency strains （BY4741 atg8：： LEU2） were construct by polymerase chain reaction （PCR） - mediated one - step gene disruption. ATGSand PMT1 double - gene disruptions （atgSApmtlA） were construc- ted by mating single - gene deleted yeast strains containing the different selectable markers （ BY4742 pmtl ： ： URA3 and BY4741 atg8 ： ：LEU2）, and further individual meiotic tetrads were dissected under optical microscope. Genomic DNA of positive clones was i- dentified by PCR. Results The 2759 bp and 980 bp bands were detected by agarose gel electrophoresis from the PCR products of the genomic DNA in ATG8 deficiency strain, and the 2759 bp, 980 bp, 1630bp and 731bp bands were detected in ATG8 and PMT1 doub- le - gene deletion strain. The ATC,8 single gene deletion strain grew in smaller monocolonies than the wild - type （ BY4742 ） and ATGSand PMTI double - gene deletion strains. Conclusion ATG8 deficiency strain, and ATG8 and PMTI double - gene deficiency strain were successfully constructed. ATC,8 deficiency could attenuat the clone - forming ability of yeast cells.

关 键 词：酿酒酵母 自噬 甘露糖基化 复制性寿命 

分 类 号：Q255[生物学—细胞生物学]