小花棘豆Alternaria oxytropis内生真菌酵母氨酸还原酶基因的Southern blot检测分析  

Southern Blot Analysis of Saccharopine Reductase Gene of the Endophytic Fungus Alternaria Oxytropis

作　　者：珠丽 卢萍[1] 席领军 高峰[1] 李玉玲[1] 姜凯 Zhu Li;Lu Ping;Xi Ling-Jun;Gao Feng;Li Yu-Ling;Jang Kai(College of Life Science and Technology,Inner Mongolia Normal University,Inner Mongolia Hohhot 010022)

机构地区：[1]内蒙古师范大学生命科学与技术学院

出　　处：《生物化工》2019年第6期16-20,共5页Biological Chemical Engineering

基　　金：国家自然科学基金项目(31460235)

摘　　要：用Southern blot技术检测小花棘豆Alternaria oxytropis内生真菌野生株(OW7.8)和酵母氨酸还原酶基因(sac)敲除株(M1),为研究基因功能奠定基础。以OW7.8和M1为材料,利用PCR法制备地高辛(DIG)标记探针,进行基因组Southern blot检测。用PvuⅡ切割M1基因组DNA后,用sac特异性探针对其进行Southern blot,杂交结果呈阴性,表明M1中sac已被敲除;用Eco RⅤ和PstⅠ切割OW7.8基因组DNA,杂交结果呈阳性,说明野生株有sac;用hph特异性探针与M1的PvuⅡ酶切产物进行Southern blot,M1杂交结果呈阳性,说明标记基因hph成功整合到M1基因组中;用hph特异性探针与Eco RⅤ和PstⅠ切割的OW7.8基因组DNA杂交,结果呈阴性。Southern blot检测到OW7.8中有sac中间序列,在M1中被hph替换,证明sac被敲除。sac基因以单拷贝形式存在于OW7.8基因组。The wild type strain of endophytic fungus(OW7.8) and the saccharopine reductase gene(sac) disruption mutant(M1) were detected by Southern blot, which laid a foundation for the study of gene function. The digoxin(DIG) labeled probes were prepared by PCR and detected by Southern blot. The genomic DNA of M1 was digested with Pvu II, then Southern blot was performed with sac specific probe, which showed negative hybridization result, indicating sac was knocked out in M1. The genomic DNA of OW7.8 was digested with Eco RV and Pst I, then Southern blot was performed with sac specific probe, which showed positive hybridization result indicating sac existed in the wild strain. The genomic DNA of M1 was digested with Pvu II, then Southern blot was performed with hph specific probe, which showed positive hybridization result. It was indicated that the marker gene hph was successfully integrated into the M1 genome. The genomic DNA of OW7.8 was digested with Eco RV and Pst I, then Southern blot was also performed with hph specific probe, which showed negative result. in OW7.8 by Southern blot, while sac intermediate sequence was replaced by hph in M1, which proved that sac was knocked out, and the sac gene exists in a single copy in OW7.8 genome.

关 键 词：地高辛 southern blot 丝状真菌 技术优化 

分 类 号：Q936[生物学—微生物学]