微波冻融法预处理用于酵母菌落PCR检测  

Application of microwave freeze-thaw pretreatment in yeast colony PCR

作　　者：李闯 彭志清 李寅琛 吴升山 林鹿 曾宪海 LI Chuang;PENG Zhiqing;LI Yinchen;WU Shengshan;LIN Lu;ZENG Xianhai(College of Energy,Xiamen University,Xiamen,Fujian 361102,China;Fujian Engineering and Research Center of Clean and High-valued Technologies for Biomass,Xiamen,Fujian 361102,China;Xiamen Key Laboratory of Clean and High-valued Utilization for Biomass,Xiamen,Fujian 361102,China)

机构地区：[1]厦门大学能源学院,福建厦门361102 [2]福建省生物质清洁高值化技术工程研究中心,福建厦门361102 [3]厦门市生物质清洁高值化利用重点实验室,福建厦门361102

出　　处：《福建农林大学学报（自然科学版）》2022年第5期705-712,共8页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家重点研发计划“可再生能源与氢能技术”专项(2019YFB1503904).

摘　　要：为实现酵母阳性转化子的高效筛选,亟需寻找一种快速、简便的菌落PCR方法用于酵母的分子遗传鉴定.以酿酒酵母为底盘微生物,转化重组质粒或染色体整合片段,挑选抗性筛选单菌落,采用不同预处理方法处理菌体以进行菌落PCR检测,并对结果进行分析.相比其他预处理方法,使用微波冻融法制备的模板进行菌落PCR检测,稳定性好、检测性能强,适用于短链DNA以及3 000 bp以上目的片段的扩增;同时,该方法可进行毕赤酵母阳性克隆的筛选.表明微波冻融法通用性较强,制备的模板适合不同遗传背景以及基因操作方式下酵母菌落PCR检测.To screen the positive yeast transformants efficiently, the establishment of a rapid colony PCR procedure for molecular genetic characterization is of great necessity. Firstly, the recombinant plasmids or chromosomal homologous recombination fragments were transformed into the chassis microorganism Saccharomyces cerevisiae. Then several single colonies on the resistant plate were treated differently prior to colony PCR. Compared with other procedures, colony PCR underwent microwave freeze-thaw pretreatment demonstrated better stability and detection performance, which was appropriate for the amplification of short targets and cloned fragments over 3 000 bp. In addition, this pretreatment was effective on Komagataella phaffii and Scheffersomyces stipitis. In conclusion, microwave freeze-thaw pretreatment is of great feasibility to yeast colony PCR detection under various genotypic backgrounds and genetic manipulations.

关 键 词：酿酒酵母 菌体预处理 菌落PCR 微波 冻融 

分 类 号：Q93-332[生物学—微生物学]