摘要
以嗜热真菌杜邦嗜热菌ThermomycesdupontiiNRRL2155为研究材料,利用同源重组原理和真菌原生质体转化方法,以潮霉素抗性基因替换嗜热真菌目标基因,获得抗潮霉素的靶向基因敲除突变菌株。优化的遗传转化体系为:用15mg/mL裂解酶,在28℃下酶解2g杜邦嗜热菌菌丝5.5h以获得原生质体,经STC缓冲液洗涤重悬后,利用PEG(polyethylene glycol)介导的遗传转化方式,将10μg线性敲除全长片段转化至杜邦嗜热菌原生质体中,通过潮霉素筛选及PCR验证得到基因替换突变菌株,同源重组率达到20%。本研究首次将原生质体转化方法应用在杜邦嗜热菌,并成功建立稳定高效的基因替换体系,为快速构建杜邦嗜热菌的遗传转化体系和研究该嗜热真菌的基因功能提供有效方法。
A strain NRRL 2155 of the thermophilus fungus Thermomyces dupontii served as recipient, and a combination of homologous recombination and fungal protoplast transformation was applied to generate the targeted gene replacement mutant with a hygromycin resistance gene. The optimum condition for targeted gene replacement was as follows: 2 g of fungal mycelia was treated with 15 mg/mL lysing enzyme at 28°C for5.5 h to yield protoplasts. After being washed twice with STC buffer, the protoplasts were treated with 10μg of linear replacement DNA fragment through PEG-mediated genetic transformation method. The putative transformants were screened out with hygromycin B marker and further confirmed with PCR analysis. The efficiency rate of this homologous recombination method was up to 20%. This was for the first time that PEG-mediated protoplast transformation method was applied to T. dupontii, and a stable and efficient gene replacement system was successfully established.
作者
何佳宁
牛雪梅
HE Jia-Ning;NIU Xue-Mei(State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,Yunnan University,Kunming,Yunnan 650091,China)
出处
《菌物学报》
CAS
CSCD
北大核心
2019年第2期230-241,共12页
Mycosystema
基金
国家自然科学基金-云南联合基金项目(U1502262)
国家自然科学基金面上项目(31470169)
云南大学首届"青年英才培育计划"项目(XT412003)~~
关键词
嗜热真菌
杜邦嗜热菌
原生质体转化
基因敲除
生物合成基因
thermophilic fungus
Thermomyces dupontii
protoplast transformation
gene disruption
biosynthesis genes
