| 一种有效的嗜热真菌杜邦嗜热菌靶向基因替代系统 |
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| 引用本文： | 何佳宁,牛雪梅.一种有效的嗜热真菌杜邦嗜热菌靶向基因替代系统[J].菌物学报,2019,38(2):230-241. |
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| 作者姓名： | 何佳宁 牛雪梅 |
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| 作者单位： | 云南大学省部共建云南生物资源保护与利用国家重点实验室 云南昆明650091;云南大学省部共建云南生物资源保护与利用国家重点实验室 云南昆明650091 |
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| 基金项目： | 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) |
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| 摘 要： | 以嗜热真菌杜邦嗜热菌Thermomyces dupontii NRRL 2155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。
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| 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 |
| 收稿时间： | 2018-08-17 |
An efficient system for targeted gene replacement in the thermophilus fungus Thermomyces dupontii |
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| Authors: | HE Jia-Ning NIU Xue-Mei |
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| Affiliation: | State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming, Yunnan 650091, China |
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| Abstract: | 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: 2g of fungal mycelia was treated with 15mg/mL lysing enzyme at 28°C for 5.5h 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. |
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| Keywords: | thermophilic fungus Thermomyces dupontii protoplast transformation gene disruption biosynthesis genes |
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