摘要
柱状田头菇(茶树菇)Agrocybeaegerita是一种美味的食用菌,具有极高的经济价值。随着其全基因组测序的完成,功能基因组学研究也逐渐展开,其中,高效的遗传转化体系作为技术基础成为研究重点。本研究以柱状田头菇原生质体为受体、潮霉素抗性基因(hph)作为筛选标记,以增强型绿色荧光蛋白基因(egfp)为报告基因,应用PEG介导法进行柱状田头菇遗传转化体系研究。结果表明,150μg/m L潮霉素可以完全抑制柱状田头菇的生长。30℃下用2%裂解酶液酶解菌丝3h,能够获得最大得率的原生质体。通过PEG介导将构建好的DNA片段转化入柱状田头菇原生质体,通过潮霉素抗性筛选获得转化子,转化得率达到7个/μg DNA。PCR验证和荧光显微镜观察,外源片段成功转入柱状田头菇中并稳定表达。本研究建立的PEG介导转化体系,为柱状田头菇基因功能研究提供了技术基础。
Agrocybe aegerita is a delicious mushroom possessing very high economic value. The functional genomic research of this mushroom has been developed progressively with the completion of genome sequencing. The efficient transformation system is the key to technical foundation of gene function research. In this study, the PEG mediated method for genetic transformation of A. aegerita was carried out. A transformed DNA fragment was constructed by employing hygromycin resistance gene hph as selection marker and enhanced green fluorescent protein gene egfp as reporting gene. The sensitivity assessment showed that the hygromycin B concentration of 150μg/mL could inhibit the mycelial growth of A. aegerita completely. The highest yield of protoplasts could be obtained by using 2% lysing enzyme to digest the fresh mycelia at 30℃ for 3 h. The constructed DNA fragments were transferred into the protoplasts by PEG-mediated method, and transformants were obtained with a transformation efficiency of 7 protoplasts/μg DNA through hygromycin resistance selection. Transformants were verified by PCR analysis and fluorescence microscopy observation. The results indicated that hph and egfp were successfully transformed into the protoplast, and they stably expressed in the strain of A. aegerita. The stable PEG-mediated transformation system provided an essential technical basis of the study on gene function of this fungus.
作者
陶南
崔祥华
柴红梅
马渊浩
赵永昌
陈卫民
TAO Nan;CUI Xiang-Hua;CHAI Hong-Mei;MA Yuan-Hao;ZHAO Yong-Chang;CHEN Wei-Min(Biotechnology and Germplasm Resources Institute,Yunnan Academy of Agricultural Sciences,Kunming,Yunnan 650223,China;Yunnan Provincial Key Lab of Agricultural Biotechnology,Kunming,Yunnan 650223,China;Key Lab of Southwestern Crop Gene Resources and Germplasm Innovation,Ministry of Agriculture,Kunming,Yunnan 650223,China;College of Life Sciences,Southwest Forestry University,Kunming,Yunnan 650224,China)
出处
《菌物学报》
CAS
CSCD
北大核心
2020年第6期1100-1108,共9页
Mycosystema
基金
国家自然科学基金(31960017)
现代农业(食用菌)产业技术体系(CARS20)。
关键词
柱状田头菇
PEG介导转化
潮霉素
绿色荧光蛋白
Agrocybe aegerita
PEG-mediated transformation
hygromycin
green fluorescent protein
