聚乙二醇介导的赭曲霉遗传转化体系的构建  

Construction of Genetic Transformation System of Aspergillus ochraceus Using Polyethylene Glycol Mediated Method

作　　者：王刘庆[1] 王龑[1,2] 王琦[1] 刘阳[1,2] 

机构地区：[1]中国农业科学院农产品加工研究所,北京100193 [2]农业部农产品加工综合性重点实验室,北京100193

出　　处：《农业生物技术学报》2016年第6期928-936,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2013CB127802);公益性行业(农业)科研专项(No.201203037);科技基础性工作专项重点项目(No.2013FY113400)

摘　　要：赭曲霉(Aspergillus ochraceus)不仅能够引起谷物霉腐,而且能够代谢产生具有强肾毒性、致癌、致畸、致突变的赭曲霉毒素A(ochratoxin A,OTA),严重危害人类健康。然而,赭曲霉也能够发酵应用于甾体转化和生产木葡聚糖酶等有益方面。为深入解析赭曲霉中OTA的生物合成与调控机制以及探索其有益的基因资源,本研究基于同源重组的原理,利用聚乙二醇(polyethylene glycol,PEG)介导的原生质体转化方法成功构建了赭曲霉的遗传转化体系。基于赭曲霉对潮霉素的敏感性,本研究选择以潮霉素抗性基因作为赭曲霉遗传转化体系的筛选标记基因,筛选浓度为70μg/m L以上,为保障筛选的稳定性和减少假阳性,选择浓度100μg/m L的潮霉素进行筛选。赭曲霉原生质体的质量和浓度是转化成功的必备条件,本研究优化2%蜗牛酶和2%纤维素酶为原生质体制备的适宜细胞壁酶解条件。本研究采用融合PCR和巢式PCR技术获得目的基因上下游和潮霉素抗性基因相融合的拟转化DNA片段,利用PEG介导的原生质体转化方法将拟转化DNA片段转化入赭曲霉原生质体,经潮霉素抗性筛选、PCR测序验证鉴定是否为阳性转化子。赭曲霉遗传转化体系的成功构建为今后赭曲霉在OTA生物合成及其分子调控机制和工业生产应用研究的开展提供了有利的技术保障。Aspergillus ochraceus not only can cause grain mildew and rot, and but also produce ochratoxin A （OTA）, which exhibits a potent nephrotoxic, carcinogenic, teratogenic and mutagenic mycotoxin. OTA is highly detrimental to human health on account of its toxicity. However, this fungus can also be fermented for steroid transformation and the production of xyloglucan enzyme in the beneficial aspect. To deeply understand OTA biosynthesis and regulation mechanisms or explore the resources of useful genes to be apply to industrial fermentation in Aspergillus ochraceus, we constructed genetic transformation system of this fungus using the method of polyethylene glycol （PEG） mediated protoplast transformation, based on the principle of homologous recombination. The hygromycin resistance gene was chosen as the screening marker gene in that this fungus was regarded to be very sensitive to hygromycin. The screening concentration was more than 70 μg/mL of hygromycin, which could inhibit the growth of this fungus completely. In order to guarantee the stability of the screening and reduce false positives, we chose the concentration with 100 μg/mL of hygromycin. Considering the quality and concentration of the protoplast was essential to the success of transformation, the protoplast preparing conditions were optimized to the most suitable condition with 2% snailase and 2% cellulase. DNA fragment was cloned and extracted by combining the upstream and downstream of the target gene with hygromycin resistant gene using fusion PCR and nested PCR technology. The DNA fragment was then transformed into the protoplast using PEG mediated protoplast transformation method. The positive transformants were identified and confirmed by hygromycin resistance screening, PCR and sequencing. The successful establishment of Aspergillus ochraceus genetic transformation system provides a favorable technical support for OTA biosynthesis, its molecular regulation mechanisms, and industrial production and application.

关 键 词：赭曲霉 遗传转化体系 原生质体 转化子 

分 类 号：Q785[生物学—分子生物学]