一种适用于真菌的高通量分子鉴定方法的建立

Development of A Protocol Suitable for Molecular Identification of Massive Fungus Strains

真菌在生物领域扮演着重要的角色,随着分子生物技术的发展,从分子水平对真菌的鉴定及对遗传转化子的验证成为必需的试验环节。但目前尚无一种比较理想的高通量分子鉴定的方法。设计一种新的高通量装置,并且建立适用于这种高通量装置的,由PCR介导的分子鉴定方法,主要通过对实验室常用菌株里氏木霉基因组上相关纤维素酶编码基因、调控基因,酿酒酵母基因组上磷酸甘油酸激酶1基因的启动子区,酿酒酵母胞内表达载体上木聚糖酶基因的克隆验证及对实验室保藏菌株草酸青霉、葡枝根霉、黑曲霉、碳黑曲霉、日本曲霉及上述两株菌株的ITS基因克隆验证,RAPD分析,确定这种方法的高效性及应用广泛性。这种方法的建立很大程度上解决了高通量分子鉴定的瓶颈问题。

Fungus plays a significant role in biology-related domains and with the molecular biology technology advancing,the key step has necessitated the identification of fungus and transformant from molecular level. By far, however,there hasn＇t remained an ideal protocol for the massive strains identification.In the research,a device used for massive identification was designed, and a novel identification protocol mediated by PCR from molecular level, suitable for the device,was developed. In the article, through the cloning of genes encoding cellulase and relative regulatory factors which are located in genome of Trichoderma reesei, the cloning of promoter of phosphoglycerate kinase gene 1 and gene encoding xylanase,respectively located in genome and expression vector of S. cerevisiae and by ITS sequences cloning RAPD analasis of the two frequently-used strains mentioned above and strains Penicillium oxalicum, Rhizopus stolonifer, Aspergillus niger, Aspergillus carbonarius, Aspergillus japonicas. the protocol turned out to be an effective one with wide application potential, To a great degree,the establishment of the method has worked out the bottleneck of high-throughput molecular identification.