引起三疣梭子蟹“牛奶病”的酵母菌18S rRNA序列测定与分析

The 18S rRNA sequencing and analysis of a pathogenic yeast,a causative agent of emulsification disease in Portunus trituberculatus

"牛奶病"是近年来秋、冬季三疣梭子蟹暂养过程中发生的一种危害较大的流行性疾病,患病蟹的体液和肌肉组织中有大量的酵母菌。根据GenBank中的假丝酵母菌的18S rRNA基因序列设计合成引物,用PCR扩增患"牛奶病"三疣梭子蟹上分离到的酵母菌的部分18S rRNA基因,PCR产物经纯化回收后克隆入pUCm-T载体,筛选出阳性克隆进行测序,测序结果与已发表的序列进行同源性比较,同时以蟹源弧菌和寄生虫的DNA样本为对照。结果,所测序列与GenBank中假丝酵母菌的18S rRNA基因的同源性为84.4%～100%。同时,仅从试验用酵母菌DNA样本中扩增出了532bp左右的特异性片段,其他对照样本均未出现扩增反应。说明本实验建立的检测酵母菌的PCR方法有效。

A pathogenic yeast strain related with emulsification disease was isolated trom Portunus trituberculatus. A pair of primers were synthesized based on C. olephila 18S rRNA sequence published on Genback. Partial 18S rRNA sequence was amplified, cloned and sequenced. Sequences were compared to the corresponding genes published by DNASTAR software. The control DNA samples of Vibrio and parasites were amplified simultaneously. The results showed that the similarity of nucleotide sequence between the yeast isolated from the diseased crab and sequence of C. olephila was between 84.4%-100%. Among the DNA samples, only a fragment of 532bp yeast nucleotide was amplified specifically, while no amplifications were found in the control DNA samples. It can be concluded that this PCR method was effective for detection of the pathogenic yeast and for molecular epidemiology study for crab yeast pathogens.