热激蛋白( HSP)是生物适应环境温度变化的重要媒介分子,本试验中克隆了多子小瓜虫Ichthyoph-thirius multifiliis HSP70(Im HSP70)基因的ORF,采用荧光定量RT-PCR法,在水温为20、25、30℃的条件下检测了HSP70基因在多子小瓜虫幼虫、滋养体和包囊3个不同发育阶段的表达情况。结果表明:Im HSP70基因的ORF为1995 bp,预测编码为664 aa; Im HSP70蛋白的二级结构主要以α-螺旋和无规卷曲为主,空间构型包括N端ATPase功能域和C端多肽结合功能域;与其他物种HSP70蛋白序列进行同源性比较发现, Im HSP70与螅状独缩虫Carchesium polypinum HSP70的同源性最高,为78.08%; Im HSP70与分类地位同为纤毛门的螅状独缩虫、嗜热四膜虫Tetrahymena thermophila、变藓棘毛虫Sterkiella histriomuscorum的HSP70聚在一个分支;3种温度下幼虫的HSP70表达量均最低,20℃和25℃时, HSP70基因在滋养体中的表达量显著高于幼虫和包囊(P<0.05),而30℃时, HSP70基因在包囊中的表达量显著高于滋养体和幼虫(P<0.05);30℃时HSP70基因在包囊中的表达量显著高于25℃(P<0.05)。研究表明,试验所用多子小瓜虫的HSP70基因在30℃时仍大量表达,且能感染试验鱼,推测该虫株为热带型多子小瓜虫, HSP70基因在多子小
In this paper, heat-shock protein(HSP70)gene ORF (open reading frame),as an important medium molecule for change in environmental temperature, was cloned in parasite Ichthyophthirius multifiliis, and the ex-pression of the gene was studied in theront,trophont and tomont of the parasite at water temperature of 20, 25, and 30 ℃ by RT-PCR. It was found that Im HSP70 gene ORF was 1995 bp encoding a putative protein of 664 amino acid residues. The gene structure analysis revealed that the Im HSP70 was primarily comprised ofα-helix and ran-don coil-based in secondary structure, and the N-terminal ATPase domain and C-terminal peptide-binding do-main in spatial configuration. Comparison with other known HSP70 protein indicated that the Im HSP70 had the maximal similarity (78. 08% identity) to that in Carchesium polypinum. Phylogenetic analysis revealed that I. mul-tifiliis was clustered into a branch with C. polypinum,Tetrahymena thermophila and Sterkiella histriomuscorum by the Im