多子小瓜虫HSP70基因ORF的克隆及表达分析

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基因在多子小瓜虫对抗外界高温环境中可能发挥着重要的作用。

Cloning and expression analysis of HSP70 ORF in Ichthyophthirius multifiliis

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 HSP70 gene. The fluorescence quantitative RT-PCR showed that the minimal expression levels of Im HSP70 were observed in theront stage at all the experimental temperature, significantly higher level in trophont than that in theront and tomont at 20℃ and 25℃（P〈0. 05）,and the maximal level in tomont at 30℃. There were higher ex-pression levels in theront and trophont stages in 30℃ than that in 25℃（P〈0. 05）. Even at 30℃,the I. multifiliis strain showed relatively higher gene expression level and had infectivity to the fish. We can speculate that HSP70 may play an important role in fighting against the external environment temperature change due to the parasite be-longing to the tropical type.