Objective: To clone and identify the heat shock factors (HSFs) of Schistosoma japonicum and analyze its molecular structure and alternative splicing pattern.
Methods: The New Zealand rabbits were infected with the cercariae of Schistosoma japonicum and were killed and dissected 42 days post-infection, and the adult worms of S. japonicum and the livers of the rabbits were harvested. Then, the total RNA was extracted by using Trizol reagent. The Sj-hsf open reading frame (ORF) and the alternative splicing fragments were amplified by RT-PCR from the female, male and egg samples, then cloned and verified by enzyme digestion and sequencing. DNAMAN 8.0, InterPro, Mega 6 combined with the Internet databases were utilized to clarify the gene structure, functional domains, alternative splicing pattern, and the homology and phylogenetic tree of HSFs.
Results: Sj-hsf ORF and the alternative splicing fragments were amplified from the female, male and egg samples of S. japonicum by RT-PCR. After cloning, the positive recombinant plasmids pBSjHSFf-F, pBSjHSFf-M, pBSjHSFf-E containing Sj-hsf ORF, pBSjHSFs-F, pBSjHSFs-M, pBSjHSFs-E with Sj-hsf alternative splicing fragments were identified by enzyme digestion and sequencing. Three alternative splicing Sj-hsf isoforms were observed through sequence analysis: Sj-hsf-isoform1 (2 050 bp), Sj-hsf -isoform2 (2 086 bp) and Sj - hsf -isoform3 (2 111 bp); the GenBank accession numbers were KU954546, KX119143 and KX119144, respectively. All the three isoforms located in the same Contig SJC_S000780 of S. japonicum genome and all expressed at female, male and egg stages, but Sj-hsf-isoform1 with a high-level expression. Sj-HSF-isoform1 (671 aa) and Sj-HSF-isoform2 (683 aa) had DBD (DNA binding domain), HR-A/B and HR-C domains, while Sj-HSF-isoform3 (282 aa) stopped in advance without HR-C domain. Phylogenetic tree analysis of HSFs illustrated that Sj - HSFs belonged to HSF1 family, with a close phylogenetic relationship to Sm-HSFs.
Conclusions: There are three alternative splicing isoforms of Sj-HSF existing in the female, male and egg stages of S. japonicum, but Sj-HSF-isoform1 expresses in a high-level. This study lays the foundation for further study on molecular mechanisms of Sj-HSFs in regulating the heat shock response system.
［摘要］目的 克隆、鉴定日本血吸虫热休克因子 (Sj-hsf), 并分析其基因结构特征和可变剪切亚型。方法 以日本血 吸虫尾蚴感染新西兰白兔, 42 d后剖杀, 以灌注法收集雌虫、雄虫及感染兔肝脏, 以Trizol法提取总RNA。以RT-PCR扩 增日本血吸虫雌虫、雄虫、虫卵总RNA, 获得Sj-hsf 开放阅读框 (ORF) 全长片段和Sj-hsf 可变剪切区域片段, 并进行克隆、 酶切及测序鉴定。用DNAMAN 8.0、InterPro、Mega 6结合日本血吸虫和曼氏血吸虫基因组数据库以及GenBank等网络分 析平台, 分析Sj-hsf 的基因结构特征、功能结构域、可变剪切模式以及不同物种间HSF序列的同源性和进化树。结果 自 雌虫、雄虫、虫卵扩增获得靶片段, 并克隆获得阳性重组质粒, 其中含Sj-hsf ORF 的pBSjHSFf-F、pBSjHSFf-M、pBSjHSFf-E 以及含可变剪切片段的pBSjHSFs-F、pBSjHSFs-M、pBSjHSFs-E, 酶切及测序鉴定正确。序列分析显示Sj-hsf 具有3种剪切 亚型：Sj-hsf-isoform1 (2 050 bp) 、Sj-hsf-isoform2 (2 086 bp) 、Sj-hsf-isoform3 (2 111 bp), GenBank登录号分别为KU954546、 KX119143、KX119144, 均位于日本血吸虫基因组SJC-S000780, 有8个外显子, 在雌虫、雄虫和虫卵阶段3种亚型均存在, 以Sj-hsf-isoform1表达量高。Sj-HSF-isoform1 (671 aa) 、Sj-HSF-isoform2 (683 aa) 具有HSF特征性DBD (DNA Binding Do- main) 、HR-A/B和HR-C结构域, Sj-HSF-isoform3 (282 aa) 提前终止, 无HR-C结构域。进化树分析表明Sj-HSF三种剪切 亚型隶属HSF1, 与曼氏血吸虫HSF近源。结论 日本血吸虫雌虫、雄虫、虫卵阶段均存在3种剪切亚型的HSF, 以Sj-HSF -isoform1表达量高。本研究为进一步阐明Sj-HSF调控Sj-HSPs等应激防御体系的分子机制奠定了基础。.
Keywords: Alternative splicing; Heat shock factor; Heat shock proteins; Heat shock response; Schistosoma japonicum.