Molecular structure and alternative splicing analysis of heat shock factors of Schistosoma japonicum

Abstract

To clone and identify the heat shock factors (HSFs) of Schistosoma japonicum and analyze its molecular structure and alternative splicing pattern. 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. 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. 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.