[Molecular structure and alternative splicing analysis of heat shock factors of ].

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

Methods: The New Zealand rabbits were infected with the cercariae of and were killed and dissected 42 days post-infection, and the adult worms of and the livers of the rabbits were harvested. Then, the total RNA was extracted by using Trizol reagent. The 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: ORF and the alternative splicing fragments were amplified from the female, male and egg samples of by RT-PCR. After cloning, the positive recombinant plasmids pBHSFf-F, pBHSFf-M, pBHSFf-E containing ORF, pBHSFs-F, pBHSFs-M, pBHSFs-E with alternative splicing fragments were identified by enzyme digestion and sequencing. Three alternative splicing isoforms were observed through sequence analysis: -isoform1 (2 050 bp), -isoform2 (2 086 bp) and -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 genome and all expressed at female, male and egg stages, but -isoform1 with a high-level expression. -HSF-isoform1 (671 aa) and -HSF-isoform2 (683 aa) had DBD (DNA binding domain), HR-A/B and HR-C domains, while -HSF-isoform3 (282 aa) stopped in advance without HR-C domain. Phylogenetic tree analysis of HSFs illustrated that - HSFs belonged to HSF1 family, with a close phylogenetic relationship to -HSFs.

Conclusions: There are three alternative splicing isoforms of -HSF existing in the female, male and egg stages of , but -HSF-isoform1 expresses in a high-level. This study lays the foundation for further study on molecular mechanisms of HSFs in regulating the heat shock response system.