Salivary gland transcriptome analysis in Hirudinaria sp. during different feeding periods and functional studies of HspHirudin

Abstract

Abstract Background Hirudinaria sp., as one of the origins of leeches used in traditional Chinese medicine, is proved clinically effective in treating cardiovascular and cerebrovascular diseases. Substances with pharmacological activity are stored in salivary glands of leeches. Among these substances, hirudin exhibits high anti-thrombin activity. However, the structure, function and expression pattern of HspHirudin in Hirudinaria sp. distributed in Guangxi are still unclear.Methods and results Illumina HiSeq platform was used to obtain the transcriptome datasets of salivary glands from Hirudinaria sp. during different feeding periods. Unigenes which were significantly up-regulated after a blood meal and had functions related to the processes of blood meal ingestion were identified. The function of HspHirudin was studied based on gene cloning, bioinformatics analysis, prokaryotic expression analysis and anti-thrombin activity analysis, and its expression pattern was characterized by quantitative real-time PCR. The reads were assembled into 167564 unigenes, and ten genes with functions related to the processes of blood meal ingestion in salivary glands of Hirudinaria sp. were identified. After alignment, transcript (TRINITY_DN14935_c0_g2) was annotated to Hirudin-P6 (2E-31) from Poecilobdella manillensis and named HspHirudin. The HspHirudin cDNA was 249 bp in length, and it encodes a mature peptide of 82 aa and a signal peptide of 20 aa. The mature HspHirudin presented typical structural characteristics of hirudins: 3 pairs of disulfide bonds, the PKP and DFxxIP motifs. HspHirudin and Hirudin-P6 shared similar structure of N-terminus 3 aa and C-terminus, and their main differences were observed in the spherical structure domain. By constructing a prokaryotic expression system, HspHirudin was proven to have anti-thrombin activity. The high expression of HspHirudin mRNA was induced by bloodmeal feeding.Conclusions Ten genes with functions related to the processes of blood meal ingestion were screened from transcriptome datasets of salivary glands from Hirudinaria sp. during different feeding periods. Among them, HspHirudin, which belongs to the family of hirudins, has anti-thrombin activity. The results of the study provide theoretical basis for further development and utilization of Hirudinaria sp..