Abstract
The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.
Highlights
From the ‡Faculty of Veterinary Science, The University of Sydney, Camperdown, New South Wales (NSW) 2006, Australia; ¶Department of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia; ʈBioinformatics Division, The Walter and Eliza Hall Institute for Medical Research, Parkville, Victoria 3050, Australia; **Department of Obstetrics and Gynaecology, Monash University, Victoria 3800, Australia; ‡‡Department of Zoology, The University of Melbourne, Victoria 3010, Australia; §§The Genome Institute, Washington University School of Medicine, Forest Park Parkway, St
CDNA Extraction—RNA was extracted from platypus venom gland tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated with Amplification grade DNase I
We used RNA-seq to compare the expression of venom transcripts in venom glands in and out of breeding season obtained from two opportunistically acquired animals from Tasmania (Fig. 1)
Summary
Transcriptome Sequencing—cDNA libraries were constructed from the venom glands from different platypuses from Tasmania. Functional term enrichment analyses were performed with Ontologizer [21] using human orthologues of platypus Ensembl genes that were expressed in the venom gland. Because of the fragmentation of proteins in the Illumina database, we considered putative proteins that fell below this cut-off but for which more than one peptide had mapped through BLAST to the same human gene These database sequences were likely to be partial fragments that could not be fully assembled because of low transcriptomic coverage. CDNA Extraction—RNA was extracted from platypus venom gland tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated with Amplification grade DNase I (Sigma-Aldrich, St. Louis, MO) to remove residual DNA. Nested RACE PCR products were run on agarose gel and the DNA was extracted as described above.
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