Characterization of Insulin-Like Growth Factor Binding Protein 7 (Igfbp7) and Its Potential Involvement in Shell Formation and Metamorphosis of Pacific Abalone, Haliotis discus hannai

Abstract

Insulin-like growth factor binding proteins (IGFBPs) are secreted proteins that play an important role in IGF regulation of growth and development of vertebrate and invertebrates. In this study, the IGFBP7 gene was cloned and characterized from mantle tissues of H. discus hannai, and designated as Hdh IGFBP7. The full-length cDNA sequence transcribed from the Hdh IGFBP7 gene was 1519-bp long with an open reading frame of 720-bp corresponding to a putative polypeptide of 239 amino acids. The molecular mass of its mature protein was approximately 23.44 KDa with an estimated isoelectric point (pI) of 5.35, and it shared significant homology with IGFBP7 gene of H. madaka. Hdh IGFBP7 has a characteristic IGFBP N-terminal domain (22–89 aa), a kazal-type serine proteinase inhibitor domain (77–128), and an immunoglobulin-like C2 domain (144–223). Furthermore, twelve cysteine residues and a signature motif of IGFBPs (XCGCCXXC) were found in its N-terminal domain. Phylogenetic analysis revealed that Hdh IGFBP7 was aligned with IGFBP7 of H. madaka. Tissue distribution analysis showed that the mRNA of Hdh IGFBP7 was expressed in all examined tissues, with the highest expression level observed in the mantle and gill tissues. The expression level of Hdh IGFBP7 mRNA was relatively higher at the juvenile stage during its metamorphosis period. In situ hybridization showed that Hdh IGFBP7 transcript was expressed in epithelial cells of the dorsal mantle pallial and mucus cells of the branchial epithelium in gill. These results provide basic information for future studies on the role of IGFBP7 in IGF regulation of shell growth, development and metamorphosis of abalone.

Effects of Cryopreservation on Gene Expression and Post Thaw Sperm Quality of Pacific Abalone, Haliotis discus hannai

“…Gene-specific primers (Table 1) were designed to performed qRT-PCR. A 2 × qPCRBIO SyGreen Mix Lo-Rox kit (PCR Biosystems, Ltd., London, United Kingdom) was used to conduct qRT-PCR as described by Sharker et al (2020a). PCR amplification was conducted using a LightCycler R 96 System (Roche, Germany) with a 20 µL reaction containing 1 µL cDNA template of each sperm sample, 10 µL SyGreen Mix, 7 µL PCR-grade water, and 1 µL (10 pmol) of each forward and reverse primer.…”

Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis discus hannai

“…Ribosomal protein L-5 (RPL-5, GenBank accession no: JX002679.1) (forward: 5 -TGTCCGTTTCACCAACAAGG-3 and reverse: 5 -AGATGGAATCAAGTTTCAATT-3 ) as an internal control was used for normalizing mRNA abundance levels of each of the samples based on its expression stability [57]. A qRT-PCR analysis was performed using a 2× qPCRBIO SyGreen Mix Lo-Rox kit (PCR Biosystems, Ltd., London, UK) as described previously [58,59]. qRT-PCR was performed with the following cycling conditions: a pre-incubation step at 95 • C for 2 min followed by 40 cycles of a three-step amplification at 95 • C for 5 s, 60 • C for 15 s, and 72 • C for 20 s. The relative mRNA abundance levels of HSP90 were quantified using the 2 −∆∆ct method [60].…”

Urinary Biomarkers for Cell Cycle Arrest TIMP-2 and IGFBP7 for Prediction of Graft Function Recovery after Kidney Transplantation

“…TIMP-2 has a molecular size of approximately 21 kDa [27], while IGFBP7 has a molecular size of more than 23 kDa [28]. High-flux dialyzers used for intermittent hemodialysis in our centers are able to remove low-and middle-sized molecules with a molecular weight of 10-15 kDa [29].…”