- Title
- Molecular cloning and characterisation of two insulin-like growth factor binding protein genes (sgIGFBP-5 AND sgIGFBP-7) from the Sydney rock oyster Saccostrea glomerata
- Creator
- Nguyen, Thi Hong Tham
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Insulin-like growth factors (IGFs) play crucial roles in regulating growth, development and metabolism in animals. IGFs are complexed with specific binding proteins known as IGF binding proteins (IGFBPs), which modulate, either stimulate or inhibit, the action of IGFs. Most of the current literature on oyster biology focuses on reproductive cycle and growth patterns as well as their relationship with their ambient environmental conditions. Compared with vertebrates, little is known about the biology of IGFBPs and the molecular mechanisms involved in the regulation of growth in invertebrates, particularly molluscs. To fill this knowledge gap, both the cDNA and genomic DNA sequences of the IGFBP-5 and IGFBP-7 homologues were cloned from the Sydney rock oyster (SRO), Saccostrea glomerata. The spatial and temporal mRNA expression patterns, single nucleotide polymorphisms (SNPs) and their consequent haplotypes of these two genes as well as their transcriptional responses to fasting and hypoxia were investigated to decipher their potential roles in the regulation of the SRO growth. The full-length cDNA and gDNA sequences of the S. glomerata IGFBP-5 and IGFBP-7 genes (designated sgIGFBP-5 and sgIGFBP-7, respectively) were cloned and characterised for the first time. For sgIGFBP-5, the ORF consists of 357 bp, encoding a polypeptide of 118 amino acid residues. The deduced amino acid sequence of sgIGFBP-5 contained conserved N- and C-terminal regions, multiple cysteine residues and two conserved motifs (GCGCCXXC and CWCV). There are multiple putative regulatory DNA elements in the promoter region of sgIGBFP-5 which might interact with specific transcription factors to drive gene transcription, including an ERE half site, a SP1 site, an AP-1 site and a HRE. The genomic DNA of sgIGBFP-5 consists of 3 exons and 2 introns. For sgIGFBP-7, its ORF comprises of 780 bp, encoding a polypeptide of 259 amino acid residues. The sgIGFBP-7 protein consists of three conserved domains, including the IGFBP N-terminal domain, the Kazal-type serine protease inhibitor domain and the immunoglobin (Ig) domain, as well as one conserved motif “xCGCCxxC”. The promoter region of sgIGFBP-7 comprises of multiple putative regulatory DNA elements, including an ERE half site, an EGR1 binding site, a NF-κB cells binding site and a HRE core sequence. Phylogenetic analysis showed that the deduced proteins of sgIGFBP-5 and sgIGFBP-7 are clustered with their corresponding homologues from other molluscan species, supporting the proper nomenclature of the SRO genes. All of the observations above suggest that sgIGBFP-5 and sgIGBFP-7 is authentic IGFBP homologues and their genomic and deduced protein structures are conserved evolutionarily. Expression of sgIGFBP-5 and sgIGFBP-7 mRNA was detected in all the tested tissues in both juvenile and adult oysters. Notably, contrasting differential expression profiles of sgIGFBP-5 and sgIGFBP-7 mRNAs between the adult slow-growing and fast-growing oysters were observed in certain tissue types, implying that IGFBPs may play potential roles in regulating the growth of SRO. DNA sequencing of 100 oysters from each of the slow and fast-growing lines revealed the presence of 183 and 76 SNPs in the genomic regions of sgIGFBP-5 and sgIGFBP-7, respectively. Among these, 7 SNPs in sgIGFBP-5 and 16 SNPs in sgIGFBP-7 are significantly different in genotype/allele frequencies between the two oyster lines. The association of the SNPs identified in sgIGFBP-5 and sgIGFBP-7 with growth traits was examined among the oyster individuals grouped according to their genotypes constituted by each SNP. For sgIGFBP-5, significant differences were found in shell length among the genotypes constituted by six SNPs and in shell height among the genotypes constituted by all seven SNPs. For sgIGBFP-7, significant differences in shell length, shell height and whole oyster weight were found among the genotypes constituted by the majority of the identified SNPs. The association of sgIGFBP-5 and sgIGFBP-7 haplotypes with growth traits was also investigated. Statistically significant differences among the haplotypes were observed with respect to shell length and shell height for sgIGFBP-5 while no significant difference among haplotypes with respect to any of the tested growth traits for sgIGFBP-7 was found. These results suggest that the SNPs/haplotypes in sgIGFBP-5 and sgIGFBP-7 may have a significant association with the growth traits in S. glomerata and hence they may provide the basis for developing marker-assisted selection strategies to improve the oyster’s growth performance in the future. An in silico protein-protein interaction analysis was performed to identify the strengths of the potential binding between the deduced protein of each nonsynonymous haplotype groups and their potential ligands. The results revealed that the orders of binding strength of the tested ligands with each of the sgIGFBP-5 and sgIGFBP-7 haplotype groups were different. This finding suggests that sgIGFBP-5 and sgIGFBP-7 may have their own binding preferences for different cellular ligands, implying that the non-synonymous SNPs presented in the haplotype groups may give rise to critical changes in the 3D structure of the encoded IGFBP proteins, which in turn alter IGFBP-ligand interaction. A short-term starvation experiment and investigation of the regulatory effect of hypoxia on mRNA expression of sgIGFBP-5 and sgIGFBP-7 were conducted. For the fasting experiment, the sgIGFBP-5 and sgIGFBP-7 expression levels in both digestive gland and mantle tissue in fasting oysters increased compared to controls at the time points of Days 3, 5 and 7. This lead to the postulation that catabolic activity was increased and IGFBPs in the starved oysters are released into the circulation to bind to IGFs, thereby restricting IGFs from binding to their cognate receptors and hence slow down the oysters’ growth. The study also pointed out that re-feeding (2 days) of fasting oysters restored the basal expression of the IGFBP mRNA. This may suggest that the downregulation of these genes could in turn release the availability of IGFs for IGF-receptor binding or growth. However, this was the short-term starvation experiment, and the changes in growth traits of S. glomerata were not measured. Thus, it remains premature to conclude the roles of the sgIGFBPs in growth regulation. For the hypoxic experiment, no significant change in HIF-1 and IGFBP mRNA expression were detected in any of the tested oyster tissues after 5 days of air exposure. These observations do not agree with a hypothesis that HIF-1 expression is increased under hypoxic conditions and the increased HIF-1 availability can in turn activate IGFBP gene transcription via binding to the HRE(s) located in the IGFBP gene promoter. Overall, the findings from this research provide information on the structures and potential functions of IGFBPs in relation to growth and stress effects. In the future, from the SNPs and/or haplotypes in sgIGFBP-5 and sgIGFBP-7, identification of growth associated genetic markers will contribute to improving the genetic stock, productivity, sustainability and profitability of the SRO (and potentially other molluscan species).
- Subject
- sgIGFBPs; Sydney rock oyster; Saccostrea glomerata; IGFs; molluscs
- Identifier
- http://hdl.handle.net/1959.13/1411170
- Identifier
- uon:36305
- Rights
- Copyright 2020 Thi Hong Tham Nguyen
- Language
- eng
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