Cloning and functional analysis of IGF2 and IGF2R genes in Chinese soft-shelled turtle（Pelodiscus sinensis）

【Objective】To explore the role of insulin-like growth factor 2 gene(IGF2) and insulin-like growth factor 2receptor gene(IGF2R) in the growth of Pelodiscus sinensis, and to lay a foundation for revealing the mechanism of IGFs system in the allometric growth and sexual growth difference of P. sinensis. 【Method】The cDNA sequences of IGF2 and IGF2R genes in P. sinensis were cloned by RACE. Bioinformatics analysis was performed using online softwares such as BioEdit, ExPASy, TMHMM Server v2.0, SignalP and SMART. The expression changes of IGF2 and IGF2R genes in different tissues, different phenotypes and different sex steroid hormones of male and female P. sinensis were analyzed by real-time fluorescence quantitative PCR. 【Result】The total length of the cDNA sequence of IGF2 gene was 1180 bp, encoding 222 amino acid residues. IGF2 protein, with a relative molecular weight of 24.84 kD and theoretical isoelectric point(pI) of 9.00, was a transmembrane protein and contained one IIGF domain. The total length of the cDNA sequence of IGF2R gene was 8765 bp, encoding 2443 amino acid residues. IGF2R protein had a relative molecular weight of 271.92 kD and a pI of 5.64. It was a transmembrane protein with fourteen repeated CIMR domains and one FN2 domain.Phylogenetic trees based on IGF2 and IGF2R amino acid sequence similarity showed that P. sinensis was most closely related to tortoises and far from fish and amphibians. IGF2 and IGF2R genes had a wide tissue expression profile in P. sinensis. The relative expression of the IGF2 gene in testis was significantly higher than that in the ovary(P<0.05, the same below), while the situation was the opposite for IGF2R gene. The relative expression of IGF2 gene in the liver of fast growing P. sinensis individuals was significantly higher than that in the slow-growing individuals, while the expression pattern of IGF2R gene was the opposite. Under estradiol(E2) treatment, except that the relative expression of IGF2R gene in the liver of female P. sinensis was significantly increased at 6 h after injection, the relative expression of IGF2 and IGF2R genes in the liver of male and female P. sinensis were decreased on the whole. MT treatment had no significant effect on IGF2 gene expression in male P. sinensis liver(P>0.05), but significantly increased the relative expression of IGF2 gene in female P. sinensis liver 48 h after injection, and significantly decreased the relative expression of IGF2R gene in female P. sinensis liver. 【Conclusion】IGF2 and IGF2R genes are involved in the regulation of the gonad development and maturation of P. sinensis, and IGF2R may negatively regulate the growth of P. sinensis by internalizing and degrading IGF2. In addition, IGF2 and IGF2R genes regulate the growth of P. sinensis by down-regulating their expression levels in response to exogenous hormone stimulation.