Expression Characteristics And Preliminary Functional Analysis Of Three Genes In The Insulin-like Growth Factor System Of Razor Clam Sinonovacula Constricta

Razor clam(Sinonovacula constricta),is one of the most important marine economic shellfish in China.Growth traits were important aims for the breeding.Insulin-like growth factor(IGF)system is well-known for its function on growth and development of animals.Micro RNA(mi RNA)was also widely involved in the process of growth and development.The gene cloned and other molecular biological techniques were used to characterize three genes in the IGF system.A high-throughput sequencing approach was used to characterize mi RNAs and their target transcripts in razor clam.Mi RNAs were screened,which are involved in IGF system regulation in razor clam.Detail results are showed as follow.Two forms of insulin-like peptide receptor(ILPR and s ILPR)had been identified in razor clam,which were produced from alternative splicing.ILPR and s ILPR respectively encoded 1502 amino acids and 1157 amino acids.There were 998 identical amino acids in the N-terminal of ILPR and s ILPR.ILPR contained all the typical features of insulin receptor(IR).s ILPR had only the extracellular domain and an additional cysteine-rich region at the C-terminal.Real-time PCR showed that the m RNA expression levels of s ILPR were lower than those of ILPR at different development stages and the levels were higher in the foot and mantle tissue but lower in gill tissue.The GST pull-down assay indicated that the LCL domain specially interacted with the pro-insulin like peptide 2 of razor clam without the signal peptide.The 10 single-nucleotide polymorphisms(SNPs)were identified in the c DNA sequences of ILPR.Four of the 10 SNPs were in the 5' UTR.Six of the 10 SNPs were in the coding region and were synonymous mutations.The analysis of associations with growth traits showed that the SNPs in the 5' UTR region were significant associations with total body weight(TW),shell length(SL),shell width(SW),and shell height(SH)in razor clam.Furthermore,one of the SNPs was used for genetic linkage mapping.The result demonstrated that ILPR was strongest linkage with the marker 26699 in linkage group 11.The LOD value of the marker 26699 in relation to growth traits ranged from 0.13 to 0.72,and did not belong to the quantitative trait locus region.The FOXO gene encoded the protein that contained 625 amino acids with a conserved DNA-binding domain.Real-time PCR analysis showed that FOXO m RNA was widely expressed in adult tissues,with higher expression in the siphons and gills.Five SNPs were identified in the coding region of FOXO.Of the five SNPs,c.543C>T(Phe181Leu),c.848A>G(Tyr283Cys),and c.1625G>C(Gly542Phe)were non-synonymous mutations.The SNPs all showed significant associations with TW,SL,SW,and SH in razor clam.The SNP c.1625G>C(Gly542Phe)was used for genetic linkage mapping.The result demonstrated the strongest linkage with the marker 96616 in linkage group 9.The LOD value of this marker in relation to growth traits ranged from 1.04 to 1.53,and did not belong to the quantitative trait locus region.IGF2BP gene in razor clam contained 610 amino acids and included typical domains of RNA binding domain.The recombinant p EGX-4T-1-IGF2 BP protein was expressed in Escherichia coli Rosetta(DE3)p Lys S cells and purified using Glutathione Sepharose 4B resin.Activity was evaluated by GST pull-down assays,and mass spectrometry results indicated that IGF2 BP protein may participate in RNA-related processes.A high-throughput sequencing approach was used to characterizted the mi RNAs and their target transcripts at eight time point of development in razor clam.1145 known mi RNAs and 53 novel mi RNAs were identified in cleavage period,483 known mi RNAs and 66 novel mi RNAs in blastula period,798 known mi RNAs and 69 novel mi RNAs in trochophore stage,742 known mi RNAs and 67 novel mi RNAs in veliger stage,735 known mi RNAs and 59 novel mi RNAs in umbo larvae,624 known mi RNAs and 37 novel mi RNAs in creeping larvae,772 known mi RNAs and 47 novel mi RNAs in single pipe larvae and 1110 known mi RNAs and 49 novel mi RNAs in juvenile.The seven mi RNAs were obtained in relation with IGF system of razor clam,based on the data analysis of target predition.These mi RNAs were formed in a complicated signal network by co-regulation genes had similar or antagonistic function with IGF system genes.