Molecular Cloning And Functional Characterization Of IGF-1R And Duplicated IGFBP-5in Grass Carp(Ctenopharyngodon Idellus) And SNP Analysis Of Different Grass Carp’s Muscle-growth Control Genes

The grass carp, Ctenopharyngodon idellus, belonging to the superorderOstariophysi, has been accepted as an important economic herbivorous freshwaterfishes. Grass carp is late sexual maturity, fast growth and a larger individual fish (>10kg), that make it as a good object for the research of growth-related genes.Insulin-like growth factor system (IGFs) is highly conserved during evolution,that play an important part in the development of the embryo, nerve and skeletalmuscle, proliferation and transformation of cells. In mammals, IGFs comprises twoligands (IGF-I and IGF-II), two receptors (IGF-IR and IGF-IIR) and six IGFBPs,which primarily exert biological function by binding to IGF-IR. After the IGF-IRand ligand binding, this caused the activation of different substrates, then startdifferent signaling pathways, which cause mitogenic effects and inhibition ofapoptosis in most cells. IGFBP-5is the most conservative members of IGFBP-family, which is a multi-purpose protein and strengthens the effect of IGF.we first report the isolation of a5741bp cDNA clone for the grass carp IGF-IRthat includes the complete5’ untranslated region(5’UTR)822bp, open readingframe(ORF) region4338bp and3’ untranslated region(3’UTR)581bp.The ORFregion encodes1445amino acid. The mature peptide contains seven structuraldomains: two Recep-L-domain, one Furin-like domain, three FN3domain and onePTKc-IGF-1R domain. PTKc-IGF-1R domain is the most conservative domain. TheORF deduced from the cDNA sequence is95%,93%and66%identical to thecorresponding regions of IGF-IRs in Cyprinus carpio, Zebrafish and Homo sapiens,respectively, which illustrate that IGF-IR gene is very conservative in the progress ofevolution and indicate that grass carp IGF-IR maybe belong to the type of A. By RT-PCR, grass carp IGF-IR expression was detected from16hpf(hours postfertilization) to larvae and almost all adult tissues except intestine. In this research,IGF-IR mRNA expression was found in heart, gonad, gill and muscle tissue. Usingwhole mount in situ hybridization, IGF-IR mRNA expression was found in severalfast-growing areas, such as brain, dorsal and tail, whereas IGF-IR mRNA expressionappeared weaker during later development.The result of the isolation of IGFBP-5a and IGFBP-5b in grass carp is asfollows：The full length of IGFBP-5b is2137bp, which contains384bp5’-UTR,792bp ORF and961bp3’UTR, which encodes263amino acids. IGFBP-5b includes19amino acids signal peptide and244amino acid mature peptide. The full length ofIGFBP-5a cDNA is2067bp, including380bp5’-UTR,807bp ORF and880bp3’-UTR. IGFBP-5a encodes268amino acids, including the21amino acids signalpeptide and247amino acid mature peptide. Like Homo sapiens and zebrafish, themature polypeptide of IGFBP-5a and IGFBP-5b in grass carp also contains tworegions: IB domain (Insulin growth factor-binding protein homologues) and TYdomain (Thyroglobulin type I repeats). IB domain contains12conserved cysteineresidues. TY domain contains six conserved cysteine residues and four protease sites.The similarity of IGFBP-5a and IGFBP-5b’coding regions in grass carp is71%. TheORF deduced from the cDNA sequence of IGFBP-5a is50%and87%identical tothe corresponding regions of IGFBP-5a in Homo sapiens and Zebrafish, respectively,while IGFBP-5b is56%and93%identical to IGFBP-5b in Homo sapiens andZebrafish. RT-PCR analysis of embryos at different times showed that grass carpIGFBP-5a and IGFBP-5b mRNAs had different expression patterns. Grass carpIGFBP-5a was not expressed in early embryos until16hpf, and expression level wasgradually rise high and then decreased from16hpf to40hpf. Grass carp IGFBP-5bwas expressed from12hpf to36hpf and the expression levels was relatively constant,but the difference was not significant. In adult fish, IGFBP-5a mRNA was expressedin all detecting tissues except slow-twitch, fast-twitch muscle, intestine and kidney,but there is a big difference in expression levels. The highest expression is in liver,and the rest of the tiusse such as gonads, eye, skin, gills, heart, spleen and brain werelower expression levels, but the expression is relatively constant. IGFBP-5b mRNAwas expressed in detecting tissues, but there is a big difference in expression levels,which expresses higher levels in the liver, gonads, eyes, skin, gills, kidney andspleen, but lower expression in slow-twitch muscle, fast-twitch muscle, intestine and brain.The single nucleotide polymorphism (SNP) was studied in fragments of fourmuscle-growth control genes, including Follistatin1(fst1), Follistatin2(fst2),Myostatin1(mstn1) and Myostatin2(mstn2). Seven groups of grass carp was used inthis study. Our results revealed that SNP existed at a relatively high frequency. Of7291base pairs sequenced, there was1SNP on average in every135base pairs. Weidentified54SNPs in9PCR fragments, among which3were in coding regions ofthe genes and51were non-synonymous. There were34transition mutations and20transversion mutations in the54SNPs. On the population frequency, there are34familiar SNPs and20rare SNPs. The SNP frequency of fst1、fst2、mstn1and mstn2was1.00%,0.64%,0.51%and0.76%respectively.3SNPs in the exons were onlyfound in fst1gene,2were non-synonymous mutations caused corresponding changeof coded amino acid and1was synonymous mutations. The number of SNPs wasdifferent in seven groups. The SNP number of Ruichang grass carp was the highest,the lowest one was Changsha grass carp. There were13-30SNPs in the seven strainsand specific SNP type was present in every groups of grass carp, so that SNP of fst1、fst2、mstn1and mstn2could be used to distinguish the groups of grass carp.The paper laid a theoretical foundation for the further exploration of the IGF-IRigfbp-5a and igfbp-5b in growth signal pathways and breeding of grass carp. What’smore, this paper also offers a theoretical basis for further study on polymorphism offst1、fst2、mstn1and mstn2in grass carp with good growth traits of breeding newspecies.