Studies On Cloning Of IGFBPs, Spationtemporal Expression Profiles Of IGF System And Correlation Analysis Of IGF-Ⅰ With Weight Trait On Sheep

Insulin like growth factorⅠand Insulin like growth factorⅡstimulate the growth of animal in embryo period and the developing of post birth period with mediating the neuroendocrine function of chondrotropic hormone.It is vital physiological effectiveness which are regulating carbohydrate metabolism,fat metabolism,protein synthesis,liver function and linear growth of all livestock.IGFs bind mainly IGFBP-1,IGFBP-2,IGFBP-3,IGFBP-4,IGFBP-5,IGFBP-6 and IGFBP-7 in circulation system of the animal organisms with the stimulating growth function throughout secreting from animal liver and carrying to the ambitus tissues.we have researched the IGF system genes regulating the growth of liangshan semi-wool sheep with Real time PCR,SSCP and cloning technique.Here we cloned mRNA of IGFBP family genes from total RNA of liver in liangshan semi-wool sheep by RT-PCR with exception for IGFBP-2.Sequence analysis indicated that the ovine IGFBP-1, IGFBP-3,IGFBP-4,IGFBP-5,IGFBP-6 and IGFBP-7 cDNA cloned were 792bp,882bp,777bp, 816bp,711bp and 849bp in length(GenBank accession number FJ589640,FJ752574,EU882037, EU727460,EU862545 and FJ589640) and the open reading frame encode a deduced protein with 263,293,258,271,236 and 282 amino acids residues respectively.The comparison of the duduced amino acids sequence of IGFBPs(including IGFBP-2) showed that the eds sequence homology were 0.334～0.548,and the animo acids homology were 0.124～0.426.Phylogenetic analysis indicates that the IGFBPs segregated into four distinct clades:IGFBP-1,IGFBP-3,IGFBP-6 and IGFBP-7.Subsequent gene duplication events within the IGFBP-1 and IGFBP-3 clades resulted in the production and divergence of IGFBP-2 and IGFBP-4 within the IGFBP-1 clade and IGFBP-5 in the IGFBP-3 clade.The primary structures of ovine IGFBPs appear to contain three distinct domains:the conserved N-terminal domain,the highly variable midregion,and the conserved C-terminal domain.the N-terminal domain of IGFBP-1,IGFBP-3,IGFBP-4,IGFBP-5,IGFBP-7 formed the six disulfide bridges(five in the case of IGFBP-6) with neighboring Cys within the conserved 12 Cys and the C-terminal domain contained the three disulfide bridges(only one of IGFBP-7) with the same modus of the N-terminal domain.there is a signal peptide within the fore 30 animo acid of N-terminal which possess a IGFBP_N functional domain and a Thyroglobulin type-1 domain(Ig-like of IGFBP-7) of the C-terminal of cloned six genes which exist large Casein kinaseⅡphosphorylation sites,Protein kinase C phosphorylation sites,Tyrosine kinase phosphorylation site,N-glycosylation sites and O-glycosylation sites.We researched the expression of IGF system genes in different tisssues with different growth period by QRT-PCR technique.The analysis indicated that all tissues we gained from liangshan semi-wool sheep were tested the expression of IGF system genes in different growth period and were different of relative expression with the different function of.the ambitus tissues.In brain tissue,there were similar to the expression mode of the eleven genes of IGF system with wave change,and were relatively high expression level at 15^th,105^th and 195^th day against the 60^th,150^th and 240^th day.In skin tissue,IGF-Ⅰ,IGF-Ⅱ,IGF-2R,IGFBP-1,IGFBP-4,IGFBP-6 and IGFBP-7 were similar to the expression mode with relatively high level at 15^th and 60^th day.However,the expression mode of IGFBP-1 and -3 were gradually lower from 15^th to 240^th day and only high expression of IGFBP-5 at 60^th day.In muslc tissue,there were similar to the expression mode of IGF-Ⅰ,IGF-Ⅱ, IGF-ⅠR,IGF-ⅡR,IGFBP-2 and IGFBP-6 and high expressional level at 150^th day,against IGFBP-1 with the function of inhibiting myocyte proliferation and differentiation,and stabilizational expression of IGFBP-4 and IGFBP-5.In liver tissue,there were similar to the expression mode of all IGF system genes during the different period with exception for IGFBP-2 at 105^th day and IGFBP-4 at 150^th day.In heart tissue,thoughout IGF system genes were similar to the expression mode with exception for IGF-ⅠR and high expressional level at 105^th day and tropesis stabilizational expression from 150^th to 240^th day.There were the positive correlation to different degree of the IGF system genes in skin,liver and brain tissues,and the same correlation with exception for negative correlation to different degree between IGF-ⅠR with IGF-Ⅰ,IGF-Ⅱ,IGF-ⅡR,IGFBP-2,IGFBP-3,IGFBP-4,IGFBP-5,IGFBP-6 and IGFBP-7 in heart tissue.In musle tissue,there were the positive correlation to different degree of the IGF system genes with exception for negative correlation to different degree between IGFBP-1 and IGFBP-3,IGFBP-4 and IGFBP-5,IGF-Ⅰand GFBP-7.It means that the expression were differentiated in tissues with the different function of the ambitus tissues.There were the two SNP site which have detected within exon 3 and exon leader region respectively with the p-1 and p-2 primers of the designed with complete exons of IGF-Ⅰin 587 individuals of liangshan semi-wool sheep by PCR-SSCP.The two SNP site were C→G and A→G mutation which the allele A and C were the predominant allele with the p-1 and p-2 primers.Polymorphism Information Content of the p-1 primer were 0.276 and 0.252～0.303,and the p-2 primer were 0.237 and 0.171～0.279 in the population and the 8 six half-sib family population respectively.The variance and relationship analysis showed that the SNP of the p-1 primer influenced significantly birth weight trait(p＜0.05) and the SNP of the p-2 primer influenced high significantly on Weaning weight and the Weaning daily weight traits and no significantly 18 months weight and 30 months weight traits.The haplotype H2 and H3 were the predominant haplotype and the combined H2H2,H1H2 and H1H3 influence significantly birth weight,18 months weight and 30 months weight traits.It may be apply to assisted selective breeding with the two SNPs in liangshan semi-wool sheep.