CDNA Cloning, Expression Of Arctic Fox GH, GHR, IGF1 And IGF1R Genes And Study Of Correlative Traits

This study was designed to clone the cDNA sequence of arctic fox GH,GHR,IGF1 and IGF1R gene based on the sequence of mammalian GH,GHR,IGF1 and IGF1R gene by comparative genomics and bioinformatics approaches; to investigate the expression characterization of GH,GHR,IGF1 and IGF1R genes by Real time PCR; to detect the polymorphisms of GH,GHR,IGF1 and IGF1R genes by DNA sequencing and PCR-SSCP methods, and to investigate the effects of genes polymorphisms on growth traits in arctic fox population from the fox farm of Da Xing An Ling Tu Qiang forest enterprise. The main results are as follows:1. The cDNA sequence of arctic fox GH gene which is 718bp were cloned and submitted to GenBank (Accesssion No. EU159584);The introns of GH gene were cloned and spliced with cDNA sequence, which had been submitted to GenBank. The length of sequence was 1692bp (Accesssion No. EU376045).2. By cloning and sequencing, the cDNA sequences of arctic fox GHR,IGF1 and IGF1R gene were obtained, which had been submitted to GenBank, the segment length was 2006bp, 1717bp and 2411bp, respectively. Accession Number was EU304325, EU301609 and EU159582, respectively.3. Real time PCR analysis indicated that the arctic fox GH gene only expressed in the pituitary among tested tissues from arctic fox of 6 month of age; The expression levels of arctic fox GH gene in the pituitary at 2,4 and 6 month of age showed that the lowest expression level was at the second month of age and the highest expression level was at the sixth month of age.4. Real time PCR analysis indicated that the arctic fox GHR gene expressed in many tissues, including liver, lung, pancreas, brain, pituitary, duodenum, testicle, heart and muscle from arctic fox of 6 month of age. The expression levels of arctic fox GHR gene in the liver and muscle tissues at 2,4 and 6 month of age showed that the expression trend of GHR gene in the two tissues were multiplicative.5. Real time PCR analysis indicated that the arctic fox IGF1 gene expressed in many tissues, including liver, spleen, kidney, lung, stomach, pancreas, brain, pituitary, testicle, heart and muscle from arctic fox of 6 month of age. The expression levels of arctic fox IGF1 gene in the liver and muscle tissues at 2,4 and 6 month of age showed that the expression trend of GHR gene in the two tissues were multiplicative. 6. Real time PCR analysis indicated that the arctic fox IGF1R gene expressed in many tissues, including liver, spleen, kidney, lung, stomach, pancreas, brain, pituitary, testicle, heart and muscle from arctic fox of 6 month of age. The expression levels of arctic fox IGF1R gene in the liver at 2,4 and 6 month of age showed that the expression trend of IGF1R gene in the tissue was multiplicative. The expression levels of arctic fox IGF1R gene in the testicle showed that the highest expression level was at the sixth month of age and the lowest expression level was at the fourth month of age. The expression levels of arctic fox IGF1R gene in the muscle showed that the highest expression level was at the fourth month of age and the lowest expression level was at the second month of age.7. Four SNPs, G81A in the second exon, C186T and T239C in second intron and C583A in the third exon, were found within arctic fox GH gene. The C583A polymorphisms of GH were associated with male fox body length (P<0.05).8. Four SNPs, C99T and A144C in the first exon, T59Cå’ŒG65A in the fifth exon, were found within arctic fox GHR gene. The C99T and A144C polymorphisms of GHR were associated with female fox body weight (P<0.05). The T59C and G65A polymorphisms of GHR were associated with male fox body weight (P<0.05), and significantly related to the skin length of the female fox (P<0.01).9. Three SNPs, G94C in 5'flanking region, C50T in the second intron and C87T in the second exon, were found within arctic fox IGF1 gene. The G94C polymorphisms of IGF1 were associated with male fox body length (P<0.05), and significantly related to abdominal circumference of female fox(P<0.01). The C50T polymorphisms of IGF1 were associated with abdominal circumference of female fox and the skin length of male fox (P<0.05), and significantly related to female fox body length (P<0.01). The C87T polymorphisms of IGF1 were associated with body length and abdominal circumference of female fox (P<0.05).10. By analysising different combination genotypes, the combination genotype of GH gene C583A and GHR gene T59C&G65A were associated with male fox body weight (P<0.05). The combination genotype of GHR gene T59C&G65A and IGF1 gene C87T significantly related to male fox body weight (P<0.01). The combination genotype of GH gene C583A and GHR gene T59C&G65A significantly related to male fox body length (P<0.01).