Genetic Effects Of MC4R And GDF9Genes And Construction Of IGF-I Eukaryotic Expression Vector In Anhui White Goat

Anhui White Goat is an excellent leather and meat dual purpose local breed. Itpossesses many benefit characteristics such as high fecundity, good adaptation, strongdisease resistance and high quality of leather and meat, etc. In order to get AnhuiWhite Goat becoming more utilization and its adevantage benefit being fμllydiscovered, in this research, the melanocortin acceptor4(MC4R) gene was cloned inthe breed, the mononucleotide polymorphism of both the melanocortin acceptor4(MC4R) and the growth differentiation factor9(GDF9) were detected by PCR-RFLPin178popμlations of Anhui White Goat, and their genetic effects were also analyzed.As well, the eukaryotic expression vector pEGFP-N1-IGF-I was successfμllyconstructed in this research.The following experimental resμlts had been gained:Experiment I，According to the related cDNA sequences of other speciesmelanocortin receptors4(MC4R) found in the NCBI, the primers of goat MC4R genewere obtained. RT-PCR was used to obtain the MC4R complete coding sequences ingoat various tissue. The mRNA content was tested in heart, liver, spleen, lung, kidney,muscle, intestine, small intestine, womb and rumen tissues of goat. The structure ofthe goat MC4R gene was analyzed by bioinformatics methods. The resμlts showedthat the fμll length of goat MC4R was2068bp（Genbank No.：JN580891）, whichcontent999bp CDS sequence and encode332amino acids. Goat MC4R geneexhibited the greatest homology to that of sheep (96.9%), cattle is the second (93.5%).MC4R was expressed in various tissues. The expression level of this gene was higherin kidney and lower in large intestine and small intestine. It was predicted that MC4Rhad seven transmembrane domains in goat. There were5serine phosphorylation sites,4threonine phosphorylation sites and one phosphorylation site of specific proteinkinase in MC4R protein. The prediction of goat MC4R tertiary structure had7αhelices and5β sheets. These resμlts indicated that the MC4R gene is conservative inthe course of animal evolution, and they have approximate biological function indifferent animals. The expression level of MC4R mRNA is tissue specific.Experiment II，With the material of Anhui White Goat blood DNA, test thepolymorphism of the candidate gene which is took from the melanocortin acceptor4(MC4R) in the Anhui White Goat by PCR-RFLP. The resμlt showed thatpolymorphism was detected in Anhui White Goat, and three genotypes (AA, AB and BB) was found in the selected Anhui White Goat; the genotype frequency of AA ishigh(0.5899), the genotype frequency of AB is low(0.1123), Allele of A ispreponderant allele; polymorphic locus of MC4R is in the u of Hardy-Weinberg;among the three gene type generating from the polymorphic site of MC4R, the bodylength of AA gene type is much longer than BB gene type (P<0.05); the values ofbody height, wipe wide and hipe length are all the biggest, but the differences to theother two types are not remarkable (P>0.05). The discovery of the polymorphic ofMC4R gene can provide theoretical foundation for the marker-assisted selectionofAnhui White Goat.Experiment III，Polymorphism of growth differentiation factor9(GDF9) gene,which as candidate genes on the prolificacy of sheep was studied in the Anhui whitegoats by PCR-RFLP. The resμlts showed that the G8mutation (C→T) of GDF9genewas not detected in the Anhui white goat breeds and these genes were all wild-type inthe Anhui white goat breeds. These resμlts indicated that the fecundity of the Anhuiwhite goat breeds was not related to the G8mutation of GDF9gene.Experiment IV，According to the RT-PCR and PCR, IGF-I gene is amplified fromthe liver of Anhui White Goat by Trizol. The gene segments are then reconstructioninto a eukaryotic expression vector pEGFP-N1through gene reconstructiontechnology, resμlting in the birth of a new eukaryotic expression vectorpEGFP-N1-IGF-I, which is later confirmed by testing the recombinant plasmids usingPCR amplification, electrophoretic analysis of the endonuclease digestion resμlts andsequencing.The research supplies the definite theoretical foundation work for marker-assistedselection and the trituration of transgenic material. It also supplies the basis forresearch of the property of Anhui White Goat and the excellent genetic resources ofAnhui White Goat.