Studies On Imprinted Gene H19, Dlk1, CLPG And Hair Follicle Development Related Gene Dkk1 In Goats

This research can be devided to two parts.Part I:Genomic imprinting is a phenomenon in which alleles of a gene are expressed differentially depending on their parental origin. The presence of imprinted gene can cause cells with a full parental complement of functional autosomal genes to specifically express one allele but not the other, resulting in monoallelic expression of imprinted loci. Genomic imprinting is a nomal precess and imprinted genes play a critical role in fetal growth and behavioral development even through they account for a little part of all genome.In human and/or mouse, H19 and Dlk1 are known to be imprinted, whereas in goats, the structure, expression and imprinting status of these two genes remains unknown.In 2002, Freking et al found a A-G single nucleotide mutation upstream 32.8kb of Gtl2 and named CLPG mutation. This A-G mutation may be the causative mutation of the Callipyge phaenotype. The objective of this study is to answer the question"is there this mutation in goat which can also results in muscular hypertriphy in goat".In experiment one, a partial DNA fragment of goat H19 in size of 1139bp was obtained, which has 96.22%, 90.51% identity with the corresponding region of sheep and bovine H19 respectively, and 72.06%, 59.64% and 51.83% identity with the corresponding region of pig, human and mouse H19 respectively. H19 was expressed in most tissues of goat fetuses; in adult goat, however, the expressions were restricted to some tissues, expressed in the liver, lung, pancreas, longissimus dorsi, spleen and kidney. One single nucleotide polymorphism for H19 was identified in the fifth exonic region. By genotyping the parents, their daughter and the transcripts in different adult tissues, we found that H19 was maternally expressed. In the second experiment, a fragment of 864bp in size for goat D1k1 was amplified and sequenced, which encodes 287 amino acids and has a high homology with those in mammalian species. Dlk1 was expressed in most tissues of goat fetuses; in adult goat, however, the expressions were restricted to some tissues, only in adrenal capsule, pancreas and thymus. One single nucleotide polymorphism for Dlk1 was identified in the fifth exonic region. By genotyping the parents, their daughter and the transcripts in different adult tissues, we found that Dlk1 was paternally expressed. In addition, two alternative transcripts of Dlk1-C and Dlk1-C2 were expressed in goat.In the third experiment, a fragment of 250bp in size for goat CLPG was amplified and sequenced, which has a high identity with the corresponding region of sheep and bovine CLPG respectively. The CLPG mutation site was analysised in Boer, Laiwu hei, Lubei white, Lubo and White cashmere goats. We did not found the CLPG mutation in this five population. There was a A-C transversion located 147bp downstream of the CLPG site named A216C site, this site were analysised in those five population. The frist four populations are all in a state of Hardy-Weinberg equilibrium (P>0.05). the White cashmere goat population was deviated from Hardy-Weinberg equilibrium(P<0.01) The frequency of A allele was the highest in Lubo goat and the lowest in Lubeibai goat population. The product of cashmere, birth weight, weanling weight among different genotypes were not different.Part II:Dkk1, a member of Dkk family, is an antagonist of Wnt signal transduction pathway, takes part in many vital process. Dkk1 may control the density of hair follicle through rivalrying the Wnt signal transduction pathway. Some reseach revealed that the down regulation of Dkk1 expression can decreasehe density of hair follicle. The objective of this study is to find the new gene which is related with goat hair follicle, understand the mechanism of the action, and provide some useful information to the molecular breeding to improve the product of cashmere.In this study, a fragment of 3491bp in size for goat Dkk1 was amplified and sequenced, its mRNA and amino acids sequences were predicted, which had high homology with those in mammalian species. We detected the SNPs in 3491bp sequence in White cashmere goat population, and found 33 single nucleotide substitute and a insertion of 4bp in intron 2. there is a missense mutation in exon1, named T1108P. we detected this site in White cashmere goat population and it is not a polymorphism. There were two mutation sites T-C and C-A site spacing 9bp named T593C-C603A site, this two sites were detected in White cashmere goat population. The cashmere product, cashmere product per body surface area, birth weight, gaining weight from birth to weanling among different genotypes were not different.