【作者】 曹贵玲；

【导师】 姜运良； 王建民；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 本研究可以分两个部分:第一部分:基因组印记是一种依赖于亲本的基因表达形式,导致功能基因单一表达一个等位基因而不表达另一个等位基因,造成印记位点的单等位表达,是一种基因表达的正常形式。虽然印记基因在哺乳动物基因组中只占一小部分,但在胚胎生长和行为发育中都起了关键作用。人和小鼠的H19和Dlk1是印记基因,但是山羊的H19和Dlk1的基因序列、基因结构和印记状态还不清楚。2002年,Freking等人发现一个位于Gtl2上游32.8kb的A-G的单碱基突变称CLPG突变。该突变可能是导致Callipyge绵羊表型的主要原因。山羊中是否也存在这样的突变,导致“肥臀山羊”成为本研究的一部分。试验一:克隆了山羊H19部分序列,得到了1139bp的序列,该序列和绵羊、牛的同源性很高,分别为96.22%和90.51%,和猪、人、小鼠的同源性较低,分别为72.06%、59.64%和51.83%。检测了山羊胎儿和成年组织中H19的表达情况。H19在胎儿的大多数组织表达,出生后的表达在有些组织中受到限制,在成年山羊中在肝脏、肺、胰脏、背最长肌、脾脏和肾脏中检测到H19转录物。在H19外显子5上找到一个SNP,通过检测杂合子基因型和转录物及其父母基因型,发现H19在成年山羊组织中是父本印记表达。试验二:克隆了山羊Dlk1部分cDNA序列,得到864bp,编码287个氨基酸残基,山羊Dlk1 cDNA和氨基酸序列与其他哺乳动物的相应序列有很高的同源性。Dlk1在胎儿大多数组织中表达,只在成年组织中的胰脏、胸腺和肾上腺表达。通过外显子5上一个SNP,发现Dlk1在成年上述三种组织中都是父本表达,是一个母本印记基因。另外Dlk1在山羊成年组织中只存在两种剪接变体Dlk1-C和Dlk1-C2。试验三:克隆了山羊CLPG区250bp的片段,该片段和绵羊、猪的相应片段有很高的同源性。用PCR-SSCP方法对波尔山羊、莱芜黑山羊、鲁波山羊、鲁北白山羊和白绒山羊进行检测,在“CLPG”突变处没有发现A→G突变,在CLPG突变下游147bp处发现一个A→C的突变,称A216C位点,用PCR-RFLP方法检测了上述5个山羊品种,其中鲁波山羊群体中A等位基因频率最高,鲁北白山羊群体中A等位基因频率最低,前四个群体中该位点处于Hardy-Weinberg平衡状态(P>0.05),白绒山羊群体偏离Hardy-Weinberg平衡状态(P<0.01)。产绒量、出生重、断奶增重等性状在该位点不同基因型之间差异不显著。第二部分:Dkk1是Dkk家族成员之一,是Wnt信号途径的拮抗物,参与多种生命过程。Dkk1可能通过抑制Wnt信号途径参与毛囊密度的控制。本研究旨在寻找新的和山羊毛囊密度相关的基因,了解其作用机制,期望能够用于提高山羊绒产量的分子育种。克隆了山羊Dkk1基因,获得3491bp序列,预测其mRNA和氨基酸序列,其mRNA和氨基酸序列和其他哺乳动物的相应序列有很高的同源性。在白绒山羊群体中对3491bp序列进行了SNP扫描,发现33处单核苷酸替代,内含子2中一处4碱基插入。外显子1中发现一处错义突变,命名为T1108P。检测了T1108P位点在白绒山羊群体中的分布,结果显示该位点在白绒山羊群体中没有多态性。启动子区有一个T-C颠换和C-A转换,二者间距9bp,命名为T593C-C603A,检测了这两个位点在白绒山羊中的基因型频率和基因频率;统计显示,产绒量、单位体表面积产绒量、出生重、断奶增重等性状在T593C位点不同基因型之间差异不显著。更多还原

【Abstract】 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.更多还原