【作者】 李密杰；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2013， 博士

【摘要】 过氧化物酶体增殖激活受体γ共激活因子1α(peroxisome proliferator-activated receptor gamma coactivator1alpha, PPARGC1A or PGC-1α),编码基因为PPARGC1A,作为转录共激活因子可以与一系列的转录因子相互作用,参与多种代谢途径,如适应性产热、线粒体的生物合成、葡萄糖代谢、肝脏糖异生、脂肪酸p氧化、脂肪细胞分化、肌肉类型转换等。在动物遗传育种中,由于PPARGC1A参与机体能量平衡和脂肪代谢调控的相关过程,PPARGC1A基因被作为影响动物产乳特性、肉质品质和动物生长的侯选基因加以研究,目前未发现该基因在中国地方黄牛品种中的相关研究。为此本研究利用基因克隆,腺病毒载体构建,细胞培养,qPCR,荧光素酶活性测定,DNA池测序,PCR-SSCP及PCR-RFLP等技术开展了秦川牛PPARGC1A基因的克隆,腺病毒介导的过表达和干扰技术对该基因的表达调控,及其在调控线粒体氧化呼吸链相关基因和肌肉细胞增殖分化相关基因的功能验证,启动子区的克隆及核心启动区的筛选分析,基因遗传变异的扫描及其与生长性状的关联分析等工作,以期为该基因在中国地方黄牛育种中的应用提供基础科学依据,主要获得了如下结果：1.克隆了秦川牛PPARGC1A基因CDS区,全长2391bp,编码796个氨基酸。并构建了腺病毒介导的过表达秦川牛PPARGC1A基因的载体Ad-PPARGC1A, TCID50法测得滴度为6×109PFU/mL,感染牛肌肉细胞的最佳MOI为150μL。Ad-PPARGC1A腺病毒感染牛肌肉细胞后,验证了PPARGC1A可促进线粒体氧化呼吸链相关基因的表达,并可促进牛肌肉细胞在含20%胎牛血清培养基中的增殖相关基因的表达及在含2%马血清培养基中的分化相关基因的表达。2.利用在线软件设计了针对牛PPARGC1A基因的3条shRNAs (shRNA-574、 shRNA-749、shRNA-1013),并通过实验筛选出shRNA-574和shRNA-749序列的干扰效果较明显。构建了针对牛PPARGC1A基因的Ad-shRNA-574和Ad-shRNA-749干扰腺病毒载体,TCID50法测得滴度分别为：3×109PFU/mL和8×108PFU/mL,感染牛肌肉细胞的最佳MOI均为200μL。干扰腺病毒Ad-shRNA574和Ad-shRNA749感染牛肌肉细胞后,在含2%马血清的分化培养基中的干扰效果较明显,干扰效率分别为41%和51%。干扰牛PPARGC1A基因后,牛肌肉细胞中的线粒体氧化呼吸链相关基因的表达量降低,同时影响肌肉细胞的正常增殖和分化相关基因的表达。3.克隆了秦川牛PPARGC1A基因启动子区2119bp片段,包括起始密码子上游-2072bp至下游+47bp处区域。构建了包含秦川牛PPARGC1A基因启动子区不同截短体的重组载体pGL3-Basic-Promoter,通过荧光素酶活性测定检测不同片段的相对启动活性并结合软件预测结果,推测该基因的启动子活性区在-2072bp至-2001bp之间。4.经DNA池测序扫描后发现牛PPARGC1A基因20处多态位点,其中位于该基因外显子和内含子区的有13处：g.64897G>A, g.72175A>G, g.72202A>G, g.84063T>C, g.84163G>A, g.85272C>T, g.85329C>T, g.85352C>T, g.85400A>G, g.85442G>A, g.85540C>T, g.85609T>C, g.85631T>G;位于基因启动子区的有7处：SNP-259T>A,-301_-298insCTTT,-915A>G,-1175T>G,-1590C>T,-1665C>T和-1690G>A。与中国部分黄牛生长性状关联分析后发现：SNP g.64897G>A与南阳牛24月龄体重和日增重相关,且GG基因型为优势基因型。更多还原

【Abstract】 Peroxisome proliferator-activated receptor gamma coactivator1alpha (PPARGC1A or PGC-la), encoded by PPARGC1A gene, was a transcription coactivator. PGC-1a participaits in many metabolic pathways through interacting with series of transcription factors, for example adaptive thermogenesis, mitochondria biogenesis, glucose metabolism, hepatic gluconeogenesis, fatty acid oxidation, adipocyte differentiation and muscle fiber conversion. As PGC-la involved in energy homeostasis and regulation of fat metabolism, PPARGC1A gene was studied as a candidate gene on milk traits, meat quality and growth characteristics of domestic animals in animal breeding. In this study, the gene cloning, construction of adenovirus vector, cell culture, real-time quantitative polymerase chain reaction(qPCR), luciferase reporter assay, DNA pool sequencing, PCR-SSCP and PCR-RFLP technologies were emploied to carry on the Qinchuan cattle PPARGC1A gene cloning, gene expression regulating through gene overexpress and RNA interference techniques mediated by adenovirus, functional verification on the regulation of mitochondrial oxidative respiratory chain genes and myocyte proliferation and differentiation related genes, PPARGC1A gene promoter cloning and the core promoter region analysis, genetic variation scanning and its associated with growth traits. The main results obtained were as follows:1. The Qinchuan cattle PPARGC1A gene codons region was cloned, which contains a total length of2391bp nucleotides, encoding796amino acids. The recombinant adenovirus vector overexpress bovine PPARGC1A gene was generated. The titer of the adenoviral stock was determined by TCID50method and reaches6×109PFU/mL. The optimum multiplicity of infection (MOI) for bovine myoctye was150μL. The PPARGC1A promoting the expression of genes related to mitochondrial respiratory chain oxidation and bovine myocyte proliferation in the growth medium and differentiation in medium containing2%horse serum was verified through the Ad-PPARGCIA adenovirus infection of bovine myocyte.2. Three shRNA sequences targeting different area of bovine PPARGC1A gene (shRNA-574, shRNA-749, shRNA-1013) were designed using online software and synthesized. After annealing, shRNA templates were constructed into pENTR/CMV-GFP/U6vecor. By cotransfecting HEK293A Cell, the result shows that entry vector expressing shRNA-574and shRNA-749sequences caused an obvious interference effect. Then we generated recombinant adenovirus vectors pAd-shRNA-574, pAd-shRNA-749by LR recombination and adenovirus were successfully packed in HEK293A Cell. The titers of the adenoviral stock were3×109PFU/mL and8×108PFU/mL for Ad-shRNA-574and Ad-shRNA-749, which were determined by TCID50method, respectively. The optimum multiplicity of infection (MOI) of adenovirus Ad-shRNA-574and Ad-shRNA-749were both200μL for bovine myoctye. The adenovirus Ad-shRNA-574and Ad-shRNA-749had more interference efficient in the differentiation culture medium than that in the growth culture medium, with the efficency of41%and51%, respectively. The expression of mitochondrial oxidative respiratory chain related genes were reduced and the normal proliferation and differentiation of myocyte related genes were affected after the bovine PPARGC1A gene was interfered through Ad-shRNA-574and Ad-shRNA-749adenovirus infection bovine myocyte.3. Qinchuan cattle PPARGC1A gene promoter region was cloned with a lengh of2119bp including-2072bp and+47bp region according to the initiation codon. The recombinant vectors pGL3-Basic-Promoter containing different truncated promoter region fragments were constructed. It was suggested that the gene promoter core activity region was in the-2072bp to-2001bp through dual-luciferase reporter assay combined with software prediction results.4. Twenty polymorphic loci of bovine PPARGC1A gene were found by the DNA pool sequencing.13loci were located in exons and introns region:g.64897G>A, g.72175A>G, g.72202A>G, g.84063T>C, g.84163G>A, g.85272C>T, g.85329C>T, g.85352C>T, g.85400A>G, g.85442G>A, g.85540C>T, g.85609T>C, g.85631T>G;7loci were located in the promoter region:-259T>A,-301_-298insCTTT,-915A>G,-1175T>G,-1590C>T,-1665C>T and-1690G>A. Association analysis between growth traits and SNPs was carried out and found that SNP g.64897G>A sites was associated with body weight and average daily gain in24months age within Nanyang cattle, and individuals with genotype GG were the dominant ones.更多还原