【作者】 王璟；

【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】 随着人们对牛肉品质的追求,提高肌间脂肪含量成为肉牛育种的重要目标,诱导细胞凋亡的DFFA样效应因子(CIDEC,又称Fsp27)作为调控脂质沉积的重要因子,小鼠等模式动物的研究表明,该基因对于脂肪细胞单房脂滴的形成、维持以及能量储存具有重要作用,同时该基因参与脂肪代谢障碍,胰岛素抵抗型糖尿病,脂肪肝等代谢综合症的调控。分析CIDEC基因在牛上的遗传变异、转录调控及其对牛脂肪细胞的脂肪沉积的调控机制,对于肉牛的标记辅助选择和转基因育种具有重要意义。本研究使用混合DNA池测序、Forced-PCR-RFLP.克隆、原核表达、真核表达、腺病毒的包装、细胞培养等技术,研究了1)CIDEC在秦川牛两个生长时期的组织表达谱,2)CIDEC基因的遗传变异与牛生长性状的关联分析,3)牛CIDEC蛋白的亚细胞定位,4)CIDEC蛋白与PPARg蛋白的互作机制,以及5)CIDEC基因对牛原代脂肪细胞凋亡及糖脂代谢的调控功能,本研究主要取得了以下结果：1. CIDEC在牛的脂肪组织中具有较高的表达水平使用实时定量PCR技术研究了CIDEC在秦川牛犊牛和成年牛不同组织的表达情况,结果显示该基因在这两阶段不同组织的表达趋势相似,都是在脂肪组织中表达量最高,肺、肠次之,而其他组织中的表达量都很低。提示CIDEC在牛的脂质代谢方面有重要作用。2. CIDEC基因启动子区的突变能够影响其转录进而影响生产性能使用混合DNA池测序在5个中国牛品种中发现了CIDEC基因的5’非翻译区存在10个新的SNPs位点,其中SNP2,3,4,6,9完全连锁,SNP8,10完全连锁,关联分析结果表明不同单倍型组合与南阳牛生长性状呈正相关状态。利用双荧光素酶报告系统检测了不同单倍型的转录活性,其中突变位点越少的单倍型转录活性越高,对应的个体生产性能越好。结合顺式作用元件的分析发现这10个SNPs位点可能通过改变转录因子的结合序列(即顺式作用元件)进而改变了该基因的转录水平,最后表现为南阳牛生长性状的改变。牛CIDEC基因的10个SNPs位点可以作为牛分子育种研究的候选位点。3. CIDEC和PPARg蛋白具有不同的亚细胞定位将构建好的真核表达载体pEGFP-C1-CIDEC和pEGFP-C1-PPARg转染到3T3-L1细胞系中发现,CIDEC蛋白位于细胞质中而PPARg蛋白位于细胞核中,说明这两个蛋白不能直接通过蛋白与蛋白的互作来发挥作用。根据其他物种的研究结果,进而推测牛PPARg也是通过结合在CIDEC的5’端的PPRE反应元件上,从转录水平调控CIDEC的表达进而调控脂质沉积。4.牛PPARg可以调控CIDEC的转录使用Genomatix软件预测得到牛CIDEC有2个启动子区(启动子1：-1936到-1336,启动子2：+3552到+4154)和3个PPRE反应元件(-1691到-1669,-1684到-1662,-1677到-1655)。对牛CIDEC启动子不同截短体进行双荧光素酶活性分析,发现启动子1为该基因的核心启动子,其主要活性区域为PPRE反应元件区,加入PPARg激动剂罗格列酮后,PPRE反应元件区转录活性显著升高。这个结果证明了我们在CIDEC细胞定位分析时的推测,牛(?)PARg能够结合在CIDEC启动子区PPRE反应元件上,诱导后者的转录表达,进而参与机体的代谢调控。5.牛CIDEC基因的克隆、序列分析以及原核表达克隆获得的牛CIDEC完整ORF片段大小为669bp,编码222个氨基酸的肽链,预测的蛋白分子量为25KDa,理论等电点为7.97。该蛋白主体结构为螺管结构。起源进化分析发现牛CIDEC蛋白与狗CIDEC蛋白首先聚为一类,再和猪的聚类后与人、黑猩猩、猴的聚为一类,然后再依次和大鼠、小鼠、马、鸡、蟾蜍聚类,与河豚和斑马鱼的距离最远。重组质粒pET-28a(+)-CIDEC表达的最佳条件为37℃,1.0mmoI/L IPTG诱导6h。经Western-Blot证实确为目的蛋白。6.过表达CIDEC会导致牛原代脂肪细胞凋亡构建了牛CIDEC腺病毒超表达载体,并包装、扩繁得到高滴度的CIDEC超表达腺病毒。根据死亡受体基因Fas的表达量变化,发现与人和小鼠上的研究结果一致,过表达CIDEC会诱导牛原代脂肪细胞发生细胞凋亡。7. CIDEC调控牛原代脂肪细胞糖脂代谢筛选出针对牛CIDEC有明显干扰效果的shRNA干扰载体(pENTR/CMV-GFP/U6-CIDEC-sh1),经包装、扩繁得到高滴度的CIDEC干扰腺病毒,滴度为3×108PFU/mL。在牛原代脂肪细胞中,Ad-siCIDEC的干扰效率可以达到72%。牛CIDEC表达量下降后,导致细胞脂质代谢(PPARg、PLIN、HSL)、葡萄糖代谢(GLUT4)、线粒体活性(UCP1)相关的5个基因表达量上升。说明牛CIDEC基因参与机体的糖脂代谢调控,当其表达量下降时,牛脂肪细胞脂解水平升高,葡萄糖平衡受到影响,线粒体数目增多、活性增强,导致脂肪细胞从储存能量向消耗能量转变。更多还原

【Abstract】 As people are Seeking after beef quality, the improvement of the content of intramuscular fat has became an important goal of the beef cattle breeding. The research in rat and other model animals show that cell death-inducing DFFA-like effector c (CIDEC/Fsp27) is required for unilocular lipid droplet formation and optimal energy storage. And it has emerged as an important regulator of metabolism associated with lipodystrophy, insulin-resistant diabetes, and hepatic steatosis. The research of genetic variats, transcriptional regulation and regulatory mechanism for the deposition of fat in bovine primary adipocytes in bovine CIDEC gene is important for marker-assisted selection and transgenic breeding of beef cattle. DNA pool sequecing, forced-PCR-RFLP, gene clone, prokaryotic expression, eukaryotic expression, packaging of the adenovirus and cell culture were used in this research to verify the function of bovine CIDEC gene. The expression profiles of CIDEC in different growth period of Qinchuan cattle, the verification of relationship between CIDEC gene genetic variation and growth traits, subcellular localization of CIDEC protein, screening of the core promoter region and PPRE reaction elements in CIDEC gene were studied, at last the function of bovine CIDEC in apoptosis and glucolipids metabolism were confirmed. The main results of this research including:1. CIDEC gene has a higher expression level in bovine adipose tissueReal-time RT-PCR was used to analyse the expression of CIDEC gene in different tissues of Qinchuan newborn and adult cattles, the results revealed that the expression of this gene in these two stages was similar, the expression level was highest in adipose tissue, then in lung and gut, in other tissues its expression was very low. These suggesting that bovine CIDEC gene has an important role in lipid metabolism.2. Variats in the promoter of CIDEC gene could affect its transcription thereby affect the growth traitsTen novel SNPs were discovered in5’regulation region of CIDEC gene by DNA pool sequencing in five Chinese cattle breeds, SNP2,3,4,6and9linked completed, SNP8and10linked completed. The association analysis revealed that different heplotypes was associated with Nanyang cattle growth traits. By detection of the transcriptional activity of different heplotypes, we found that the haplotype with less variats show higher transcriptional activity, the individuals with this haplotype also show better growth traits. Combined with the analysis of the cis-acting elements, we can speculate that these10SNPs could changed the cis-acting elements, then haplotypes of the cis-acting elements may affect transcriptional activity, thus affecting the growth traits of livestock. These10SNPs loci could be used as candidate loci in bovine molcular breeding.3. CIDEC and PPARg have different subcellular localizationThe recombinant eukaryotic expression vector pEGFP-C1-CIDEC and pEGFP-C1-PPARg were transfected in3T3-L1cell, CIDEC protein located in cytoplasm and PPARg protein located in nucleus, suggesting that these two protein can’t interacted with each other by directed protein-protein contact. According to the research in other species, we speculated that bovine PPARg gene may regulate the transcription activity of CIDEC gene by binding to its PPRE reaction element, which located in the5’regulation region of CIDEC gene.4. Bovine PPARg could regulate the transcription activity of CIDEC geneBy searching the Genomatix software, there are2promoter region (promoter1:-1936to-1336, promoter2:+3552to+4154) and3PPRE response elements (-1691to-1669,-1684to-1662,-1677to-1655) in bovine CIDEC gene. The results of dual luciferase activity analysis shown that the promoter1was the core promoter, and its active region was PPRE response elements, the activity of the PPRE response elements elevated by adding the agonist of PPARg gene:rosiglitazone. So this result confirmed our inference, bovine PPARg gene regulates the transcription activity of CIDEC gene by binding to PPRE reaction element, which located in the5’regulation region of CIDEC gene.5. Cloning, sequence analysis and prokaryotic expression of bovine CIDECThe ORF region of bovine CIDEC contain696bp, encodes peptide with222amino acids, the deduced molecular weight of CIDEC protein was25KDa, the isoelectric point was7.97. The main secondary structure of this protein was spiral tube. Phylogenetic analysis revealed that cattle exhibit greater sequence similarity with dogs, chimpanzees, humans, monkeys, pigs and mice than chicken or fish. The optimal condition for the expression of the recombinant plasmid pET-28a(+)-CIDEC were:37℃,1.0mmol/L IPTG and induced6h. Western-blot confirmed the products were the target protein.6. Overexpression of CIDEC inducing apoptosis of the bovine primary adipocytesAdenovirus vector of bovine CIDEC gene was constructed, after packaging and amplification successfully, high-titer of Ad-CIDEC was infected with bovine primary adipocytes. According to the expression of the Fas gene we found that overexpression of CIDEC gene inducing apoptosis of bovine primary adipocytes, which was consistent with the research in human and mouse.7. CIDEC gene regulate the glucolipid metabolism of bovine primary adipocytes We obtained one shRNA sequence for bovine CIDEC gene which could cause obvious interference effect (pENTR/CMV-GFP/U6-CIDEC-shRNAl). High-titer of Ad-siCIDEC was obtained after packaging and amplification (3×108PFU/mL). In bovine primary adipocytes, the RNA interference efficiency of Ad-siCIDEC was up to72%. The reduction of CIDEC gene can result in the up-regulation of PPARg, PLIN, HSL (lipoprotein metabolism), GLUT4(glucose metabolism), UCP1(mitochondrial activity). Bovine CIDEC gene participated in the glucolipids metabolism, when the expression of CIDEC was decreased, the level of lipolysis was up regulated, the balance of glucose was affected, the number and activity of mitochondrial was increased, in brief, the bovine adipocytes was changed from energy storage to energy consumption.更多还原