【作者】 陈超；

【导师】 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 脂肪是人和动物体内必须的营养成分,也是构成生物体的重要组分,其代谢调控受众多因素的影响,主要包括遗传因素、营养水平、环境因素等,它们形成复杂的调控网络共同调节脂肪的代谢。猪作为人类动物蛋白的重要来源,其体内脂肪的过度沉积直接影响瘦肉率的提高,因此寻找影响脂肪代谢的主效基因成为当前动物遗传育种专家研究的热点。ATF4 (activating transcription factor 4)即激活转录因子4属于碱性亮氨酸拉链蛋白家族。最近的研究发现其与脂肪的代谢和能量消耗有关,鉴于其在脂肪代谢中的重要作用,而在猪中的研究报道较少,本文选取其作为影响猪脂肪性状的候选基因进行研究,探讨瘦肉型和脂肪型猪差异形成的分子机制,为从分子水平上培育瘦肉率高肉质好的猪种提供理论依据。本实验对ATF4基因进行克隆,遗传效应分析和表达模式分析,取得了如下结果：1.克隆了大白猪和梅山猪ATF4基因的CDS序列,通过测序、酶切验证发现在翻译起始密码子ATG下游159bp处存在A159G转换。2.对大白猪、长白猪、梅山猪、通城猪和大白猪×梅山猪F2进行了AluI-RFLP分析,并对大白猪×梅山猪的F2群体195个体与生产性状进行关联分析,结果表明,ATF4的多态性与臀部平均膘厚存在极显著相关(P<0.01),与胸腰椎间膘厚、平均膘厚、眼肌高、眼肌面积存在显著相关(P<0.05)。3.通过RT-PCR方法对ATF4基因在心、肝、脾、肺、肾、胃等11个组织中的表达量进行定量分析,发现ATF4基因在肌肉中表达量较高。4.以背最长肌为材料,对ATF4基因在大白猪和梅山猪不同发育阶段的表达情况进行了定量分析,发现在胚胎期65d、出生后3d在大白猪和梅山猪中基因表达量较低,两个品种之间没有明显的差异,在60d和120d时期基因表达量均增加,但是梅山猪表达量显著高于大白猪。5.通过PCR技术扩增出ATF4基因启动子序列2007bp,并利用生物信息学在线软件对启动子区域的顺式作用元件和转录结合位点进行了预测,根据预测的结果设计缺失引物,构建了7个含有不同长度启动子缺失片段的双萤光素酶报告基因表达载体。6.将构建7个缺失片段重组质粒瞬时转染PK-15和C2C12细胞,检测双萤光素酶的相对活性,结果构建的载体都具有活性,其中pGL3-1280的相对活性最高在C2C12细胞中,为进一步鉴定ATF4基因的转录调控机制奠定实验基础。7.对大白猪和梅山猪启动子区域进行序列比对发现存在11处潜在的SNP位点和插入/缺失,对其中一处20bp的缺失进行了验证发现,在梅山猪中为缺失型,在大白猪为插入型。8.在ATG上游2540bp和2570bp处测序发现存在一个5bp的缺失(TCTTA)和碱基突变(C/T),其中2570bp处碱基转换可以被BstXI酶识别,进行了BstXI-RFLP分析,结果发现检查的梅山猪均为TT型,大白猪均为CC型,对136头F2进行酶切分型。9.克隆猪ATF4基因CDS,构建pcDNA3.1(+)-ATF4真核超表达载体,为下一步在细胞水平和个体水平研究该基因功能奠定基础。更多还原

【Abstract】 Fat is not only essential nutrients to human and animals but also an important component of living organisms. Regulation of its metabolism is affected by many factors, including genetic, nutrition, and environmental factors, which form a complex regulatory network in the regulation of fat metabolism. Pigs as an important source of animal protein whose over fat deposition directly impact on the improvement of lean meat, Therefore, research on genes related with fat deposition become hotspot for experts in animal genetics and breeding.Activating transcription factor 4 (ATF4), belongs to the family of basic zipper-containing proteins. Reccently study identifies a possible new function for ATF4 in regulating lipid metabolism and thermogenesis.Given its important role in fat metabolism and the study in porcine reported less, we select ATF4 as a candidate gene for fat deposition study the molecular mechanism of the differences between lean and fat-type pigs in order to provide more information on this gene for molecular marker-assisted selection breeding.Based on these we clone ATF4 and studied its genetic effects.And the main results are as follows:1 Sequence comparison revealed that an mutation occurred A159G substitution at downstream of initiation codon (ATG).2. We carried out PCR-AluI-RFLP analysis followed by association analysis in F2 "Large white×Meishan" resource family. In all the individuals tested, Large White and Landrace pigs possessed AA genotype, while Meishan and Tongcheng pigs possessed GG genotype. Association analysis in F2 resource family showed that this site was highly associated with buttock fat thickness(BFT) (P<0.01) and had significant effect on thorax-waist fat thickness(TFT), average backfat thickness(ABT), loin eye height(LEH) and loin eye area (LEA)(P<0.05).3.RT-PCR was applied to detect the tissue distribution of the porcine ATF4 gene in different tissues (heart, liver, spleen, lung, kidney, stomach and so on). Result show that the ATF4 gene was the highest expressed in muscle.4. Real-time RT-PCR analysis showed that the expression level of ATF4 gene in the skeletal muscle of Large White and Meishan pigs were no significant difference before postnatal 3 days. And at postnatal 60 and 120 days, the expression level was dominantly higher in Meishan pigs than in Large White pigs.5. Cloning 2007bp in ATF4 promoter region by PCR and using the online bioinformatics software, we predict core promoter region and find cis-acting elements and transcription binding sites, we use this predicted result to desgin primer and. construct 6 repoorter gene with different length of promoters.6. The recombinant constructs were transfected into PK-15 and C2C12 cell. The promoter activity was evaluated by dual-luciferase reporter assay. The 7 promoters all were transcriptional activated and pGL3-1280 had the high activity in C2C12 cell. All the result laid the experimental foundation for further study on the transcriptional regulation of ATF4.7. Sequence comparison revealed that there are 11 sites of potential SNP and insertion/ deletion.we find an 20bp insertion/deletion mutation occurred at 1984 bp of the of initiation codon (ATG). We then carried out PCR analysis.In all the individuals tested, Large white pigs pigs possessed the insertion, while Meishan possessed the deletion.8. Sequence comparison revealed that a mutation occurred C/T substitution at 2570bp downstream of initiation codon (ATG). We carried out BstXI-RFLP analysis.In all the individuals tested, all Meishan pigs possessed the TT type, Large white pigs possessed CC type.9. We construct an eukaryotic expression vector pcDNA3.1 (+)-ATF4 by cloning porcine ATF4 gene,which provide us advantage for further study of gene function at the cellular and individual level.更多还原