【作者】 王明辉；

【导师】 潘玉春；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2011， 博士

【摘要】 猪的生长发育影响增重速度、饲料转化效率以及胴体组成等诸多性状,进而影响经济效益。然而至目前为止,对于猪的生长发育调控机理仍不是十分清楚,以致严重制约了猪种的遗传改良。近些年来,候选基因分析、全基因组扫描以及全基因组关联分析等均被用于揭示生长发育等复杂性状的遗传机制,但是这些方法可以找出影响数量性状的DNA片段(QTL)或SNP位点,却不能反映从基因到性状的生物学过程。为此,本项目首先提出了候选基因集法,进而将这种方法运用到了猪生长发育遗传机制的研究中。一、候选基因集法的建立:候选基因法具有一定的盲目性并易受环境因素影响,QTL定位需要特定的群体资源并且区间跨度太大,全基因组关联分析则面临费用高昂等问题。有鉴于此,本文提出了候选基因集法,包含基因集的定义、QTL上的映射、SNP点的推断、SNP检测分型、表型分型、关联分析以及功能验证等规范化的步骤与流程。该方法具有较强的目的性、针对性、操作简单、费用合理等优点。二、骨骼肌再生时间序列芯片的转录因子分析:骨骼肌在受到创伤后具有很强的再生能力。这一过程在诸多转录因子的精细调控下完成。这些调控进程往往通过调控网络发挥其功能。我们利用已有的时间序列芯片及启动子区域结合位点信息研究了骨骼肌再生过程的转录调控网络。通过对共表达基因启动子区域结合位点信息的分析,我们获得了一系列转录因子与靶基因的关系。时间延迟分析通过相关系数及调控时间延迟量进一步探究了真正的转录因子与靶基因间的调控关系。通过分析我们获得了13个在骨骼肌再生过程中发挥重要作用的转录因子,其中的6个转录因子已经有文献报道过。这些转录因子中的FoxO3富集于胰岛素信号通路(IGF1-FoxO),该信号通路曾被报道与生长发育密切相关。因此,鼠的骨骼肌再生芯片分析提供的IGF1-FoxO可以作为影响猪生长发育的候选通路。三、IGF1-FoxO通路进化分析:IGF1-FoxO信号转导通路在内分泌调控进程中发挥着重要作用,该信号通路在脊椎动物物种中保持一定的一致性。虽然该信号通路成员组成在模式生物物种中已经被广泛研究,但是通路的进化模式还有待于进行深入的研究。我们首先对通路成员组成即在不同的物种中信号通路成员的组成是否存在差异开展了研究,进而对在这一复杂的系统中各个成员组成在进化历程中是否具有相似的选择约束等问题做了探索。我们的结果表明大多数的通路成员存在于所有的待研究的物种中,他们遭受较强的选择约束。通路成员中的2个基因受到正选择的影响。我们发现整个通路的选择强度呈梯度变化,即上游基因相对于下游基因具有相对较强的选择约束。我们的研究也发现,通路成员的选择压力影响因素较为复杂,除受密码子偏性、蛋白长度影响外,还有其它的待研究因素。四、猪IGF1-FoxO通路成员的确定及通路成员SNP信息的推断:通过对模式生物进行进化研究,我们获得了通路成员的保守组成。我们利用人、鼠模式生物的27个保守基因分别到猪的基因组数据库进行blast搜索,用来获取猪的通路成员。因为猪的基因组注释信息的不完整性,所以我们同时也采用搜索通路成员的侧翼序列,用来尽可能获取猪的通路成员。最终我们获得保守通路27个成员中的22个基因。我们将这些基因定义为要研究的基因集。这些通路成员基因序列与NCBI Trace Repository和GenBank中的鸟枪序列进行blast搜索及序列比对操作,进而推断通路成员的SNP位点信息。五、IGF1-FoxO通路SNP的检测分型及性状的关联分析:在对猪IGF1-FoxO通路SNP推断分析的基础上,我们运用SNaPshot与PCR-RFLP技术对猪部分通路基因多态位点进行检测分型,同时运用奥斯本自动喂料记录系统对111头杜长大猪做了生长育肥试验,进而对多态位点与生长发育相关性状进行关联分析,以期阐明通路中的哪些基因、哪些SNP位点决定了性状的不同表型,这些基因和多态位点是如何发挥作用的,即其生物学功能及作用模式如何,从而在通路水平上揭示猪的生长发育机制。研究结果表明,FoxO3可能是控制生长发育的一个主效基因或者与控制生长发育的主效基因紧密连锁。我们所开展的这些研究,为揭示复杂性状的遗传机制尤其是进行猪生长发育性状的遗传机制分析及标记辅助选择,进而对猪进行遗传改良提供了坚实的基础。更多还原

【Abstract】 The growth and development of porcine will influence the growth rate, feed efficiency, body composition and carcass traits, and so on, which last influence economic efficiency. However, studying the mechanism of controlling growth and development confronts significant challenges at present and it restricts genetic improvement of pigs. Candidate genes analyzing, whole genome scanning, and whole genome association analysis were widely used to study the mechanism of controlling growth and development and obtained some candidate QTLs and SNPs, but it could not reflect biological process from genes to traits. Thus, this project first put forward a Candidate Gene Set Approach and used it to study the the mechanism of controlling growth and development of pig.1. The establishment of Candidate Gene Set ApproachThere is some blindness and arbitrariness for candidate gene method and this method is vulnerable to the influence by environment. Moreover, QTL used to location by linkage analysis spans tens of map units, or hundreds of genes, and this method need special population. Further, it costs much by GWAS method. In this case, we present the Candidate Gene Set Approach, and the standardize process includes the gene set selection, QTL mapping, candiadted SNP prediction, genotyping and associated analysis. This method has the virtue of motivated, easy for performance, reasonable for costs, etc.2. Uncovering the transcriptional circuitry in skeletal muscle regenerationSkeletal muscle has a remarkable ability to regenerate after repeated and complete destruction of the tissue. The healing phases for an injured muscle, including degeneration, inflammation, regeneration and remodeling, undergo an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network confronts significant challenges and requires the integration of multiple experimental data types. In this work, we focus on getting insights into the transcriptional circuitry during skeletal muscle regeneration using time-course expression data and motif scanning information. A set of→TtFarget associations was derived from predicted TF binding sites in promoter regions of co-expressed genes. Time-lagged correlation analysis was utilized to evaluate the TF→target associations. Our analysis identified 13 TFs that potentially play a central role throughout the regeneration process. Six of which have previously been described to be important for muscle regeneration and differentiation. The transcriptional factor FoxO3 was enrichment into the insulin pathway, and this pathway was involved into growth and development reported by previous literature. Therefore, mouse skeletal muscle regeneration analysis offered the clues that IGF1-FoxO was a candidate pathway that influence pigs’growth and development.3. The molecular evolutionary patterns of the insulin/FOXO signaling pathwayThe insulin/insulin growth factor-1(IGF1)/FOXO (IIF) signal transduction pathway plays a core role in endocrine system and maintains remarkable consistency across all vertebrate species. Although the components of this pathway have been best-characterized, the evolutionary pattern remains poorly understood. Here, we performed a comprehensive analysis to study whether the differences of signaling transduction elements exist among different animal species and to determine whether the genes are subject to equivalent evolutionary forces and how natural selection shapes the evolution of proteins in an interacting system. Our results demonstrate that most IIF pathway components are present throughout all animal phyla investigated here, and they are under strong selective constraint but two of which show evidence for positive selection. Remarkably, we detected a gradient in the strength of purifying selection along the pathway, increasing from the upstream to the downstream genes. We also found that the dN/dS may be influenced by quite complicated factors including codon bias, protein length and other factors.4. The identification of the porcine components of IGF1-FoxO pathway and SNP inference of pathway component membersWe got conserved pathway members though evolutionary analysis of model animals. We make use of 27 conserved members from model animals of human and mouse to blast pig genome to get pathway components. Because of incomplete annotation informations of pig’s genome, flanking sequences of pathway componences were also used to blast pig’s genome to get whole pathway members. At last, we got 22 genes from 27 pathway members. These members were then blast to shotgun sequences from NCBI Trace Repository and GenBank to mine SNP informatics.5. Experimental validation of candidate SNPs from IGF1-FoxO pathway and the association between polymorphism and traits of pig The polymorphism (SNP) of IGF1-FoxO pathway genes mining by previous researchwere chosen for experimental validation by NaPshot sequencing and PCR-RFLP. We then performed association analysis between polymorphisms and growth and development traits to illustrate how different phenotypes were influenced by different genes and SNPs and how different genes and SNPs excerting their roles. Namely, knowing the biological function and regualatory mode, we could recovery the growth and development mechanism of pig from pathway perspective. Our result demonstrated that FoxO3 may be one or linkage with one of the major genes that influence the growth and development of pig. The above results help to lay a foundation for the further research of molecular genetic mechanisms of pig growth and development performance and provide evidence for pig marker assistant selection and marker-assisted introgression.更多还原