黑线仓鼠PPARβ的部分序列的克隆及SNP分析

【作者】 郑莹莹；

【导师】 徐来祥； 薛慧良；

【作者基本信息】 曲阜师范大学 ， 动物学， 2012， 硕士

【摘要】 黑线仓鼠（Cricetulus barabensis）繁殖力强，分布范围广，破坏性大，是我国北方农田的主要害鼠之一。通过研究影响其繁殖力的PPARβ基因（Peroxisomeproliferator-activated receptors β），从分子水平上揭示害鼠种群数量的波动机制，可为对鼠害的综合防控提供新的思路和方法。PPARβ基因作为核受体超家族成员之一，早在胚胎发育的早期阶段就已出现，与动物胚胎着床、胎盘的形成、胚胎发育、卵巢功能的维持等有着密切的关系。此外，在个体生长过程中，PPARβ分布广，对个体正常的生长发育有着重要的调节作用。本研究首次采用基因克隆的方法分离黑线仓鼠PPARβ部分序列，同时对黑线仓鼠种群内部分序列进行了单核苷酸多态性（SNP）分析，为黑线仓鼠种群暴发机制的研究从分子水平上提供理论依据。本研究以捕获于山东省的黑线仓鼠为研究对象，用RT-PCR方法克隆PPARβ mRNA外显子4-7的部分序列480bp（GenBank登录号：JN662960），并对其核苷酸和氨基酸序列、二级结构、空间结构进行了生物学分析和预测。在其结构分析过程中发现了特殊的结构区域：锌指结构。在蛋白质二级结构中Alpha螺旋占很大比例，但锌指结构则位于较小的疏水性区域中。将得到的黑线仓鼠PPARβ mRNA序列与其他物种PPARβ中相对应的mRNA序列进行同源性比对，并利用不同的方法构建了系统进化树，结果显示黑线仓鼠与啮齿目的金仓鼠(Mesocricetus auratus)、大鼠(Rattus norvegicus)、小鼠(Mus musculus)亲缘关系最近，与野猪(Sus scrof)、牛(Bos Taurus)、兔(Oryctolagus cuniculus)等哺乳动物亲缘关系较远，与人亲缘关系最远，这一结果与以上物种之间的传统分类进化关系相一致，说明PPARβ在进化上相对比较保守。本研究还首次用PCR法克隆并检测了山东地区30个黑线仓鼠的PPARβ基因部分序列（297bp）的多态性，该序列包括外显子6（175bp），内含子6（104bp）和外显子7（18bp）。利用DnaSPv5.0软件分析发现，该区段共有11种单倍型，10个多态位点，其中外显子6中有6个，内含子中有4个。分析该区段发现，转换有9个，颠换1个。PPARβ基因外显子区域碱基发生突变的概率较大，引起编码的相应的氨基酸发生改变。这与从人上研究该区域的结果相一致，也许与其功能有着密切的关系。丰富的单核苷酸多态性为种群遗传多样性提供了可能性，进而使物种能够更好的适应外界环境。本研究对黑线仓鼠繁殖相关基因——PPARβ进行了分子生物学方面的研究，研究结果将为从分子生物学角度寻找控制黑线仓鼠数量及其危害性的有效方法提供借鉴。更多还原

【Abstract】 Cricetulus barabensis，wide distribution, the destructive wait for a reason, be in the north ofthe advantage of the farmland against one of the rat. Begin with breeding genes, from molecularlevel reveals harmful rat population fluctuation mechanism, provide new ideas forcomprehensive prevention and control the rat. This paper is mainly on the reproducive genePPARβ for the molecular biology study.PPARβ gene (Peroxisome proliferator-activated receptors beta) as one of the nuclearreceptor superfamily members, already appeared as early as in an early stage in embryonicdevelopment, and has close relationship with animal embryo implantation, the formation of theplacenta, the embryonic development, the maintenance of ovarian function, in addition inindividual growth process, PPAR β distribution is quite widespread and has important regulatoryrole to the normal growth and development of the individual. This is the first time amplify thePPAR β gene sequence of C.barabensis by cloning method, and made single-nucleotidepolymorphisms (SNPS) analysis in part sequence of C. barabensis, to provide the theory basis atthe molecular level for the C. barabensis.This study to capture in shandong province C. barabensis as the research object, withRT-polymerase chain reaction (PCR) cloning get PPAR β mRNA exon4-6part of the sequence480bp (GenBank registration number: JN662960), and make the biology analysis and forecastthe nucleotide, amino acid sequence, secondary structure and space structure. In its structureanalysis process found the special structure area: zinc finger structure. In the secondary structureof protein Alpha helix of accounting for a large scale, but zinc finger structure is located in thesmaller hydrophobic areas. To make the homology compared the corresponding PPAR β mRNAsequence of C. barabensis with Mesocricetus auratus, Mus musculus, Rattus norvegicus, Susscrof, Bos Taurus, Oryctolagus cuniculus,et al. and make the system evolutionary tree bydifferent methods. Results show that the C. barabensis has closest genetic relationship withMesocricetus auratus, Mus musculus, Rattus norvegicus, and further with Sus scrof, Bos Taurus,Oryctolagus cuniculus, and furthest with Homo sapiensc, this result with more species oftraditional classification relationship between evolution is consistent, explain PPAR beta can beevolutionary relatively conservative.With the first time use PCR method get the PPAR β gene297bp of thirty C.barabensis inShandong province, including exon6(175bp), introns6(104bp) and exon7（18bp）.UseDnaSPv5.0software analysis found that, in this section, the number of Haplotypes:11; Variable(polymorphic) sites:10, among them, there are6in the exon6, and4in the intron. This sectionanalysis found that conversion has9; Transversion is1. PPAR β gene exon area the probability of mutations bases is larger, the cause of the amino acids encoding corresponding change. PPARβ gene rich SNP offers possibility for population genetic diversity and species can better adapt tothe external environment.更多还原