【作者】 李文学；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 生殖质是在许多物种卵子发生中形成的一种特殊细胞质成分，含有这种成分的细胞才能发育为生殖细胞。对果蝇研究表明，mago nashi基因是生殖质组装和形成的一个关键基因，其编码的蛋白质能识别并结合某些生殖质决定基因的RNA，形成复合体，穿过核膜进入核质区域，指导生殖质的正确组装形成。从低等的真核生物酵母到高等的哺乳动物人，都存在mago nashi的同源基因并且相似性非常高。为进一步阐明家蚕生殖发育的调控机制，在家蚕基因组、EST数据以及芯片数据分析的基础上，本论文对果蝇中已报道的生殖质决定相关基因在家蚕中的同源基因进行了鉴定，并对其表达模式进行了分析，然后对高度保守的mago nashi基因进行了克隆和原核表达，以期为以后深入了解该基因的功能研究奠定基础，获得结果如下：1．果蝇生殖质决定相关基因在家蚕中的同源基因分析从FlyBase下载果蝇生殖质形成相关基因18个(aubergine，spire，nanos，torso，tudor，vasa，pumilio，par-1，cappuccino，staufen，oskar，valois，germ-cell less，polar-granule component，mtlrRNA，mago nashi，tsunagi，exuperantia)，通过与家蚕基因组数据进行BLAST比对分析，发现除oskar，valois，germ-cell less，polar granule component，mtlrRNA 5个基因外，其余13个基因在家蚕中均有同源基因，并根据基因芯片数据分析了预测的家蚕生殖质形成相关基因的表达模式。发现在这些同源的生殖质形成相关基因中，有5个基因(Bmcapu，Bmnos，Bmvas，Bmaub，Bmexu)在雌雄发育后期有显著差异表达；有4个基因(Bmtud，Bmmago，Bmtsu，Bmstau)在雌雄发育过程中无差异表达；有3个基因(Bmnos，Bmvas，Bmaub)在生殖腺中特异表达；有6个基因(Bmcapu，Bmmago，Bmtsu，Bmexu，Bmstau，Bmtud)除在生殖腺中表达外，还在其它组织有高表达。2．家蚕mago nashi基因的克隆及mRNA表达检测根据预测序列设计引物，进行RT-PCR扩增和克隆测序验证，成功地克隆到家蚕mago nashi基因完整的编码区序列。分析发现该基因位于nscaf2655上，只有一个完整的拷贝，在基因组上有1611bp，共有两个外显子，分别为258bp和183bp，并且外显子、内含子边界符合典型的GT-AG结构。克隆的mago nashi基因完整的编码区序列为441 bp，编码146个氨基酸残基。RT-PCR检测了该基因在发育各时期各组织器官的表达情况，发现该基因在所检测的所有发育时期、组织器官均有表达，是一个非特异表达基因。3．家蚕mago nashi基因的原核表达将家蚕mago nashi基因的编码区序列克隆到原核表达载体pET28-a中，并将构建成的pET-mago nashi重组质粒转化表达宿主菌Escherichia coli BL21(DE3)细胞，IPTG诱导后进行SDS-PAGE检测分析。结果表明，同对照相比，重组质粒在23.5KD处有一条十分明显的新增蛋白带，与预测的蛋白质大小一致，说明该基因体外重组表达成功。进一步的分析发现该重组蛋白主要以包涵体的形式存在。更多还原

【Abstract】 Germ plasm in many species formed during oogenesis ,it is a special cytoplasmic component. cells without the germ plasm can’t develop into germ cells. Mago nashi ,a biological reproductive gene in Drosophila, plays an important role in assembling and formation of germ plasm. The protein encoded by Mago nashi gene can recognize and bind some RNA which decide the fate of germ cells .This RNP complex can guide germ plasm assembled correctly. From the lower eukaryotic like yeast to the higher mammals such as homo-sapiens,in which Mago nashi gene has the homology which has very high similarity with it. Mago nashi gene plays a conservative and important role in biological evolution.To further clarify the Silkworm reproductive and developmental mechanisms, based on the analysis of the silkworm genome ,the large scale EST datas and microarray data ,this paper analyzed the homology and Spatio-temporal expression of the key gene which had been reported to determine the formation of germ plasm; The cloning and expression of Mago nashi gene have been done with a view to understand the function of the gene, the results obtained are as follows :1 The homology of reproductive genes playing essential roles in Drosophila germ plasm formation in the silkworm:Downloaded 18 reproductive genes playing essential roles in germ plasm formation in the Drosophila from FlyBase (aub, spir,nos, tor, tud,vas,pum,par-1,capu,stau,osk,valois,germ-cell less,polar- granule component,mtlrRNA, mago nashi, tsunagi,exuperantia). Used BLAST to compare them with silkworm genome data, excepting oskar, valois,germ-cell less,polar granule component, mtlrRNA, the remaining 13 genes in Silkworm have homologous gene. According to the gene array and the gene expression microarray data to analyz the expression patterns of the predicted genes which may have influence on the formation of germ cells. Five of these genes have significant differences in expression of paroecious development in later stage, the expression in femina is significantly stronger than maleness; in the paroecious development process four of these genes have no difference in expression; Three of the genes differentially expressed only in gonad; six genes expressed not only in gonad,but also in other tissues and organs.2 The cloning and expression pattern of Bmmago gene in Bombyx moriFor the expression analysis of Bmmago gene, we cloned the coding sequence of the Bmmago gene by RT-PCR,then the clone sequenced. The Bmmago gene, a single copy locating on the genome, spans 1611bp and is comprised of 2 exons and 1 intron. The gene comprised an open reading frame of 441bp,and the encoded protein was 146 amino acids. The expression of Bmmago gene was found in all kinds of tissues,organs and all stages,thus the gene is a non-specific expression gene.3 The expression of Bmmago gene in E.coliIn order to further research of the characteristics and function of the encoding protein, a prokaryote express vector, pET-Bmmago, containing the complete ORF of Bmmago, had been constructed. The recombinant plasmid was induced by IPTG ,then performed the SDS-PAGE, we found the recombinant protein expressed. Comparing with the reference, between the marker 31KD-20.1KD, recombinant protein shown a clearly new band. This molecular weight correspond the predicted protein size. Further analysis revealed that the recombinant protein in the form of inclusion bodies.更多还原