【作者】 周静；

【导师】 袁榴娣；

【作者基本信息】 东南大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 前体mRNA的选择性剪接发生频率很高,它在人类基因组中广泛存在,是增加蛋白质多样性及基因表达复杂程度的主要原因。近来研究显示,一些富含丝氨酸和精氨酸蛋白(SR蛋白)对于剪接的顺利完成有着至关重要的作用。SR蛋白是具有帮助识别正确剪接位点并促进剪接体的成熟的一类重要剪接因子,为组成性剪接所必需,在选择性剪接中更起着不可替代的作用。Dxl6基因是2000年克隆的一种新基因。根据氨基酸序列和核酸序列推测它可能是剪接因子的候选基因。原位杂交显示,Dxl6基因主要在后期胚胎的神经系统表达,与神经发育相关。诱变剂EMS(甲基磺酸乙酯)所致Dxl6的2核甘酸缺失突变导致果蝇胚胎后期或1龄幼虫致死。P因子插入突变(插入内含子区域)引起三龄幼虫或后期蛹致死,但约有5%的逃逸者,这些逃逸者发育延迟并发有刚毛、翅膀、眼睛、腿等器官的发育异常。为了深入研究Dxl6蛋白的功能,了解其在果蝇胚胎和胚胎后期及神经系统发育中的作用,本文用SELEX方法筛选到了Dxl6可能的作用靶序列。首先,将Dxl6氨基端含两个RNA识别基序的基因片段构建入原核表达载体,通过摸索各种条件,成功地以可溶的形式表达了融合蛋白,并用GST亲合层析柱进行了纯化,给后续的实验提供了较好的工具。其次,经分离纯化的Dxl6N-GST融合蛋白与RNA文库在一定条件下孵育结合,共进行六轮SELEX筛选,得到了Dx16可能作用的靶序列,并用EMSA验证了它们之间的相互作用。最后,用生物信息学分析了筛选到的Dx16可能作用的靶序列的二级结构、一致基序及所在的候选基因,并用RT-PCR检测部分候选靶基因在Dx16功能缺陷的突变果蝇中的表达状态,以及Dx16对这些底物的剪接方式和表达水平的影响,为下面的体外剪接实验奠定了基础,从而也为探讨Dx16的调控机制及其在发育中的作用提供数据。更多还原

【Abstract】 Alternative splicing of pre-mRNA, which happens in high frequency and generally exists in genome, is the main reason that increases the diversity of proteins and multiplicity of gene expression. Recent research shows that some Ser and Arg amino acid rich proteins (SR protein) play an essential role in alternative splicing. SR protein is a kind of splicing factors that help recognition splicing site correctly and promote mature of splicesome,they are essential to constitutive splicing and have pivotal contribution to alternative splicing.Dxl6 gene, a novel gene encoding SR protein, has been cloned in 2000. According to its amino acid and nuclear sequence, Dxl6 is considered as the candidate gene of splicing factors. In situ hybridization shows that Dxl6 expresses mainly in nervous system in later embryo stage and is thought to associate with nervous development. The 2-nucleotide deletion mutation induced by EMS results in lethal to drosophila in later embryo stage or first instar-larvae. The insertion mutation (inserted in intron region) caused by P element results in lethal to drosophila in later pupae, although there is 5 percent escaper of P element k00230 insertion, these escapers have abnormity in bristles, wings, legs, eyes and the like.The aim of the research is to study the role that Dxl6 plays in fly development in central nervous system in embryo as well as after embryo stage, SELEX is used to find the substrates interacting with Dxl6 during splicing procedure.First of all, clone the gene fragments including the two RRM regions into prokaryotic expression vector, and finally the protein is expressed in E.coli in a soluble condition and purified successfully. It provides a good tool for the next experiment.Dxl6N and a random RNA library are hatched together in a certain condition, after six rounds of SELEX selections, the sequences that interact with Dxl6 are gotten. With the help of biological information analysis, the consensus sequence of candidate genes and their common motif and second structure are acquired. RT-PCR analysis the mRNA expression level of the target genes in Dxl6 mutation fly lines and wild type flies. The results lay a basis for studying splicing in vitro and more regulation mechanism of Dxl6 in fly development.更多还原