果蝇新型碱性磷酸酶相关基因CG6236的敲除及表型分析-遗传学专业论文.docxVIP

温少tl医学院硕士学位论文果蝇新型碱性磷酸酶相关基因CG6236的敲除及表型分析 温少tl医学院硕士学位论文 果蝇新型碱性磷酸酶相关基因CG6236的敲除及表型分析 摘 要 目的：碱性磷酸酶广泛存在于人体各器官中，其水平异常与一系列疾病有关，但 其在发育中的具体作用机制尚不明确。硫酸酯酶是一种重要的硫酸基团水解酶， 在信号通路、细胞代谢等个体发育过程中具有重要的作用。本文以果蝇为模式生 物，研究一个新型碱性磷酸酶样蛋白基因CG6236在发育中的功能。 方法：利用P．转座子介导的不精确剪切来获取CG6236突变体果蝇；用嵌合体细 胞克隆进行基因功能缺失分析；用分子克隆的方法构建CG6236全长过表达质 粒；用Gal．UAS系统进行基因过表达分析；用Hela细胞染色确定CG6236蛋白 的表达位置。 结果：果蝇中有4蛋白与CG6236蛋白具有相同的碱性磷酸酶样结构域，经预测 保守性高于30％；获得三个CG6236突变体果蝇品系，发现CG6236纯合突变体 半致死，存活的幼虫及成蝇腹部出现黑色素瘤样表型；CG6236融合蛋白与内质 网蛋白calnexin共定位，提示CG6236蛋白定位于内质网中；另外，缺失或过表 达CG6236均不影Ⅱ向Wingless和Hedgehog信号通路。 结论：基因CG6236纯合突变体成蝇和幼虫腹部出现黑色素瘤样表型，提示 CG6236对果蝇发育具有重要的作用；CG6236与Wg，Hh信号通路无关，并且其 表达的蛋白很可能不具有硫酸酯酶的活性。 关键词 果蝇；CG6236；碱性磷酸酶；硫酸酯酶； 基因缺失； 黑色素瘤 温州医学院硕士学位论文Drosophila 温州医学院硕士学位论文 Drosophila Novel Alkaline Phosphatase Related Gene CG6236 Deletion and Phenotype Analysis Abstract Objective：Alkaline phosphatase(ALP)is present in all tissues through the entire body in human，the abnormal ALP level is related to many diseases．However,the molecular mechanism of ALP during development remains unclear．Sulfatase is hydrolytic enzyme to cut sulfate groups from substrates，it has an important role in cell signaling nd cell metabolism during development．Here，we report and analyze the function of gene CG6236,an important ALP—like gene，using Drosophila model system．Methods：CG6236 mutant alleles were generated by P element mediated imprecise excision；Loss—of-function analysis is used by mosaic clone；Molecular clone techniques were utilized to get pUAS-attB．CG6236-V5 construct； Gain。of-function analysis is used by Gal-UAS system；Hela cell staining is used to observe the localization of CG6236 protein．Results：There are four proteins which possess the same alkaline phosphatase-like domain with CG6236 protein，we found that sequences identity is over 30％：We generated three mutant alleles for CG6236, and found that deletion of CG6236 in Drosophila is semi．1ethal，and there are melanotic tumors in the abdomen of homozygous mutant larvae and adult flies； More