兰州大学机构库 >生命科学学院
果蝇中waw基因的敲除及其蛋白表达
Alternative TitleKnock out of waw in drosophila and its protein purification
马克君
Thesis Advisor谢小冬
2009-05-22
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword果蝇 基因waw 基因敲除 P因子 蛋白纯化
Abstract果蝇是一种模式生物,有着丰富的遗传学表型,在基因功能的研究中常被使用。waw基因是E. coli中LepA在果蝇中的同源基因。LepA是翻译延伸因子,在核糖体翻译蛋白质过程的延伸步骤中与核糖体作用,引起核糖体“反转运”,从而使得翻译过程中不正确配对的氨基酸退回,纠正错配。为了研究果蝇中waw基因的功能,我们构建了一个含有突变ATG,并且有移码突变片段的外源质粒pTARG-∆waw。通过质粒大提的方法获得用于注射要求的质粒,与helper gene一并提纯,显微注射的方法注射到果蝇的卵中,利用同源重组的原理,在果蝇中进行基因打靶,敲除目的基因,使用了P因子跳出的方法敲除了目的基因。同时,利用UAS-GAL4系统构建了一个用于过表达和一个带GFP用于定位的质粒pUAST-waw, pUAST-waw-GFP, 同样使用显微注射的方法将提纯的质粒与helper gene一起注射的果蝇的卵中。为了研究该基因在体外的功能,我们利用His,GST,MBP融合的方法,分别构建pET22b-waw,pET28a-waw, p-waw, pMal-waw等质粒用于表达蛋白,使用亲和层析,离子交换,分子筛等蛋白纯化方法纯化蛋白。结果表明:在突变了的果蝇中雌蝇尾部明显黑于野生型雌果蝇,有一定雄蝇的特征,可能说明waw基因与果蝇的性别决定有一定关系。
Other AbstractDrosophila is a kind of model animals which can be used in gene function research for its abundant genetic phenotypes. waw is homologous gene of E. coli LepA. LepA, an elongation factor, has been reported that it can interact with ribosome in elongation of protein synthesis, which makes back-translocation happen, and correct the mismatched Amino acid. To study the function of waw, we constructed a plasmid, pTARG-∆waw, which contained ATG mutanted and frame shift mutation. Purified the plasmid with helper gene by plasmid maxi extraction. Transferred the plasmid into fly eggs by microinjection to knock out waw by gene targeting. And also we did a P-element jump-out experiment to get the mutant fly. Meanwhile, by using the UAS-GAL4 system, we constructed plasmids of pUAST-waw and pUAST-waw-GFP to overexpress the gene in vivo, and identify its location. To study the gene’s function in vitro, we constructed plasmids of pET22b-waw, pET28a-waw, p-waw, pMal-waw and transformed them into E. coli to express the protein, such purification methods like iron exchange, were used to purify it. The results show that the mutant female flies have black rumps, which are different from wildtype female and alike to the male flies, suggest that waw may relate to sex determination.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221281
Collection生命科学学院
Recommended Citation
GB/T 7714
马克君. 果蝇中waw基因的敲除及其蛋白表达[D]. 兰州. 兰州大学,2009.
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