家蚕BmNAT基因的克

发布时间：2018-03-27 18:57

本文选题：家蚕　切入点：N-乙酰转移酶　出处：《浙江理工大学》2016年硕士论文

【摘要】：N-乙酰基转移酶(N-acetyltransferase,NAT,EC 2.3.1.5)是一种催化乙酰基团在乙酰辅酶A和胺之间转移的酶。昆虫NAT主要与色素沉积、生理节律、几丁质的合成、杀虫药物的设计等有关。家蚕(Bombyx mori)是继果蝇之后又一典型的鳞翅目模式生物,还能作为生物反应器生产一些重要的药用重组蛋白。目前对家蚕N-乙酰转移酶基因(Bombyx mori NAT,BmNAT)的研究还比较少,关于BmNAT基因在家蚕中的基因调控机制还没有相关报导。我们根据已公布的家蚕N-乙酰基转移酶基因(Bombyx mori NAT,BmNAT)序列进行了生物信息学分析,该基因序列全长1690 bp,ORF为1065 bp,编码355个氨基酸残基,分子量大小为39.894kDa(GenBank登录号:XP_004932777.1),等电点pI为5.609;多序列比对的同源性分析表明,BmNAT与同是家蚕微管组织的其他BmNAT蛋白同源性较高,而与其他昆虫或者高等动物的NAT同源性较低,与BmNAT(GenBank登录号:XP_004932776.1)的同源性最高;与玉带凤蝶(Papilio polytes)同源蛋白同源性最低。通过构建原核表达重组质粒pET28a-BmNAT,在大肠杆菌中表达融合蛋白,将重组质粒转化BL21(DE3),IPTG诱导表达后,经SDS-PAGE分析,在43 kDa左右有一条特异性蛋白条带,与预期大小(融合标签3.56 kDa,BmNAT 39.894 kDa)相符。经纯化蛋白并免疫新西兰兔制备多克隆抗体,间接ELISA测定抗体效价达到1:12800以上,Western blotting检测发现抗体特异性较高。分别提取家蚕各发育时期和五龄幼虫各组织的总RNA和总蛋白,qRT-PCR和Western blotting检测BmNAT在家蚕各时期和各组织的表达情况,分析BmNAT的表达谱。结果表明,BmNAT在家蚕头部、蛾期、卵期的表达量较高,初步推断BmNAT可能在这些组织或时期中发挥一定的功能。亚细胞定位显示BmNAT主要存在于细胞质和细胞核中。通过构建重组真核表达载体pIEx-1-BmNAT,转染进入BmN细胞,过表达BmNAT蛋白,流式细胞术检测结果显示BmNAT对细胞周期可能存在一定的影响。前期研究表明,BmNAT存在乙酰化修饰,我们通过定点突变、免疫沉淀和Western blotting检测,进一步证实了BmNAT的乙酰化及其修饰位点。本研究首次证实家蚕乙酰化酶本身的乙酰化修饰,为认识家蚕新的基因翻译后修饰调控机理打下基础。
[Abstract]:N-acetyltransferase (N-acetyltransferase) is an enzyme that catalyzes the transfer of acetyl groups between acetyl coenzyme A and amine. Insect NAT is mainly associated with pigment deposition, physiological rhythm, and chitin synthesis. Bombyx morii (Bombyx morii) is another typical Lepidoptera model organism after Drosophila. It can also be used as a bioreactor to produce some important pharmaceutical recombinant proteins. At present, studies on the silkworm N-acetyltransferase gene, Bombyx mori nat, BmNATs, are relatively rare. There is no related report on the gene regulation mechanism of BmNAT gene in silkworm. We have carried out bioinformatics analysis based on the published sequence of silkworm N-acetyltransferase gene Bombyx mori nat. The total length of the gene is 1065 BP, encoding 355 amino acid residues, the molecular weight is 39.894kDa(GenBank accession number 0: XP 003 2777.1, and the isoelectric point Pi is 5. 609. The homology analysis of multiple sequence alignment shows that BmNat has high homology with other BmNAT proteins in the same microtubule of Bombyx mori. The homology of NAT with other insects or higher animals was lower, and the homology with BmNAT(GenBank accession number XP0049327761 was the highest, and the homology with Papilio polytes was the lowest. The fusion protein was expressed in Escherichia coli by constructing the prokaryotic expression plasmid pET28a-BmNAT. After the recombinant plasmid was transformed into BL21DE-3 SDS-PAGE, there was a specific protein band about 43 kDa, which was consistent with the expected size (fusion label 3.56 kDa BmNat 39.894 kDa). The purified protein was then immunized to prepare polyclonal antibodies from New Zealand rabbits. Indirect ELISA assay showed that the antibody titer was more than 1: 12800 and the specificity of the antibody was higher. Total RNA and total protein were extracted from the tissues of silkworm and fifth instar larva respectively. The expression of BmNAT was detected by RT-PCR and Western blotting in all stages and tissues of Bombyx mori. The expression of BmNAT was analyzed. The results showed that the expression of BmNat was higher in the head, moth and egg stage of Bombyx mori. The subcellular localization showed that BmNAT mainly existed in cytoplasm and nucleus. The recombinant eukaryotic expression vector pIEx-1-BmNATA was transfected into BmN cells and overexpression of BmNAT protein. The results of flow cytometry showed that BmNAT might have an effect on cell cycle. Previous studies have shown that there is acetylation of BmNat. We detected it by site-directed mutagenesis, immunoprecipitation and Western blotting. The acetylation of BmNAT and its modification site were confirmed for the first time in this study, which laid a foundation for the understanding of the regulation mechanism of the new gene post-translational modification in silkworm, Bombyx mori (Bombyx mori).
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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