家蚕N-乙酰基转移酶基因BmNAT的克隆与表达分析

发布时间：2017-12-27 00:33

本文关键词：家蚕N-乙酰基转移酶基因BmNAT的克隆与表达分析　出处：《蚕业科学》2016年02期 　论文类型：期刊论文

【摘要】：N-乙酰基转移酶(N-acetyltransferase,NAT)是昆虫体内一种催化乙酰基团在乙酰辅酶A和胺之间转移的酶,主要与色素沉积、生理节律等有关。对已公布的家蚕(Bombyx mori)N-乙酰基转移酶基因Bm NAT(Gen Bank登录号:NP_001040401.1)进行生物信息学分析:该基因序列全长763 bp,ORF为564 bp,编码187个氨基酸残基,分子质量21.4 k D;多序列比对表明Bm NAT与脐橙螟(Amyelois transitella)同源蛋白质的序列相似性最高。通过原核表达的方法制备了融合Bm NAT蛋白,经蛋白质纯化并免疫新西兰兔获得多克隆抗体。亚细胞定位显示Bm NAT主要存在于细胞质和细胞核中。利用Western blotting检测Bm NAT在家蚕不同发育时期和5龄幼虫各组织器官的表达谱,结果表明Bm NAT在蛾、卵期以及5龄幼虫的头部表达量较高,与Bm NAT的qRT-PCR检测结果基本一致,初步推断Bm NAT可能在家蚕的这些组织或发育时期发挥一定的生物学功能。流式细胞实验显示Bm NAT对细胞周期可能存在一定的影响。
[Abstract]:N- N-acetyltransferase (NAT) is an enzyme that catalyzes the transfer of acetyl groups between acetyl coenzyme A and amine in insects, which is mainly related to pigment deposition, physiological rhythm and so on. Published on the silkworm (Bombyx mori) N- acetyltransferase gene (Bm NAT Gen Bank accession number: NP_001040401.1) by bioinformatics analysis: the gene sequence was 763 BP, ORF 564 BP, encoding 187 amino acid residues, the molecular weight of 21.4 K D; multiple sequence alignment showed that Bm NAT and navel orange moth (Amyelois transitella) the highest similarity of homologous protein sequences. The fusion Bm NAT protein was prepared by prokaryotic expression, and the polyclonal antibody was obtained by protein purification and immunization with New Zealand rabbits. Subcellular localization showed that Bm NAT was mainly found in cytoplasm and nucleus. Using Western blotting to detect Bm NAT expression in different developmental stages of silkworm and 5 instar larva tissues and organs of the spectrum, the results show that the Bm NAT in the moth, egg stage and 5 instar larvae of relatively high expression in the head, consistent with the Bm NAT qRT-PCR test results, it is concluded that Bm NAT may play a certain biological function in silkworm these tissues or developmental stages. Flow cytometry showed that Bm NAT might have a certain effect on the cell cycle.
【作者单位】：浙江理工大学生命科学学院;浙江省家蚕生物反应器和生物医药重点实验室;
【基金】：国家重点基础研究发展计划“973”项目(No.2012CB1146-00) 浙江省自然科学基金项目(No.LY16C170002) 国家高技术研究发展计划“863”项目(No.2011AA100603)
【分类号】：S881.2;Q78
【正文快照】： 鳞翅目昆虫的体色形成模式与生理节律对其生存和繁衍具有重要意义。研究家蚕体色形成及生理节律的分子调控机制不仅有助于理解鳞翅目昆虫色素及形态发育的代谢和调控网络,还能够为家蚕功能基因的利用(如家蚕育种或鳞翅目害虫防治)提供新思路[1]。N-乙酰基转移酶(N-acetyltrans

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