家蚕glial cell missing(BmGcm)基因鉴定、表达、亚细胞定位和功能  

Identification,Expression,Subcelluar Localization,and Function of glial cell missing(gcm) in Silkworm(Bombyx mori)

作　　者：张奎[1] 潘光照[1] 苏晶晶[1] 谈娟[1] 徐曼[1] 李钰添 崔红娟[1] ZHANG Kui;PAN GuangZhao;SU JingJing;TAN Juan;XU Man;LI YuTian;CUI HongJuan(State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716)

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716

出　　处：《中国农业科学》2018年第7期1401-1411,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31672496);中国博士后科学基金(2017M620408);重庆市自然科学基金(cstc2016jcyj A0425);重庆市高校创新团队建设计划(CXTDX201601010)

摘　　要：【目的】鉴定、克隆家蚕(Bombyx mori)glial cell missing(Bm Gcm)基因,分析其m RNA表达及亚细胞定位特征。制备多克隆抗体,同时在细胞水平进行过表达,检测Gcm对细胞增殖和周期的影响,为探究Bm Gcm功能打下基础。【方法】利用RACE方法克隆获得Bm Gcm全长c DNA序列,利用ORF Finder和SMART等在线工具对Bm Gcm基本序列特征和结构信息进行分析,运用Clustalx和MEGA 6.0等软件对多物种Gcm蛋白进行同源序列比对和进化分析。采用RT-PCR和q RT-PCR方法检测Bm Gcm的表达情况。利用原核表达系统获得重组蛋白,通过蛋白纯化和免疫小鼠制备多克隆抗体,运用Western blot对抗体进行检测。构建Bm Gcm表达载体,转染家蚕胚胎细胞系,分析其亚细胞定位情况,同时利用EDU细胞增殖标记和流式细胞仪对其功能进行探索。【结果】Bm Gcm(BGIBMGA006182)定位于4号染色体的nscaf2847上,其基因全长4 046 bp,包含4个外显子和3个内含子。其c DNA全长1 734 bp,包含166 bp的5′UTR、227 bp的3′UTR和1 341 bp的完整开放阅读框(ORF)。该基因编码446个氨基酸残基,预测蛋白分子量为50.61 k D,等电点5.557,含有典型的GCM结构域。多重比对结果显示GCM结构域在不同物种间具有高度的保守性,进化分析显示昆虫Gcm蛋白单独聚为一支,其中Bm Gcm蛋白与帝王蝶同源蛋白亲缘关系最为接近。表达分析结果显示Bm Gcm在胚胎发育第4天表达达到峰值,随后表达水平逐渐下调,而在幼虫阶段,Bm Gcm主要表达于中肠、精巢和卵巢。将Bm Gcm完整的开放阅读框序列构建至原核表达系统,经IPTG诱导和亲和层析纯化获得高纯度重组蛋白,通过免疫小鼠获得了多克隆抗体,Western blot检测该抗体可以特异性识别重组蛋白。在家蚕细胞系中过表达Bm Gcm蛋白,结果显示其定位于细胞核。在细胞水平,过表达Bm Gcm会明显抑制细胞增殖,将细胞周期阻滞于G1/S期。【结论】克隆鉴定得到Bmgcm�【Objective】The objective of this study is to identify and clone Bombyx mori glial cell missing（Bm Gcm）, analyze its expression and subcelluar localization, prepare polyclonal antibody and overexpress Bm Gcm at the cellular level to explore its function in proliferation and cell cycle, and to provide a theoretical basis for further studying the function of Bm Gcm in B. mori.【Method】The full-length of Bm Gcm was acquired by RACE technology. Basic sequence and structural information of Bm Gcm were analyzed by using several online softwares, including ORF Finder and SMART. Multiple sequence alignment and evolutionary analysis were acquired by Clustalx and MEGA 6.0, respectively. The expression profile was investigated by RT-PCR and real-time PCR. The recombinant protein of Bm Gcm was obtained using prokaryotic expression system, and purified with nickel affinity chromatography, then the antibody was prepared. Bm Gcm over-expression vector was transfected into B. mori cell line to analyze the subcellular location of Bm Gcm, and EDU and flow cytometry were used to study its function. 【Result 】 The Bm Gcm（BGIBMGA006182） was clustered on nscaf2847 which was located on chromosome 4. The genomic DNA total length of Bm Gcm is 4 046 bp, which contains 4 exons and 3 introns. The full-length of c DNA is 1 734 bp, with a 166 bp 5′ UTR, a 227 bp 3′ UTR and a 1 341 bp open reading frame（ORF）. This gene encodes 446 amino acid residues, the predicted molecular mass is 50.61 k D and the p I is 5.557, which contains a typical GCM-motif. Multiple sequence alignment demonstrated that GCM motif was highly conserved. Phylogenetic analysis revealed that Gcm of insect was clustered alone, and Bm Gcm had the highest relationship withGcm from Danaus plexippus. The expression profile revealed that Bm Gcm reached peak at the 4 th day of embryonic period, and then gradually decreased during embryonic stage. Bm Gcm mainly expressed in larval midgut, testis and ovary. The complete ORF sequence of Bm Gcm was inserted in

关 键 词：家蚕glial cell missing基因(BmGcm) 克隆 表达分析 抗体制备 过表达 

分 类 号：S881.2[农业科学—特种经济动物饲养]