家蚕翅原基发育关联基因BmHMGS的鉴定与表达特征分析  

Identification and Expression Characteristics of 3-Hydroxy-3-methylglutaryl-Co A Synthase Gene Related to Bombyx mori Wing Disc Development

作　　者：蒋诗梦[1,2] 钟杨生 林碧敏[1] 陈芳艳[1] 严会超[1] 林健荣[1] Jiang Shimeng;Zhong Yangsheng;Lin Bimin;Chen Fangyan;Yan Huichao;Lin Jianrong(College of Animal Science, South China Agricultural University, Guangzhou 510642, China;The Sericultural Research Institute of Hunan Province, Changsha 410127, China)

机构地区：[1]华南农业大学动物科学学院,广州510642 [2]湖南省蚕桑科学研究所,长沙410127

出　　处：《蚕业科学》2018年第2期242-248,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.31272368);现代农业产业技术体系建设专项(No.CARS-22);公益性行业(农业)科研专项(No.201403064)

摘　　要：家蚕幼虫的翅原基最终发育为成虫的翅,翅原基发育与家蚕的变态发育紧密关联。为了研究激素作用下家蚕翅原基发育的分子调控机制,以经过蜕皮激素20E体外诱导培养和未经诱导培养的家蚕5龄幼虫翅原基为材料分别提取总蛋白质,利用Shotgun质谱分析在20E诱导组的翅原基中获得132种差异蛋白,在未经20E诱导组的翅原基中获得76种差异蛋白。进一步通过生物信息学技术及GO分类法在20E诱导组的翅原基的差异蛋白中,筛选到1个具有生物调节作用的酶类蛋白基因Bm HMGS作为翅原基发育相关的候选基因,设计引物并以上蔟1 d的蚕体翅原基总RNA反转录得到的c DNA为模板,克隆了该基因。通过qRT-PCR检测到Bm HMGS基因在幼虫5龄1 d、上蔟3 d、蛹期1 d蚕体内的表达量较高,其中在上蔟3 d的表达量达到峰值。构建重组载体进行Bm HMGS的原核表达并制备抗体后,采用Western blot技术在5龄1～7 d和上蔟1～3 d的蚕体翅原基、脂肪体中及上蔟1～3 d的生殖腺中都可以检测到Bm HMGS的表达,并且在翅原基中的表达没有发育时期特异性。研究结果表明,从20E诱导组的家蚕翅原基中鉴定的Bm HMGS与翅原基发育相关,其表达在5龄至上蔟阶段没有时期和组织的特异性,推测Bm HMGS是一个具有多种生物学功能的基因。The wing discs of silkworm（ Bombyx mori） larvae eventually develop into adult wings,and the development of wing disc is closely related to the metamorphosis of silkworm. In order to explore the mechanism of molecular regulation of wing disc development in silkworm under hormone control,total proteins were extracted from the wing disc of 5 th instar larvae induced by ecdysone 20 E in vitro and the wing disc without induction respectively. Totally 132 differentially expressed proteins were obtained from the wing disc of 20 E induction group and 76 differentially expressed proteins were obtained from the group without 20 E induction using Shotgun mass spectrometry. An enzyme named Bm HMGS（ Bombyxmori 3-hydroxy-3-methylglutaryl-Co A synthase） with biological regulation function was screened out in the differential proteins of wing disc group induced by ecdysone20 E as a candidate gene related to wing disc development using bioinformatic and GO taxonomy approaches.The coding gene was cloned by PCR using templet obtained through reverse transcription of the total RNA of silkworm larvae wing disk on the 1 st day of wandering period. qRT-PCR results showed high expression of Bm HM-GS gene on the 1 st day of 5 th instar,the 3 rd day of the wandering period and the 1 st day of the pupal period,among which the expression on the 3 rd day of the wandering period was the highest. After construction of the recombinant vector for prokaryotic expression and preparation of antibody,Bm HMGS protein was detected in wing disc,fat body on the1 st to 7 th day of 5 th instar and the 1 st to 3 rd day of wandering period,and in gonad on the 1 st to 3 rd day of wandering period. Besides,the expression of Bm HMGS protein in wing disc was not dependent on developing period. The results show that Bm HMGS identified from the wing disc of the 20 E induction group is associated with the development of wing disc,and its expression has no temporal and spatial specificity from the 5 th instar to wandering period,suggesting that Bm HMGS is a

关 键 词：家蚕翅原基 体外培养 蜕皮激素 Shotgun质谱 BmHMGS基因 表达特征 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q786[农业科学—畜牧兽医]