家蚕BmYippee蛋白的表达和亚细胞定位分析.pdf

浙江理工大学硕士学位论文摘要在对家蚕蛹期eDNA文库测序分析中,我们获得一条eDNA序列长度为 1272bp,该基因的开放阅读框(ORF)全长366 bp,编码121个氨基酸残基, ,。我们将它命名为家蚕Yippee蛋白BmYippee(Yippee from Bombyx mori), Genbank登录号为DN236885,我们将其ORF克隆到原核表达载体pET-28a中, 构建重组质粒,双酶切鉴定及序列测定正确后转化大肠杆菌BL21(DE3),经 IPTG诱导表达的重组蛋白以包涵体形式存在,,以该重组蛋白作为抗原免疫新西兰大白兔制各多克隆抗体,经ELISA检测,抗体效价可达1:8000,Western blotting结果显示抗体的特异性较好。提取家蚕各个时期和五龄幼虫各组织的总RNA分别进行荧光定量PCR分析结果表明,家蚕发育时期在蛹期转录水平最高,卵中最低;五龄幼虫时期在生殖腺中转录水平较高,丝腺和脂肪较低。提取家蚕各发育时期以及家蚕五龄幼虫各组织蛋白进行Westernblotting分析,结果表明,BmYippee蛋白在家蚕蛹中及五龄幼虫的***、气管、马氏管中表达较高。亚细胞定位结果表明,在Bm5细胞的细胞间期BmYippee在细胞核和细胞质中均有分布,而细胞分裂期时在某些区域有相对集中的分布。关键词l;ombyx mori;Yippee;亚细胞定位;RT-PCR 浙江理工大学硕+学位论文 Abstract Yippee was firstidentified by yeast interaction trap screen as aprotein thatphysicaily interactswithhemolin ofHyalophora was found aconserved gene family ofproteins present indiverse anisms containing a putative zinc-finger-like metal binding may play anovel function involved inthecelldivision. A pupae eDNA library ofsilkworm hasbeenconstructed in identified agene with all open reading frame(ORE)of 366bp which encodes 121 amino acids containing a conservative domain nominated thisgene asBmYippee(Yippee gene from Bombyx mori),and ession number inGenbank ORF ofthegene Was cloned into theprokaryotie expression vector pET-28a and binantplasmid wastransformed into (DE3).The fusion protein Was essfully expressed,purified and thenimmuned New Zealand rabbit to get titerofthe polyclonal antibodies reaches 1:8000 measured by thequantity of BmYippee mRNA indifferent tissues ofthe fifthinstarlarvaand differentstages ofsilkworm usingRT- subcellular localization of BmYippee shows that Was localized inbothcytoplasm and nucleus during interphase and atsevemlregions ofcytoplasm during mitotic phase ofBm5 cells. Key words:Bombyx mori;、Flppee;subcellular localization;RT-PCR 浙江理T大学硕士学位论文 ACN AP aeetonitrile