家蚕富含半胱氨酸的酸性分泌蛋白基因BmSPARC的表达及亚细胞定位  

Expression and Subcellular Localization of Bombyx mori Secreted Protein Acidic and Rich in Cysteine Gene

作　　者：朱玉娟[1] 吕正兵[1] 陈琴[1] 聂作明[1] 王丹[1] 陈健[1] 刘立丽[1] 郑青亮[1] 陈剑清[1] 沈红丹[1] 盛清[1] 全滟平[1] 徐杰[1] 张文平[1] 吴祥甫[1] 张耀洲[1] 

机构地区：[1]浙江理工大学生物化学研究所,浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《蚕业科学》2009年第2期253-263,共11页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究发展计划“973”项目(编号2005CB12-1006);国家高技术研究发展计划“863”项目(编号2007-AA021703,2007AA120402)

摘　　要：富含半胱氨酸的酸性分泌蛋白(secreted protein acidic and rich in cysteine,SPARC)是一种小分子酸性糖蛋白,在有机体内广泛分布,通过与胞外基质蛋白相互作用而表现出多种生物活性。从家蚕蛹cDNA文库中获得SPARC基因(BmSPARC)的cDNA序列,GenBank登录号为FJ602783。利用生物信息学方法对此序列进行分析表明,BmSPARC的cDNA序列全长1 626 bp,含有1个954 bp的开放阅读框(ORF),编码317个氨基酸残基;同源性比较显示该基因编码蛋白的氨基酸序列在无脊椎动物中具有较高的保守性。将BmSPARC片段在大肠杆菌BL21中表达并纯化后,免疫新西兰兔制备多克隆抗体,通过亚细胞定位研究发现该基因表达蛋白主要存在于细胞质中。利用实时荧光定量PCR的方法,对BmSPARC在家蚕不同发育时期、不同组织中的mRNA转录水平进行比较,并采用Western blotting方法对BmSPARC蛋白的表达进行分析,结果表明:BmSPARC在家蚕卵期和5龄幼虫丝腺组织中无表达,而在5龄幼虫、蛹、蛾发育时期及5龄幼虫的头部、脂肪体、中肠、马氏管、卵巢、表皮和气门等7个组织中均有不同程度的表达,其中在蛾期和5龄幼虫卵巢中的表达量较高。研究结果为进一步探讨BmSPARC在家蚕生长发育过程中的生物学功能打下了基础。Secreted protein acidic and rich in cysteine（SPARC）, a small molecule acidic glycoprotein, exists widely in higher organisms. SPARC interacts with extracellular matrix proteins to show a variety of biological activities. According to the large-scale sequencing of cDNA library from silkworm pupa, a cDNA which encodes BmSPARC gene was identified （GenBank accession number FJ602783）. Sequence analysis revealed that the full length cDNA of BmSPARC was 1 626 bp, and the ORF was 954 bp long and encoded 317 amino acids. Homology analysis indicated that the amino acid sequence encoded by this gene had high conservatism in invertebrates. After BmSPARC gene was expressed in E. coil BL21, the expressed protein was purified and used to immune New Zealand rabbits to prepare high titer polyclonal antibodies, Subcellular localization with polyclonal antibody indicated that BmSPARC was mainly distributed in cytoplasm. We compared the quantity of BmSPARC mRNA in different stages and different tissues of silkworm by real-time RTPCR. BmSPARC expression was also analyzed by Western blotting, It was observed that different levels of expression of BmSPARC existed in all developmental stages and organs tested except for egg stage and silk gland. It was especially higher in moth stage and in ovary. The present study laid a good foundation for further studies on biological functions of BmSPARC in the growth and development of silkworm.

关 键 词：家蚕 富含半胱氨酸的酸性分泌蛋白基因 亚细胞定位 实时荧光定量PCR 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]