家蚕含RRM保守结构域蛋白基因(Bmrrm)的克隆及序列分析与表达研究  

Cloning,Sequence Analysis and Expression Profiles of Bmrrm Gene That Contains RRM Conserved Domain From Silkworm,Bombyx mori

作　　者：涂雯婷[1] 郑青亮[1] 聂作明[1] 陈健[1] 于威[1] 张耀洲[1] 

机构地区：[1]浙江理工大学生物化学研究所,杭州310018

出　　处：《蚕业科学》2009年第3期499-507,共9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;2007AA100504)

摘　　要：RRM(RNA recognition motif)是RNA结合蛋白中广泛存在的一种保守结构域。从构建的家蚕蛹cDNA文库中筛选得到一条含有RRM保守结构域的cDNA,命名为Bmrrm(GenBank登录号:DQ534196)。Bmrrm基因全长1086 bp,开放阅读框大小为894 bp,编码297个氨基酸残基,蛋白理论分子质量33.2 kD,等电点9.69,预测的三级结构由2个α螺旋和4个β折叠组成。将该基因的PCR扩增产物于原核表达载体pET-28a(+)中表达后经SDS-PAGE检测,在36 kD处的特异性蛋白条带与预期值相符,重组蛋白以可溶的形式表达。将纯化的重组蛋白免疫新西兰大白兔制备多克隆抗体,ELISA检测效价大于1∶6400。采用荧光定量PCR方法对家蚕卵、5龄幼虫、蛹、蛾4个发育时期以及5龄幼虫各组织中Bmrrm的mRNA转录水平进行检测比较,发现在蛹期和5龄幼虫生殖腺、表皮中的转录水平较高。采用免疫印迹方法在家蚕的卵期、5龄幼虫、蛹期,以及5龄幼虫的生殖腺、表皮、脂肪体中检测到BmRRM蛋白有明显表达,但在蛾期及5龄幼虫的其他组织中未检测到该蛋白。以家蚕Bm5细胞进行免疫细胞化学实验,BmRRM蛋白在整个细胞周期主要分布于细胞核,仅少量分布于细胞质。初步推测BmRRM蛋白参与蚕体内RNA前体的剪接、细胞定位及维持稳定等转录后调控过程。RRM（RNA recognition motif） is a conserved domain which exists widely in RNA binding protein.The cDNA,which encodes the protein containing RRM conserved domain,was identified from the cDNA library constructed from Bombyx mori pupae.We nominated it as Bmrrm（Bombyx mori rrm） and the GenBank accession number is DQ534196.Full length of this gene is 1 086 bp.It contains an ORF of 894 bp and encodes 297 amino acids.According to the ORF of Bmrrm,two primers were designed to obtain the coding region of Bmrrm gene.The ORF of the gene was cloned into pET-28a（＋） vector and expressed.SDS-PAGE analysis showed that the fusion protein His-BmRRM was expressed highly in BL21 cells with a molecular weight of 36 kD which is consistent with the predicted value.Most of the recombinant protein was soluble and was purified with metal-chelating affinity chromatography.The purified protein was used to generate polyclonal antibody by immuning New Zealand rabbit and the titer of the obtained antibody was higher than 1∶6 400 measured by ELISA.We analyzed the transcription level of Bmrrm mRNA in different developmental stages of silkworm and different tissues of the fifth instar larva.The results suggested that BmRRM was highly expressed in silkworm′s pupae and seminal glands,epidermis respectively.The total proteins were extracted from brain,epidermis,silk gland,midgut,Malpighian tubule,fat body,and stigma of the fifth instar larvae and were analyzed by Western blotting.The results showed that BmRRM only existed in seminal gland,epidermis and fat body.Immunocytochemistry in Bm5 cells showed that the protein mainly existed in the nucleus.These results laid a good foundation for further studies on BmRRM.

关 键 词：家蚕 Bmrrm基因 荧光定量PCR 组织表达 亚细胞定位 

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]