家蚕微孢子虫半胱氨酸脱硫酶基因的克隆及表达和亚细胞定位  

Cloning,Expression and Subcellular Localization of Cysteine Desulfurase of Nosema bombycis

作　　者：林立鹏[1] 潘国庆[1] 李田[1] 马成[1] 边茂飞[1] 党晓群[1] 罗洁[1] 邓远洪[1] 周泽扬[1,2] 

机构地区：[1]西南大学蚕学与系统生物学研究所,重庆400716 [2]重庆师范大学动物生物学重点实验室,重庆400047

出　　处：《蚕业科学》2012年第1期82-91,共10页ACTA SERICOLOGICA SINICA

基　　金：教育部高校基本业务费重大孵育项目(No.XDJK2009-A005);国家自然科学基金重点项目(No.30930067);重庆市科技攻关项目(No.CSTC2010AA1003);西南大学博士基金项目(No.SWU109039)

摘　　要：半胱氨酸脱硫酶是一类活性依赖于磷酸吡哆醛的酶类,可催化L-半胱氨酸转化为L-丙氨酸和硫烷硫原子,为铁硫簇组装提供硫原子。基于家蚕微孢子虫基因组数据,克隆了家蚕微孢子虫半胱氨酸脱硫酶(NbNfs)基因。序列结构分析显示NbNfs含有其活性所需的保守氨基酸位点,但不具有线粒体型N端导肽;系统发育分析表明微孢子虫半胱氨酸脱硫酶与真菌线粒体型半胱氨酸脱硫酶聚为一类,属于类群Ⅰ,起源于α-变形细菌;共线性分析表明半胱氨酸脱硫酶基因在不同微孢子虫之间其基因座位保守,具有共线性。构建重组质粒pET30a(+)-NbNfs,并转化到E.coli Rosetta(DE3),经IPTG诱导表达NbNfs融合蛋白,用Ni-NTA亲和层析柱进行纯化后,将融合蛋白免疫昆明小鼠制备多克隆抗体,Western blotting检测NbNfs在成熟家蚕微孢子虫具有表达。胶体金免疫定位显示NbNfs主要定位于成熟家蚕微孢子虫的细胞质中。研究结果为家蚕微孢子虫铁硫簇组装途径的研究奠定了基础。Cysteine desulfurases are pyridoxal-5’-phosphate（PLP）-dependent enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur,and serve as a sulfur donor in Fe-S cluster biogenesis.Based on the genomic data of Nosema bombycis,the gene of Nosema bombycis cysteine desulfurase（NbNfs） was cloned.Sequence structure analysis revealed that NbNfs possesses the typical conserved regions implicated in cysteine desulfurase activity but does not contain a recognizable transit peptide targeting to mitochondrion.Phylogenetic analysis showed that microsporidian cysteine desulfurase clusters with fungal mitochondrial cysteine desulfurases,which belong to groupⅠ and are derived from α-proteobacteria.Syntenic analysis indicated that gene loci of Nfs are conserved and syntenic among different microsporidian species.We constructed the prokaryotic expression vector pET30a（＋）-NbNfs.The recombinant plasmid was transformed into competent E.coli Rosetta（DE3） cells and expression of NbNfs fusion protein was induced with IPTG.After purification with Ni-NTA affinity chromatography,the fusion protein was used to immunize Kunming mice for preparing polyclonal antibody.Western blotting analysis showed that NbNfs was expressed in mature spores of Nosema bombycis.Colloidal gold immunolocalization displayed that NbNfs was mainly distributed in cytoplasm of the spore.These results provide experimental data for elucidating the assembly of Fe-S cluster in Nosema bombycis.

关 键 词：家蚕微孢子虫 半胱氨酸脱硫酶 基因克隆 表达 亚细胞定位 

分 类 号：S884.21[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]