海带3个着丝粒蛋白编码序列的克隆及特征分析  

Molecular cloning and characterization of 3 centromere specific proteins(CENPs) genes from Saccharina japonica

作　　者：徐梅[1] 周志刚[1] 

机构地区：[1]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2016年第7期1028-1038,共11页Journal of Fisheries of China

基　　金：国家高技术研究发展计划(2012AA10A406);国家自然科学基金(41376136)~~

摘　　要：本研究根据海带基因组及糖海带转录组数据库中获得的编码着丝粒相关蛋白CENH3、Nuf2和Ndc80的contig序列设计特异性引物,利用PCR技术自海带中克隆得到它们的开放阅读框(ORF)序列,分别命名为Sj CENH3、Sj Nuf2和Sj Ndc80,大小分别为432、1365和2172 bp,相应地编码由143、454和723个氨基酸组成的蛋白。同源序列比对表明,Sj CENH3由氨基端尾巴和羧基端的组蛋白折叠域组成,后者包括4个α螺旋(αN、α1、α2和α3)和2个环(Loop1和Loop2);但与组蛋白H3相比,Sj CENH3的氨基端略长且序列不相似,同时组蛋白折叠域的α1、α2螺旋及Loop1序列更多变;Sj Nuf2主要的结构域有Nuf2 domain、Spc7和SPT2;Sj Ndc80主要的结构域除了具有与Sj Nuf2相似的SPT2和Spc7结构域以外,还有Ndc80_Hec、Fo P_duplication和Kin17_mid。基于海带和其他物种的CENH3、H3、Nuf2和NCD80蛋白序列所构建的Neighbor-Joining系统进化树显示,Sj CENH3和Sj H3分别被显著地聚类在CENH3和H3两支中,H3在物种间是高度保守的,而CENH3在物种间的差异相对较大;来自不同生物的Nuf2和NCD80均可显著地分为被子植物和藻类2支,它们可能存在着共同演化的关系。本研究首次报道了海带着丝粒蛋白Sj CENH3、Sj Nuf2和Sj NCD80编码序列的特征,为制备特异抗体通过免疫荧光技术来定位海带染色体着丝粒的具体位置,通过染色质免疫共沉淀获得海带染色体着丝粒DNA序列及进行染色体核型分析打下了良好的基础。Based upon contigs coding for centromere specific proteins（CENPs） from a pyrosequencing transcriptome of Saccharina japonica, gene-specific primers were designed for the three centromere specific protein gene cloning from S. japonica. Three CENPs genes, named as Sj CENH3, Sj Nuf2 and Sj NCD80 were cloned by using the technique of Polymerase Chain Reaction. The open reading frame of the cloned full-length c DNAs were 432, 1365 and 2172 bp in length, encoding putative proteins composed of 143, 454 and 723 amino acids, respectively. Homologous sequence alignments show： Sj CENH3 include the diversity of the N-terminal region and C-terminal histone fold domain（HFD）, and HFD including four αhelixes（αN helix, α1 helix, α2 helix and α3 helix） and two loops（Loop1 and Loop2）. But compared to H3, Sj CENH3 amino terminus is slightly longer and there is no similarity between them, while the sequence of histone fold domain α1-helix, α2-helix and Loop1 are more variable. The main domains of Sj Nuf2 are Nuf2 domain, Spc7 and SPT2. The main domains of Sj Ndc80 are SPT2, Spc7, Ndc80_Hec, Fo P_duplication and Kin17_mid. Neighbor-Joining（NJ） phylogenetic tree inferred from the putative proteins of CENH3, Nuf2 and NCD80 genes of S. japonica and other species indicated that Sj CENH3 and Sj H3 genes were divided two clades： CENH3 and H3 clade. H3 is highly conserved across species,while CENH3 differences among species are relatively large. Nuf2 and Ndc80 from different organisms could be significantly divided into two clades, angiosperms and algae. They both may have relations of co-evolution. This study first reported the characteristics of three centromere protein-coding genes Sj CENH3, Sj Nuf2 and Sj Ndc80,providing a good basis to locate the centromere position by immunofluorescence technique and to get the centromeric DNA sequence by chromatin immunoprecipitation and to analyze chromosome karyotype using preparation of antibodies specific for the corresponding proteins of S. japonica.

关 键 词：海带 CENH3 动粒蛋白 基因克隆 生物信息学 

分 类 号：Q785[生物学—分子生物学]