家蚕CDK11与RNPS1和9G8相互作用的鉴定

doi:10.3864/j.issn.0578-1752.2017.22.016

摘要/Abstract

摘要： 【目的】鉴定家蚕（Bombyx mori）CDK11与RNPS1和9G8的相互作用，为解析其是否参与前体RNA的剪接打下基础。【方法】利用家蚕基因组数据库SilkDB找到本研究克隆的家蚕基因序列，采用Primer 5.0进行引物设计，通过PCR技术克隆获得家蚕的两个重要剪接因子RNPS1和9G8，并构建具有不同抗原标签的过表达载体，采用NCBI检索并获得其他物种的相关序列，利用在线预测软件SMART进行结构域预测，采用ClustalX 1.83和GENEDOC 3.2预测同源序列，利用软件MEGA 6.0构建系统进化树，通过免疫荧光验证家蚕CDK11两种剪切体CDK11A和CDK11B分别与RNPS1和9G8的共定位情况，进一步通过免疫共沉淀验证CDK11A、CDK11B分别与RNPS1和9G8的相互作用。【结果】RNPS1的开放阅读框长为882 bp，编码293个氨基酸；9G8的开放阅读框长为531 bp，编码176个氨基酸；RNPS1和9G8都属于SR蛋白家族，具有该蛋白家族成员的一个富含丝氨酸/精氨酸（S/R）重复序列的RS结构域。同源序列比对表明，RNPS1和9G8都具有典型的RRM结构域，此外9G8还含有一个锌指结构域。系统进化分析显示，RNPS1聚类于无脊椎动物一支，与同为鳞翅目昆虫的斑点木蝶、黑脉金斑蝶等亲缘关系较近；9G8也聚类于无脊椎动物一支，与同为鳞翅目昆虫的黑脉金斑蝶、金凤蝶等关系较近。荧光共定位证明，RNPS1分别与CDK11A和CDK11B共定位于细胞核中，且具有点状共聚集现象；同时发现，RNPS1还具有胞质定位，点状聚集于核外周。而9G8分别与CDK11A、CDK11B在细胞核中也具有共定位现象。进一步通过免疫共沉淀发现，在所有的总蛋白和细胞裂解离心后的上清液样品中，均能检测到对应目的条带的表达，表明共转染的细胞均能正常表达目的蛋白，并且蛋白溶于上清。同时，在所有的共沉淀条带中，均能检测到对应目的条带，以上结果表明CDK11A、CDK11B均与RNPS1和9G8具有相互作用。【结论】家蚕RNPS1和9G8具有典型的RRM结构域，属于SR家族。CDK11A和CDK11B能够与RNPS1和9G8相互作用。

关键词: 家蚕, CDK11, RNPS1, 9G8, pre-RNA剪接

Abstract: 【Objective】 The objective of this study is to identify the interactions of silkworm (Bombyx mori) CDK11 with RNPS1 and 9G8, and to lay a foundation for the analysis of whether it is involved in the splicing of precursor RNA. 【Method】 The B. mori gene sequences in this study were found in silkworm genome database SilkDB, and Primer 5.0 was used for primer design. Firstly, two important splicing factors RNPS1 and 9G8 were cloned by PCR and the overexpression vector with different epitope tags was constructed. Then, NCBI was used to retrieve and obtain the relevant sequence of other species. The online prediction software SMART was used for domain prediction. ClustalX 1.83 and GENEDOC 3.2 were used to predict the homologous sequence, and the phylogenetic tree was constructed by software MEGA 6.0. Next, immunofluorescence was used to verify the co-localization of CDK11A and CDK11B with RNPS1 and 9G8, respectively. Finally, the interaction of CDK11A and CDK11B with RNPS1 and 9G8 was further confirmed by immunoprecipitation. 【Result】 The open reading frame of RNPS1 is 882 bp, encoding 293 amino acids. And the open reading frame of 9G8 is 531 bp, encoding 176 amino acids. Both RNPS1 and 9G8 belong to the SR protein family, which has RS domain that contains rich of serine/arginine (S/R) repeats. Homozygous sequence alignment showed that both RNPS1 and 9G8 had a typical RRM domain, and 9G8 also contained a zinc finger domain. Phylogenetic analysis showed that RNPS1 was clustered in an invertebrate, which possessed high homology with other Lepidoptera insects like Pararge aegeria and Danaus plexippus. 9G8 also clustered in an invertebrate, and had close relationship with other Lepidoptera insects like D. plexippus and Papilio machaon. Fluorescence co-localization showed that RNPS1 was co-located in the nucleus with CDK11A and CDK11B, respectively, and had dot-like co-aggregation. While, 9G8 also had a co-localization with CDK11A and CDK11B in the nucleus, respectively. Furthermore, by using immunoprecipitation, it was found that the expression of the corresponding band was detected in all total protein and supernatant samples after cell lysis, indicating that the co-transfected cells were able to express the protein of interest and the protein was dissolved in the supernatant. At the same time, in all the coprecipitation bands, the corresponding target bands could be detected. The above results indicated that both CDK11A and CDK11B interacted with RNPS1 and 9G8.【Conclusion】Both RNPS1 and 9G8 have a typical RRM domain which belong to the SR family. CDK11A and CDK11B have interaction with RNPS1 and 9G8.

Key words: Bombyx mori, CDK11, RNPS1, 9G8, pre-RNA splicing

张倩，刘太行，董小龙，吴云飞，杨基贵，周亮，潘彩霞，潘敏慧. 家蚕CDK11与RNPS1和9G8相互作用的鉴定[J]. 中国农业科学, 2017, 50(22): 4398-4407.

ZHANG Qian, LIU TaiHang, DONG XiaoLong, WU YunFei, YANG JiGui, ZHOU Liang, PAN CaiXia, PAN MinHui. Identification of the Interactions of CDK11 with RNPS1 and 9G8 in the Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2017, 50(22): 4398-4407.

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