家蚕CDK11与RNPS1和9G8相互作用的鉴定

doi:10.3864/j.issn.0578-1752.2017.22.016

Abstract

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

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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Identification of the Interactions of CDK11 with RNPS1 and 9G8 in the Silkworm (Bombyx mori)

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