Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (7): 1401-1411.doi: 10.3864/j.issn.0578-1752.2018.07.017

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Identification, Expression, Subcelluar Localization, and Function of glial cell missing (gcm) in Silkworm (Bombyx mori)

ZHANG Kui, PAN GuangZhao, SU JingJing, TAN Juan, XU Man, LI YuTian, CUI HongJuan   

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716
  • Received:2017-08-13 Online:2018-04-01 Published:2018-04-01

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Abstract: 【Objective】The objective of this study is to identify and clone Bombyx mori glial cell missing (Bmgcm), analyze its expression and subcelluar localization, prepare polyclonal antibody and overexpress Bmgcm at the cellular level to explore its function in proliferation and cell cycle, and to provide a theoretical basis for further studying the function of Bmgcm in B. mori.【Method】The full-length of Bmgcm was acquired by RACE technology. Basic sequence and structural information of Bmgcm were analyzed by using several online softwares, including ORF Finder and SMART. Multiple sequence alignment and evolutionary analysis were acquired by Clustalx and MEGA 6.0, respectively. The expression profile was investigated by RT-PCR and real-time PCR. The recombinant protein of Bmgcm was obtained using prokaryotic expression system, and purified with nickel affinity chromatography, then the antibody was prepared. Bmgcm over-expression vector was transfected into B. mori cell line to analyze the subcellular location of Bmgcm, and EDU and flow cytometry were used to study its function. 【Result】 The Bmgcm (BGIBMGA006182) was clustered on nscaf2847 which was located on chromosome 4. The genomic DNA total length of Bmgcm is 4 046 bp, which contains 4 exons and 3 introns. The full-length of cDNA is 1 734 bp, with a 166 bp 5′ UTR, a 227 bp 3′ UTR and a 1 341 bp open reading frame (ORF). This gene encodes 446 amino acid residues, the predicted molecular mass is 50.61 kD and the pI is 5.557, which contains a typical GCM-motif. Multiple sequence alignment demonstrated that GCM motif was highly conserved. Phylogenetic analysis revealed that gcm of insect was clustered alone, and Bmgcm had the highest relationship with gcm from Danaus plexippus. The expression profile revealed that Bmgcm reached peak at the 4th day of embryonic period, and then gradually decreased during embryonic stage. Bmgcm mainly expressed in larval midgut, testis and ovary. The complete ORF sequence of Bmgcm was inserted into prokaryotic expression system, and the recombinant protein of Bmgcm was induced by IPTG and purified using affinity chromatography. Finally, mice were immunized with purified proteins to produce polyclonal anti-gcm antibody. Western blot results indicated that the antibody could specifically recognize the target protein. Moreover, Bmgcm was overexpressed in B. mori cell line, and the result suggested that Bmgcm was located in nucleus. Furthermore, Bmgcm could inhibit cell proliferation and arrested cell cycle in G1/S period. 【Conclusion】 The Bmgcm was identified and cloned, its expression profile and subcelluar localization were analyzed. The available polyclonal antibody was generated by prokaryotic expression, affinity chromatography, and animal immunization. Overexpressed Bmgcm could inhibit cell proliferation and affect cell cycle progression.

Key words: Bombyx mori glial cell missing (Bmgcm), cloning, expression analysis, antibody preparation, overexpression

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