家蚕细胞周期蛋白E(Cyc E)基因的2种剪切体克隆及其蛋白质的亚细胞定位

细胞周期蛋白E(Cyc E)是细胞从G1期转换到S期的重要调节因子。为了解家蚕Cyc E的功能,从家蚕BmN细胞中克隆了2种不同剪切方式的cyc E片段,分别命名为cyc E1173和cyc E837(GenBank登录号:HQ619696.1,HQ619697.1),将2种剪切体分别融合eGFP后利用杆状病毒系统在BmN和Sf9细胞中进行表达产物的亚细胞定位实验。家蚕cyc E的2种剪切方式的变化主要集中在第5～6外显子处。Cyc E1173和Cyc E837在2种细胞系中都定位于细胞核,但在Sf9细胞中存在Cyc E1173的荧光逐渐聚集到核膜及核心荧光弱化的现象,而在BmN细胞中,Cyc E1173和Cyc E837的荧光分布均匀,这种差异的原因可能与Cyc E出核降解相关。Western blotting检测在Sf9细胞中表达的Cyc E1173出现2条降解条带,而Cyc E837的融合蛋白并无这种降解情况,其可能的原因是cyc E1173和cyc E837之间存在表达产物降解水平上的差异,Cyc E1173更容易降解,推测其与细胞周期调控的关系更为密切。

Cloning and Subcellular Localization of Two Splicing Variants of Bombyx mori Cell Cycle Protein E(Cyc E) Gene

Cell cycle protein E(Cyc E) is an important regulation factor of G1 to S phase transition in the cell cycle.To gain an insight into the function of Bombyx mori Cyc E,two splicing variants of cyc E,termed BmcycE1173 and BmcycE837(GenBank accession No.HQ619696.1 and HQ619697.1) were cloned from B.mori BmN cells.Using baculovirus expression system,subcellular localization of the expressed products of BmcycE1173 and BmcycE837 fused with eGFP were observed in BmN and Sf9 cells.The results revealed that these two kinds of Bmcyc E splicing variations were mainly from variation in the 5th to 6th exons.Both of the expressed products were shown to be localized in nucleus of Sf9 and BmN cells.Interestingly,in Sf9 cells,the fluorescence of BmCyc E1173 proteins was gradually accumulated on the nuclear membrane and weakened in the core of nucleus,while in BmN cells,the fluorescence of both BmCyc E1173 and BmCyc E837 was evenly distributed in the nucleus.The reason of this difference may be related to the degradation of BmCyc E outside nucleus.Western blotting showed that BmCyc E1173 expressed in Sf9 cells were degraded into two bands and the fusion protein BmCyc E837 did not degrade.It was suggested that degradation level of the expressed products was different between Bmcyc E1173 and Bmcyc E837.BmCyc E1173 was easier to degrade,suggesting that it is more closely related to the regulation of cell cycle.