家蚕生存素基因在BmN4细胞有丝分裂中的功能分析

Functional analysis of survivin gene in the mitosis of BmN4 cells of Bombyx mori

【目的】阐明生存素基因在家蚕Bombyx mori BmN4细胞有丝分裂中的功能。【方法】qRT-PCR分析家蚕生存素基因BmSurvivin在家蚕5龄第3天幼虫不同组织(丝腺、中肠、马氏管、精巢、卵巢、脂肪体、皮细胞层和表皮)中的表达量;构建pIZT/V5-His-BmSurvivin-GFP(BG)融合载体并转染BmN4细胞,以免疫荧光标记检测BmSurvivin和第10位丝氨酸(Ser10)磷酸化的组蛋白H3(H3Ser10ph)在BmN4细胞有丝分裂分裂不同时期的定位。【结果】BmSurvivin在家蚕5龄第3天幼虫马氏管中表达量最高,其次是在丝腺和中肠中。成功构建pIZT/V5-His-BmSurvivin-GFP载体。免疫荧光结果显示,在BmN4细胞间期核中可以看到明显的GFP信号,涵盖了细胞核和细胞质区域,指示BmSurvivin的定位;随着细胞进入分裂期,GFP信号指示BmSurvivin与染色质共定位,待细胞形成明显的双取向时在纺锤体区域也可以看到GFP信号;后期随着姐妹染色单体分开,GFP信号指示BmSurvivin定位在染色质及胞质分裂区;末期随着两个新的子细胞形成GFP信号指示BmSurvivin仅定位在胞质分裂区。H3Ser10ph定位在BmN4细胞前中期染色质凝缩处,至中期信号最强,与整条染色体重合,后期信号消失。【结论】BmSurvivin与有丝分裂周期中染色质和纺锤体的动态变化相关。

【Aim】To clarify the function of survivin gene in the mitosis of BmN4 cells of Bombyx mori.【Methods】The expression levels of BmSurvivin in different tissues(silk gland,midgut,Malpighian tubules,testis,ovary,fat body,epidermis and cuticle)of the day-35th instar larvae of B.mori were detected by qRT-PCR.The fusion vector pIZT/V5-His-BmSurvivin-GFP(BG)was constructed and transfected into BmN4 cells.The localization of BmSurvivin and Ser10 phosphorylated histone H3(H3Ser10ph)in different mitotic stages of BmN4 cells was detected by immunofluorescence.【Results】The expression level of BmSurvivin was the highest in the Malpighian tubules of the day-35th instar larvae of B.mori,followed by that in the silk gland and midgut.pIZT/V5-His-BmSurvivin-GFP vector was successfully constructed.The immunofluorescence results showed that obvious GFP signal could be seen in the interphase nucleus of BmN4 cells,covering the nuclear and cytoplasmic regions indicating the location of BmSurvivin.At the mitotic phase,BmSurvivin was co-localized with chromatin indicated by GFP signal.When the BmN4 cells formed obvious double orientation,GFP signal was also visible in the spindle region.At the anaphase stage,with the separation of sister chromatids,BmSurvivin was located in the chromatin and cytokinesis region indicated by GFP signal.At the telophase stage,with the formation of two new daughter cells,BmSurvivin was located only in the cytokinesis region indicated by GFP signal.H3Ser10ph was located at the site of chromatin condensation at the prometaphase stage of BmN4 cells,and the signal intensity was the strongest at the metaphase,coinciding with the whole chromosome.The signal disappeared at the anaphase stage.【Conclusion】BmSurvivin is associated with the dynamic changes of chromatin and spindle during mitotic cycle.