Caspases,which play key roles in cell apoptosis,undergo alternative splicing to form different splicing variants that can regulate the apoptotic process.Lepidopteran insect caspases undergo alternative splicing,althou...Caspases,which play key roles in cell apoptosis,undergo alternative splicing to form different splicing variants that can regulate the apoptotic process.Lepidopteran insect caspases undergo alternative splicing,although the functions of their splicing variants are still unclear.The Spodoptera exigua caspase-5(SeCaspase-5)gene was cloned and found to produce four different splicing variants with different gene sequences and protein functional domains,which were named SeCaspase-5a,SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Overexpression of these variants in S.exigua cells(Se-3)showed that SeCaspase-5a had a proapoptotic function,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not.Semi-qPCR analysis revealed that the expression of the SeCaspase-5 variants significantly differed during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.Furthermore,the SeCaspase-5 variants were constructed into the AcMNPV bacmid and transfected into Se-3 cells,which revealed that SeCaspase-5a promoted cell apoptosis and reduced virus production,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not promote cell apoptosis but instead increased virus production.Moreover,an analysis of the interactions between the SeCaspase-5 variants revealed that SeCaspase-5a directly interacted with SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Coexpression of these variants in Se-3 cells also revealed that SeCaspase-5b,SeCaspase-5c and SeCaspase-5d inhibited the proapoptotic function of SeCaspase-5a,resulting in a reduction in the percentage of apoptotic cells by about 20%.These results indicate that SeCaspase-5 undergoes alternative splicing and is involved in regulating the apoptosis induced by baculovirus infection.These findings increase our understanding of the functions of lepidopteran insect caspases and provide new insights into the mechanism of host-cell apoptosis induced by baculoviruses.展开更多
为了研究Bm K IT提高Ac MNPV抗虫活性的作用机制,将Bm K IT基因插入到Ac MNPV中形成重组病毒。采用重组单价病毒Ac MNPV-Bm K IT(IE1)、Ac MNPV-Bm K IT(P10)、Ac MNPV-Bm K IT(PH)和一种重组双价病毒Ac MNPV-Bm K IT(P10)-vcath(PH),...为了研究Bm K IT提高Ac MNPV抗虫活性的作用机制,将Bm K IT基因插入到Ac MNPV中形成重组病毒。采用重组单价病毒Ac MNPV-Bm K IT(IE1)、Ac MNPV-Bm K IT(P10)、Ac MNPV-Bm K IT(PH)和一种重组双价病毒Ac MNPV-Bm K IT(P10)-vcath(PH),通过四唑盐比色法(MTT)和蛋白质印迹法(Western Blot)分析了Bm K IT在Ac MNPV的3个启动子调控下对Sf9细胞增殖和细胞凋亡的影响,结果显示,感染病毒36,48 h Bm K IT在不同启动子调控下表达量从高到低依次为PH、P10、IE1。同时分析了Ac MNPV介导的Bm K IT与组织蛋白酶的协同表达对昆虫Sf9细胞增殖和调亡的机制,结果表明,Ac MNPV-Bm K IT(P10)-vcath(PH)处理组对Sf9细胞抑制率比Ac MNPV-Bm K IT(P10)处理组平均提高了14.5%,凋亡相关蛋白c-Myc、cleaved-Caspase3、Bax表达量增加,Bcl-2表达量减少。展开更多
基金funded by the National Natural Science Foundation of China(32202393)the Natural Science Foundation of Shandong Province,China(ZR2021QC190)+1 种基金the Science and Technology Benefiting the People Demonstration Project of Qingdao,China(24-1-8-xdny-10-nsh)the Qingdao Agricultural University High-level Talent Fund,China(663/1120101)。
文摘Caspases,which play key roles in cell apoptosis,undergo alternative splicing to form different splicing variants that can regulate the apoptotic process.Lepidopteran insect caspases undergo alternative splicing,although the functions of their splicing variants are still unclear.The Spodoptera exigua caspase-5(SeCaspase-5)gene was cloned and found to produce four different splicing variants with different gene sequences and protein functional domains,which were named SeCaspase-5a,SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Overexpression of these variants in S.exigua cells(Se-3)showed that SeCaspase-5a had a proapoptotic function,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not.Semi-qPCR analysis revealed that the expression of the SeCaspase-5 variants significantly differed during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.Furthermore,the SeCaspase-5 variants were constructed into the AcMNPV bacmid and transfected into Se-3 cells,which revealed that SeCaspase-5a promoted cell apoptosis and reduced virus production,whereas SeCaspase-5b,SeCaspase-5c and SeCaspase-5d did not promote cell apoptosis but instead increased virus production.Moreover,an analysis of the interactions between the SeCaspase-5 variants revealed that SeCaspase-5a directly interacted with SeCaspase-5b,SeCaspase-5c and SeCaspase-5d.Coexpression of these variants in Se-3 cells also revealed that SeCaspase-5b,SeCaspase-5c and SeCaspase-5d inhibited the proapoptotic function of SeCaspase-5a,resulting in a reduction in the percentage of apoptotic cells by about 20%.These results indicate that SeCaspase-5 undergoes alternative splicing and is involved in regulating the apoptosis induced by baculovirus infection.These findings increase our understanding of the functions of lepidopteran insect caspases and provide new insights into the mechanism of host-cell apoptosis induced by baculoviruses.
基金supported by grants from the National Natural Science Foundation of China(No.31272100,31372199)the Natural Science Foundation of Shanxi Province,China(No.2014011038-1)+1 种基金the National High Technology Research and Development Program of China(863 Program,No.2012AA020809)the Program for the Top Young Academic Leaders of Higher Learning Institutions of Shanxi Province,China
文摘为了研究Bm K IT提高Ac MNPV抗虫活性的作用机制,将Bm K IT基因插入到Ac MNPV中形成重组病毒。采用重组单价病毒Ac MNPV-Bm K IT(IE1)、Ac MNPV-Bm K IT(P10)、Ac MNPV-Bm K IT(PH)和一种重组双价病毒Ac MNPV-Bm K IT(P10)-vcath(PH),通过四唑盐比色法(MTT)和蛋白质印迹法(Western Blot)分析了Bm K IT在Ac MNPV的3个启动子调控下对Sf9细胞增殖和细胞凋亡的影响,结果显示,感染病毒36,48 h Bm K IT在不同启动子调控下表达量从高到低依次为PH、P10、IE1。同时分析了Ac MNPV介导的Bm K IT与组织蛋白酶的协同表达对昆虫Sf9细胞增殖和调亡的机制,结果表明,Ac MNPV-Bm K IT(P10)-vcath(PH)处理组对Sf9细胞抑制率比Ac MNPV-Bm K IT(P10)处理组平均提高了14.5%,凋亡相关蛋白c-Myc、cleaved-Caspase3、Bax表达量增加,Bcl-2表达量减少。
