摘要
家蚕质型多角体病毒(Bombyx mori cypovirus,BmCPV)是家蚕的重要病原之一,也是典型的RNA病毒,为探索其在侵染过程中与家蚕免疫防御系统的互作机制,选择由BmCPV基因组编码的miRNA BmCPV-miR-10进行研究。通过stem-loop qRT-PCR在感染BmCPV的家蚕幼虫中肠中检测到BmCPV-miR-10的表达;靶基因预测发现,家蚕R2D2蛋白基因(BmR2D2)为BmCPV-miR-10的候选靶基因,靶位点位于BmR2D2 mRNA序列的CDS区域。进一步验证miRNA对靶基因的调控作用,分别构建BmCPV-miR-10及其靶基因BmR2D2的慢病毒表达载体,借助包装质粒共转染HEK293T细胞,同时合成了BmCPVmiR-10 mimics转染BmN细胞,qRT-PCR检测结果均证明BmCPV-miR-10能下调靶基因BmR2D2的表达。对感染BmCPV的家蚕幼虫体腔注射BmCPV-miR-10 mimics,分别检测靶基因和BmCPV基因组RNA片段的表达变化,亦证明了BmCPV-miR-10对靶基因BmR2D2的调控作用,并且促进BmCPV基因组的复制。上述结果表明,BmCPV在侵染家蚕的过程中,可能通过编码BmCPV-miR-10下调靶基因BmR2D2的表达,影响宿主RNAi途径在抵抗病毒过程中的作用,从而促进病毒自身的复制。
Bombyx mori cypovirus(BmCPV)is one of the major pathogens of silkworm,and also a typical RNA virus.In order to explore its interaction mechanism with silkworm immune defense system in the process of infection,a BmCPVencoded miRNA,BmCPV-miR-10,was selected for study.Expression of BmCPV-miR-10 was detectable in virus-infected silkworm midgut by stem-loop qRT-PCR.It was found by target gene prediction that Bombyx mori R2D2 protein gene(BmR2D2)was a candidate target gene for BmCPV-miR-10.Its target site was located in CDS region of BmR2D2 mRNA.In order to further verify the regulation effect of BmCPV-miR-10 on BmR2D2,lentiviral expression vectors of BmCPV-miR-10 and its target gene BmR2D2 were constructed respectively,and transfected into HEK293 T cell with package plasmids.Meanwhile,synthesized BmCPV-miR-10 mimic was transfected into BmN cell.qRT-PCR results showed that BmCPV-miR-10 could down-regulate BmR2D2 expression.According to detection results of expression level of target gene and BmCPV genome after injection of BmCPV-miR-10 mimic into the silkworm larvae infected with BmCPV,it was further proved that BmCPV-miR-10 could regulate BmR2D2 expression and promote BmCPV genome replication.These results demonstrated that BmCPVmiR-10 facilitate virus replication by down-regulating BmR2D2 expression and affecting effectiveness of host RNAi pathway.
作者
王永升
林苏
赵泽
徐平
侯成香
高坤
钱荷英
郭锡杰
Wang Yongsheng;Lin Su;Zhao Ze;Xu Ping;Hou Chengxiang;Gao Kun;Qian Heying;Guo Xijie(School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212018,China;Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212018,China)
出处
《蚕业科学》
CAS
CSCD
北大核心
2020年第5期594-604,共11页
ACTA SERICOLOGICA SINICA
基金
国家自然科学基金项目(31572463)
