家蚕核型多角体病毒Bm11基因对病毒基因转录及ODV病毒粒子包埋的影响

Effect of Bombyx mori Nucleopolyhedrovirus Bm11 Gene on Transcription of Viral Genes and the Embedding of Occulusion-Derived Virus

家蚕核型多角体病毒(BmNPV)Bm11是一个高度保守的基因,但其在病毒感染过程的作用尚不清楚。利用λ-Red同源重组技术构建Bm11敲除型病毒,并利用Bac-to-Bac系统重新构建恢复型及野生型重组病毒。转染试验及荧光定量PCR(qPCR)结果表明,Bm11基因缺失并不影响出芽型病毒粒子(BV)的产生及病毒基因组DNA复制,但对晚期和极晚期基因的转录水平影响显著(P<0.01)。经皮注射虫体实验结果显示,敲除型病毒的致死中时(LT50)明显长于恢复型病毒和野生型重组病毒。经口感染实验表明,添食106个野生型重组病毒或恢复型病毒多角体(OB)的家蚕死亡率均达到100%,而添食敲除型病毒多角体的家蚕死亡率只有64.89%,且死亡时间明显延长。透射电子显微镜观察发现,与恢复型病毒和野生型重组病毒相比,Bm11敲除型病毒感染细胞内多角体包埋的包涵体衍生型病毒粒子(ODV)数量显著减少(P<0.01)。综上所述,Bm11虽不是BmNPV复制必需基因,但该基因的缺失对病毒晚期和极晚期基因转录以及ODV粒子包埋效率均有重要影响。

Bombyx mori nucleopolyhedrovirus(BmNPV)Bm11 gene is a highly conserved gene,whose role in viral infection process is still unknown.In this experiment,Bm11 knockout Bacmid was constructed throughλ-Red recombination system,and repair Bacmid and wild type recombinant Bacmid were constructed using the Bac-to-Bac system.Transfection experiment and qPCR revealed that deletion of Bm11 did not affect the production of budded virus(BV)or DNA replication of viral genome,but significantly affected the transcription level of late and very late genes(P<0.01).Moreover,median lethal time(LT50)of Bm11 knockout virus was significantly longer than those of repair and wild type recombinant virus according to the experimental results of direct injection.Results of per os infection showed that the silkworm fed with 106 occlusion bodies(OBs)of repair or wild type recombinant virus had a mortality of 100%,while that fed with Bm11 knockout virus had a mortality of only 64.89%with significantly prolonged death time.It was suggested that the number of occlusion-derived virus(ODV)embedded in OB was significantly less(P<0.01)than those in repair and wild type recombinant virus under electron microscopy observation.In summary,though Bm11 is not an essential gene for viral replication,its deletion has great influence on the transcription of late and very late genes and the embedding of ODVs.