摘要39K同源基因存在于所有鳞翅目昆虫杆状病毒基因组中，其编码产物为一种磷蛋白。迄今的研究表明，39K基因与病毒基因表达调控有关，但家蚕核型多角体病毒(Bombyx mori Nucleopolyhedrovirus, BmNPV) 39K基因对病毒复制和转录的具体调控作用尚不清楚。本研究利用Red重组技术和Bac-to-Bac系统分别敲除和回补39K基因，构建了39K缺失型病毒39K-ko-Bacmid和修复型病毒39K-re-Bacmid，并分别转染家蚕(Bombyx mori)细胞系BmN细胞，发现二者均能产生具有感染活力的病毒粒子，表明39K基因不是BmNPV复制的必需基因，但是该基因的缺失可显著降低病毒滴度。进一步利用qPCR发现，39K基因缺失对病毒基因组早期基因组的复制没有显著影响，但在后期会降低病毒基因组的复制水平，并导致病毒各时期基因转录水平的极显著下降(P＜0.01)。为了进一步了解39K基因对BmNPV极晚期基因多角体启动子的表达调控作用，本研究构建了由BmNPV多角体启动子驱动的萤火虫萤光素酶和家蚕胞质肌动蛋白A3启动子驱动海肾萤光素酶的双萤光素酶报告系统，通过定量检测荧光素酶活性的变化情况，证明了39K基因对多角体启动子具有显著的负调控作用。综上所述，可知39K基因不是BmNPV复制的必需基因，但该基因的缺失可显著降低病毒各时期基因的表达水平，并导致多角体基因启动子的启动活性增强。本研究结果将为深入了解BmNPV 39K基因对病毒基因组复制、转录的影响奠定基础，同时也将为家蚕杆状病毒(Bombyx mori baculovirus)表达系统高效转录机制的研究提供资料。
Abstract：Bombyx mori Nucleopolyhedrovirus (BmNPV) 39K gene which encodes a phosphoprotein associated with viral gene expression, exists in genomes of all Lepidopteran Baculoviruses. The results of existing studies show that the 39K gene is related to the regulation of viral gene expression, but the specific regulation effects of BmNPV 39K gene on viral replication and transcription is still unknown. Red recombination technique and Bac-to-Bac system were respectively utilized to delete and recover 39K gene to construct a knockout bacmid (39K-ko-Bacmid) and 39K- rescued bacmid (39K-re-Bacmid). Then the constructed bacmids DNA were transfected into BmN cells. The results showed that both types of viruses could yield infectious virion in cells, indicating that 39K gene was not essential for the replication of BmNPV, however, the deletion of 39K gene could reduce virus titer significantly. In late transfection, the replication level of the viral genome was related to infectious budded virus (BV) which was generated by virus. Possibly, the reduced BV led to decreased replication capacity of deficient virus in late transfection. Furthermore, the resultes of qPCR showed that the deletion of 39K gene had no effect on virus early replication but would decrease the transcription level of viral late genes and caused extremely significant reduction of viral gene transcription level (P＜0.01) in different phases. In order to explore the role of 39K gene in the expression regulation of polyhedral promoter, we constructed a recombinant bacmid with BmNPV polyhedral promoter drived-firefly luciferase and Bombyx mori cytoplasm action A3 promoter drived-renilla luciferase. Quantitative detection of luciferase activity proved that 39K gene had significant regulation effect on polyhedral gene promoter, the deletion could enhance the activity of polyhedral gene promoter. In conclusion, 39K gene was not essential for the replication of BmNPV, but the deletion could reduce virus gene transcription level in different phases and enhance the activity of polyhedral promoter. We speculated that the 39K protein which may act as a repressor protein bound to polyhedron gene promoter, so that affected the polyhedron gene promoter combining with other transcription factors, and then resulted in decreasing of polyhedron gene promoter activity. This study will lay the foundation for further investigating the effect of BmNPV 39K gene on viral genome replication and transcription, also provides information on the efficient transcription mechanism of Bombyx mori baculovirus expression system.
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