Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang, 212013, China.
Curr Microbiol. 2011 Jan;62(1):191-7. doi: 10.1007/s00284-010-9691-5. Epub 2010 Jun 22.
Orf68 (ac68) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is identified to be an early gene, but its transcription start site remains unknown. The coding sequence of ac68 overlaps 280-bp leading sequence and 159-bp coding sequence of lef3 (ac67). In this study, the transcription start site of ac68 was determined by 5' RACE analysis to be 18 nucleotides upstream from the start codon. In order to investigate the effect of ac68 deletion on virus propagation, we generated a bacmid with an ac68 knockout by deleting 360-bp inside the ac68 gene, which also deleted 220-bp leading sequence of lef3. Production of infectious budded virus and formation of nucleocapsids and occlusion bodies exhibited wild-type patterns of virus propagation in Sf-9 cells infected with the mutant bacmid. The result demonstrated that ac68 was not an essential gene for viral propagation which was confirmed by further deletion of ac68, and disruption of the lef3 leading sequence did not affect viral propagation. Ac68 was the second auxiliary gene discovered besides Ac133 (alk-exo) among the 30 core genes of AcMNPV.
美洲棉铃虫多核多角体病毒（AcMNPV）的 Orf68（ac68）被鉴定为早期基因，但它的转录起始位点仍不清楚。ac68 的编码序列与 lef3（ac67）的 280bp 前导序列和 159bp 编码序列重叠。在本研究中，通过 5' RACE 分析确定了 ac68 的转录起始位点，位于起始密码子上游 18 个核苷酸处。为了研究 ac68 缺失对病毒繁殖的影响，我们通过在 ac68 基因内缺失 360bp 来生成带有 ac68 缺失的 bacmid，这也删除了 lef3 的 220bp 前导序列。突变 bacmid 感染 Sf-9 细胞后，产生了有感染力的芽生病毒，形成了核衣壳和包含体，显示出病毒繁殖的野生型模式。结果表明，ac68 不是病毒繁殖所必需的基因，这一结果通过进一步缺失 ac68 得到了证实，并且 lef3 前导序列的破坏并不影响病毒繁殖。ac68 是除 Ac133（alk-exo）之外在 AcMNPV 的 30 个核心基因中发现的第二个辅助基因。
