摘要
对杆状病毒ie0/ie1启动子区域甲基化测序和甲基化特异性PCR,发现ie0区域甲基化程度较高,且随着感染过程发生变化,在感染后8h有明显下降.为了研究启动子区域内甲基化对其活性及表达调控的影响,构建了表达AcMNPV极早期蛋白IE0和IE1的质粒p402-ie1、p402-ie0M-A及报告质粒pie0p-luc、pie1p-luc,采用体外甲基化的方式,研究DNA甲基化对IE0、IE1调控自身启动子作用的影响.结果证实ie0p受IE0蛋白激活,而受到IE1蛋白抑制,发现DNA甲基化极大降低ie0p、ie1p活性.验证了IE0、IE1蛋白对于ie1p的激活作用,而甲基化后IE1/IE0对其激活作用依然保持.不同比例的p402-ie1/p402-ie0M-A转染实验,发现在IE0>IE1时,甲基化能够逆转IE0/IE1对于ie0p的调控作用.这些结果表明,DNA甲基化状态可以影响杆状病毒IE0和IE1蛋白对ie0启动子对基因表达的调控.
The results of bisulfite sequencing and methylatiowspecific PCR showed that the promoter of AcMNPV immediate-early gene （COp） is CpG methylated at a relatively high level, and its methylation level changed during infection reaching the lowest around 8 h post infection. To study the effect of DNA methylation on the regulation of ie0 and id promoters, plasmids p402 iel and p402 ieOM-A, which can express IE0 and IE1 protein, respectively, and reporter plasmids pie0p-luc and piell〉lue were reconstructed. Methylated reporter plasmid DNA was obtained by treating the plasmid DNA with bacterial methyltransferase M. Sss I in vitro. The results verified that IE0 activated/cOp, whereas IE1 inhibited it. DNA methylation was found to reduce the activity of icO and id promoter greatly. By contrast, both IE0 and IE1 increased id p activity, and maintained such activation after methylation. By cotransfecting different ratio of p402-iel and p402 ie0M A with reporter plasmid into Sf9 cells, we demonstrated that DNA methylation could reverse the regulation by IE0 and IE1 on icOp when IE0〉IE1. These results suggest that DNA methylation can affect baculovirus gene expression and regulation.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
2013年第6期717-723,730,共8页
Journal of Fudan University：Natural Science
基金
国家自然科学基金(31170143)资助项目
