orf60基因对家蚕杆状病毒复制和转录的调控作用

发布时间：2018-07-07 21:29

  本文选题：BmNPV + 复制　； 参考：《浙江理工大学》2017年硕士论文

【摘要】：众所周知,家蚕杆状病毒(BmNPV)多角体启动子具有超高效的启动功能,在昆虫杆状病毒表达系统中常被用来启动外源基因的表达。通过酵母单杂交技术筛选出多种与多角体启动子存在相互作用的蛋白因子,其中ORF60蛋白被认为是Bm NPV的DNA结合蛋白,且可能具有调控病毒基因转录的功能,但其调控机制还不是很清楚。Red同源重组技术是研究杆状病毒基因功能常用的基因敲除的方法,运用此技术我们成功的构建了orf60缺失型病毒orf60-ko-Bacmid。Bac-to-Bac技术将敲除的片段再次补回到家蚕杆状病毒(BmNPV)基因组中,通过此技术,我们又成功的构建了orf60补回型病毒orf60-re-Bacmid。然后将三种病毒(wtBacmid、orf60-ko-Bacmid和orf60-re-Bacmid)分别转染家蚕Bm N细胞,病毒滴度和荧光定量PCR(qPCR)实验结果表明,orf60是BmNPV基因组复制的必需基因,也是包装成成熟病毒的必需基因。反转录实时定量PCR(RT-qPCR)分析orf60基因的转录时相,结果表明orf60基因是BmNPV的一个晚期基因。进一步通过qPCR技术分析orf60基因缺失对野生型病毒(wtBm NPV)3个不同时期(早期、晚期和极晚期)基因转录的调控作用,结果表明orf60基因的缺失导致病毒各个时期基因的转录水平均显著降低。最后,构建了一个由Bm NPV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启动子驱动的海肾萤光素酶构成的双萤光素酶检测系统,实验结果表明orf60基因在BmNPV多角体启动子启动转录的过程中起正调控作用。
[Abstract]:It is well known that the polyhedrosis promoter of Bombyx mori baculovirus (BmNPV) has super efficient function and is often used to activate the expression of foreign genes in insect baculovirus expression system. A variety of protein factors interacting with polyhedrosis promoter were screened by yeast single hybrid technique. The ORF60 protein is considered to be the DNA-binding protein of BmNPV, and it may have the function of regulating the transcription of virus gene. However, its regulatory mechanism is not very clear. Red homologous recombination technology is commonly used to study the gene function of baculovirus gene knockout method, Using this technique, we successfully constructed the orf60 deletion virus orf60-ko-Bacmid.Bac-to-Bac technology to repair the knockout fragment back into the genome of Bombyx mori baculovirus (BmNPV). By using this technique, we successfully constructed the orf60 complementary virus orf60-re-Bacmid.Using this technique, we successfully constructed orf60-re-Bacmidvirus. Then three kinds of viruses (wtBacmidorf60-ko-Bacmid and orf60-re-Bacmid) were transfected into Bombyx mori BmN cells, respectively. The results of virus titer and fluorescence quantitative PCR (qPCR) showed that the virus titer and the fluorescence quantitative PCR (qPCR) showed that the three viruses (wtBacmida orf60-ko-Bacmid and orf60-re-Bacmid) were essential genes for the replication of BmNPV genome and for packaging them into mature viruses. The transcriptional phase of orf60 gene was analyzed by reverse transcription real-time quantitative PCR (RT-qPCR). The results showed that orf60 gene was a late gene of BmNPV. Furthermore, the effects of orf60 gene deletion on the transcription of wild type virus (wtBm NPV) at three different stages (early, late and very late) were analyzed by qPCR. The results showed that the deletion of orf60 gene resulted in a significant decrease in transcription level at all stages of the virus. Finally, a double luciferase detection system was constructed, which was driven by Bm NPV polyhedrosis promoter and silkworm cytoplasmic actin A3 promoter. The results show that orf60 gene plays a positive role in the initiation of transcription of BmNPV polyhedrosis promoter.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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