杆状病毒RNA聚合酶（RNAP）由病毒自身编码，由4个亚基组成，是已知结构组成最简单的RNAP，负责转录病毒晚期和极晚期基因。杆状病毒RNAP如何参与启动子的特异性识别及转录起始是一个重要科学问题。 LEF-9是杆状病毒RNAP第二大亚基，包含一个所有生物的RNA聚合酶活性中心的保守motif。LEF-9被认为与RNA聚合酶II β’亚基同功，推测其参与催化作用以及转录起始位点的选择，但其实际功能未知。本项目已完成lef-9基本特性研究，已构建lef-9缺失和回复重组bacmid并制备了LEF-9抗体，计划从以下四个方面进行研究：对LEF-9保守区进行缺失或点突变，并与18个其他LEF进行体外共转染实验，分析晚期基因的转录起始和转录水平；将突变引入重组病毒进行体内实验；研究LEF-9与10个晚期转录直接相关因子的相互作用；以NMR方法分析研究LEF-9的结构。本研究将解析LEF-9的结构和功能及分子机制, 将帮助阐明杆状病毒RNA聚合酶特异性识别启动子和转录起始的机制，加深对其他RNAP的转录机制及病毒基因表达调控机制的认识，为杆状病毒表达系统的改造利用和构建高效体外转录系统奠定基础。

Baculovirus RNA polymerase (RNAP) is encoded by the virus itself, is composed of only 4 subunits and known as the structure of the simplest RNAP. It is responsible for the transcription of viral late and very late genes. How does the baculovirus RNAP function in the specific recognition and transcription initiation of the viral late promoter is an important scientific question. LEF-9 is the second largest subunit of the virus’ RNAP, containing a motif conserved in all RNA polymerase activity centers of all organisms. Lef-9 is considered to play the same role as the β’ subunit of RNAP II, suggesting its involvement in catalysis and transcription start site selection, but its actual function remains unknown. Previous work studied the basic characteristics of lef-9 and constructed lef-9 Del and Rec recombinant bacmids and prepared anti-LEF-9 antibody. To elucidate the function of LEF-9 involved in the transcriptional start site selection and catalysis of RNA synthesis, the study will be carried out in the following four aspects: to mutate LEF-9 by deletion or point mutation and to analyze the transcription initiation and transcription level of the viral late genes by co-transfection of wt LEF-9 or LEF-9 mutants with 18 other LEFs; to introduce mutation of lef-9 in the context of the viral genome and to analyze in vivo the transcription initiation and transcription level of the viral late genes of recombinant virus; to study the interaction between LEF-9 and 10 other LEFs directly involved in late gene transcription; to analyze the structure of LEF-9 by NMR method. This project will elucidate the structure and function of LEF-9 and its molecular mechanism, will help to clarify the mechanism of RNA polymerase functioning in specific recognition of the viral late promoter and transcription initiation, deepen the understanding of the transcription mechanism of other RNAPs and the mechanism of viral gene expression regulation. This study will also provide a theoretical basis for further modifying the baculovirus expression system and building in vitro transcription system of baculovirus RNAP.