Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will benefit studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.源自草地贪夜蛾（fall armyworm, FAW，*Spodoptera frugiperda*）的细胞系，已被广泛用于生产重组蛋白，服务于基础研究、医药与农业领域。核型多角体病毒（nucleopolyhedrovirus, NPV）的启动子是此类表达系统中的常用元件，但此类启动子存在一定局限，利用草地贪夜蛾内源基因的启动子或可克服上述缺陷。然而，目前关于此类内源启动子的公开信息仍较为有限。本研究从草地贪夜蛾的中肠、脂肪体、表皮、卵巢细胞系Sf9以及中肠细胞系Sf17的转录组中，筛选出多个高表达基因。随后在Sf9与Sf17细胞中，对21个高表达基因的潜在启动子活性展开了检测。结果显示，SfHSC70-P1780与SfPub-P2009这两个启动子的活性，高于常规使用的hr5/ie1启动子（即杆状病毒增强子元件hr5与即刻早期基因ie1的组合）。值得注意的是，添加hr5增强子元件后，这两个启动子的活性均有所提升。研究人员借助质粒表达系统在Sf9细胞中表达外源性细胞色素P450（P450）蛋白，对hr5/SfPub-P2009启动子的性能进行了验证；同时利用杆状病毒表达系统在草地贪夜蛾体内表达绿色荧光蛋白（green fluorescence protein, GFP），以检测该启动子的活性。两项实验均证实，hr5/SfPub-P2009启动子的表现优于常规使用的hr5/ie1启动子。上述高效内源启动子，将为草地贪夜蛾及其他鳞翅目昆虫的相关研究提供有力支撑，可应用于蛋白表达、基因组编辑以及转基因昆虫构建等多个领域。