Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States.
Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States.
Insect Biochem Mol Biol. 2020 Nov;126:103455. doi: 10.1016/j.ibmb.2020.103455. Epub 2020 Aug 19.
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 be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.
从秋粘虫（FAW），Spodoptera frugiperda 中衍生出的细胞系已被广泛用于生产重组蛋白，用于基础研究和医学及农业应用。核多角体病毒（NPV）的启动子通常用于这些表达系统。这些启动子有一些局限性，可以通过使用来自 S. frugiperda 的基因的启动子来克服。然而，关于这些启动子的信息尚不可用。我们从 S. frugiperda 中肠、脂肪体、表皮、卵巢细胞系（Sf9）和中肠细胞系（Sf17）的转录组中鉴定了几个高表达基因。评估了 21 个高表达基因的潜在启动子在 Sf9 和 Sf17 细胞中的活性。这两个启动子之一 SfHSC70-P1780 和 SfPub-P2009 的活性高于常用的 hr5/ie1（杆状病毒增强子元件，hr5 和早期基因 1，ie1）启动子。有趣的是，添加 hr5 增强子后，这两个启动子的活性增加。通过使用基于质粒的表达系统在 Sf9 细胞中表达外源 P450 蛋白，评估了 hr5/SfPub-P2009 启动子的性能。还通过使用杆状病毒表达系统在 FAW 中表达绿色荧光蛋白来评估该启动子的性能。在这两种情况下，hr5/SfPub-P2009 启动子的性能均优于常用的 hr5/ie1 启动子。这些强内源性启动子将有助于 S. frugiperda 和其他鳞翅目昆虫的多项研究，包括蛋白质表达、基因组编辑和转基因昆虫。
