Resistance to BmNPV via Overexpression of an Exogenous Gene Controlled by an Inducible Promoter and Enhancer in Transgenic Silkworm, Bombyx mori

Abstract

The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 105 occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.

Screening and optimization of an efficient Bombyx mori nucleopolyhedrovirus inducible promoter

“…The 39K promoter within this region had been previously used to express recombinant protein in baculovirus-insect expression system, and was capable of augmenting the level of foreign gene expression (Lin and Jarvis, 2013;Toth et al, 2014). However, this region of 39K was missing when the promoter was used for pathogen induction (Jiang et al, 2012a;Zhang et al, 2014b).…”

“…In our previous study, controlled by p39K (p-151) promoter, the RNAi expression system was activated by BmNPV infection, which in turn blocked virus proliferation. Recently, studies on BmNPV resistance in silkworm demonstrated that expression of an anti-pathogen factor controlled by hr3-39k can be activated upon infection (Jiang et al, 2012a;Zhang et al, 2014b), while the hr3-39k promoter shows weaker activity that causes lower expression level of foreign genes following infection in transgenic pears. In this study, the RNAi system was regulated by an efficient BmNPV-inducible HP39K promoter.…”

“…Moreover, Lin and Jarvis found that the 39K promoter has the higher inducible activity by compared with the four AcMNPV promoters (ie1, 39k, p6.9 and polh) in insect cells (Lin and Jarvis, 2013). Furthermore, the p39K promoter has been applied to drive the expression of exogenous hycu-ep32 gene and regulate artificial microRNA precursors to inhibit the proliferation of the BmNPV in the silkworm (Jiang et al, 2012a;Zhang et al, 2014b). However, the low promoter activity of p39K remains a hindrance in the application of pathogen-inducible promoters.…”

Selection of reference genes for analysis of stress-responsive genes after challenge with viruses and temperature changes in the silkworm Bombyx mori

“…Previous studies have used actin-1 (A1) (Li et al 2014), actin-3 (A3) (Gao et al 2014b;Wang et al 2014Wang et al , 2015Kolliopoulou et al 2015), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bao et al 2009), translation initiation factor 3 (TIF-3) (Zhou et al 2013), TIF-4A (also known as sw22934) (Jiang et al 2012a(Jiang et al , b, 2013b, and 18S rRNA (Xue et al 2012) as housekeeping genes in silkworms. Importantly, because use of unsuitable RGs may lead to incorrect interpretation of gene expression patterns and functions (Velada et al 2014), the correct selection of RGs is required.…”

Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis

“…BmNPV is a serious viral pathogen in sericulture industry, and no effective drug or specific prevention is available to date. Researchers gave major attention to inhibition of the virus replication by over-expression of antiviral exogenous gene [2] or RNA interference [3,4]. However, mechanisms of BmNPV BV entry into the host cell have not been addressed.…”