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

Liang Jiang,Yang Xiao,Tingcai Cheng,Shengkai Jin,Ping Zhao,Qiong Yang,Genhong Wang,Qingyou Xia,Ping Lin,Y Adam Yuan

doi:10.1371/journal.pone.0041838

Liang Jiang, Yang Xiao + Show 8 more

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https://doi.org/10.1371/journal.pone.0041838

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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.

Highlights

Bombyx mori nucleopolyhedrovirus (BmNPV) is a member of the Baculoviridae family
The nucleotide sequences of hycu-ep32 and 39 K promoter of BmNPV (39 KP) were cloned from the genomic DNA of Hyphantria cunea NPV (HycuNPV) and BmNPV, respectively
The overexpression vectors pb-EKG, pb-HEKG, pb-EAG, and pb-HEAG (Fig. 1) were constructed using the transgenic plasmid piggyBac [36p3 EGFP afm], where hycu-ep32 was driven by 39 KP, 39 KP combined with hr3, A4 promoter (A4P), and A4P combined with hr3, respectively

Summary

Introduction

Two virion phenotypes occur during the NPV infection cycle, i.e., occlusion-derived virus (ODV) and budded virus (BV). ODV causes contagion among individual silkworms (Bombyx mori), whereas BV induces systemic infection throughout the host body [1,2]. NPV infects host larvae mainly via the oral route by ODV. After the ODV nucleocapsids have invaded the host cells, the viral genes begin transcription in a temporal manner to initiate a primary infection [3]. The expression of the immediate early genes of Autographa californica multiplenucleopolyhedrovirus (AcMNPV), such as ie-1 and ie-2, begins within the first 6 hours post-infection (hpi) [4]. The BV virions are produced which cause secondary infection at approximately 20 hpi [4].

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