Figure 4
Expression and immunohistochemical analysis in transgenic silkworms. (A) RT-PCR of BmDV-derived transcripts and the + nsd-1 transgene in the midgut of transgenic silkworms. Transgenic silkworms carrying the nonspliced form of the susceptibility gene + nsd-1 [glutamic acid (E) at 110 and arginine (R) at 118], the spliced form of + nsd-1 (E at 85 and R at 93), and the point mutated spliced form of + nsd-1 [lysine (L) at 85 and R at 93] were generated. K (blue) and R (red) indicate the amino acid residue conserved in resistant and susceptible strains, respectively. −/−, −/G, U/−, and U/G indicate the wild-type, GAL4, UAS, and GAL4/UAS lines, respectively. The midgut was dissected from day 3 fifth instar following exposure to BmDV on day 0 of the fourth instar stage. RT-PCR was performed for 30 cycles with primer sets for + nsd-1, the NS1 gene of BmDV20, and ribosomal 18S as a positive control. (B) Immunohistochemical analysis of BmDV in the midgut of transgenic silkworms expressing the nonspliced form of the susceptibility gene + nsd-1. Sections were incubated with anti-BmDV capsid antibody (1:100) followed by a secondary antibody labeled with AlexaFluor488 (1:200) (green) and counterstained with DAPI (1:1000) (red). DAPI staining (left); BmDV signal (center); merge (right). Scale bar: 200 μm.
