Table 1.
List of primer sequences used in this study.
Fig 1.
The structure of pBHSA and pESNT-PBase plasmids.
(A). Schematic representation of the pBHSA plasmid used in our transgenic experiment. pBL and pBR: the sequence of the left and right arms of the piggyBac transposon plasmid; FL promoter, the promoter sequence of the fibroin light chain gene; FLSP, the signal peptide sequence of the fibroin light chain gene; His6 tag, the sequential 6×His-tag; DDDDK, enterokinase recognition site; HSA CDS, the HSA coding sequence; FL polyA, the polyA signal sequence of the fibroin light chain gene; 3×P3 promoter, the artificial promoter specifically driving the marker gene expression in the eyes and nervous system; DsRed CDS, the coding sequence of the red fluorescent protein gene; SV40 polyA, the SV40 polyA signal sequence. (B). Schematic representation of the pESNT-PBase plasmid used in this transgenic experiment, which was constructed in our previous study [26].
Fig 2.
The fluorescence phenotypes of the DsRed-specific expressed in the eyes of the transgene-positive silkworm.
A and C are the wild-type silkworm larvae and moths viewed under the normal light; B and D are transgene-positive silkworm larvae and moths viewed under the normal light. A' and C' are wild-type silkworm larvae and moths viewed under the red fluorescence; B' and D' are transgene-positive silkworm larvae and moths viewed under the red fluorescence. White arrows indicate the position of the eye.
Fig 3.
Locations of the insertion site of the transgenic silkworm pedigrees on the chromosome.
Fig 4.
Identification of rHSA gene expression in transgenic silkworm pedigrees using qRT-PCR, SDS-PAGE and Western blot, and purification of rHSA protein from the cocoon shells.
(A) The relative expression levels of the rHSA gene in the PSGs of the transgenic silkworm pedigrees on the 3rd day of the 5th instar were measured by qRT-PCR. WT: the wild-type silkworm Lan 10. HSA-1, HSA-2, HSA-3, HSA-4 and HSA-5: the transgene-positive silkworm pedigrees. (B) SDS-PAGE analysis of rHSA derived from the soluble protein of cocoon shells and (C) Western blot analysis of the cocoon layer. WT: the protein samples from the wild-type silkworm Lan 10. HSA-1, HSA-2, HSA-3, HSA-4 and HSA-5: the protein samples from the transgene-positive silkworm pedigrees. (D) The results of purification of the rHSA protein. S: the protein sample. W1, W2: recovered solutions after adding binding buffer. E1-E4: recovered solutions after adding elution buffer.