Amino acid sequence analysis of DAP3.

<p>(<b>a</b>) The conserved SOD domain of DAP3 revealed by BLASTp analysis online (<a href="http://www.ncbi.nlm.nih.gov/BLASTp" target="_blank">http://www.ncbi.nlm.nih.gov/BLASTp</a>). (<b>b</b>) Specific motifs of DAP3 protein. NIT in the box is the glycosylation site, the shading characters are SOD domain and the asterisks indicate amino acids of oxidative modification sites. Cu = copper binding, Zn = zinc binding, Cu, Zn = copper and zinc binding, S-S = disulfide bond.</p

Alignment of amino acid sequences of DAP3 and Cu/Zn-SODs.

<p>The amino acid sequence alignment of <i>A.pernyi</i> DAP3 with <i>Papilio polytes</i>, <i>Aedesa egpyti</i>, <i>Bombyx mori</i>, <i>Bombyx mandarina</i>, <i>Hyphantria cunea</i>, <i>Homo sapiens</i>, <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i> Cu/Zn-SOD.</p

SOD activity detection of purified protein HIS-DAP3.

<p>(<b>a</b>) Change in absorbance at 450 nm with time for a DAP3 dilution series. A linear regression analysis of the reaction rates during the first 6 minutes of incubation is shown. (<b>b</b>) Inhibition curve for the DAP3 dilution series shown in figure 7-a. Results shown are the averages from three separate experiments. (<b>c</b>) Calculation of the SOD activity.</p

Transcription spectrum and Expression profile analysis of <i>A.pernyi DAP3</i> gene.

<p>(<b>a</b>) qRT-PCR of transcription level of <i>DAP3</i> in different developmental stages. 1: diapause pupae; 2: non-diapause pupae; 3–6: pupae in the period of diapause development for 4days, 8days, 11days and 14days; 7: moth; 8: egg; 9: developed egg; 10–14: 1st to 5th instar larvae <i>of A.pernyi</i>. Date represent mean ± SEM from three independent experiments. *p<0.01 vs diapause pupaereference, **p<0.01 vs diapause pupae reference. (<b>b</b>) Western blot of expression level of DAP3 in different developmental stages. Lane 1: diapause pupae; lane 2: non-diapause pupae; lane 3–6: pupae in the period of diapause development for 4days, 8days, 11days and 14days; lane 7: moth; lane 8: egg; lane 9: developed egg; lane 10–14: 1st to 5th instar larvae of <i>A.pernyi</i>. **p<0.01 vs diapause pupae.</p

Effects of UV irradiation on the transcription and expression level and the SOD activity of DAP3 in <i>A.pernyi</i> pupae.

<p>(<b>a</b>) Relative mRNA levels of DAP3 were detected by qRT-PCR with β-actin as an internal control. Results are expressed as mean ± SEM of three independent experiments. **p<0.01 vs control. (<b>b</b>) Protein levels of DAP3 revealed by Western blot. *p<0.05 vs control, **p<0.01 vs control. (<b>c</b>) Effects of UV irradiation on SOD activity of <i>A.pernyi</i>pupae. * p<0.05 vs control.</p

Transcription spectrum and Expression profile analysis of <i>DAP3</i> gene of <i>A.pernyi</i> 5th instar larvae.

<p>(<b>a</b>) qRT-PCR of transcription level of <i>DAP</i>3 in different tissues of 5th instar larvae <i>of A.pernyi</i> with β-actin as an internal control. Results are represented as mean ± SEM of three independent experiments. *p<0.05 vs midgut value, **p<0.01 vs midgut value. (<b>b</b>) Western blot of expression level of DAP3 in different tissues of 5th instar larvae <i>of A.pernyi</i>. **p<0.01 vs midgut value.</p

Construction of recombinant plasmid and production of DAP3 recombinant protein.

<p>(<b>a</b>) TAE agarose gel electrophoresis of PCR and double digestion analysis. Lane M:Gene Ruler 100 bp plus DNA Ladder; lane 1: PCR with the recombinant plasmid as template; lane 2: Digestion with <i>Bam</i>H I and <i>Hind</i> III. (<b>b</b>) SDS-PAGE analysis of DAP3 recombinant protein in <i>E.coli</i> BL21 cells. Lane M: protein moleculai mass marker; lane 1: <i>E.coli</i> BL21 containing expression vector pET28a(+) induced by IPTG; lane 2: <i>E.coli</i> BL21 cells containing expression vector pET28a(+)-<i>DAP</i>3 without IPTG; lane 3: <i>E.coli</i> BL21 cells containing expression vector pET28a(+)-<i>DAP</i>3 induced by IPTG; lane 4: Supernatant solution after ultrasonic; lane 5: Precipitation after ultrasonic; lane 6: purified protein by Ni-NTA affinity column.</p

The putative 3D structure of <i>A.pernyi</i> DAP3 and alignment with <i>B.mori</i> TIME-EA4.

<p>(<b>a</b>) The putative tertiary structure of <i>A.pernyi</i> DAP3. (<b>b</b>) Tertiary structure of <i>B.mori</i> TIME-EA4. (<b>c</b>) The alignment of <i>A.pernyi</i> DAP3 with <i>B.mori</i> TIME-EA4. (<b>d</b>) The 3D structure with metal ions and glycosylation modification.</p