Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance
Abstract
:1. Introduction
2. Results
2.1. β-Cypermethrin Intake Slows Larval Development
2.2. Effect of β-Cypermethrin on Protease Activities
2.3. Amino Acid Similarities
2.4. Phylogenetic Analyses
2.5. Developmental Expression Profiles of BdSPs
2.6. Temporal Expression of BdSPs in Response to β-Cypermethrin
3. Discussion
4. Experimental Section
4.1. Insects
4.2. Exposure to β-Cypermethrin
4.3. Preparation of Enzyme Extracts
4.4. Protein Concentration and Protease Activity
4.5. RNA Isolation and First-Strand cDNA Synthesis
4.6. Sequence Analysis and Phylogenetic Tree Construction
4.7. Analysis of SP Gene Expression
4.8. Statistical Analysis
5. Conclusions
Acknowledgments
Conflicts of Interest
References
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Groups | Specific activity (nmol/mL/min/mg) | |
---|---|---|
BApNA | L-TAME | |
Control | 302.67 ± 79.93 | 201.33 ± 16.92 |
Treatment | 450.00 ± 32.97 * | 266.00 ± 13.89 * |
Species | Serine protease | GenBank Accession number | Conserved regions a | Length | Enzyme specificity b | ||
---|---|---|---|---|---|---|---|
TAAHC | DIAL | GDSGGP | |||||
Bactrocera. dorsalis | BdSP1 | GAAP01000017 | SATHC | DIGL | 265 | T(DGG) | |
Bactrocera. dorsalis | BdSP2 | GAAP01000019 | DIAI | 259 | T(DGG) | ||
Bactrocera. dorsalis | BdSP3 | GAAP01000020 | DLAL | 247 | E(GVD) | ||
Bactrocera. dorsalis | BdSP4 | GAAP01000021 | 258 | T(DGG) | |||
Bactrocera. dorsalis | BdSP5 | GAAP01000022 | 257 | E(GVD) | |||
Glossina morsitans morsitans | GmmTR1 | ACB98719.1 | DVAV | 256 | T(DGG) | ||
Glossina morsitans morsitans | GmmTR2 | ADD19605.1 | DYSL | 281 | T(DGG) | ||
Aedes aegypti | AaSP1 | XP_004520098.1 | DLAL | 248 | E(GVD) | ||
Aedes aegypti | AaSP2 | XP_001659492.1 | 247 | E(GVD) | |||
Aedes aegypti | AaSP3 | XP_001659851.1 | DIAV | 274 | E(GVD) | ||
Ceratitis capitata | CcSP | XP_004520096.1 | DVAL | 254 | E(GVD) | ||
Ceratitis capitata | CcTR | XP_004517776.1 | DIAV | 257 | T(DGG) | ||
Drosophila melanogaster | DmTR1 | AAA17449.1 | DIVI | 253 | T(DGG) | ||
Drosophila melanogaster | DmTR2 | NP_525112.1 | DIAI | 256 | T(DGG) | ||
Drosophila erecta | DeTR1 | XP_001976081.1 | DIAV | 256 | T(DGG) | ||
Drosophila erecta | DeTR2 | XP_001976082.1 | DVGI | 262 | T(DGG) | ||
Drosophila yakuba | DyTR | XP_002091228.1 | DIAI | 256 | T(DGG) | ||
Phlebotomus papatasi | PpTR | AAM96943.1 | DFSL | 268 | T(DGW) | ||
Manduca sexta | MsTR | P35046.1 | DIAI | 256 | T(DGG) | ||
Hypoderma diana | HdSP | ACF98290.1 | DVAI | 256 | T(DGG) | ||
Stomoxys calcitrans | SsSP | AAC39131.1 | DVAV | 254 | T(DGG) |
Genes | Relative expression | |||
---|---|---|---|---|
Egg | Larva | Pupa | Adult | |
BdSP1 | 1.0 | 7.48 ± 0.93 | 0.05 ± 0.02 | 1.61 ± 0.51 |
BdSP2 | 1.0 | 189.62 ± 23.11 | 0.47 ± 0.16 | 118 ± 28.69 |
BdSP3 | 1.0 | 4.04 ± 0.86 | 0.45 ± 0.22 | 2.21 ± 0.03 |
BdSP4 | 1.0 | 6.71 ± 1.72 | 0.67 ± 0.25 | 1.10 ± 0.19 |
BdSP5 | 1.0 | 7.86 ± 1.48 | 0.04 ± 0.02 | 3.56 ± 0.10 |
Genes | GenBank accession number | Primer name and sequence (5′-3′) | PCR efficiency (%) | R2 |
---|---|---|---|---|
BdSP1 | GAAP01000017 | S: ACACACTCGGGTTTTAGCGT A: GAGGCGCAATCTTCACGTTG | 106.25 | 0.981 |
BdSP2 | GAAP01000019 | S: AGAGGACGTATTGTTGGCGG A: CGGCACGGATTCTCAAAACC | 96.17 | 0.993 |
BdSP3 | GAAP01000020 | S: ATCCCACGGGTCGTGTAGTA A: GTTGTGCCTGTTTCGACCAC | 108.43 | 0.986 |
BdSP4 | GAAP01000021 | S: CAACGTGAAGATTGCGCCTC A: CCTTTTTGGCACAGCCTTCC | 91.95 | 0.989 |
BdSP5 | GAAP01000022 | S: TTCCCCCATCAGGTCTCAC A: CCGAGTGTGCGTTGGATAC | 94.61 | 0.988 |
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Hou, M.-Z.; Shen, G.-M.; Wei, D.; Li, Y.-L.; Dou, W.; Wang, J.-J. Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance. Int. J. Mol. Sci. 2014, 15, 3272-3286. https://doi.org/10.3390/ijms15023272
Hou M-Z, Shen G-M, Wei D, Li Y-L, Dou W, Wang J-J. Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance. International Journal of Molecular Sciences. 2014; 15(2):3272-3286. https://doi.org/10.3390/ijms15023272
Chicago/Turabian StyleHou, Ming-Zhe, Guang-Mao Shen, Dong Wei, Ya-Li Li, Wei Dou, and Jin-Jun Wang. 2014. "Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance" International Journal of Molecular Sciences 15, no. 2: 3272-3286. https://doi.org/10.3390/ijms15023272