Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori)
Abstract
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Silkworm, B. mori
2.2. Construction of the BmTrpA1−/− Mutant and Prediction of the Three-Dimensional Structure of the Protein
2.3. Mutant Screening
2.4. Investigation of Larval Growth and Development and Cocoon Economic Traits
2.5. Extraction of Total RNA from Silkworm Tissues and RT-qPCR Analysis
2.6. Embryonic RNA Interference
3. Results
3.1. Construction of a BmTrpA1−/− Mutant Strain
3.2. No Obvious Differences Were Observed in the Growth, Development, or Diapause Phenotype between BmTrpA1−/− and wt
3.3. BmPyrexia, BmPainless, and BmUpf2 Showed Upregulated Expression in BmTrpA1−/− Mutant
3.4. RNA Interference of the UPF2 Gene Alters the Mutant’s Diapause Phenotype
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Primer Name | Primer Sequence (5′–3′) | Primer Purpose |
---|---|---|
TrpA1-582-F | GATAACCGATCTGGACGCTGAATG | Mutation site sequencing detection |
TrpA1-880-R | CTTGAGCGCAAGCTAAGTGCACAG | |
gRNA-771-793 | TtctaatacgactcactatagCGTTCATGGAGGTGATA TCAgttttagagctaga | Synthesis of sgRNA in vitro |
Primer Name | Primer Sequence (5′–3′) | Gene Accession No. |
---|---|---|
BmPainless-F | CGGTCTTGCGGTTAGTGACA | NM_001309624.1 |
BmPainless-R | GCTACCGATAAGCACGCTCT | |
BmPyrexia-F | ATGATGGCCGCTTACGACAT | NM_001309607.1 |
BmPyrexia-R | TCCGAGTCCTGAGTAACCGT | |
BmUpf1-F | GCGAGAGGCAATGGAGTCTT | XM_004929604.4 |
BmUpf1-R | CACCGGCTTCTTCCTTGAGT | |
BmUpf2-F | CATTGCTGTCCCGATGACCT | XM_038010585.1 |
BmUpf2-R | AACGAATTCCACGCCCTCTT | |
BmUpf3a-F | GGATCGGAAGAGACAGACACA | NM_001046855.2 |
BmUpf3a-R | TTCTTTCGCGAGACGCTGTT | |
BmActin3-F | CGGCTACTCGTTCACTACC | NM_001126254.1 |
BmActin3-R | CCGTCGGGAAGTTCGTAAG | |
BmRp49-F | TCAATCGGATCGCTATGACA | NM_001098282.2 |
BmRp49-R | ATGACGGGTCTTCTTGTTGG |
Primer Name | Primer Sequence (5′–3′) |
---|---|
dsGFP-F | ACGTAAACGGCCACAAGTTC |
T7dsGFP-F | TAATACGACTCACTATAGGGACGTAAACGGCCACAAGTTC |
dsGFP-R | TGTTCTGCTGGTAGTGGTCG |
T7dsGFP-R | TAATACGACTCACTATAGGGTGTTCTGCTGGTAGTGGTCG |
dsUpf1-F | CTCGCAATCGCTCACGTTTC |
T7dsUpf1-F | TAATACGACTCACTATAGGGCTCGCAATCGCTCACGTTTC |
dsUpf1-R | ACATTCCGAGCACCACATGA |
T7dsUpf1-R | TAATACGACTCACTATAGGGACATTCCGAGCACCACATGA |
DsUpf2-F | TCATCAAAACTGCGGGTGGAT |
T7dsUpf2-F | TAATACGACTCACTATAGGGTCATCAAAACTGCGGGTGGAT |
DsUpf2-R | TGAGAGTTCTCCTCTGGTGTG |
T7dsUpf2-R | TAATACGACTCACTATAGGGTGAGAGTTCTCCTCTGGTGTG |
Group | Whole Cocoon Weight of Females (g) | Whole Cocoon Weight of Males (g) | Cocoon Shell Weight of Females (g) | Cocoon Shell Weight of Males (g) |
---|---|---|---|---|
WT (n = 30) | 1.673 ± 0.1385 | 1.300 ± 0.06543 | 0.3189 ± 0.03326 | 0.2973 ± 0.03197 |
TRPA1−/−(n = 30) | 1.744 ± 0.1559 | 1.479 ± 0.1320 | 0.3014 ± 0.02375 | 0.299 ± 0.02042 |
p Value | 0.0683 | <0.0001 | 0.0225 | 0.8145 |
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Wang, D.-Y.; Zhu, J.; Zhang, Y.-Z.; Cui, Q.-Y.; Wang, S.-S.; Ning, Y.-W.; Shen, X.-J. Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori). Insects 2024, 15, 313. https://doi.org/10.3390/insects15050313
Wang D-Y, Zhu J, Zhang Y-Z, Cui Q-Y, Wang S-S, Ning Y-W, Shen X-J. Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori). Insects. 2024; 15(5):313. https://doi.org/10.3390/insects15050313
Chicago/Turabian StyleWang, Dong-Yue, Juan Zhu, Yi-Zhong Zhang, Qian-Yi Cui, Shan-Shan Wang, Yang-Wei Ning, and Xing-Jia Shen. 2024. "Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori)" Insects 15, no. 5: 313. https://doi.org/10.3390/insects15050313
APA StyleWang, D.-Y., Zhu, J., Zhang, Y.-Z., Cui, Q.-Y., Wang, S.-S., Ning, Y.-W., & Shen, X.-J. (2024). Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori). Insects, 15(5), 313. https://doi.org/10.3390/insects15050313