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