Knocked-Out Bombyx mori Protein Disulfide Isomerase Decreases Silk Yields and Mechanical Properties by Affecting the Post-Translational Modification of Silk Proteins
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Silkworm Rearing and Sample Collection
2.2. Bioinformatics Analysis
2.3. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
2.4. sgRNA Design and Vector Construction
2.5. Embryonic Microinjection and Screening
2.6. Phenotypic and Economic Trait Analysis
2.7. Scanning Electron Microscopy of Cocoon Silk
2.8. Tensile Properties of Cocoon Silk
2.9. Fourier Transform Infrared Spectroscopy
2.10. Extraction Analysis of Silk Proteins
2.11. Sample Preparation for LC-MS/MS Detection
2.12. LC-MS/MS Analysis
2.13. Statistical Analysis
3. Results
3.1. BmPDI Is Evolutionarily Conserved with the Highest Expression in PSG of Silkworm
3.2. CRISPR/Cas9-Mediated BmPDI Knock-Out
3.3. Deletion of BmPDI Inhibits the Development of Silk Glands and Decreases the Silk Yields
3.4. Deletion of BmPDI Reduces the β-Sheet Crystal of Silk and Decreases the Silk’s Mechanical Properties
3.5. BmPDI Knock-Out Decreases the Silk Yields and Mechanical Properties by Regulating Post-Translational Modification of Silk Proteins
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations
ASG | Anterior silk gland |
MSG | Middle silk gland |
PSG | Posterior silk gland |
Nd | Naked pupa |
qRT-PCR | Real-time quantitative-PCR |
FibH | Fibroin heavy protein |
FibL | Fibroin light protein |
P25 | Protein 25 kDa |
Ser1 | Sericin-1 |
Ser2 | Sericin-2 |
Ser3 | Sericin-3 |
PDI | Protein disulfide isomerase |
ER | Endoplasmic reticulum |
ERS | Endoplasmic reticulum stress |
UPR | Unfolded protein response |
Bip | Immunoglobulin-binding protein |
IRE1 | Inositol-requiring protein 1 |
ATF6 | Activating transcription factor 6 |
PERK | Protein kinase RNA-like ER kinase |
eIF2α | eukaryotic translation initiation 2 |
gRNA | guide RNA |
PAM | Protospacer-associated motif |
CDS | Coding sequence |
WT | Wild type |
aa | Amino acids |
Dronc | Drosophila Nedd2-like caspase |
Dredd | Death related ced-3/Nedd2-like protein |
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Bombyx mori Line | G0 Eggs Microinjected | G0 Eggs Hatched | G1 Moth Broods | Positive G1 Moth Broods | Positive Ratio |
---|---|---|---|---|---|
D9L | 300 | 57 | 20 | 2 | 10% |
Category Name | Number of Disulfide-Linked Peptides in WT Lines | Number of Disulfide-Linked Peptides in BmPDI-KO Lines |
---|---|---|
FibH protein | 1 | 0 |
FibL protein | 56 | 41 |
P25 protein | 70 | 53 |
Total | 127 | 94 |
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Yang, S.; He, M.; Li, X.; Dong, H.; Lei, H.; Wang, F.; Deng, H.; Zhou, H.; Chen, S.; Zhou, Y.; et al. Knocked-Out Bombyx mori Protein Disulfide Isomerase Decreases Silk Yields and Mechanical Properties by Affecting the Post-Translational Modification of Silk Proteins. Insects 2025, 16, 684. https://doi.org/10.3390/insects16070684
Yang S, He M, Li X, Dong H, Lei H, Wang F, Deng H, Zhou H, Chen S, Zhou Y, et al. Knocked-Out Bombyx mori Protein Disulfide Isomerase Decreases Silk Yields and Mechanical Properties by Affecting the Post-Translational Modification of Silk Proteins. Insects. 2025; 16(7):684. https://doi.org/10.3390/insects16070684
Chicago/Turabian StyleYang, Shifeng, Mengyao He, Xian Li, Huan Dong, Hexu Lei, Fangyu Wang, Hanxin Deng, Hongji Zhou, Siyu Chen, Yujuan Zhou, and et al. 2025. "Knocked-Out Bombyx mori Protein Disulfide Isomerase Decreases Silk Yields and Mechanical Properties by Affecting the Post-Translational Modification of Silk Proteins" Insects 16, no. 7: 684. https://doi.org/10.3390/insects16070684
APA StyleYang, S., He, M., Li, X., Dong, H., Lei, H., Wang, F., Deng, H., Zhou, H., Chen, S., Zhou, Y., Meng, Z., Tu, D., Wang, H., Xia, Q., & Wang, F. (2025). Knocked-Out Bombyx mori Protein Disulfide Isomerase Decreases Silk Yields and Mechanical Properties by Affecting the Post-Translational Modification of Silk Proteins. Insects, 16(7), 684. https://doi.org/10.3390/insects16070684