A Comparative Analysis of Bombyx mori (Lepidoptera: Bombycidae) β-fructofuranosidase Homologs Reveals Different Post-Translational Regulations in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Insects, Vectors, and Cell Lines
2.2. Enzymatic Determination of β-FFase in Larval Tissues
2.3. Degenerate Polymerase Chain Reaction (Degenerate PCR)
2.4. Reverse Transcription PCR (RT-PCR) and Rapid Amplification of cDNA Ends (RACE)
2.5. Expression of Recombinant Proteins in E. coli and Preparation of GpSUC1a Polyclonal Antibody
2.6. Expression and Purification of Recombinant Proteins through the Bac-to-Bac System
2.7. SDS-PAGE and Western Blotting Analysis
2.8. Immunohistochemistry Assay
2.9. Purification of GpSUC1a and BmSUC1 by Immunoprecipitation (IP) and β-FFase Activity Confirmation
2.10. Liquid Chromatograph-Mass Spectrometer (LC-MS) Analysis
3. Results
3.1. GpSuc1a Is a Homologous Gene of BmSuc1 Identified in G. pyloalis
3.2. Recombinant GpSUC1a Showed Lower Activity Compared to BmSUC1
3.3. GpSuc1a Highly Expressed in the Midgut of G. pyloalis and Displayed obvious β-FFase Activity
3.4. Identification of Post-Translational Modifications (PTMs) of GpSUC1a and BmSUC1
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Assay | Primer Name | Primer Sequence (5′ to 3′) |
---|---|---|
Degenerate PCR | SUCdgpF1 | TGGATIAAYGAYCCIAAYGG |
SUCdgpR1 | GTGIGTIGCRTARAAITC | |
SUCdgpF2 | TGGGGICCIATGCAYTGGGGICA | |
SUCdgpR2 | GCRTARAARTCRTGICCRTRRTC | |
RACE | GpSuc1a-5RAgsp | AGCACCACCGGAGAAACACATTTCTT |
GpSuc1a-5RAngsp | GGGCTGGACACGTGGCCCCAGTGCAT | |
GpSuc1a-3RAgsp | ACAGTTTACATGTGGGAATGCCCTGA | |
GpSuc1a-3RAngsp | GGCATGGAACCCAAAGGCGATCGGTT | |
GpSuc1b-5RAgsp | TCCACTGCCCGAGAAGCACTGTTCTT | |
GpSuc1b-5RAngsp | GCGCTGGACGAGTGCCCCCAATGCAT | |
GpSuc1b-3RAgsp | ATGGGGTACATGTGGGAGTGCCCTGA | |
GpSuc1b-3RAngsp | GGAATGGCACCTCAGGGTGACAGGTA | |
GPSuc2a-5RAgsp | AGCAATTGGTAGATGCTTCCAATGGAA | |
GpSuc2a-5RAngsp | ATGCCCCCAATGAGCTATTCCAGGTT | |
GpSuc2a-3RAgsp | GATGGCGTCACTACTAAAAAGTATCGG | |
GpSuc2a-3RAngsp | CGCCTCAGGGTATAGAACCGGAAGGA | |
GpSuc2b-5RAgsp | CTCCACTTTTGTCATATGCCTTGTCC | |
GpSuc2b-5RAngsp | TCGCTGCTCATGGTCGGGCTTTTTG | |
GpSuc2b-3RAgsp | CTTGGATACATGTGGGAATGTCCAGATT | |
GpSuc2b-3RAngsp | CGCCTCAAGGTGTGAAACCTGAAGGA | |
GpSuc2c-5RAgsp | TCTGTGACTTATGTGCCAAAATGCTG | |
GpSuc2c-5RAngsp | TTAAGGGAATGCCATTCTCATCCGTT | |
GpSuc2c-3RAgsp | CTTGGMTAYATGTGGGAATGTCCAGATT | |
GpSuc2c-3RAngsp | CAAATGACAAAACAAACTGGCAAGAA | |
RT-PCR | GpSuc1a Fw | ATTGTTGTACACTGGGCGACTAACC |
GpSuc1a Re | TGGTGAGTCGTTCGGAGTGTACGTA | |
GpSuc1b Fw | CGCAGCTATGAACAACTTGAGGC | |
GpSuc1b Re | CTGACCACCACCATTTGTGCCAGT | |
GpSuc2a Fw | GATGGTACAGGTGGGGATGATG | |
GpSuc2a Re | CAGTGCTCCTTATTCCATCATGG | |
GpSuc2b Fw | TGTCAGGGAACTTCGGTTGAAC | |
GpSuc2b Re | GCCATTGAAAATACTTTTGGCGACT | |
GpSuc2c Fw | CGAAGAGCACCAAGCTCTTGGTGTG | |
GpSuc2c Re | TTGATTCATATAGTAGAACCTGAC | |
BmSuc1 Fw | CGGACCCGTTTTACAACGAA | |
BmSuc1 Re | CACGTAGGAGAGGACTGGAT | |
Genomic PCR And pET-24b Expression Vectors Construction | GpSuc1a F | ATAGGATCCGATGGGGCTCCTAAGGCTAATCG |
GpSuc1a R | CCGCTCGAGTTCAGGTATACTTCTTCTTAAATG | |
GpSuc1b F | CTAGATCTCATATGATGGCGGTCTTCACATTCACTG | |
GpSuc1b R | CCGCTCGAGCAATGTTGGGATAACTGG | |
GpSuc2a F | GGACTTCCATATGATGACGAGAGATTTACTGAGGC | |
GpSuc2a R | TATCTCGAGGGGCAAAACGGAACGCTTGAG | |
GpSuc2b F | CGCGGATCCGATGAATTGATCCGAACGGCTTCAG | |
GpSuc2b R | TATCAAATGCGGCCGCAACAACAGAACGTGTCAATG | |
GpSuc2c F | ATAGGATCCGATGACTAAAACTTTTTTAGGGC | |
GpSuc2c R | CCGCTCGAGATTTACTTCTTGCCAGTTTG | |
BmSuc1 F | CAGCTGTACATATGTTCGCCTGGAGCACAC | |
BmSuc1 R | CGGCTCGAGAGCGGGTACACTTCTTCTCAATC | |
pFastBac Dual Expression Vectors Construction | GFP F | CCGCTCGAGATGGTGAGCAAGGGC |
GFP R | CGGGGTACCTTACTTGTACAGCTC | |
pFB-BmSuc1 F | ATTTGCGGCCGCTATGTTCGCCTGGAGC | |
pFB-BmSuc1 R | CCCAAGCTTTTAATGATGATGATGATGATGAGCGGG | |
TACACT | ||
pFB-GpSuc1a F | ATAGGATCCGATGGGGCTCCTAAGGCTAATCG | |
pFB-GpSuc1a R | CCCAAGCTTTTCAATGATGATGATGATGATGGGTATA | |
CTTCTTCTTAAATG |
Protein | Mass (Da) | PI | Homology (%) | |||
---|---|---|---|---|---|---|
BmSUC1 BmSUC2 | M. sexta | B. amyloliquefaciens | S. tuberosum | |||
GpSUC1a | 56,400.10 | 4.48 | 64.90 | 65.24 | 37.86 | 22.26 |
35.98 | ||||||
GpSUC1b | 58,635.53 | 5.31 | 54.89 | 60.27 | 35.74 | 21.70 |
35.03 | ||||||
GpSUC2a | 55,794.92 | 5.47 | 37.43 | 41.27 | 37.90 | 18.68 |
35.26 | ||||||
GpSUC2b | 50,642.75 | 6.40 | 37.50 | 38.97 | 37.20 | 19.62 |
34.50 | ||||||
GpSUC2c | 23,868.99 | 7.78 | 16.73 | 16.57 | 16.70 | 10.19 |
13.28 |
Protein | Position (aa) | Amio Acid Sequences | Modifications |
---|---|---|---|
BmSUC1 | 21–47 | ALRQQNETTKRELEEYIADKKA | Methyl [K20]; Deamidated [R11] |
EINPR | |||
GpSUC1a | 20–45 | SFKQQFDNVADLEEYIAQKRTE | Deamidated [Q5; Q18;R20;R26] |
INPR | |||
BmSUC1 | 48–73 | YRPHYHISPPVGWMNDPNGFS | Methyl [R2; K24; K26]; |
YYKEK | Deamidated [R2; N18] | ||
GpSUC1a | 46–71 | YRLQYHVTPPVGWMNDPNGFS | Deamidated [Q4] |
FYKGE | |||
BmSUC1 | 162–183 | KYEGNPVLSYVPDNSADFRDPK | Methyl [R19; K22] |
GpSUC1a | 160–181 | KYEGNPVLTYTPRPDFNDSDPK | Methyl [R19; K22]; |
Deamidated [N13] | |||
BmSUC1 | 187–202 | FKDHWYVVIGSSSNK · R | Methyl [K2]; Deamidated [N14] |
GpSUC1a | 185–201 | HEDHWYVVIGSKTVDGR | No sites |
BmSUC1 | 283–307 | TDKYFQELDYGHDFYAT | Methyl [K3; K25] |
QTIQGDGK | |||
GpSUC1a | 282–306 | PETEFQELDYGHDIYATQSLEK | No sites |
DGT | |||
BmSUC1 | 337–344 | ELQLIGTR | No sites |
GpSUC1a | 336–343 | EIKLEGDR | Methyl [K3] |
BmSUC1 | 360–376 | SVHNGDLEPQQAIEFGP | No sites |
GpSUC1a | 359–375 | SLFDGDLLPEQSIEFEK | Deamidated [Q11] |
BmSUC1 | 424–436 | QVEWVPIGKTSWR | No sites |
GpSUC1a | 359–375 | QVEWNPIGSQSWR | Deamidated [Q1] |
BmSUC1 | 467–483 | VKNSSPQTLSVEAYRLR | Deamidated [Q7] |
GpSUC1a | 467–483 | LTNLSPQNLSVEAYHLR | Deamidated [N3; N8] |
BmSUC1 | 484–488 | RSVPA | No sites |
GpSUC1a | 484–505 | RSIPEMDVFVTITENRLNSGFK | Deamidated [R1; N18] |
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Share and Cite
Zhao, Y.; Yang, L.; Chen, Y.; Zhang, X.; Li, J.; Liang, D.; Jiang, S.; Gao, J.; Meng, Y. A Comparative Analysis of Bombyx mori (Lepidoptera: Bombycidae) β-fructofuranosidase Homologs Reveals Different Post-Translational Regulations in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae). Insects 2022, 13, 410. https://doi.org/10.3390/insects13050410
Zhao Y, Yang L, Chen Y, Zhang X, Li J, Liang D, Jiang S, Gao J, Meng Y. A Comparative Analysis of Bombyx mori (Lepidoptera: Bombycidae) β-fructofuranosidase Homologs Reveals Different Post-Translational Regulations in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae). Insects. 2022; 13(5):410. https://doi.org/10.3390/insects13050410
Chicago/Turabian StyleZhao, Yue, Liangli Yang, Yu Chen, Xinwei Zhang, Jing Li, Dan Liang, Song Jiang, Junshan Gao, and Yan Meng. 2022. "A Comparative Analysis of Bombyx mori (Lepidoptera: Bombycidae) β-fructofuranosidase Homologs Reveals Different Post-Translational Regulations in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)" Insects 13, no. 5: 410. https://doi.org/10.3390/insects13050410