Identification and Functional Analysis of E3 Ubiquitin Ligase g2e3 in Chinese Tongue Sole, Cynoglossus semilaevis
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Animal Euthanasia and Ethics Statement
2.2. Animals and Samples
2.3. Cloning and Phylogenetic Analysis of Cs-g2e3
2.4. Expression Pattern of Cs-g2e3 in Different Tissues and Stages of C. semilaevis
2.5. In Situ RNA Hybridization
2.6. Cell Culture
2.7. The Knockdown Effect of Cs-g2e3 siRNA in C. semilaevis Gonad Cells
2.8. In Vivo RNAi-Mediated Cs-g2e3 Knockdown in the Gonads of C. semilaevis
2.9. Construction of Promoter Plasmids, the Interaction between Cs-g2e3 and Transcription Factors, and Co-Transfection and Dual Luciferase Assay in C. semilaevis
2.10. Statistical Analysis
3. Results
3.1. The Structural and Phylogenetic Analyses of g2e3 in C. semilaevis
3.2. Expression Pattern of Cs-g2e3 in C. semilaevis
3.3. Localization of Cs-g2e3 mRNA in the Gonads of C. semilaevis
3.4. The Knockdown Effects on Cs-g2e3 and Other Related Genes by siRNA Transfection in Gonadal Germ Cell Lines
3.5. In Vivo RNA Interference in C. semilaevis
3.6. Detection and Analysis of the Activity of the Cs-g2e3 Promoter and the Regulation of Transcription Factors
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence (5′ to 3′) | Product Length (bp) | Tm Values (°C) | Purposes |
---|---|---|---|---|
g2e3-F | GCCACGTAACCCCATAT | 2869 | 52 | sequence validation |
g2e3-R | AACGCAAGCAGTAGAACAG | |||
β-actin-RT-F | TTCCAGCCTTCCTTCCTT | 124 | 53 | qRT-PCR |
β-actin-RT-R | TACCTCCAGACAGCACAG | |||
g2e3-RT-F | GTGTTCGAGGGTCCAGAAGG | 126 | 58 | qRT-PCR |
g2e3-RT-R | GAAGCCAATCAGGGCTCCAT | |||
Y-g2e3-F | ATTTAGGTGACACTATAGAA CTGCAGGAAGTCCGTTCACT | 556 | 65 | ISH |
Y-g2e3-R | TAATACGACTCACTATAGGG GGCCCAGTCTTTGATGTCCA | |||
sox9-RT-F | AAGAACCACACAGATCAAGACAGA | 150 | 57 | qRT-PCR |
sox9-RT-R | TAGTCATACTGTGCTCTGGTGATG | |||
cyp19a-RT-F | GGTGAGGATGTGACCCAGTGT | 230 | 56 | qRT-PCR |
cyp19a-RT-R | ACGGGCTGAAATCGCAAG | |||
tesk1-RT-F | GCAGAAACTCTCTCACCCCAACA | 290 | 59 | qRT-PCR |
tesk1-RT-R | CCAGACCAAAGTCCGTCACCA | |||
piwil2-RT-F | CGTCACCTTCGCTCCAAAT | 171 | 56 | qRT-PCR |
piwil2-RT-R | TCTTCGTCGTCCGTTCGC | |||
g2e3-P-F | AGATCTGCGATCTAAGTAAGCT GCGTCCTCCAGTTTGGCTA | 1540 | 68 | Promoter cloning |
g2e3-P-R | CAACAGTACCGGAATGCCAAGCT CGCTTTTCTTCCGTTCCG | |||
Cs-sex-F | CCTAAATGATGGATGTAGATTCTGTC | 169/134 | 56 | sex determination |
Cs-sex-R | GATCCAGAGAAAATAAACCCAGG | |||
g2e3-835-F | GCAACAAUCAGGACAACUUTT | siRNA | ||
g2e3-835-R | AAGUUGUCCUGAUUGUUGCTT | |||
g2e3-1562-F | CCGUGAAGAUCUCUACUUUTT | siRNA | ||
g2e3-1562-R | AAAGUAGAGAUCUUCACGGTT | |||
g2e3-1928-F | GCAGACGUUGGGUGUCUUUTT | siRNA | ||
g2e3-1928-R | AAAGACACCCAACGUCUGCTT |
Groups | Firefly Luciferase | Renilla Luciferase | The Relative Luciferase Value (±SD) |
---|---|---|---|
pGL3-g2e3-pro | 640,038 | 1,102,383 | 0.56 ± 0.05 |
571,785 | 954,903 | ||
511,223 | 1,016,504 | ||
pGL3-g2e3-p + C/EBPα | 640,123 | 586,912 | 0.99 ± 0.18 |
706,702 | 644,511 | ||
391,717 | 499,763 | ||
pGL3-g2e3-p + POU1F1a | 941,729 | 395,459 | 2.38 ± 0.02 |
786,581 | 327,948 | ||
984,677 | 417,072 | ||
pGL3-g2e3-p + myogenin | 812,538 | 1,023,484 | 0.83 ± 0.07 |
868,884 | 950,887 | ||
883,088 | 1,132,129 | ||
pGL3-g2e3-p + YY1 | 365,876 | 1,897,689 | 0.18 ± 0.02 |
305,774 | 1,580,501 | ||
321,856 | 1,940,210 | ||
pGL3-g2e3-p + JunB | 143,5884 | 1,269,235 | 1.21 ± 0.10 |
134,7471 | 1,020,311 | ||
1,462,650 | 1,236,206 | ||
mu-g2e3_myogenin + myogenin | 841,408 | 1,980,682 | 0.42 ± 0.02 |
851,162 | 1,927,374 | ||
742,405 | 1,831,696 | ||
mu-g2e3_YY1 + YY1 | 1,565,732 | 3,620,141 | 0.45 ± 0.04 |
1,682,182 | 3,401,520 | ||
1,014,972 | 2,447,655 | ||
mu-g2e3_JunB + JunB | 339,348 | 854,085 | 0.42 ± 0.05 |
293,453 | 623,706 | ||
356,566 | 937,653 | ||
pGL3-control | 7,175,562 | 766,433 | 9.65 ± 0.47 |
9,262,670 | 909,082 | ||
12,162,782 | 1,294,003 | ||
pGL3-Basic | 105,484 | 765,987 | 0.14 ± 0.02 |
107,500 | 648,147 | ||
139,058 | 1,061,613 |
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Cui, Z.; Luo, J.; Cheng, F.; Xu, W.; Wang, J.; Lin, M.; Sun, Y.; Chen, S. Identification and Functional Analysis of E3 Ubiquitin Ligase g2e3 in Chinese Tongue Sole, Cynoglossus semilaevis. Animals 2024, 14, 2579. https://doi.org/10.3390/ani14172579
Cui Z, Luo J, Cheng F, Xu W, Wang J, Lin M, Sun Y, Chen S. Identification and Functional Analysis of E3 Ubiquitin Ligase g2e3 in Chinese Tongue Sole, Cynoglossus semilaevis. Animals. 2024; 14(17):2579. https://doi.org/10.3390/ani14172579
Chicago/Turabian StyleCui, Zhongkai, Jun Luo, Fangzhou Cheng, Wenteng Xu, Jialin Wang, Mengjiao Lin, Yuqi Sun, and Songlin Chen. 2024. "Identification and Functional Analysis of E3 Ubiquitin Ligase g2e3 in Chinese Tongue Sole, Cynoglossus semilaevis" Animals 14, no. 17: 2579. https://doi.org/10.3390/ani14172579