Prediction of the Effect of Methylation in the Promoter Region of ZP2 Gene on Egg Production in Jinghai Yellow Chickens
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Sample Collection
2.3. Primer Synthesis and Quantitative Real-Time PCR (qRT-PCR)
2.4. ZP2 Gene Core Promoter Region Prediction and Deletion Vector Construction
2.5. Cell Transfection and Determination of Luciferase Activity
2.6. DNA Extraction from Ovarian Tissue and Sulfite Treatment
2.7. CpG Island Analysis of Promoter Region of ZP2 Gene and BSP Primer Design
2.8. PCR Amplification and Product Recovery
2.9. BSP Sequencing
2.10. Statistical Analysis
3. Results
3.1. Differential Expression Analysis of ZP2 Gene in Ovarian Tissues
3.2. Prediction of the Core Promoter Region of ZP2 Gene and Assay of ZP2 Activity in DF-1 Cells Transfected with Deletion Vector
3.3. ZP2 Gene CpG Island Prediction and PCR Amplification
3.4. BSP Sequencing Results of CpG Island Methylation of the ZP2 Gene
3.5. Pearson Correlation Analysis between Methylation Level and mRNA Expression
3.6. Prediction of the Potential Transcription Factor Binding Sites of the ZP2 Gene
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Accession Number | Primer Sequence (5′-3′) | Product Size (bp) |
---|---|---|---|
ZP2 | NM_001039098.1 | F: TGATGTCTTGTGAGCACGCTGTAG R: GTGCCATTCAGCAGCAGGAG | 121 |
β-actin | X00182 | F: CAGCCATCTTTCTTGGGTAT R: CTGTGATCTCCTTCTGCATCC | 169 |
Fragment Name | Primer Sequence (5′-3′) | Product Size (bp) |
---|---|---|
pGL3-P7 | CAGCTGTGGCTTCTCTGAGCG | 1999 |
pGL3-P6 | CTTTACTCTGTTTCCCCCCTCC | 1552 |
pGL3-P5 | GTGCCCCTGGGAGCGTACAGA | 1348 |
pGL3-P4 | TTCAGCTGTCAGTAGCCTGCC | 1042 |
pGL3-P3 | TCCCCTGTGAGGGAAGGCTTG | 594 |
pGL3-P2 | GCCTACAGGAAAGCTGGGGAG | 266 |
pGL3-P1 | GCAGTGAGACACTGGAACAGG | 150 |
ZPDR | TTAAGACCACCTCGTTCCACCC |
Gene | Primer Sequence (5′-3′) | Product Size (bp) |
---|---|---|
ZP2-1 | F: AAGTTATTTAGATTGAAAATATAAATTAGGGGAT R: CATTCATTTCCACCACAACAACAAC | 304 |
ZP2-2 | F: GGAGTTATAGGTTTTAAGAGAGGGAGG R: CCACATTACTCCATCTATACCCAAAAAC | 417 |
Core Region | Start (bp) | End (bp) | Primer Sequence (5′-3′) | Score (0–1) |
---|---|---|---|---|
1 | −1516 | −1466 | GAATCACGGCATAAAACATCAGCCCCGGAGGGAGCGCTGCAGCGATGGGG | 0.96 |
2 | −249 | −198 | GGAGGGACTTTTTATAGGGGCAGGTAGTGACCAGATGGCTTTAAACTGGA | 0.95 |
Fragment | Overall Methylation Level (%) | Number of Methylated Sites | |
---|---|---|---|
High Laying Group | Low Laying Group | ||
ZP2-1 | 81.1 | 85.0 | 19 |
ZP2-2 | 83.1 | 84.0 | 30 |
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Zhang, J.; Zhang, X.-Q.; Ling, X.-Z.; Zhao, X.-H.; Zhou, K.-Z.; Wang, J.-Y.; Zhang, G.-X. Prediction of the Effect of Methylation in the Promoter Region of ZP2 Gene on Egg Production in Jinghai Yellow Chickens. Vet. Sci. 2022, 9, 570. https://doi.org/10.3390/vetsci9100570
Zhang J, Zhang X-Q, Ling X-Z, Zhao X-H, Zhou K-Z, Wang J-Y, Zhang G-X. Prediction of the Effect of Methylation in the Promoter Region of ZP2 Gene on Egg Production in Jinghai Yellow Chickens. Veterinary Sciences. 2022; 9(10):570. https://doi.org/10.3390/vetsci9100570
Chicago/Turabian StyleZhang, Jin, Xiang-Qian Zhang, Xuan-Ze Ling, Xiu-Hua Zhao, Kai-Zhi Zhou, Jin-Yu Wang, and Gen-Xi Zhang. 2022. "Prediction of the Effect of Methylation in the Promoter Region of ZP2 Gene on Egg Production in Jinghai Yellow Chickens" Veterinary Sciences 9, no. 10: 570. https://doi.org/10.3390/vetsci9100570