Characterization of miR-206 Promoter and Its Association with Birthweight in Chicken
Abstract
:1. Introduction
2. Results
2.1. The Identification of the Promoter Region of gga-miR-206
2.2. MyoD up-Regulates the Transcriptional Activity of gga-miR-206 in Vitro
2.3. MyoD Induces Muscle-Specific miR-206 Expression in Fibroblast and Myoblast Cells
2.4. Polymorphisms in the Promoter Region of gga-miR-206
2.5. The Mutation G-419C Affects the miR-206’s Promoter Activity
2.6. Genetic Effects of Variants in the Promoter Region of miR-206 on the Birth Weight
2.7. miR-206 Partly Induced Myogenesis by Myog and MCK
3. Discussion
4. Experimental Section
4.1. Ethics Statement
4.2. Animals
4.3. Cell Culture
4.4. RNA Extraction and RT-PCR
4.5. Real Time RT-PCR
4.6. Over-Expressing miR-206 and MyoD in Vitro
4.7. SNP Identification
4.8. Luciferase Reporter Assay for miR-206 Promoter Activity
4.9. Association Analysis and Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Maker | Position | Chr Location | Type | Sequences |
---|---|---|---|---|
SNP1 | −1020 bp | 107215055 | AT | AACCAAAATGACTGGGTGTGA |
SNP2 | −964 bp | 107214999 | CG | AGTTGCGAAACAGAAAGTTTCT |
SNP3 | −908 bp | 107214943 | AG | GGTAGGATGAAGAATCCCATG |
SNP4 | −804 bp | 107214839 | GT | TGGGACCTCTGGGTCCATCTG |
SNP5 | −713 bp | 107214748 | CG | AGAAGAGATACTCCACTACCT |
SNP6 | −674 bp | 107214709 | CA | ATGAAGAAGACACAGGAGATA |
SNP7 | −615 bp | 107214650 | GA | TGGTTGCAATGCATCAGGTTA |
SNP8 | −607 bp | 107214642 | CT | ATGCATCAGGCTAACTTCCCT |
SNP9 | −569 bp | 107214604 | CT | AAGACATTTCCACTTAGGACA |
SNP10 | −515 bp | 107214550 | GT | ACTCCAACCGGTTTTTTCCCA |
SNP11 | −470 bp | 107214505 | AT | ATCTCCAGCAACCCTGAGTGT |
SNP12 | −454 bp | 107214489 | CT | AGTGTAGCTGTCCCATCAAGA |
SNP13 | −419 bp | 107214454 | GC | CAAACCAGGTGCTCCAGTAGA |
SNP14 | −390 bp | 107214425 | CG | AAGGGAGGGACAGTGGTGCCA |
SNP15 | −356 bp | 107214391 | TC | CCAATTGACCTAAGCTTGACC |
SNP16 | −327 bp | 107214362 | GT | GGCAAAGAGAGGGACAAGAGG |
SNP17 | −244 bp | 107214279 | GA | GAACCAGACCGGGCTCCAGTG |
SNP18 | −145 bp | 107214180 | AG | CTTCCTGATCAGGACATTTGT |
SNP19 | −140 bp | 107214175 | GA | TGATCGGGACGTTTGTACCAA |
SNP20 | −120 bp | 107214155 | CT | ATAATAATAACATTTTGGTGT |
SNP21 | −114 bp | 107214149 | GA | ATAATATTTTGGTGTTCCTGC |
SNP22 | −83 bp | 107214118 | AG | AGGAGAAAGCAGATCACCAGC |
SNP23 | −32 bp | 107214067 | CT | TCTCCAGGAGCGCCCAGAGGT |
SNPs | Position | Mutation | Birthweight (Least Squares Mean, g) | p Value | ||
---|---|---|---|---|---|---|
SNP7 | −615 bp | G/A | 30.1424 (GG/66) | 31.7435 (GA/23) | 29.4868 (AA/189) | 0.0035 |
SNP10 | −515 bp | G/T | 29.5055 (GG/183) | 30.8324 (GT/34) | 30.2292 (TT/72) | 0.0355 |
SNP11 | −470 bp | A/T | 31.0194 (AA/36) | 30.9600 (AT/15) | 29.6075 (TT/240) | 0.0232 |
SNP12 | −454 bp | C/T | 30.1040 (CC/151) | 30.5220 (CT/41) | 29.2430 (TT/100) | 0.1726 |
SNP13 | −419 bp | G/C | 30.8111 (CC/36) | 29.1286 (CG/27) | 29.0522 (GG/249) | 0.0229 |
SNP15 | −356 bp | T/C | 30.1899 (TT/69) | 31.0147 (TC/34) | 29.5439 (CC/189) | 0.0138 |
SNP17 | −244 bp | G/A | 30.5969 (GG/129) | 30.555 (AG/40) | 28.8797 (AA/123) | 0.1202 |
SNP18 | −145 bp | A/G | 31.0194 (AA/36) | 31.25 (AG/16) | 29.6203 (GG/241) | 0.0163 |
SNP19 | −140 bp | G/A | 31.0194 (AA/36) | 31.25 (AG/16) | 29.6203 (GG/241) | 0.0163 |
SNP20 | −120 bp | C/T | 28.9643 (CC/126) | 30.5429 (CT/42) | 30.6032 (TT/126) | 0.2140 |
SNP21 | −114 bp | G/A | 31.0194 (AA/36) | 31.25 (AG/16) | 29.6203 (GG/241) | 0.0163 |
Primer Name | Primer Sequences (5′→3′) | Accession Number | Product Size (bp) | Tm (°C) | Notes |
---|---|---|---|---|---|
miR206-pro-F1 | ggggtaccggcatcaccttgctaccctaaa | NR_031431.1 | 1592 | 65 | pGL-vector |
miR206-pro-R1 | ccgctcgagagaggcagcatttctcctcatc | ||||
miR206-pro-F2 | ggggtaccgcccatttccttcaacctttcc | NR_031431.1 | 1121 | 65 | pGL-vector |
miR206-pro-R2 | ccgctcgagagaggcagcatttctcctcatc | ||||
miR206GT-F | cataaatcgtggaacaacgcataa | NR_031431.1 | 1198 | 62 | SNP |
miR206GT-R | gcagtagctggaagcagaggac | ||||
myoD-F2 | ggggtaccactccgacgttcccagtc | NM_204214.2 | 1213 | 62 | pcDNA-MyoD |
myoD-R2 | cggaattccaggttccctattctccaaa | ||||
MyoD-F | ggaaggaggaaacctgagtga | NM_204214.2 | 129 | 60 | qPCR |
MyoD-R | ctggacctgcctttatagcac | ||||
MYOG-F | cggaggctgaagaaggtgaa | NM_204184.1 | 219 | 60 | |
MYOG-R | cggtcctctgcctggtcat | ||||
MHC-F | ctcctcacgctttggtaa | NM_001319304 | 218 | 60 | |
MHC-R | tgatagtcgtatgggttggt | ||||
MCK-F | ctgggcttctcggaggtgga | NM_205507.1 | 109 | 60 | |
MCK-R | cgtctatgggctggttctgct | ||||
TMEM8C-F | atcgaccttcatcatgtttgg | NM_001318457.1 | 161 | 60 | |
TMEM8C-R | ttgtctggatacagccctttc | ||||
gga-miR-206 | tggaatgtaaggaagtgtgtgg | NR_031431.1 | 65 | 58 | |
U6-qiagen-F | cgatacagagaagattagcatgg cccctgc | EU240275 | 109 | 58 | |
β-Actin-F | ctcccccatgccatcctccgtctg | NM_205518.1 | 179 | 60 | |
β-Actin-R | gctgtggccatctcctgctc |
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Jia, X.; Lin, H.; Abdalla, B.A.; Nie, Q. Characterization of miR-206 Promoter and Its Association with Birthweight in Chicken. Int. J. Mol. Sci. 2016, 17, 559. https://doi.org/10.3390/ijms17040559
Jia X, Lin H, Abdalla BA, Nie Q. Characterization of miR-206 Promoter and Its Association with Birthweight in Chicken. International Journal of Molecular Sciences. 2016; 17(4):559. https://doi.org/10.3390/ijms17040559
Chicago/Turabian StyleJia, Xinzheng, Huiran Lin, Bahareldin Ali Abdalla, and Qinghua Nie. 2016. "Characterization of miR-206 Promoter and Its Association with Birthweight in Chicken" International Journal of Molecular Sciences 17, no. 4: 559. https://doi.org/10.3390/ijms17040559