Influence of reference genes on expression of TYR, MITF and ASIP genes in tissues of Silky Fowl
Silky Fowl is a precious local breed containing a large amount of melanin. Therefore, it is considered to have high edible and medicinal value. Study on the related gene expression is helpful to reveal the internal regulation mechanism of melanin deposition. TYR (tyrosinase), MITF (microphthalmia-as...
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| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2015-11-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2015.04.131 |
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| Summary: | Silky Fowl is a precious local breed containing a large amount of melanin. Therefore, it is considered to have high edible and medicinal value. Study on the related gene expression is helpful to reveal the internal regulation mechanism of melanin deposition. TYR (tyrosinase), MITF (microphthalmia-associated transcription factor) and ASIP (agouti signaling protein) are important candidate genes for the formation of pigment. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) has been widely applied to analyze gene expression as a convenient approach, and the selection of appropriate reference genes according to specific samples or conditions is critical to ensure the accuracy and reliability of gene expression.The objectives of this study are to screen the ideal reference genes and to provide a necessary basis for further study on gene expression regulation of melanin deposition and other important traits in Silky Fowl through investigating the expression stability of reference genes (ACTB, GAPDH and 18S rRNA) and their effects on the expression of melanin genes (TYR, MITF and ASIP) in tissues of Silky Fowl.RT-qPCR was adopted to investigate mRNA expression levels of the related genes in the muscle, kidney, liver, gizzard and skin of Silky Fowl, and the ultraviolet spectrophotometry was used to measure the melanin content in tissues of Silky Fowl. GeNorm and NormFinder softwares were applied to evaluate the expression stability of reference genes, and 2<sup>-△△C</sup>T method was applied to calculate the relative expression of target gene. The results from RT-qPCR showed that the melting curve displayed an obvious single peak and standard curves had good linear relationship. Comprehensive evaluation of geNorm and NormFinder indicated that the expression stability of GAPDH was the highest, while ACTB was the lowest among the three reference genes in tissues. Relative quantity and melanin content analysis demonstrated that taking GAPDH as a reference gene, the expression level of TYR, ASIP and MITF genes with the melanin content in tissues had the same trend: skin > kidney > gizzard > liver > muscle, but the expression level of ASIP gene was opposite to melanin deposition content, which implied that each target gene expression was matched with their respective function on regulating melanin deposition. For instance, high expression of TYR and MITF genes promoted melanin deposition, while the ASIP gene inhibited melanin deposition. Taking 18S rRNA and ACTB as reference genes, the expression of TYR, MITF and ASIP genes were not related to the melanin deposition, and an ideal calibration result could not be obtained.In conclusion, reference gene plays an important role in the expression of target gene. In this paper, GAPDH gene not only had highest expression stability in tissues, but also could truly reflect the regulation effect of the target genes (TYR, MITF and ASIP) on melanin deposition. Thus, GAPDH gene is the most suitable reference gene for normalizing the mRNA expression of genes in tissues of Silky Fowl. |
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| ISSN: | 1008-9209 2097-5155 |