| Animal sciences |
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| Influence of reference genes on expression of TYR, MITF and ASIP genes in tissues of Silky Fowl. |
| Zheng Nenzhu1, Li Li1, Xin Qingwu1, Miao Zhongwei1, Zhu Zhiming1, Liu Fenghui2, Wu Jianfei2, Lu Lizhi3* |
| (1. Institute of Animal Science and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 2. Animal Science Station of Licheng District of Putian City in Fujian Province, Putian 351100, Fujian, China; 3. Institute of Animal Science and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China) |
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Abstract 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-△△CT 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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Published: 20 November 2015
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Cite this article:
Zheng Nenzhu, Li Li, Xin Qingwu, Miao Zhongwei, Zhu Zhiming, Liu Fenghui, Wu Jianfei, Lu Lizhi. Influence of reference genes on expression of TYR, MITF and ASIP genes in tissues of Silky Fowl.. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(6): 732-740.
URL:
http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2015.04.131 OR http://www.zjujournals.com/agr/Y2015/V41/I6/732
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内参基因对TYR、MITF和ASIP基因在白绒乌骨鸡各组织表达水平的影响
以健康白绒乌骨鸡肌肉、肾、肝、肌胃和皮肤为试验素材，应用实时荧光定量聚合酶链反应技术探讨β-肌动蛋白（beta-actin,ACTB)、3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)和18S核糖体RNA（18S ribosomal RNA, 18S rRNA）3个内参基因的组织表达稳定性及其对酪氨酸酶（tyrosinase，TYR）、小眼畸形相关转录因子(microphthalmia-associated transcription factor，MITF)和刺鼠信号蛋白(agouti signaling protein，ASIP)等目的基因在白绒乌骨鸡各组织表达水平的影响，并采用紫外分光光度法分析各组织黑色素沉积规律，从中筛选出理想的内参基因。geNorm和NormFinder软件分析结果显示，内参基因GAPDH在白绒乌骨鸡各组织表达稳定性最高，ACTB表达稳定性最低。相对定量和黑色素沉积结果表明，GAPDH作为内参基因时，各组织MITF、TYR基因的相对表达量与黑色素沉积规律基本一致，表现为皮肤>肾>肌胃>肝>肌肉；ASIP基因的相对表达量与黑色素沉积规律正好相反，表现为肌肉>肝>肌胃>肾>皮肤，这与各目的基因对黑色素沉积的调控功能相符，即MITF和TYR基因高表达有利于黑色素沉积，ASIP基因高表达则抑制黑色素沉积。而18S rRNA和ACTB作为内参基因时，各目的基因表达量未表现出上述规律，与其各自对黑色素沉积的调控功能不相吻合，没有达到理想的校正效果。表明GAPDH基因稳定性最好，且能真正体现目的基因对黑色素沉积的表达调控，是校正目的基因在白绒乌骨鸡不同组织中mRNA表达量的最优内参基因。
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