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| Identification and Promoter Activity Analysis of Porcine miR-181 and miR-1 |
| ZHANG Hai-xin, LIU Yi-nan, ZHAO Yan-he, REN Zhu-qing, XIONG Yuan-zhu |
1.Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture/Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction, Ministry of Education/College of Animal Science, Huazhong Agricultural University, Wuhan 430070, P.R.China
2.College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R.China |
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摘要 Since its discovery a decade ago, microRNA has been identified as one of the major regulatory gene families in eukaryotic cells. Many functions of microRNAs have been revealed both in flora and fauna in recent years, but the transcriptional regulation of microRNA genes is not well-understood. In the present study, a series of primers were designed in the 2 000 nt upstream regions of porcine miR-181 and miR-1 and then the sequences were cloned into pGL3-basic vector to test their transcriptional activity. Dual-luciferase reporter assays showed that, the activity of 5´-flanking sequence of miR-181 started on construct -51, decreasing with the length of the fragment up to -444. The upstream 590 bp confer maximal transcriptional activity and the basal promoter activity is located within the -82 to +16 bp region. For miR-1, the activity starts on construct -50, decreasing with the length of the fragment up to -1 254 in despite of a bit of fluctuation, and the basal promoter activity is located within the -50 to +47 bp region. Furthermore, some putative regulation elements of both miR-181 and miR-1 were located. In addition, tissue distribution revealed that miR-181 is expressed at a relatively low level.
Abstract Since its discovery a decade ago, microRNA has been identified as one of the major regulatory gene families in eukaryotic cells. Many functions of microRNAs have been revealed both in flora and fauna in recent years, but the transcriptional regulation of microRNA genes is not well-understood. In the present study, a series of primers were designed in the 2 000 nt upstream regions of porcine miR-181 and miR-1 and then the sequences were cloned into pGL3-basic vector to test their transcriptional activity. Dual-luciferase reporter assays showed that, the activity of 5´-flanking sequence of miR-181 started on construct -51, decreasing with the length of the fragment up to -444. The upstream 590 bp confer maximal transcriptional activity and the basal promoter activity is located within the -82 to +16 bp region. For miR-1, the activity starts on construct -50, decreasing with the length of the fragment up to -1 254 in despite of a bit of fluctuation, and the basal promoter activity is located within the -50 to +47 bp region. Furthermore, some putative regulation elements of both miR-181 and miR-1 were located. In addition, tissue distribution revealed that miR-181 is expressed at a relatively low level.
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Received: 18 January 2011
Accepted:
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| Fund: This work was funded by the National Natural Science Foundation of China (31000996), the International Foundation for Science (B/4534-1), and the Specialized Research Fund for the Doctoral Program of Higher Education, China (200805041012). |
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Corresponding Authors:
REN Zhu-qing, Tel: +86-27-87284285, Fax: +86-27-87287139, E-mail: renzq@mail.hzau.edu.cn
E-mail: renzq@mail.hzau.edu.cn
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| About author: ZHANG Hai-xin, E-mail: catchacold.student@sina.com; |
Cite this article:
ZHANG Hai-xin, LIU Yi-nan, ZHAO Yan-he, REN Zhu-qing, XIONG Yuan-zhu .
2012.
Identification and Promoter Activity Analysis of Porcine miR-181 and miR-1. Journal of Integrative Agriculture, 12(6): 986-992.
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