simple artificial microRNA vector based on ath-miR169d precursor from Arabidopsis

• Published in: Molecular biology reports 2010-02, Vol.37 (2), p.903-909
• Main Authors: Liu, Chong, Zhang, Lan, Sun, Jie, Luo, Yanzhong, Wang, Ming-Bo, Fan, Yun-Liu, Wang, Lei
• Format: Article
• Language: English
• Subjects: Animal Anatomy; Animal Biochemistry; Arabidopsis; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Base Sequence; Biomedical and Life Sciences; Botany; Cloning; Cloning, Molecular; Genetic engineering; Genetic Vectors - chemical synthesis; Genetic Vectors - genetics; Glucuronidase - genetics; Green Fluorescent Proteins - genetics; Histology; Life Sciences; MicroRNAs - genetics; Models, Biological; Molecular biology; Molecular Sequence Data; Morphology; Recombinant Proteins - genetics; Ribonucleic acid; RNA; RNA Interference; RNA Precursors - genetics; RNA, Plant - genetics; Sequence Homology, Nucleic Acid
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-009-9713-1

Artificial microRNA (amiRNA) is becoming a powerful tool for silencing genes in plants, and several amiRNA vectors have recently been developed based on the natural precursor structures of ath-miR159a, ath-miR164b, ath-miR172a, ath-miR319a and osa-miR528. In this study we generated a simple amiRNA vector (pAmiR169d) based on the structure of Arabidopsis miR169d precursor (pre-miR169d). Two unique restriction sites were created inside the stem region of pre-miR169d, which allows for the artificial miRNA sequences to be cloned as either ~80 bp synthetic oligonucleotides or PCR products. A β-glucuronidase:green florescent protein fusion gene (GUS-GFP) was efficiently silenced in transient assays using a pAmiR169d-derived construct targeting the GUS-GFP sequence. 5′-RACE showed that the target GUS-GFP transcript was cleaved precisely at the expected position across nucleotides 10 and 11 of the artificial miRNA. Thus, pAmiR169d allows for both easy construction of artificial miRNA constructs and efficient silencing of target genes in plants.