A simple artificial microRNA vector based on ath-miR169d precursor from Arabidopsis.
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 beta-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.
Liu
Chong
C
Biotechnology Research Institute/The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, 100081, Beijing, China.
Zhang
Lan
L
Sun
Jie
J
Luo
Yanzhong
Y
Wang
Ming-Bo
MB
Fan
Yun-Liu
YL
Wang
Lei
L
D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D034622:RNA Interference; D012322:RNA Precursors / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D029681:Arabidopsis Proteins; D035683:MicroRNAs; D012322:RNA Precursors; D018749:RNA, Plant; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005966:Glucuronidase
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