Abstract: DUF647 (domain of unknown function 647) protein family is found in diverse eukaryotic organisms and highly conserved in eukaryotes. It has 6 members in Arabidopsis genome. So far, the function of 4 members of Arabidopsis DUF647 family is unknown. In this report, using an endogenous Arabidopsis MIR319a precursor as the backbone, we constructed two artificial microRNAs (amiRNAs) to knock down the expression of two DUF647 family genes At1g13770 and At2g23470. Using the WMD (Web microRNA Designer) platform, we designed two amiRNAs targeting At1g13770 and At2g23470 genes, respectively. Both amiRNAs sequences were engineered into the MIR319a precursor using overlapping PCR and the amiRNAs backbones were transferred into the binary vector pCHF3. The resulting plasmids that harbor amiRNAs stem loop fragments were transformed into Arabidopsis by Agrobacterium-mediated floral diping. Upon constitutive expression of these two amiRNAs, the target genes were efficiently down-regulated in transgenic line. The decreased level of At2g23470 transcript in At2g23470-amiRNA transgenic plants resulted in severe sterility. This work will facilitate the functional analysis of At1g13770 and At2g23470 genes in Arabidopsis growth and development.

Key words: Arabidopsis, DUF647 gene family, artificial microRNAs, gene silencing