Abstract
Chlamydomonas reinhardtii is a useful organism to analyze unidentified gene functions because of its haploid nucleus. Into the genome of a Chlamydomonas strain 19-P(1030) that possesses an aadA transgene conferring spectinomycin resistance, we introduced a aadA mRNA-targeting silencer-construct, which consists of an inverted repeat of the aadA ORF and a ble marker gene to obtain a transformant (RNAi-37). In this transformant aadA mRNA was reduced to about 20% of the 19-P(1030). A tag-aphVIII DNA was introduced into the RNAi-37 to obtained a transformant 92-12C, in which RNAi efficiency was apparently reduced and a 14-kb genome region was deleted along with the tag insertion. This genome region contains three putative ORFs encoding an unidentified protein, a thioesterase-like protein and a protein with PWI/zinc-finger motives. We have obtained three BAC-subclones containing each of the three genes with promoter region and one for the entire deletion region, and are transforming the 92-12C with the plasmids to identify the gene that is involved in the reduced RNAi efficiency.
