Abstract
We have succeeded to induce RNAi against aadA mRNA that confers spectinomycin resistance in a unicellular green alga C. reinhardtii. More than 80% of aadA mRNA was degenerated by the expression of inverted repeat silencer DNA construct. To disrupt any RNAi related gene, we executed random tag insertion to the RNA induced cell. Sp far about 31,000 tag inserted cells were obtained and screened for its sensitivity change for spectinomycin. 114 showed notable decrecement. Highly likely some of their new phenotypes were result of disruption on the RNAi related genes.
Up- and down-stream genome sequences of a tag were determined by RESDA-PCR method, then based on the inform disrupted gene was deduced by Blast search against whole Chlamydomonas genome. From RNAi response enhanced tag-lines, histone, APG5 genes were identified; while from RNAi response repressed lag lines, protein kinase, ATPase, were identified.
