A robust protocol for site-directed mutagenesis of the D1 protein inChlamydomonas reinhardtii: A PCR-splicedpsbA gene in a plasmid conferring spectinomycin resistance was introduced into apsbA deletion strain

Abstract

In this paper, we describe a protocol to obtain a site-directed mutants in thepsbA gene ofChlamydomonas reinhardtii, which overcomes several drawbacks of previous protocols, and makes it possible to generate a mutant within a month. Since the large size of the gene, and the presence of four large introns has made molecular genetics of thepsbA gene rather unwieldy, we have spliced all of the exons of thepsbA gene by PCR to facilitate genetic manipulation and sequencing of the gene. The resultant construct (plasmid pBA153, with several unique restriction sites introduced at exon boundaries) carried 1.2 and 1.8 kb intact sequences from the 5′- and 3′-flanking regions, respectively. The plasmid was used to transform a D1-deletion mutant and was found to complement the deletion and restore photosynthetic activity. In addition, a bacterialaadA gene conferring spectinomycin resistance (spe ^r) was inserted downstream of the intron-freepsbA gene, to give construct pBA155. This allowed selection of mutant strains deficient in photosynthesis by using spectinomycin resistance, and eliminated the possibility of selection for revertant strains which is a consequence of having to use photosynthetic activity as a selection pressure. Finally, pBA155 was used to construct pBA157, in which additional restriction sites were inserted to facilitate cassette mutagenesis for generation of mutations in spans thought to be involved in donor-side interactions. AllpsbA deletion strains transformed with intron-freepsbA-aadA constructs encoding the wild-type D1 sequence, and screened on spectinomycin plates for thespe ^r phenotype, were able to grow photosynthetically, and all showed identical kinetics for electron transfer from primary (Q_A) to secondary quinone (Q_B) in Photosystem II, as assayed by the decay of the high fluorescence yield on oxidation of the reduced primary acceptor (Q_A ⁻).