Development of a stable transformation system of Dunaliella salina by using homologous recombination

Abstract

Nitrate reductase (NR) catalyzes reduction of nitrate to nitrite metabolism. However, NR converts chlorate to chlorite which is toxic to microalgae when concentration of chlorate is excessed concentration of nitrate in media. Because of the NR gene present in Dunaliella salina as haploid type, the gene has been used for genetic engineering of homologous recombination. In this report, transformation system of D. salina was established to improve the transformation efficiency by using homologous recombination in NR gene. The constructed plasmid vector pSK-HR-fGH is consisted of flounder growth hormone (fGH) gene expression cassette between nitrate reductase gene fragments. A PCR product containing the CaMV 35S promoter, fGH and Rbcs2-3’UTR that is flanked by the NR gene fragment was introduced into D. salina by using glass beads agitation. Treated cells were plated to 100 mM potassium chlorate media for selection and formed visible colonies after 2 weeks later. PCR and RT-PCR showed that the fGH gene was exactly integrated to nitrate reductase gene and transcribed in the transformed D. salina. This result of the present study proposed that transformation system using homologous recombination in NR gene may be used to efficient tool.