Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.
Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed.
Sun
Yu
Y
Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, The Chinese Academy of Science, Shanghai, People's Republic of China.
Gao
Xiaoshu
X
Li
Qiyun
Q
Zhang
Qingqi
Q
Xu
Zhengkai
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics*; D048028:DNA, Algal / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D050901:Nitrate Reductase / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D048028:DNA, Algal; D050901:Nitrate Reductase