Nelson J A, Lefebvre P A
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108, USA.
Mol Cell Biol. 1995 Oct;15(10):5762-9. doi: 10.1128/MCB.15.10.5762.
We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.
我们利用同源重组技术破坏了莱茵衣藻中的核基因NIT8。这是关于莱茵衣藻内源性位点靶向基因破坏的首次报道，也是光合真核生物中的第二篇此类报道。NIT8编码莱茵衣藻同化硝酸盐和亚硝酸盐所必需的一种蛋白质。通过将赋予吐根碱抗性的CRY1-1选择标记基因置于NIT8编码区内构建了一个破坏载体。nit8突变体不能以硝酸盐作为唯一氮源生长（Nit-），并且对氯酸盐杀伤具有抗性。在吐根碱-氯酸盐培养基上筛选后获得的2000个转化体中，有一个转化体包含五个破坏质粒拷贝同源插入到NIT8基因中，产生了抗吐根碱、抗氯酸盐的Nit-表型。野生型NIT8基因转化后挽救了突变体表型。在nit8位点还鉴定出另外七个突变，推测是与破坏质粒同源重组的结果，但显示这些突变伴随着周围基因组区域的缺失。
