Three-piece-ligation PCR and application in disruption of chlorophyll synthesis genes in Synechocystis sp. PCC 6803.
Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chlL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chlL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light.
Kong
Renqiu
R
Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan, Hubei 430072, P.R. China.
Xu
Xudong
X
D001426:Bacterial Proteins / Q000235:genetics; D002734:Chlorophyll / Q000096:biosynthesis / Q000235:genetics*; D000458:Cyanobacteria / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000096:biosynthesis; D017353:Gene Deletion; D005798:Genes, Bacterial; D005819:Genetic Markers; D008190:Lyases; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000235:genetics; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005819:Genetic Markers; D011506:Proteins; C095441:chlL protein, bacteria; D002734:Chlorophyll; D008190:Lyases; C100609:ChlH protein, Synechocystis