Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA.
We introduced plasmid pCmVCAT containing a chloramphenicol acetyltransferase (CAT) gene, flanked by the cauliflower mosaic virus 35S promoter and nopaline synthase polyadenylation sequences, into Chlamydomonas reinhardtii by electroporation; chloramphenicol (CAM) resistance was used for selection. Several mutants with aberrant response to cadmium (Cd) toxicity were obtained by screening CAM resistant transformants. Southern blot analysis showed random integration of pCmVCAT sequence into the nuclear genome. Expression of CAT gene was confirmed by the detection of CAT gene transcript in Northern blot analysis and the detection of CAT enzyme by ELISA assay. This study demonstrated the feasibility of transforming Chlamydomonas reinhardtii with heterologous DNA by electroporation, and the expression of heterologous gene, in this alga.
Tang
D K
DK
Dept. of Biology, Hong Kong University of Science and Technology, Kowloon.
Qiao
S Y
SY
Wu
M
M
D000818:Animals; D002104:Cadmium / Q000633:toxicity; D019256:Cadmium Chloride; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics*; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002702:Chloramphenicol Resistance; D002712:Chlorides / Q000633:toxicity; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000235:genetics*; D018274:Electroporation; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D014162:Transfection / Q000379:methods*
D002712:Chlorides; D016054:DNA, Protozoan; D004274:DNA, Recombinant; D012333:RNA, Messenger; D016053:RNA, Protozoan; D002104:Cadmium; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D019256:Cadmium Chloride