The Construction Of Chloroplast Site-specific Integration Expression Vector And Genetic Transformation In Phaeodactylum Tricornutum

To establish chloroplast expression system of Phaeodactylum tricornutum, the sequences rns-trnl and trnA-rnl from P. tricornutum chloroplast genome were cloned and used as homologous recombination elements by PCR using specific primers,chloramphenicol acetyltransferase (CAT) expression cassette and green fluorescent protein GFP expression cassette from plasmid pLysS and pBI121-GFP were amplified respectively, CAT gene conferring chloramphenicol resistance was employed as selection marker, and green fluorescent protein GFP as reporter gene. Based on the TA cloning vector pMD19-T, CAT and GFP expression cassette were cloned in between the two homologous recombination elements by a series of restriction enzymes digestion and T4 DNA ligase.The resultant chloroplast transformation vector pPtC-GFP was transferred into the chloroplast of P. tricornutum by electroporation. Transplastomic P. tricornutum cells were obtained upon chloramphenicol selection.Expression of CAT and GFP gene were detected by CAT gene amplification and laser scanning confocal microscope respectively.Sequences rns-trnl/trnA-rnl, chloramphenicol acetyltransferase (CAT) expression cassette and green fluorescent protein GFP expression cassette have been cloned. The results of restriction enzymes digestion showed chloroplast transformation vector was constructed successfully. Afer different concentration of chloromycetin(200μg ml-1,300μg ml-1 and 400μg ml-1), algae moss in solid medium could be observed clearly. DNA from Transplastomic P. tricornutum cells was extracted and been used for amplification of CAT gene, the sequencing results showed this gene is CAT. After eliminated the interference of green fluorescent of chloroplast, reporter GFP could be detected in the chloroplasts,suggesting that P. tricornutum chloroplast transformation system has been established successfully.