Abstract
Plasmid pBI221aadAGUS which carried both of GUS (β-glucuronidase) and aadA (aminoglyoside transferase) genes besides of the 35S cauliflower mosaic virus promoter was constructed and used for stable nuclear transformation of Chlamydomonas reinhardtii. The vector was transformed into the alga by particle gun bombardment and two positive colonies were selected on spectinomycin–containing medium. The restriction analysis of the DNA of the positive colonies showed that aadA was inserted in two orientations. The presence of introduced genes in the transformed colonies was confirmed by (PCR) using primers specific to GUS and aadA genes. The expression of aadA and GUS genes was revealed in all colonies that were grown on spectinomycin in liquid culture for 3–4 generations. The usefulness of this vector, differing in the orientation of the aadA cassette, was manisfested by transforming C. reinhardtii to spectinomycin resistance in the stable expression. This constructed plasmid-based expression vector system would help to unravel the functions of various genes in the green alga.
Highlights
Unlike bacteria, the transformation of algae is not accomplished and poses various technical problems [1]
The results indicated that the transformed DNA contained aadA genes of 800 bp (A), and GUS gene of 1.8 kb (B)
The constructed plasmid pBI221aadAGUS, which carries the GUS gene as a reporter gene and aadA as selectable gene was successfully transformed into Chlamydomonas cells
Summary
The transformation of algae is not accomplished and poses various technical problems [1]. The major problem is the design of vectors (i.e. plasmids or viruses) that could be successfully incorporated into, accepted by and expressed in the algal cells. The green alga Chlamydomonas reinhardtii and some other algal species have been used as model systems to standarise transformation protocols in algae [2,3,4,5,6,7]. Walker et al [26] indicated that the heterologous Dunaliella tertiolecta RbcS (ribulose1,5-bisphosphate carboxylase/oxygenase) promoter and 3 untranslated could be used to mediate the expression of bleomycin resistance gene (ble) in C. reinhardtii. Hallmann and Wodniok [27] showed that a construction of aminoglycoside 3 phosphotransferase (aphVIII)-based gene with 3 and 5 untranslated flanking sequences (including promoters) from the green alga Volvox worked in Chlamydomonas
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