Abstract：The cut-and-paste piggyBac (PB) transposon system has been shown to be a useful tool for genetic modification in a number of organisms including mammals, insects, and yeast. However, little is known whether it is active in the green microalga Chlamydomonas reinhardtii. In this study, we constructed stable PB element-containing C. reinhardtii strains bearing either a single copy or multiple copies of PB element. Subsequently, we transiently expressed TPase or codon-optimized crTPase in the PB element-containing strains to assay for transposition of PB element using restriction fragment length polymorphism (RFLP) methodology. Our analyses show that initial RFLP patterns of PB element are altered in cells one week and two weeks after plasimds containing TPase or crTPase expression cassette. The size of PB element-containing fragment remained unchanged in TR1 cells after transformation of the pJR38 plasmid containing no TPase or crTPase sequences. This result indicated that PB element was stable without TRpase or crTPase activity in cells. On the contrary, RFLP patterns of the PB element were altered 1 week and 2 weeks after transient expression of TPase or crTPase in TR1 or TR3 cells. For example, a fragment of 1.5 kb containing PB element was present in TR1 prior to transformation of TPase-containing plasmid. One week after transformation, one initial fragment of 1.5 kb and 3 new fragments of 2.8, 4.2 and 6.5 kb disappeared and appeared, respectively, suggesting transposition of the PB element occurred in TR1 cells. Two weeks after transformation, the size of all 3 fragments changed again, implying that transposition persists. In TR3, 1 week after the transformation, 3 fragments of 1.5, 3 and 4 kb and 1 fragment of 6.5 kb remained unchanged and disappeared, respectively. Meanwhile, a new fragment of 2.8 kb appeared. The RFLP pattern was hardly altered again 2 week after the transformation. These results indicated that the sizes of PB element-containing fragments changed, implying the PB element transposed after introducing the TPase and crTPase activities in C. reinhardtii TR1 and TR3 strains. Furthermore, to test whether PB element had been transposed in these cells, we examined the presence of a new joint generated by excision of the PB element in the genome. To this end, primers flanking to the new joint were applied for PCR analysis using the genomic DNA derived from the colonies. Clearly, excision event of the PB element was detected. This result supports the results that PB transposon system is active in C. reinhardtii. To find out the reason that the cells could survive the transposition of PB element, PCR fragments containing the PB element excision joint were subjected to nucleotide sequence analysis. Alignment of the sequences indicated that cells from the colonies suffered from illegitimate excision of PB element. The study shows that illegitimate excision of PB element occasionally occurs at the short repeat sequences 5'-TTT-3' and 5'-ACGCAG-3', but the standard excision site 5'-TTAA-3'. This study shows that the TPase and crTPase are active in transposition of PB element in C. reinhardtii. Hence, we propose that PB transposon systems can be adopted for genome modification in C. reinhardtii.
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