Establishment of a chloroplast transformation system in Tisochrysis lutea

Tisochrysis lutea is a haptophyte microalga commonly used as a commercial feed for juvenile fish and shellfish larvae. Genetic modification is of considerable importance for developing the potential economic value of T. lutea. However, the genetic transformation system of T. lutea has not yet been established, which limits functional genomic studies and strain improvement of this important microalgal species. In the current study, a chloroplast transformation vector harboring the phosphinothricin resistance gene (bar) as a selectable marker was established, and two short peptide-encoding genes (ant1 and ant2) driven by the endogenous psbA and rbcL promoters were cloned in this vector. The recombinant plasmid was transformed using a biolistic method into the trnI/trnA locus of the chloroplast genome via homologous recombination. After continuous selection on phosphinothricin, the integration of foreign genes and the expression of specific products in the transformants were detected using polymerase chain reaction (PCR), Southern blotting, and western blot analysis. This is the first report of establishment of a stable transformation system in T. lutea, which is a prerequisite for functional genomics and applied research on this species.