The chloroplast genetic engineering of a unicellular green alga Chlorella vulgaris with two foreign peptides co-expression

The purpose of this study was to establish a chloroplast transformation system in Chlorella vulgaris for expressing recombinant proteins. The 16S-trnI/trnA-23S in the inverted repeat region of C. vulgaris chloroplast genome was used as the flanking fragments in the homologous recombination vector. Meanwhile, the selectable marker aadA gene was promoted by the endogenous 16S promoter, and two codon-optimized antimicrobial peptide NZ2114 and Piscidin-4 genes linked by endogenous ribosome binding site (RBS) in a polycistron were regulated by the rbcL promoter of C. vulgaris. The plasmid was transformed into the chloroplast of C. vulgaris via microparticle bombardment and the transformants were screened using spectinomycin. After multiple rounds' selection, the homoplastic transformants were identified using polymerase chain reaction (PCR), Southern blotting, and Western blotting. This research provided an efficient chloroplast genetic system for C. vulgaris, and will be benefit for the algal further research and application.