The pea (Pisum sativum L.) rbcS transit peptide directs the Alcaligenes eutrophus polyhydroxybutyrate enzymes into the maize (Zea mays L.) chloroplasts

With a concept of producing polyhydroxybutyrate (PHB), a biodegradable thermoplastic polymer in maize chloroplasts, over 200 independent transgenic maize plants were produced. Five different constructs, pPHBA, pPHBB, pPHBC and pBY520 or pJS 101, were used in transformation experiments. PHB constructs contained phb genes from Alcaligenes eutrophus fused with the pea Rubisco small subunit (rbcS) transit peptide (TP) plus the first 24 amino acids of the mature rbcS protein. Plasmids BY520 and JS 101 contained the selectable marker bar gene linked with abiotic stress-related gene either hva 1 or mtl D. Southern blots on putative transgenic plants confirmed that the transgenic plants harbor transgenes from all five constructs. Transcription of transgenes was confirmed by Northern blot analyses. Western blot analyses using total cellular protein and protein from isolated chloroplasts confirmed that all the three genes produced their corresponding enzymes, and all PHB enzymes were targeted into maize chloroplasts. Immunofluorescent localization on both transgenic and non-transgenic maize chloroplasts treated with polyhydroxybutyrate synthase (PHBC)-antiserum also confirmed the targeting of PHBC enzyme to the chloroplasts of transgenic maize.