AbstractMedium-chain-length polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHA biosynthesis is type II PHA synthase. The genephaC2 encoding type II PHA synthase was placed under the control ofpsbA-pro andpsbA-ter of rice (Oryza sativa) to construct aphaC2 cassette, which was ligated with the screening marker geneaadAcassette (prrn–aadA–TpsbA-ter). These recombined fragments were cloned between the plastidrbcLandaccDgenes for targeting to the large single copy region of the chloroplast genome. A chloroplast transformation vector, pTC2, was constructed and introduced into the tobacco (Nicotiana tobacum) chloroplast genome by particle bombardment. PCR and Southern blot analysis confirmed stable integration ofphaC2 into the chloroplast genomes of T0and T1transgenic plants, and T1transgenic plants exhibited homoplasmy. The expression ofphaC2 at transcription level was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation ofphaC2 encoding type II PHA synthase in tobacco via chloroplast genetic engineering.
Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection