Construction and Evaluation of Chloroplast Expression Vectors in Higher Plants

Plastid transformation has a number of advantages in comparison with nuclear transformation. Currently, only tobacco (Nicotiana tabacum) is routinely used in plastid transformation. Here we constructed a series of chloroplast expression vectors specific for spinach (pCEV1), tomato (pCEV2 and pCEV3), and N. benthamiana (pCEV4). Selection marker aminoglycoside 3′-adenyltransferase (aadA) conferring spectinomycin resistance was used in pCEV1, pCEV2, and pCEV4, while selection marker neomycin phosphotransferase II (nptII) was used in pCEV3. The expression cassette in these vectors was integrated in the intergenic spacer between trnI and trnA of plastid genome via homologous recombination. Several transgenes, including a reporter gene encoding GFP:GUS fusion protein and genes from tomato (lycopene b-cyclase, z-carotene desaturase) and bamboo mosaic virus satellite RNA (encoding coat protein CP20), were independently cloned into some of these vectors. Transient GUS expression was detected in spinach leaves bombarded by pCEV1/GFP-GUS. Functional expression of selection markers aadA and nptII was demonstrated for spinach, tomato, and N. benthamiana. Seedling assay from T0 self-pollinated plant of transplastomic N. benthamiana confirmed maternal inheritance of transgenes, and genomic PCR analysis confirmed integration of transgenic expression cassette into the plastid genome of N. benthamiana. Moreover, auxiliary vectors pECaad and pECnpt are also reported.