Transplastomic Plants
Homoplasmic for Foreign Transgenes
ZHANG Zhong-Lin1* ,QIAN Kai-Xian2,
SHEN Gui-Fang1
( 1Biotechnology Research Institute, Chinese Academy of
Agricultural Sciences, Beijing 100081, China；2Department of Bioscience and
Technology, Zhejiang University, Hangzhou 310027, China )
Abstract Scientists
pay more and more attention to the research on plastid engineering for its
following advantages： foreign genes can be integrated
site-specifically into the plastid genome (plastome)； there are no position effects
as experienced with random insertion of transgenes in nuclear transformation； pollen-mediated dispersion of
transgenes can be avoided because chloroplasts are maternally transmitted； gene silencing does not occur
in plastids and therefore transgene expression is stable in progeny of
transplastomic plants； and the high ploidy level of the
plastome in leaf cells makes high levels of transgene expression feasible. At
the same time, however, the highly polyploid plastome makes it difficult to get
transplastomic plants homoplasmic for foreign transgenes. In this work,
chloroplast transforming vector pTRCH205, which carries two psbA5′-nifH–psbA3′and
Prrn-aadA-TpsbA cassettes flanked by plastid DNA sequence to
target their insertion between psbA and trnK operons, was
constructed. Plastid transformation of Nicotiana tabacum was carried
out by the biolistic delivery of transforming plasmid pTRCH205 DNA into leaf
cells. Integration of nifH and aadA by two homologous
recombination events via the flanking ptDNA sequences, and selective
amplification of the transplastomes on MS medium with high concentration of
spectinomycin, yielded resistant cell lines. All the independent transplastomic
lines were subjected to three additional rounds of regeneration and were
subcultured for 6―10 times through stem sections on MS medium containing 500
mg/L spectinomycin, to obtain homoplasmic tissues. The results of PCR assay and
Southern blot hybridization, probed with 0.9 kb BglII/SnaBI
homologous fragment, indicated that foreign genes had been integrated into the
plastomes of transgenic plants, which finally became homoplasmic for foreign
transgenes.
Key words plastid transformation；homoplastome；aadA gene；spectinomycin
*Corresponding author：Tel,86-10-68919854；Fax,86-10-68975402；e-mail,[email protected]