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Plastome Engineering of Ribulose-1,5-Bisphosphate
Carboxylase/Oxygenase in Tobacco to Form a Sunflower Large Subunit and
Tobacco Small Subunit Hybrid1
Ivan Kanevski,
Pal Maliga,
Daniel F. Rhoades, and
Steven Gutteridge*
Waksman Institute, Rutgers, The State University of New Jersey,
Piscataway, New Jersey 08855-0759 (I.K., P.M.); and Central
Research and Development Department, Experimental Station, DuPont
Company, Wilmington, Delaware 19880-0402 (D.F.R., S.G.)
Targeted gene replacement in plastids
was used to explore whether the rbcL gene that codes for
the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase,
the key enzyme of photosynthetic CO2 fixation, might be
replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that
contained the rbcL gene from either sunflower
(Helianthus annuus) or the cyanobacterium
Synechococcus PCC6301, along with a selectable marker.
Three stable lines of transformants were regenerated that had altered
rbcL genes. Those containing the
rbcL gene for cyanobacterial ribulose-1,5-bisphosphate
carboxylase/oxygenase produced mRNA but no large subunit protein or
enzyme activity. Those tobacco plants expressing the sunflower large
subunit synthesized a catalytically active hybrid form of the enzyme
composed of sunflower large subunits and tobacco small subunits. A
third line expressed a chimeric sunflower/tobacco large subunit arising
from homologous recombination within the rbcL gene that
had properties similar to the hybrid enzyme. This study demonstrated
the feasibility of using a binary system in which different forms of
the rbcL gene are constructed in a bacterial host and
then introduced into a vector for homologous recombination in
transformed chloroplasts to produce an active, chimeric enzyme in vivo.
1
This research was supported by the National
Science Foundation (grant no. MCB 93-05037) and by DuPont Science and
Engineering (grant to P.M.).
*
Corresponding author; e-mail steven.gutteridge{at}usa.dupont.com;
fax 1-302-366-5738.
Plant Physiol. (1999) 119: 133-142
Copyright Clearance Center: 0032-0889/99/119//10
© 1999 American Society of Plant Physiologists
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