ЭлБиб

PLASTID TRANSFORMATIONINHIGHERPLANTS: NEW

OPPORTUNITIESFORBASICSCIENCEANDBIOTECHNOLOGICAL

APPLICATIONS

PALMALIGA

WaksmanInstitute, Rutgers, TheState University o fNewJersey, Piscataway, N J08854-

8010;fax: (732)445-5329; E-Mail: m

aliga@mbcLrulgers.edu

The circular, 120- to 180-kb circular genome of plastids is present in

500 to 10,000 copies per cell. Introduction of foreign genes into the plastid

genome is achieved by targeted gene insertion. The gene of interest is cloned

next to a selectable marker gene which is flanked by plastid DNA targeting

sequences in a plasmid vector. Biolistic transformation is followed by

integration o f the linked transgenes and elimination of the wild-type plastid

genome copies during repeated cell divisions. Efficient transformation

protocols rely on selection for a chimeric aadA gene encoding spectinomycin

resistance (1, for review see ref. 2).

Plastid transformation has been applied to study plastid biology (3),

gene regulation (4,5) and RNA editing (6). The plastid genome is an attractive

target for engineering since: proteins in plastids may be expressed at a

high-level (7,8); genes for pathways may be encoded in polycistronic mRNAs;

transgenes are uniformly expressed due to targeted insertion into the plastid

genome; the transgenes don't spread via pollen. A good example for high

level protein expression is accumulation of the

Bacillus thuringiensis

protoxin

to 3-5% of the soluble protein in tobacco leaves from a plastid-encoded

crylA(c) gene (8).

Expression of plastid transgenes in different cell types from tissue-

specific promoters would be desirable. So far foreign genes in plastids have

been expressed from promoters transcribed by the plastid-encoded

E.coli-

like RNA polymerase (PEP). PEP transcribes photosynthetic and

housekeeping genes. Housekeeping genes typically have a second promoter

recognized by a nuclear-encoded plastid RNA polymerase (NEP; ref. 5)

which share a 10 nucleotide consensus sequence around the transcription

initiation site. Most NEP promoters are inactive in photosynthetic cells, and

their pattern of expression is unknown. Our efforts are focused on the

identification of NEP promoters which may be suitable for the expression of

foreign proteins in a tissue and/or cell-type specific manner.

Plastid engineering is routine only in tobacco. Adaptation of the

technology to other species is a challenge for the coming years. We are

currently working on plastid transformation in

Arabidopsis

and rice.

References

(1) Svab Z, Maliga P (1993) PNAS 90:913; Zoubenko et al. (1994) NAR

22:3819.

(2) Maliga, P (1993) Trends in Biotechnology 11:101.

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