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PLANT PHYSIOLOGY , Vol 103, Issue 4 1285-1290, Copyright © 1993 by American Society of Plant Biologists
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MOLECULAR BIOLOGY AND GENE REGULATION |
Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco
T. Brears, C. Liu, T. J. Knight and G. M. Coruzzi
Department of Biology, New York University, 1009 Main Building, Washington Square East, New York, New York 10003 (T.B., C.L., G.M.C.)
Here, we monitor the effects of ectopic overexpression of genes for pea
asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The
AS genes of pea and tobacco are normally expressed only during the dark
phase of the diurnal growth cycle and specifically in phloem cells. A
hybrid gene was constructed in which a pea AS1 cDNA was fused to the
cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore
ectopically expressed in all cell types in transgenic tobacco and
constitutively expressed at high levels in both the light and the dark.
Northern analysis demonstrated that the 35S-AS1 transgene was
constitutively expressed at high levels in leaves of several independent
transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold
increase in free asparagine in leaves of transgenic 35S-AS1 plants
(construct z127) compared with controls. Plant growth analyses showed
increases (although statistically insignificant) in growth phenotype during
the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1
construct was further modified by deletion of the glutamine-binding domain
of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we
reasoned that inhibition of glutamine-dependent AS activity might enhance
the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine
levels in the transgenic plants expressing gln[delta]AS1 compared with wild
type suggests that the novel AS holoenzyme present in the transgenic plants
(gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These
data indicate that manipulation of AS expression in transgenic plants
causes an increase in nitrogen assimilation into asparagine, which in turn
produces effects on plant growth and asparagine biosynthesis.
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