To engineer the pathway toward -carotene formation, it is
necessary to complement four plant enzymes, namely phytoene
synthase, phytoene desaturase, -carotene desaturase
(the desaturases catalyzing the introduction of two double
bonds, each), and lycopene -cyclase. Alternatively, the transformation
effort can be simplified by reducing the number of
enzymes required and using a bacterial carotene desaturases
capable of introducing all four double bonds required (Fig. 1).
Initially, we sought to introduce all genes into immature
rice embryos (TP 309) stepwise, i.e., singly by particle bombardment,
aiming at subsequently unifying all transgenes into
a single plant by subsequent crossing. However, this approach
was not successful, mainly due to the deleterious integration
pattern frequently produced by this transformation technique,
as revealed by Southern hybridization analysis. Therefore,
Agrobacterium-mediated transformation of precultured rice immature
embryos was used, designed so as to install the entire
-carotene biosynthetic pathway into rice endosperm in a
single transformation effort. Three vectors, schematically depicted
in Figure 2, were constructed. pB19hpc combines the
sequences for a plant phytoene synthase (psy) originating from
daffodil (Narcissus pseudonarcissus; accession no. X78814) (4)
with the sequence coding for a bacterial phytoene desaturase
(crtI) originating from Erwinia uredovora (accession no.
D90087), the two being placed under the control of the
endosperm-specific glutelin (Gt1) and the constitutive CaMV
35S promoter, respectively. The phytoene synthase cDNA
contained a 5-sequence coding for a functional transit peptide,
as was demonstrated previously (5), while the crtI gene
was fused to the transit peptide sequence of the pea Rubisco
small subunit (tp), as constructed by Misawa et al. (6). This
plasmid, thus, should direct the formation of lycopene in the
endosperm plastids, the site of GGPP formation.
To engineer the pathway toward -carotene formation, it isnecessary to complement four plant enzymes, namely phytoenesynthase, phytoene desaturase, -carotene desaturase(the desaturases catalyzing the introduction of two doublebonds, each), and lycopene -cyclase. Alternatively, the transformationeffort can be simplified by reducing the number ofenzymes required and using a bacterial carotene desaturasescapable of introducing all four double bonds required (Fig. 1).Initially, we sought to introduce all genes into immaturerice embryos (TP 309) stepwise, i.e., singly by particle bombardment,aiming at subsequently unifying all transgenes intoa single plant by subsequent crossing. However, this approachwas not successful, mainly due to the deleterious integrationpattern frequently produced by this transformation technique,as revealed by Southern hybridization analysis. Therefore,Agrobacterium-mediated transformation of precultured rice immatureembryos was used, designed so as to install the entire-carotene biosynthetic pathway into rice endosperm in asingle transformation effort. Three vectors, schematically depictedin Figure 2, were constructed. pB19hpc combines thesequences for a plant phytoene synthase (psy) originating fromdaffodil (Narcissus pseudonarcissus; accession no. X78814) (4)with the sequence coding for a bacterial phytoene desaturase(crtI) originating from Erwinia uredovora (accession no.D90087), the two being placed under the control of theendosperm-specific glutelin (Gt1) and the constitutive CaMV35S promoter, respectively. The phytoene synthase cDNAcontained a 5-sequence coding for a functional transit peptide,as was demonstrated previously (5), while the crtI genewas fused to the transit peptide sequence of the pea Rubiscosmall subunit (tp), as constructed by Misawa et al. (6). Thisplasmid, thus, should direct the formation of lycopene in theendosperm plastids, the site of GGPP formation.
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