Contents - Faperta

Genetic Transformation of Crops for Resistance to Insect Pests 213
FIGURE 7.1 Selection of putative transgenic plants of sorghum with the herbicide glufocinate (BASTA) as a
selection marker.
an identifi able protein product to indicate DNA incorporation. Construction of DNA
sequence for incorporation into vectors consists of several components, for example, in
toxin genes from Bt, the gene should be fi rst converted from AT-rich (typical of bacteria) to
GC-rich (typical of higher plants) to increase toxin expression. Most changes are made to
the third codon, thereby minimizing changes in the amino acid sequence, and increasing
the expression of Bt toxin by 10- to 100-fold. For expression of the Bt gene in the higher
plants, a recognizable promoter and a terminator sequence must bracket the Bt gene.
Popular constitutive promoters as described above include CaMV35S, ubiquitin, PEPC, and
maize pollen specifi c promoter (Koziel et al., 1993). The size of the vectors ranges from
5,000 to 11,000bp, depending on the Bt gene and the promoter incorporated into the vector.
Delivery of the vectors into the nucleus can be achieved by using Agrobacterium-mediated
transformation and biolistic methods (Koziel et al., 1993).
Premature polyadenylation at times may lead to poor expression of the transgene
(Haffani et al., 2000). Transformation of potato with the cry3Ca1 gene resulted in transformants
with poor expression of the transgene (Kuvshinov et al., 2001). No full-length
transcript (2,300 nucleotides) was detected, but short transcripts of approximately 1,100
nucleotides were observed. The sites at which premature polyadenylation took place were
not those that showed the highest degree of identity to the canonical AAUAAA motif, suggesting
that premature polyadenylation may contribute to poor expression of transgenes
in a foreign host. Expression of the transgene may also depend on the plant species in
which the gene has been deployed. Expression level of the synthetic gene cry9Aa under the
control of double 35S promotor has been observed to be three to ten times lower in potato,
caulifl ower, and turnip as compared to that in tobacco (Kuvshinov et al., 2001). In tobacco
plants transformed with a truncated native cry9Aa gene and with a translational fusion
construct of the truncated native cry9Aa and uidA (GUS) gene, the expression level of the
native cry9Aa gene ranged from 0.03 to 1 pg of cry9Aa mRNA per 1 μg of total RNA, while
the expression level of the synthetic cry9Aa gene was fi ve to ten times higher at the mRNA
level, and at least 50 times higher at the translational level. Therefore, considerable care
should be exercised while selecting a gene construct, the promotor, and the marker genes
for genetic engineering of plants for resistance to insects.