BIOTECHNOLOGY
Applications of Biotechnology
PRODUCTIVE FACT BITESl The plants obtained through genetic engineering containing a gene,
usually from an unrelated organism are known as transgenic plants.
l Transgenic plants have proved to be extremely valuable tools in
studies on plant molecular biology, regulation of gene action and
identification of regulatory/promotory sequences, e.g. T-DNA and
transposable elements produce mutations by becoming inserted
within genes and thereby act as molecular tags for gene
identification and isolation.
l Transgenic plants also permit the analysis of metabolic pathways,
studies of cis and trans-acting factors in gene function and the
elucidation of plant responses to environment stresses, etc.
l A typical plant gene has regions beginning with 5 ′-end-promoter,
enhancer/silencer, transcriptional start or cap site, an untranslated
region or leader sequence, initiation codon, exons, introns, a second
untranslated region and lastly a poly-A tail.
l The 5 ′ leader sequence of mRNA is the untranslated sequence lying
between cap site and ATG. The end of gene coding sequence is
specified by chain termination codon TAG or one of the other two
nonsense codons.
l A variety of promoter sequences have been used to drive genes in
plant cells, such as nos (nopaline synthase), ocs (octopine synthase)
and mas (mannopine synthase) promoters from Agrobacterium.
l 35 S-RNA gene pro moter of Cau li flower Mo saic Vi rus (CMV) is the
most commonly used constitutive promotor both in dicots and
monocots. It is 10-40 folds more efficient than the nos promoter.
l The maize alcohol dehydrogenase 1 (Adh 1) promoter shows
anaerobic induction, i.e. expression in roots. The Adh 1 promoter
activity in monocots is either comparable to or higher than that of
35 S promoter.
l Expression of many genes is confined to specific tissues or induced
by specific stimuli, such genes are called tissue specific or
stimulus-responsive genes respectively.
l Such specificities in gene expression are due to certain DNA
sequence called enhancers or silencers, either lying within or at a
considerable distance (upto several kbs) away from the promoters
they affect.
l An enhancer may be defined as a DNA sequence which increases the
activity of a promoter gene while DNA sequences suppressing
promoter activity are called silencers. Thus, enhancer and silencer
sequences regulate the activity of promoters but are not themselves
involved in promoter activity.Applications in Plant Biotechnology
l The silencer sequence associated with the gene for alcohol
dehydrogenase 1 (Adh 1) of maize is an example of negative
regulating element. This gene is expressed only under anaerobic
conditions, and hence only in roots. The Adh 1 enhancer sequence
seems to suppress Adh 1 expression under aerobic conditions. Adh 1
is expressed because the enhancer is unable to suppress its
expression in the absence of O 2 stimulus.
l Several varieties of insect resistant transgenic crops have been
successful. Such resistant plants contain either a gene from the
bacterium B. thuringiensis or cowpea trypsin inhibitor gene.
l These genes produce crystal proteins that form crystalline
inclusions and thus are responsible for insecticidal activities. The cry
genes have been grouped into 16 distinct groups which either code
for a 130 k Da or 70 kDa protein.
l The toxic function of these proteins is localised in the N-terminal
half of 130 kDa proteins. The C-terminal half of these proteins is
highly conserved and most likely involved in crystal formation.
l None of the truncated proteins crystallises in the typical
bipyramidal shape of most of the 130 kDa proteins.
l The Cry I proteins are insecticidal to Lepidopteran insects, all the
proteins even the Cry IA subfamily, have a distinctive insecticidal
spectrum.
l The Cry II A proteins are active against both Lepidoptera and Diptera,
while Cry II B is specific to Diptera. Their homology with other Cry
proteins is rather limited.
l The Cry III proteins are active against Coleoptera species, while
Cry IV proteins are specific to Diptera.
l The Cyt A protein does not show any insecticidal activity but is
cytolytic for a variety of vertebrate and invertebrate cells. It does
not exhibit any homology with other Cry proteins.
l Truncated cry genes are used for the production of transgenic
plants since, the level of expression of complete genes in transgenic
plants is extremely low.
l The Cry I protein A genes have been successfully transferred into
tobacco, potato and tomato.
l The transgenic tobacco plants expressing Cry protein at about
0.004% of their total leaf protein killed all M. sexta larvae within
6 days. Similar results were obtained with transgenic tomotoes for
insects Manduca sexta and Heliothis virescens.