Title of Invention


Abstract The present invention relates to the isolation of plant nucleic acids and DNA encoding Glutamate decarboxylase, which, emerge in the plants subjected to environmental stress conferring thereby conferring valuable pharma and agro properties.
Full Text

US2003110530 patent application describes a novel transgenic plant having enhanced resistance to invertebrate pests is provided. The plant is transformed with a recombinant nucleic acid encoding a functional glutamate decarboxylase (GAD).
The gabaergic pathways represent the main group inhibiting the nervous system in vertebrates. Impairment of the activity of the GABAergic neurons manifests itself immediately in the whole body by dyskinesias or convulsions. Furthermore, the role of cerebral gamma-amino-butyric acid (GABA) is not limited to neurotransmission. A neurotrophic action during development has indeed been attributed to it, in particular in the neuroretina. Moreover, GABA is also present in the beta cells of the islets of Langerhans, where it seems to play a role in the regulation of the production of insulin.
The possibility of restoring or installing de novo a GABA synthesis in a precise region of the body therefore has a major therapeutic advantage both for conditions directly linked to the degeneration of GABAergic neurons and for those which respond to GABA agonists. The present invention thus describes a new approach for treating conditions linked to a GABA deficiency, consisting in inducing the in vivo synthesis of GABA by targeted release of a biologically active enzyme.
Glutamate decarboxylase activity (GAD) is an enzyme which catalyses, in a relatively specific manner, the conversion of glutamate to gamma-amino-butyric acid (GABA) with the aid of a cofactor, pyridoxal phosphate (vitamin B6). In the native state, this enzyme exists in the form of a dimer of 120 kilodaltons. After reduction of the disulphide bridges, examination by specific electroimmunoanalysis (Western blotting) reveals two bands at 65 and 67 kilodalton. It was recently demonstrated that these two monomers correspond to two different proteins encoded by distinct genes (Eriander et al., Neuron 7 (1991) 91). The two molecules, hereinafter called GAD 67 and GAD 65, differ from an enzymatic point of view, the short form having a lower affinity for pyridoxal phosphate, and by their subcellular localization, the short form being better represented in the neuronal extensions whereas the long form accumulates in the perikaryon. The long form, by virtue of its lower dependence on the concentration of pyridoxal phosphate, is therefore the one most capable of being expressed in various types of cells. GAD 65 on the other hand has the feature of becoming rapidly inactivated in the absence of pyridoxal phosphate by becoming converted to an apoenzyme, lacking catalytic activity. The reverse conversion, from apoenzyme to active holoenzyme, is relatively slow.
GABA is a naturally-occurring inhibitory neurotransmitter which has ready access to the nervous system of invertebrates, but not that of vertebrates such as man, and has been shown to deter insect grow and development (Ramputh and Bown, 1996 Plant Physio. 111:1349). Typically, GABA levels are low in plants (ranging from 0.03 to 2.00 [mujmol/g fresh weight (FW)), but increase several fold in response to many diverse stimuli such as insect walking and feeding (i.e. biotic stress) and temperature shock (i.e. abiotic stress). This result a be attributed to increases in cytosolic H+ or calcium/calmodulin levels which directly affect the activity of the enzyme responsible for the synthesis of GABA, namely glutamate decarboxylase (GAD) (FIG. 1; Shelp et al., 1999 Tr. Plant Sci. 4:446). In particular, calcium/calmodulin binds to a carboxyl-

terminal domain on the GAD gene, thereby relieving the autoinhibition of GAD activity. Although the endogenous synthesis of GABA appears to serve as a plant defense mechanism, further increases in GABA levels may lead to corresponding reductions in damage due to invertebrate pests Transgenic plants which overexpress GAD and thereby cause GABA accumulation have been prepared; however, these plants do not express invertebrate pest-resistant root GABA levels and have questionable utility in that they exhibit severe morphological abnormalities (Baum et al., 1996 EMBO Journal, 15:2988).
1. mRNA purification was performed by first, isolating high quality total RNA from 6 day old RASI seedlings and, subsequently by isolating mRNA from total RNA using oligo (dT) cellulose in a filter syringe by making use of a double purification method.
2. mRNA was converted into first and second strand cDNA followed by Sal I adapter addition, Not I digestion, cDNA vector ligation and transformation to obtain the cDNA library.
3. The superscript plasmid system with Gateway for cDNA cloning and synthesis was employed throughout.
4. The clones obtained were picked, digested using Not I and Sal I enzymes, to obtain the inserts and these were further sequenced and checked for homology.
5. The sequencing of the selected clones was done on ABI Prism, 377, DNA Sequencer (Perkin Elmer).
The usefulness of the present invention lies in the fact that the Glutamate decarboxylase permits a high level of production of gamma aminobutyric acid, which, apart from being a nerve inhibitory mediator, is also useful in the development of medicines or food materials.
Further molecular studies reveal the mechanism by which the gene is controlled in the plants, which could thereby increase the efficiency of nitrogen metabolism in plants and thus improve, crop quality and production.

We claim
1. The method of developing transgenic plants overexpressing Glutamate
decarboxylase thereby showing high levels of production of gamma
aminobutyric acid, which, apart from being a nerve inhibitory mediator, is
also useful in the development of medicines or food materials.
2. The method of transforming plants with nucleic acids encoding a functional
glutamate decarboxylase, thereby generating a transgenic plants having
enhanced resistance to pests.
3. A claim as in claim 1 & 2, wherein, the transgenic plants could apply to all
varieties of plants.
4. A claim as in claim 2, wherein the transgenic plants confer increased tolerance
to pests.
5. A claim as in claim 1 & 3, wherein the transgenic plants have increased
efficiency of nitrogen metabolism in plants and thus irnproved, crop quality and production.
6. A claim as in claim 1, wherein, the product of Glutamate decarboxylase
gene expression, GABA, would be useful in the development of medicines
or food materials.


771-che-2003 abstract-06-07-2009.pdf

771-che-2003 claims 24-08-2009.pdf

771-che-2003 claims-06-07-2009.pdf

771-che-2003 correspondence others-06-07-2009.pdf

771-che-2003 correspondence others-11-08-2009.pdf

771-che-2003 description(complete)-06-07-2009.pdf

771-che-2003 drawings-06-07-2009.pdf

771-che-2003 form-1-06-07-2009.pdf




771-che-2003-form 1.pdf

Patent Number 241630
Indian Patent Application Number 771/CHE/2003
PG Journal Number 30/2010
Publication Date 23-Jul-2010
Grant Date 16-Jul-2010
Date of Filing 24-Sep-2003
# Inventor's Name Inventor's Address
PCT International Classification Number C12N15/82
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA