Title of Invention


Abstract The present invention relates to the isolation of a nucleic acid sequence, the product of which, confers protection to plants, animals and human beings against environmental stress, improves oxidative stress tolerance, enhances root development and increases the post harvest shelf life of the plant or plant parts thereof. The invention relates primarily to the field of plant genetic engineering.
Full Text

to pinkish greyish bark has been used in India's native Ayurvedic medicine for over three centuries, primarily as a cardiac tonic. A clinical evaluation of this botanical medicine indicates that it can be used in the treatment of coronary artery disease, heart failure, hypercholestrolemia and provide relief from anginal pain. It is also reported to have anti bacterial and antimutagenic properties (Miller, 1998). Kumar and Prabhakar, 1987, reported on its ability to cure hepatic, urogenital, venereal and viral diseases. The active constituents in Terminalia arjuna include tannins, triterpenoids, saponins (arjunic acid, arjunolic acid, arjungenin, arjunglycosides), flavonoids (arjunone, arjunolone, luteolin), gallic acid, ellagic acid, oligomeric proanthocynadins (OPCs), phytosterols, calcium, magnesium, zinc and copper. The mechanism of action involves improvement of cardiac muscle function and the subsequent improved pumping activity of the heart and this seems to be the primary benefit of Terminalia arjuna. Saponins Glycosides are considered to be responsible for the inotropic effects of Terminalia arjuna, whereas the flavonoids and OPCs provide free radical antioxidant activity and vascular strengthening. The constituents of Terminalia arjuna have not been extensively studied. A study of this would be beneficial in understanding this botanical plant.
The bark is astringent, sweet, acrid, cooling, and acts as an aprhodisiac, demulcent, cardiac tonic, styptic, anti dysenteric, urinary, astringent, expectorant, alexiteric, lithontriptic tonic. It is useful in the treatment of fractures, ulcers, leucorrhoea, diabetes, anaemia, caridopathy etc. The bark contains a crystalline compound, aijunine, a lactone arjunetin, essential oils, tannin, reducing sugar and colouring matter (Nayar & Chopra, 1956). Besides Terminalia arjuna's four active constituents, Arjunic acid, a trihydroxyrtiterpene, along with P-sitosterol ellagic acid, and the glucoside-Arjunetin is isolated from the bark and sapogenin arjungenin is isolated and characterised as 2a, 2P, 19a, 23 tetrahydroxyolean-12-en-28-oic acid; two saponins, arjunglucoside-I and arjunglucoside-II, have been isolated and characterised (Rastogi and Mehrotra, 1990). The genus is chemically characterised by thepresence of tannins and related compounds (Tanaka et al, 1986, Okuda et al., 1981, Haseam et al., 1967, Lin et al, 1990, Tanaka et al., 1991).
In a highly competitive environment of contemporary pharmaceutical research, natural products (secondary metabolites) provide a unique element of molecular diversity and biological functionality, which is indispensable for drug discovery. Plants have evolved through an immense variety of biochemical pathways. The plasticity of plant metabolic activity is most evident in the variety of secondary metabolites accumulated by plants in their leaves, roots and other organs. The use of the whole plant preparation or the extracts thereof for medicinal purposes goes a long way back, in fact much before history was recorded. In recent times many plant derived products have reached the market as useful drugs for treating human disorders including atropine, hyoscyamine, scopolamine, taxol (anticancer), artemisin, quinine (antimalarial). Plants are now considered as metabolite factories for production of a variety of compounds that are of medicinal, nutritional and industrial values. On a more functional level, the application of molecular techniques has permitted the manipulation of biosynthetic pathways for a generation of novel chemical species.
The role of secondary metabolites has been much debated upon. Terpenoids are the largest family of natural products and they play a vital role in plants. They are widespread in nature, the building block of terpene is hydrocarbon isoprene CH =

C(CH3)-CH = CH2. What is common in these substances are the isoprene units of the 5-carbon atoms, which, after the subsequent polymerisation reaction, give rise to numerous isoprenoids (Chappell, 1995; Weissenborn et al., 1995).
Terpene hydrocarbons have molecular formula (C5 H8)n. They are classified according to the number of isoprene units. Monoterpenes (Camphor, menthol, limonene), diterpene (phytol and vitamin A), triterpene (squalene) and tetraterpene (carotene) are also widely prevalent in plants. Compounds like alcohol, aldehydes or ketoses group containing oxygen are called terpenoids. The enzymes involved in their biosynthesis are hard to isolate. Modem methods for recombinant DNA technology bye pass this difficulty and allow direct isolation of the genes. A further study of these genes will lead to an understanding of the various metabolic steps and regulation of this complex but economically significant metabolic pathway and this knowledge will assist in manipulating the genes in enhancing the production of these metabolites.
The first step in the synthesis of the triterpenoid starts from the mevalonic acid (MVA) which is catalysed by the 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR), Mevalonate, is a six carbon unit sequentially phosphorylated and decarboxylated to produce isopentylpyrophosphate (IPP) by mevalonate kinase and mevalonate 5-diphosphate decarboxylase enzyme. The IPP and Dimethylallyl Pyrophosphate (DMAPP), isomers of IPP from the conversion reaction catalysed by the IPP isomerase are the building blocks for the synthesis of all other isoprenoid compounds. The condensation of DMAPP with IPP produces Geranyl Pyrophosphate (GPP) and the addition of another IPP molecule will give rise to Famesyl Pyrophosphate (FPP) and the subsequent addition of IPP to this produces Geranylgeranyl Pyrophosphate (GGPP) (Chappell, 1995). Poulter and Rilling, 1981, reported that the isoprenoid metabolism is catalysed by Prenyl Transferases. Famesyl Diphosphate Synthases (B.C. which is the control enzyme in the r-4 chain elongation process. It catalyzes the sequential condensation of DMAPP and Geranyl Diphosphate with IPP. The product. Famesyl Diphosphate, gives rise to several branches in the pathway which supply C 15 precursors for several classes of essential metabolites including sterols, dolichols, ubiquinone and triterpenoids. Squalene monxygenase (Squalene epoxidase) (B.C. is involved in sterol and triterpenoid biosynthesis in almost all the eukaryotes (Gaik et al., 1983).
The properties and corresponding DNA sequences of squalene monoxygenase from mammal and fungi have been investigated into (Favre & Ryder, 1997; Jandrosite et al., 1991, Kosuga et al., 1995: Sakakibara et al, 1995; Satoh et al, 1993). Three sequences of Arabidopsis thaliana and two of Brassica napus cDNAs encoding Squalene Monoxygenase homolog (Sqp 1 and Sqp2) have been reported by Schafer et al, 1999.
A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from the Citrus sinensis salt-treated library. A sequence homology of the same revealed a considerable homology to mammalian glutathione peroxidase.
There are reports of the presence of Glutathione peroxidase in pharmaceutical compositions for use in the treatment of cancer as an antioxidant.

The compound has also been reported to be useful in drugs for the prevention of cataracts. It is also known to prevent Hyperglycemia, induced free radical cell damage and may thereby help in reducing the vascular complications associated with diabetes.

The present invention relates to the isolation of a nucleic acid sequence, the product of which, confers protection to plants, animals and human beings against environmental stress, improves oxidative stress tolerance, enhances root development and increases the post harvest shelf life of the plant or plant parts thereof. The invention relates primarily to the field of plant genetic engineering.
Plants which express genes in either or both sub cellular compartments have revealed increased glutathione content in the leaves and/or in the developing fruit. The increased anti oxidant capacity resulting from sustained, elevated glutathione content is expected to be beneficial to the plant including resistance to a number of potentially damaging oxidative events, arising from both, biotic and abiotic stress.
The methods and vectors of the present invention may play a significant role in increasing tolerance to abiotic and biotic stress, enhance plant capacity for anti oxidant regeneration and leading to improvements in water use and nutrient uptake.
According to the present invention, there is provided a method of manipulating the oxidative status of a plant, wherein the genes are differentially expressed under the control of different promoters. In the alternative, the construct also includes one gene encoding an enzyme involved in the redox cycling of glutathione between its reduced and oxidized forms. Preferably the gene encoding an enzyme involved in the redox cycling of glutathione is a gene encoding the glutathione peroxidase. The nucleic acid construct will most preferably comprise of the glutathione peroxidase gene. The alteration in the oxidative status may be assessed by comparing with a plant in which the nucleic acid has not been introduced. Preferably the construct is comprised within the vector. Wherever reference is made to genes encoding an anti oxidant enzyme, capable of reducing oxidized glutathione, it should be understood that except where the context demands otherwise, variants, both, artificial and natural, may be used as long as the variant forms retain the ability to encode a polypeptide with an appropriate corresponding enzymatic capability. Variants may used to alter the oxidative stress resistance characteristics of the Terminalia arjuna plant. In the alternative they may include a sequence which interferes with the expression or activity of the polypeptide, namely, the sense or anti sense suppression.
Variants may be naturally occurring nucleic acids or they may be artificial. They may include orthologues, alleles, isoalleles or homologues of the gene. The variants may include only a distinctive part or fragment corresponding to a portion of the relevant gene, encoding at least functional parts of the polypeptide. The vectors will be stable and capable of modifying the production and/or redox cycling of the glutathione in organisms in which they are expressed.
Homology may be at the nucleotide sequence and/or at the amino acid sequence level, around 70% homology. Homology may be over the full length of the relevant sequence.
Changes to a sequence may produce a derivative by way of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid leading to the

addition, deletion, insertion or substitution of one or more amino acids in the encoded polypeptide. Such changes may alter sites required for post translation alteration in the encoded polypeptide. Altering the primary structure of a polypeptide may not significantly alter the activity of that peptide since the side chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted. Substitutions to regions of a peptide which are not critical in determining its confirmation because they may not significantly alter the three dimensional structure of the peptide. In regions that are critical in determining the confirmation or activity of the peptide, such changes may confer advantageous properties on the peptide, e.g. altered stability or specificity.
Vector is defined to include any plasmid, cosmid, phage or agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilisable and which can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally, namely, autonomous replicating plasmid with an origin of replication. Preferably the vector is a plasmid. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences including promoter sequences, polyadenylation sequences, enhancer sequences, marker genes, and other appropriate sequences. Shuttle vector refers to a DNA vehicle capable, naturally or by design, of replication in two different host organisms, e.g. higher plant, yeast or fungal cells.
The nucleic acid in a vector is under the control of and operably linked to two appropriate promoters or other regulatory elements for transcription in a host cell such as a plant cell, operably linked to a different promoter. The vector may be a bi-functional expression vector which functions in multiple hosts and in the case of the genomic DNA, it may contain its own promoter or other regulatory elements and in the case of cDNA, it may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
Promoter is a sequence of nucleotides from which the transcription may be initiated of DNA operably linked downstream. Operably linked means, joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is under transcriptional initiation regulation of the promoter. In a preferred embodiment, at least one of the promoters is an inducible promoter and the expression refers to the 'switching on' or increase in response to an applied stimulus. The nature of the stimulus varies between promoters. Expression from any inducible promoter is increased in the presence of the correct stimulus.
If desired, select genetic markers may be used in the construct, namely, those that may be used to confer select phenotypes such as resistance to antibiotics or herbicides.
In a further aspect of this invention, there is disclosed a host cell containing a heterologous construct, especially a plant cell. The term heterologous is used broadly to indicate that the gene/sequence of the nucleotide have been introduced into the said cell of the plant or an ancestor thereof using genetic engineering, i.e., by human intervention. A heterologous gene may replace an equivalent, endogenous gene, i.e., one which performs normally the same or similar function or the inserted sequence

may be additional to the endogenous gene or other sequence. Nucleic acid heterologous to a plant cell may be non naturally occurring in cells of that type, variety or species and may comprise a coding sequence of or derived from a particular type, variety or species. The nucleic acid sequence could also be placed within a cell in which it or a homologue is found naturally but where the nucleic acid sequence is linked and/or adjacent nucleic acid sequence which does not occur naturally within the cell or cells of that type or specie or variety of plant, such as operably linked to one or more regulatory sequences such as a promoter sequence, for control of expression.
The host cell, namely the plant cell, is preferably transformed by the construct, i.e. the construct becomes established within the cell altering one or more of the cell's characteristics and hence the phenotype, e.g., with reference to the anti oxidant capacity due to the elevated glutathione content.
Nucleic acid can be introduced into plant cells by using any suitable technology.
The invention also encompasses a host cell, especially a plant cell transformed with a vector and the gene under the control of different promoters to enable differential expression.
In addition to the regenerated plant, the invention also covers the following, namely, clone of the plant, seed or hybrid progeny and descendants. The invention also provides a plant propagule from the plant, i.e., any part which may be used in reproduction or propagation, sexual or asexual including cuttings, seed etc. It also provides for any part of the plant including the plant cell heterologus to the gene involved in the cycling of the glutathione between the reduced and the oxidized form.
The plant involved in the present invention has been found to have improved root weight and development in comparison to control plants, enabling improved water and nutrient uptake. Further, the plant has been found to have enhanced glutathione level.
In addition, the invention enhances the tolerance of the plant to oxidative stress.

We claim:
1. A nucleic acid sequence which comprises of a gene encoding glutathione
peroxidase, possessing agronomic properties of utility and methods of arriving
at the same from Terminalia arjuna.
2. A claim as in claim 1 wherein the construct encodes the gene.
3. A claim as in claim 2 wherein the construct comprises of the gene being
operably linked to a promoter to enable differential expression.
4. A claim as in claim 3 wherein the gene operably linked to a promoter encodes
an enzyme involved in the redox cycling of the glutathione between its
reduced and oxidised forms.
5. A claim as in claim 4 wherein the construct involved in the redox cycling is
glutathione peroxidase.
6. A claim as in claim 4 wherein the enzyme involved in the redox cycling is glutathione peroxidase.
7. A claim as in claim 4 wherein the promoter is an inducible promoter.
8. A claim as in claims 4 and 7 wherein the promoter is heterologous to the gene
with which it is operably linked.
9. A claim as in claim 5 wherein the construct is a plant binary vector.
10. A claim as in claim 9 wherein the vector comprises of suitable genetic
11. A method of producing a transgenic plant.
12. A method of providing for a plant having reduced levels of glutathione.
13. A method of improving oxidative stress tolerance of the plant.
14. A method of enhancing root development of a plant
15. A claim as in claims 1, 11, 12, 13 and 14 wherein the said method can be
applied to all plants.


0777-che-2003 complete specification as granted.pdf

777-che-2003 abstract 22-07-2009.pdf

777-che-2003 abstract 28-07-2009.pdf

777-che-2003 claims 22-07-2009.pdf

777-che-2003 claims 28-07-2009.pdf

777-che-2003 correspondence others 22-07-2009.pdf

777-che-2003 correspondence others 28-07-2009.pdf

777-che-2003 description(complete) 22-07-2009.pdf

777-che-2003 description(complete) 28-07-2009.pdf

777-che-2003 form-1 28-07-2009.pdf





777-che-2003-form 1.pdf


Patent Number 237254
Indian Patent Application Number 777/CHE/2003
PG Journal Number 51/2009
Publication Date 18-Dec-2009
Grant Date 11-Dec-2009
Date of Filing 24-Sep-2003
# Inventor's Name Inventor's Address
PCT International Classification Number C12P15/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA