|Title of Invention||
UTILITY OF THIONIN GENE POSSESSING SIGNIFICANT AGRO AND PHARMA PROPERTY USES THEREOF
|Abstract||The present invention relates to the temporal dynamics of salinity stress responses, providing insight into the response mechanisms that contribute to survival, following exposure to high salinity stress. The gene includes within its ambit, a higher degree of resistance to fungal infection. This invention relates to the emergence of the thionin gene from the RASI variety of rice seedlings under environmental stress conditions.|
DNA construct, vectors and transformed plants each comprising the gene or part thereof
Drought and soil salinity, in both, dry land as well as irrigated agricultural settings are the most important factors limiting modem agricultural production system (Cushman & Bohnert, 2000). Plants, being sessile organisms, have developed a sophisticated and complex set of adaptive responses allowing them to withstand abiotic and environmental insults. This complexity has stymied traditional selection based breeding approaches to isolate germ plasm with improved salinity or drought tolerance (Flowers & Yeo, 1995).
Genetic and molecular studies have determined that many gene products contribute to salinity and drought tolerance (Hasegawa et al., 2000). Major classes of salt or drought tolerant determinants include osmo-protectants, ion carriers and channels, transporters and symporters, water channels, reactive oxygen scavengers, heat shock proteins, various stress proteins of unknown functions, transcriptional activators and signalling moleculars (Hasegawa et al, 2000; Cushman, 2001).
Genome sequencing and cDNA clone analysis promise to rapidly isolate and identify all the genes of the 'osmome', 'xerome', or, the 'thermome', the complement essential for tolerance to osmotic dessication and heat or cold stress tolerance respectively (Cushman & Bohnert, 2000).
Thionins are highly abundant polypeptides with anti fungal activities. These polypeptides are located in cell walls of leaf cells and the synthesis of thionine mRNA was increased after fungal attack (Bohlmann et al., 1988). Expression of the a-thionin gene from barley in transgenic tobacco has been shown to confer resistance to bacterial pathogens (Carmona et al., 1993).
The in vitro toxicity against plant pathogenic bacteria and fungi indicates the role of thionin in the resistance of plants to the said bacteria and fungi. The induction of the thionin gene in response to salt stress identifies this gene with the osmome of the complement essential for tolerance towards osmotic dessication. The usefulness of the emergence of the thionin gene under salt stress lies in the fact that the rice plant harbouring one or several anti fungal disease genes can control the fungal diseases, thereby minimising the use of chemical fungicides.
Thionins are small, highly basic, Cys-rich proteins that show antimicrobial activity and seem to have a role in plant defense against fungi and bacteria. The overexpression of the THI2.I thionin in Arabidopsis enhanced resistance to a phytopathogenic fungus (Epple et al., 1997). The overexpression of alpha -hordothionin in tobacco also enhanced resistance to a phytopathogenic bacterium (Carmona et al,, 1993). In addition, during barley and powdery mildew interactions, the accumulation of thionins was higher in the incompatible interaction than in the compatible one (Ebrahim-Nesbat et al., 1993).
The thionins contain a signal sequence, the thionin domain and an acid polypeptide domain as well as the conserved Cys residues (Bohlmann et al., 1994), A new class of
Cys-rich antimicrobial protein, gamma -thionin, has a similar size (5 kD) and the same number of disulfide bridges as thionins. However, since gamma -thionins do not have significant sequence homologies with thionins, they have been described as plant defensins (Terras et al., 1995). Both defensin and thionin genes in Arabidopsis are inducible via a salicylic acid-independent pathway different from that for PR proteins (Epple et al, 1995; Penninckx et al., 1996).
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).
1. A nucleic acid sequence and/or a fragment of the same thereof encoding a salt inducible or salt responsive protein encoding thionine gene from the RASI variety of Indica rice or a functionally active fragment or variant thereof and use of the said nucleic acid and/or fragment thereof and/or nucleotide sequence information thereof and/or single nucleotide polymorphism thereof as a molecular genetic marker and applicable to all crops.
2. A claim as in claim 1, wherein the nucleic acid or its fragment thereof includes a salt like protein.
3. A construct including a nucleic acid or nucleic acid fragment according to claim 1.
4. A vector including a nucleic acid or nucleic acid fragment according to claim 1.
5. A vector according to claim 5 including a promoter, with the said promoter, nucleic acid or nucleic acid fragment being operatively linked.
6. A plant, plant cell, plant seed or other plant part including a construct according to claim 3.
7. A plant, plant cell, plant seed or other plant part including a vector according to claims 4 and 5.
8. A plant, plant cell, plant seed or other plant part according to claims 6 and 7.
9. A method of modifying plant tolerance to environmental and/or osmotic stress.
10. A method of transgenic plant which comprises an expression cassette including a thionin gene and being capable of expressing the thionin gene, and which exhibits resistance to at least one disease.
11. A method according to Claim 9 wherein the said environmental and/or osmotic stress includes salt stress.
12. A method of modifying the plant capacity to withstand the salt shock, according to claim 1.
13. A method of modifying plant recovery after exposure to salt stress according to claim 1.
14. A method of modifying plant metabolism to salt stress according to claim 1.
15. Use of nucleic acid or its fragment thereof and/or nucleotide sequence information thereof and/or single nucleotide polymorphisms thereof as a molecular genetic marker, according to claim 1.
16. A substantially isolated or purified polypeptide and functionally active fragments and variants thereof.
17. A claim as in claim 15 wherein the polypeptide includes an amino acid and/or its sequence thereof and its functionally active fragments and variants thereof.
18. A claim as in claim 1 wherein the invention can be applied to all crops.
AVESTHA GENGRAINE TECHNOLOGIES PVT. LTD.
:"DISCOVERER" 9th FLOOR
INTERNATIONAL TECHNOLOGY PARK
TO BANGALORE - 560 066
The Controller of Patents The Patent Office Chennai
|Indian Patent Application Number||767/CHE/2003|
|PG Journal Number||31/2010|
|Date of Filing||24-Sep-2003|
|Name of Patentee||Avestha Gengraine Technologies Pvt Ltd|
|Applicant Address||'DISCOVERER' 9TH FLOOR, UNIT 3, INTERNATIONAL TECH PARK WHITEFIELD ROAD BANGALORE 560 066|
|PCT International Classification Number||C12N15/82|
|PCT International Application Number||N/A|
|PCT International Filing date|