|Title of Invention||
ARABINOGALACTAN PROTEIN GENES ISOLATED FROM RICE UNDER ENVIRONMENTAL STRESS POSSESSING AGRO/PHARMA PROPERTIES
|Abstract||The present invention relates to the isolation of Arabinogalactan protein gene ("AGP") from the RASI variety of rice seedlings under environmental stress conditions. The expression of the gene is in response to various stress conditions like wounding, pathogen infection and drought.|
amino acid sequences of isolated AGP fragments were isolated. The invention presents for the first time an intaTct AGP amino acid sequence derived from a corresponding AGP gene. he instant invention further provides methods useful in obtaining AGP genes encoding an AGP peptide comprising a specific isolated hydroxyproline-rich (OAST-rich) sequence or a specific isolated hydroxyproline-poor sequence.
AGPs are found in flowering plants from every taxonomic group tested. These proteoglycans are widely distributed in most higher plants occurring in almost all tissues including leaves, stems, roots, floral parts, seeds and in many of their secretions. The AGPs are a family of structurally related glycosylated molecules containing high proportions of carbohydrate and usually less than 10% by weight of protein.
AGPs function in several biological processes including plant development, cell-cell adhesion, pollen-stigma recognition, water retention, and disease resistance. AGPs may serve as glues or provide nutrients for growing pollen tubes. It has been suggested [Fincher et al. (1983) supra] that AGP proteins may interact with lectins or other proteins in the extracellular spaces and may be involved in the cellular response to extracellular oligosaccharide signal molecules [Norman et al. (1990) Planta 181:365-373]. Since AGPs interact with Yariv antigens and fiavonol glycosides [Jermyn (1978) J. Plant Physiol. 5:563-571], they have been thought to have lectin-like properties. The molecular structure of AGPs has been proposed [Randall et al. (1989) Food Hydrocolloids 3:65-75] to resemble a type of block copolymer wherein carbohydrate blocks are covalently linked to a central polypeptide chain, thus explaining its abihty to sterically stabilize emulsions and dispersions.
The process of obtaining an AGP clone has been found to be complex and problematic. Two of the problems associated with the AGPs and their genes are - (1) the very high redundancy associated with the characteristic amino acid sequence of an AGP peptide, i.e., a high hydroxyproline content and regions containing a high content of hydroxyproline, alanine, serine and threonine (OAST) and (2) the GC-richness of corresponding oligonucleotides leading to problems with the specificity of hybridisation, indistinct and imprecise alignment during nucleic acid hybridisation, for e.g., the PCR technique, which has resulted in a lack of success in the ability to obtain an AGP clone. This results in the amplification of incorrect sequences. Plants are also known to contain a variety of glycine rich proteins, which are also encoded by GC-rich DNA.
Two approaches to the isolation of the AGPs from plant extracts have been used in previous studies. One approach consists of classical fractionation of plant extracts [Fincher et al. (1974) Aust. J. Biol. Sci. 27:117-132; Aspinall (1969) Adv. Carbohydrate Chem. 24:333-379]. A convenient initial fractionation of extracts is treatment to saturation with (NH4)2 S04, which does not usually precipitate AGPs. Subsequent ion-exchange and affinity chromatography can be used to isolate the AGPs.
Another approach to the isolation of AGPs from plant extracts is precipitation with a
class of dyes prepared by coupling diazotized 4-aminophenyl glycosides to phloroglucinol [Jermyn et al. (1975), supra]. These dyes were first prepared by Yariv et al. (1962) Biochem. J. 85:383-388) as precipitating antigens for antibodies to glycoside determinants, and the .beta.-glycosyl artificial carbohydrate antigen was shown to precipitate an arabinose-and-galactose-containing polymer from soya bean, jack bean and maize [Yariv et al. (1967) Biochem. J. 105:lc-2c]. Since then, this precipitation reaction has been widely used to isolate AGPs from extracts of seeds of every taxonomic group of flowering plants, as well as leaf extracts and callus-culture filtrates [Jermyn & Yeow (1975) Aust. J. Plant Physiol. 2:501-531; Anderson et al. (1977), supra; and review by Clarke et al. (1979), Phytochemistry 18:521-540].
The most striking feature of arabinogalactan is that of all the sugars in the furanose conformation. Galactofuronase is rarely found in nature and not at all in mammals, therefore targeting its synthesis and incorporation in to the wall should yield agents which are highly specific for mycobacteria, and not toxic. The arabinogalactan is linked to the 6-positoin of the muramic acids forming a fairly typical bacterial peptidoglycan layer via a unique diglycosylphosphoryl bridge, L-rhamnose-(l->3)-D-N-acetylglucosamine-( 1 ->P).
The introduction of immunochemistry has made a great impact on AGP research and has revitalised interest in the functional significance of AGP and AGP regulation during growth and development. It has already been postulated that AGPs as a soluble and diffusible component of the extracellular matrix and of the plasma membrane, play a role as messangers in cell-cell interactions during differentiation.
The usefulness of the present invention lies in the fact that the emergence of the AGP gene in rice is significant since the AGP's synthesis can be targeted and incorporated into the cell wall and in turn yield agents which are highly specific for the tuberculosis mycobacteria and not harmful to the patient. Any such agent with low toxicity will exhibit improved pharmacokinetic properties in comparisin with the drugs presently available. It is also an important tool in the food processing industry.
1. mRNA purification was performed by first, isolating high quality total RNA from 6 day old RASI seedlings and, subsequendy 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 instant invention provides a source of AGP that is not dependent upon its isolation from plant exudates, e.g., gum arabic, guar gum, etc. The availability of
natural sources of AGP-containing gums, e.g., from trees, roots, seeds, seaweed, microbes, etc., present problems associated with harvesting, climate, man-power, fermentation, isolation, purity, and high costs. The production of AGPs using recombinant gene technology ensures (a) a method of supplying AGP that is independent of harvesting or fermentation requirements and problems, (b) that enables high levels of quality control, (c) that provides a supply of substantially pure AGP product, (d) that permits an overproduction of AGP in a host cell, and (e) that can be adapted to produce a specifically engineered AGP having desired properties. Thus, this invention provides a means for supplying the functions and utiUties of plant gums, e.g., gum arable, etc., without the need for finding renewable but shrinking natural sources of plant gums. These functions find wide applications as thickening, gelling, emulsifying, dispersing, suspending, stabilizing, encapsulating, flocculating, film-forming, sizing, adhesive, binding and/or coating agents, and/or as lubricants, water-retention agents, and coagulants
1. A cloned DNA fragment encoding a protein backbone of a Arabinogalactan
protein gene ("AGP") from the RASI variety of rice seedlings under
environmental stress conditions.
2. A method of developing transgenic plants by expressing Arabinogalactan
protein gene, functioning in several biological processes including plant
development, cell-cell adhesion, pollen-stigma recognition, water retention,
and disease resistance.
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 1, 2 & 3, wherein, the product of gene expression in the
transgenic plants, a soluble and diffusible component of the extracellular
matrix and of the plasma membrane, plays a role as messangers in cell-cell
interactions during differentiation.
5. A method of targeting the synthesis of AGP's and its incorporation into the
cell wall, yielding agents which are highly specific for the tuberculosis
mycobacteria and not harmful to the patient
6. The production of AGPs using recombinant gene technology ensuresing (a)
a method of supplying AGP that is independent of harvesting or
fermentation requirements and problems, (b) that enables high levels of
quality control, (c) that provides a supply of substantially pure AGP product,
(d) that permits an overproduction of AGP in a host cell, and (e) that can be
adapted to produce a specifically engineered AGP having desired properties.
7. The method providing a means for supplying the functions and utilities of
plant gums, e.g., gum arabic, etc., without the need for finding renewable but
shrinking natural sources of plant gums; the functions find wide applications
as thickening, gelling, emulsifying, dispersing, suspending, stabilizing,
encapsulating, flocculating, film-forming, sizing, adhesive, binding and/or
coating agents, and/or as lubricants, water-retention agents, and coagulants.
|Indian Patent Application Number||770/CHE/2003|
|PG Journal Number||43/2010|
|Date of Filing||24-Sep-2003|
|Name of Patentee||M/S AVESTHA GENGRAINE TECHNOLOGIES PRIVATE LIMITED|
|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|