EP1397502A2 - Sequen age d'adn du transporteur abc ou d'une proteine resistant a de multiples medicaments pour des plantes cultivees tolerant mieux le stress environnemental - Google Patents

Sequen age d'adn du transporteur abc ou d'une proteine resistant a de multiples medicaments pour des plantes cultivees tolerant mieux le stress environnemental

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Publication number
EP1397502A2
EP1397502A2 EP02715621A EP02715621A EP1397502A2 EP 1397502 A2 EP1397502 A2 EP 1397502A2 EP 02715621 A EP02715621 A EP 02715621A EP 02715621 A EP02715621 A EP 02715621A EP 1397502 A2 EP1397502 A2 EP 1397502A2
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EP
European Patent Office
Prior art keywords
gene
stress
abc transporter
rice
environmental stress
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02715621A
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German (de)
English (en)
Inventor
Patell M. Avestha Gengraine Tech Pvt. Ltd VILLOO
Azaghiri K. Avestha Gengraine Tech Pvt. Ltd ARUN
Nirmal Avestha Gengraine Tech Pvt. Ltd RASHMI
Sanjeev Avestha Gengraine Tech Pvt. Ltd KUMAR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Avesthagen Ltd
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Avestha Gengraine Technologies Pvt Ltd
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Application filed by Avestha Gengraine Technologies Pvt Ltd filed Critical Avestha Gengraine Technologies Pvt Ltd
Publication of EP1397502A2 publication Critical patent/EP1397502A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Definitions

  • the present invention relates to polynucleotides and polypeptides belonging to the ABC Transporter family in respect of stress resistance in rice, from the Indica rice variety.
  • the invention is based on an ABC transporter gene cloned from the indica rice variety (RASI) through RNA differential display revealing its implications and role in salinity stress tolerance.
  • Rice is one of the most important cereal crops in the world. Rice is one of the few crops, which, is grown in varied and diverse ecosystems. It is cultivated most extensively in the tropical regions and forms the staple diet of a vast majority of the population in South and South East Asian countries. However the current production of rice is well short of the demand and it is but imperative that steps be taken to improve both, the quality as well as the productivity of the crop.
  • Biotic stress factors namely, pests and diseases considerably affect the productivity in rice.
  • Abiotic stress has been known to cause more harm than biotic stress.
  • the significant abiotic stress factors include drought, salinity, floods, extremes of temperature and metal toxicity, which have played no less a significant role in diminishing the rice yield.
  • steps have to be taken to minimise the crop losses caused on account of abiotic stress especially in relation to stress caused due to drought and salinity, in order to bring about improvements to the rice yield and tolerance towards stress.
  • Salinity in this context per se refers to the presence of the various salts in soil and irrigation water in varying concentrations that contribute in no small measure in affecting the growth and yield of rice.
  • Sodium chloride (Common salt) is a dominant salt present in saline soil. Saline-alkaline and sodic soils apart from possessing inorganic ions also have an excess of chlorides, sulphates and biocarbonates of sodium, calcium and potassium.
  • the quality of the irrigation water is generally found to be sub-standard, EC of 2-5ds/m or more.
  • the said water is not available for the benefit of the plant on account of an alteration in the osmotic potential of the soil.
  • the water needs to be imported against a free energy gradient, but, if the water is taken up freely, the endogenous salt concentration too increases.
  • Macromolecular assembly and enzyme activity are known to be associated with the shaping and maintaining of each cell but proceed only within a properly constituted ionic environment.
  • the inorganic ions selectively neutralise charges on macromolecular surfaces and at the same time permit the formation of intramolecular bridges that finally determine the confirmation of many proteins.
  • the said ions also determine the availability of free water around the enzymes and their substrates and thus, the rate of catalysis. Ionic gradients that are set up at a considerable cost to the plant cell constitute free energy gradients that can be tapped to direct the flow of organic molecules within and between the cells.
  • Salt imposed stress has been shown to have an impact even prior to the ions entering the cell.
  • Extracellular sodium ions can leach calcium ions from the root cell plasmlemma, and, thereby, increase the efflux of potassium ions as a result of membrane destabilisation. If the stress is prolonged, the normal maintenance processes are impaired due to general protein synthesis and metabolism decline. Denatured proteins may form inactive complexes with otherwise functional proteins and the enzymes are likely to be poisoned when inorganic factors are displaced by incoming salts.
  • Rice cultivation in tropical areas is mostly dependent on seasonal rainfall. The vagaries of tropical monsoon render the growth and yield of rice crop uncertain. The high yielding varieties of rice are incapable of attaining their full genetic potential in the absence of adequate and good quality water.
  • Drought occurs when there is insufficient soil water to be taken up by the plants over a period of time to meet its transcriptional requirements. Sustained drought results in complete loss of free water, which, in turn results in dessication and dehydration. Concentration of solutes in the cell leads to a reduction in the potential of cellular water. Loss of turgor leads to changesin the cell volume and membrane area. The crucial cell wall plasma membrane continuum is lost. Osmotic shock can cause extensive damage to the cell on account of disruption of membrane integrity and leakage of the cellular contents. Cellular water deficit produces extensive damage to the functional proteins and increases the formation of misformed proteins. An impairment in the normal metabolic pathways leads to formation of toxic and highly reactive bye-products, particularly in the reactive oxygen species. A lot of other cellular changes similar to those occurring during salt stress are observed during drought.
  • drought affects several of the developmental processes. The germination of the seed does not take place in a uniform manner. At the vegetative stage, the photosynthetic rates decrease drastically, thus, affecting the root growth. Moreover, leaf rolling and leaf scorching are observed. In the reproductive stage, drought causes pollen sterility consequently resulting in small, thin and deformed anthers. Drought during anthesis causes inhibition of anther dehiscence and pollen germination, reduces pollen viability, causes failure in the panicle to exert the flag leaf and ultimately resulting in loss of grain. A restriction in the water at the time of ripening causes incomplete grain filling.
  • the present invention relates to those DNA sequences which exhibit a high degree of homology to the ABC Transporter super family.
  • the ABC Transporters i.e. the ATP Binding Cassette Transporters have a similar, basic structure and design but are variable and dynamic with regard to their functions.
  • the ABC Transporters utilise the release of phosphate bond energy by ATP hydrolysis to carry out their specific functions. These transporters play a role in toxic heavy metal transport, excretion of a range of organic ions, extrusion of cytotoxic substances from the cell and are known to regulate the activity of heterologous ion channels.
  • MDR 1 type Multi Drug Resistance Gene, a member of the ABC gene family
  • MDRl-Pgp is also detected clearly in human CD34+ haematopoietic progenitor cells and in several other sub-classes of bone marrow and peripheral leukocytes, particularly in CD56+ natural killer (NK) cells and in CD8 cytotoxic T cells.
  • the importance of the present invention lies in the fact that salinity stress mechanisms are linked to the ABC super family of transporters. This invention also exhibits the relation between the ABC transporter and rice (Oryza sativa). The invention could be extended to address salinity stress in other crop plants as well.
  • Salinity and water deficit both, are known to induce the expression of a number of genes.
  • the gene products either have a regulatory role in gene expression or else a functional role in the adaptive responses of plant cells to stress.
  • Several protein kinases, calmodulins, G-proteins and transcription factors have been identified in different transduction pathways in plants.
  • the AtPLCl, ATCDPK1, ATCDPK2, components of the MAPK cascades and other proteins have been found to be induced by a variety of environmental stress factors, namely, light, freezing, drought, high salinity, heavy metals, mechanical stimuli etc.
  • the transport protein that mediate ion flux can be generally classified as pumps, carriers and channels. Pumps directly utilise metabolic energy for vectorial tansport, viz, Na+ ATPase, Ca2+-ATPase etc.
  • the channels mediate passive transport, i.e., movement down a free energy gradient (K+ inward), although, the movement may be electrophoretic flux resulting from an energy dependent process.
  • the gene has been chosen from a stressed rice plant.
  • the approach has resulted in the expression of the genes that are required to help the plant survive stress.
  • the DNA sequence has been picked up vide this method.
  • the approach to relate stress to the genes expressed during stress solve much of the problems as enumerated supra.
  • the present invention deals with the ABC Transporters displaying stress resistance in rice.
  • the ABC Transporter gene has been cloned from the indica rice variety (RASI) through RNA differential display exhibiting its implications and role in salinity stress resistance. It is the first gene that has been cloned from rice.
  • the active efflux of sodium ion from cytosol across the plasma membrane and out of the cell or across the tonoplast and into the vacuole has been linked to the ⁇ pH cell generated by antiporters located in these membranes.
  • the gene expression and the physiological responsiveness of the plasma membrane H+ ATPase to sodium chloride is positively correlated with salt tolerance, since, halotolerant cells and plants exhibit higher transcript levels and/or pump activities than their intolerant counter parts.
  • the plasma membraneH+ APTase mRNA accumulation is induced only during sodium chloride adaptation and low mRNA levels have been observed after the adaptation.
  • a 70-kD (sub unit B) mRNA of the vacoular H+ ATPase accumulated in response to the sodium chloride treatment and the salt induced tonoplast H+ ATPase activity has been linked to stress adaptation.
  • vacoular compartmentalisation of chloride ions is mediated by channels with transport by the electrophoretic flux generated by proton pumps across the protoplast under saline conditions.
  • the efficient vacoular compartmentalisation of chloride ions is an essential adaptation for sodium chloride tolerance.
  • CHEM 8 a transcriptionally activated cDNA clone, designated as CHEM 8 (345 bp) and sharing a high, structural homology to an Arabidopsis protein has been identified in maize.
  • the gene has also been known to be present in tobacco, mammals, drosophillia, bacteria and yeast and is homologous to an evolutionarily conserved gene family of the membrane channel protein. This protein, it is believed, is involved in the uptake and transport of ions by plant cells.
  • the CHEM 8 has been constitutively expressed in maize but its transcript levels increased upon heat stress, mercuric chloride treatment, salinity, polluted rain water and ultra violet radiation.
  • This protein is believed to maintain ion homeostasis in plants during abiotic stress, but, its exact role is yet to be unravelled. Therefore the movement of the sodium and chloride ions into the cell, particularly during sub cellular compartmentation is attributed to be a function occurring due to the presence and activity of membrane channel proteins.
  • the ABC transporters to which MDR belongs are present in small organisms ranging from bacteria to humans, the substrates vary from the smallest of ions like arsenite to complex molecules such as proteins.
  • the ABC Transporters have 12 transmembrane segments and two ATP binding sites present in a single, polyopeptide chain assembled from half or quarter molecule.
  • ABC Transporters can be utilised for a wide range of functions. These transporters are so versatile that they can even find new solutions to old problems, if required.
  • Arsenite for instance is a toxic compound encountered by most living creatures. Since bacteria possesses an efficient arsenite pump, detailed studies have revealed that this pump translolcates oxyanions. In contrast to this, there is considerable evidence on record that arsenite pumps of the protozoal parasite, Leishmania and of the mammalian cells use ABC Transporters that convey complexes of arsenite with glutathione.
  • the prototype of this GS-X pump is MRP1, the multi drug resistance protein, which exudes cytotoxic substances from the cell and are known to regulate the activity of heterologous ion channels. It prevents uptake of toxic substances and simultaneously stimulates excretion of the compounds. The transport occurs against a concentration gradient and is independent of the electrochemical trans membrane potential or proton gradient.
  • RNA display has been developed as a tool to detect and characterise altered gene expression in eukaryotic cells (P. Liang & A.B. Pardee, 1992).
  • the method relied on has been the use of the reverse transcription from an anchored olig- dT primer which anneals to the beginning of a sub-population of the poly (A) tails of mRNA.
  • the anchored olig-dT primers consist of 11 to 12 T's plus to 3' bases to provide specificity, which, are used in conjunction with a decamer oligodeoxynucleotide randomly selected sequences for subsequent PCR amplification.
  • the amplified cDNA fragments of 3 'termini of mRNAs are separated by size up to 500bp on a denaturing polyacrylamide gel. This method also permits an estimate of the genes that are differentially expressed under various circumstances. When many experimental treatments are compared, the intersecting regulatory pathways are reflected in the genes that are perturbed by more than one treatment.
  • RNA was extracted (Chomezynski P. & Sacchi N, Anal. Biochem, 162, 156-159, 1987) from control and seedlings of the rice lines IR-64 and Rasi were dehydrated.
  • a combination of the Laing P. and the modified Genehunter RNAmap kit protocols were adopted.
  • the resulting cDNA was amplified with the same anchored oligo(dT) primer and a short arbitrary primer labelled dATP was included in the reaction.
  • the labelled products were segregated and placed on a DNA sequencing gel and visualised by autoradiography. It was observed that each pair of the primers produced a distinct pattern of bands. The band pattern obtained for each cell type with each primer pair was compared.
  • differentially expressed bands were excised from the gel, the DNA was eluted and reamplified.
  • the amplified products were used to probe an RNA blot to confirm that they represent differentially expressed mRNA species and also that they can be used to probe a cDNA or genomic DNA library to recover a full sized clone of the gene of interest.
  • this technique has been used to study the regulation of gene expression at the cellular level in drought/salinity susceptible cultivars like RASI and IR64 respectively.
  • Salinity Stress RASI and IR64 rice variety seeds were placed on moist filter paper for germination. The four day old germinated seedlings were then subjected to 150 M NaCl solution for 16 hours. The said seedlings were harvested at the end of the 16 hour period and thereafter frozen in liquid nitrogen and stored.
  • RNA Isolation The total RNA isolation was carried out as per the Guanidium Thiocyanate method of Sacchi et al., 1997. The RNA was checked for degradation. The chromosomal DNA contamination was removed from RNA. 50 ⁇ g of total cellular RNA was incubated for about 30 minutes at 37°C with 10 units of human placental ribonuclease inhibitor (BRL, Gaithersburg MD), 10 units Dnase I (BRL or Worthington Biochemical Corp., Freehold N.J.) in 100 mM Tris-Cl, pH 8.3, 50 mM KCI, 1.5 mM MgC12. After the extraction with phenol/CHC13 (3:1), the supernatant was precipitated with ethanol in the presence of 0.3M NaOAC and RNA was re- dissolved in diethyl pyrocarbonate-treated water.
  • the mRNA differential display was performed using 2 ⁇ g of total RNA as the template.
  • the various anchored oligo (dT) primers along with the random primers were used to amplify the different sub-populations of mRNA.
  • the cycling parameters were as follows: 94 (C, 30 sec(40(C,2 min(72 ⁇ C, 5 min (4 ⁇ C.
  • the amplified cDNAs were then separated onto a 6% DNA sequencing gel.
  • Recovery And Reamplification of cDNA Probes The DNA sequencing gel was blotted on to a piece of Whatman 3mm paper and dried without methanol/acetic acid fixing. The autoradiogram and dried gel were oriented with either radioactive link or needle punches. After developing the film, the cDNA bands of interest were located either by marking the same with a clean pencil or else by cutting through the film.
  • the gel slice along with the 3mm paper was incubated in lOO ⁇ L dH20 for about 10 minutes.
  • the cDNA was diffused by boiling the gel portion for about 15 minutes in a tightly capped microfuge tube.
  • the cDNA was recovered by precipitating the same in ethanol in the presence of 0.3M NaOAC, 5 ⁇ L of lO ⁇ g/ml glycogen as a carrier and re-dissolved in lO ⁇ L of H20.
  • 4 ⁇ L of the eluted cDNA probe was reamplified in a 40 ⁇ L reaction volume using the same primer set and the PCR conditions as had been used in the mRNA display excepting for the dNTP concentrations which showed a reading of 20 ⁇ M and minus the addition of an isotope.
  • 30 ⁇ L of the PCR samples were run on 1.5% agarose gel and stained with ethidium bromide. The remaining samples were stored at -20ooC for sub-cloning.
  • a uniquely expressed fragment, A-9 from the RASI rice variety was isolated.
  • the reamplified cDNA probe was cloned into pGEM-T vector (Promega, Madison, USA).
  • the cloned fragment was detected and confirmed by colony hybridisation by using the amplified fragment as a probe.
  • 5'RACE is a technique used to obtain the 5 ⁇ end of an mRNA.
  • 'GSP' Gene Specific Primer
  • the synthesis of the first strand cDNA was done using an SP1 primer. This was followed by degradation of the mRNA template by the RNase H activity of the AMV. The purified cDNA was tailed with dATP and TdT. It was further amplified with the GSP and the oligo dT-anchor primer.
  • the 5 RACE product obtained was 0.8Kb fragment of cDNA. The said fragment was named as A-98 and was cloned in pGEM- T vector and sequenced using SP-6 and T-7 primers.
  • a BLASTn search of the A-98 fragment was done and it showed a very high homology with Arabidopsis ihaliana putative MRP-like, the ABC Transporter gene, with a high score of 866 and the N value as low as 7 e ⁇ 33 , enclosed herein as Fig. 1.
  • the high density Japonica BAC library filter was screened and a few positive BACs were obtained.
  • One of the BAC clones was J-14, which, was further sub-cloned and sequenced.
  • the J-14 BAC DNA was isolated and subcloned onto the pCR4Blunt-TOPO vector and screened for blue (Non-Recombinant) and white (Recombinant) colonies on Luria Broth Agar(LBA) plates using Ampicillin and Kanamycin as antibiotic markers with X-gal as the colouring substrate.
  • the white colonies were replica plated in 15 micro- titre plates (MTPs) containing LBA with Ampicillin and Kanamycin. The plates were then stored at 4°C.
  • the PCR was set up by making use of the following protocols with the T7 and T3 primers set.
  • the MJ Research Tetrad PCR machine was used.
  • PCR data is enclosed depicting therein Plate No. J14_6 and J14 10, enclosed herein as Fig.s 2 A & 2B.
  • the PCR product was cleaned using the Millipore vacuum manifold, MANU 030.
  • Step 2 50°C for l0sec. 1 35 cycles
  • Step 4 60°C for 5 min.
  • the importance of the present invention lies in the fact that it is for the first time, that salinity stress mechanism have been shown as linked to the ABC super family of transporters.
  • the invention is the first of its kind to show the relation between the ABC transporter and rice (Oryza sativ ⁇ ). This invention could also be extended to address salinity stress in other crops as well.
  • AtMRP2 an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites : functional comparisons with AtMRPl. Plant Cell, 10, 267 - 282

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Abstract

L'invention se rapporte à des polynucléotides et des polypeptides appartenant à la famille du transporteur ABC et se référant à la tolérance au stress du riz, notamment de variétés de riz indica. L'invention se rapporte également à un gène du transporteur ABC cloné à partir de la variété de riz indica (RASI) par le biais de la technique du DD d'ARN qui a mis en lumière ces implications et son rôle en matière de tolérance au stress environnemental. Ledit gène peut être utilisé de manière efficace pour conférer à d'autres variétés de plantes cultivées une tolérance au stress environnemental.
EP02715621A 2001-01-17 2002-01-14 Sequen age d'adn du transporteur abc ou d'une proteine resistant a de multiples medicaments pour des plantes cultivees tolerant mieux le stress environnemental Withdrawn EP1397502A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IN48CH2001 2001-01-17
INMA004801 2001-01-17
PCT/IB2002/000169 WO2002057406A2 (fr) 2001-01-17 2002-01-14 Sequençage d'adn du transporteur abc ou d'une proteine resistant a de multiples medicaments pour des plantes cultivees tolerant mieux le stress environnemental

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CN102393416B (zh) * 2011-08-25 2013-08-21 北京农业智能装备技术研究中心 基于非损伤微测技术的水稻生理性青枯病评价方法
CN102507701B (zh) * 2011-11-16 2014-05-07 北京农业智能装备技术研究中心 基于非损伤微测技术的动态离子流筛选抗旱水稻的方法
CN116003553A (zh) * 2022-10-25 2023-04-25 齐齐哈尔大学 一种辣椒腺苷三磷酸结合盒转运蛋白及基因和表达载体构建方法

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US5689039A (en) * 1994-03-16 1997-11-18 The University Of Tennessee Research Corporation Plant peptide transport gene
GB9614274D0 (en) * 1996-07-06 1996-09-04 Univ Manchester Treatment and diagnosis of infections of gram positive cocci
WO1998021938A1 (fr) * 1996-11-18 1998-05-28 The Trustees Of The University Of Pennsylvania Transport induit par des conjugues s de glutathion dans des vegetaux

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