Patent Application
20110126312
The field of the present invention is related to the molecular biology area, particularly in the implication of plants' genes in sodium detoxification processes, the use of transformed organisms expressing these genes in constitutive or induced form, and bio-remedy methods to recuperate the medium. Thus, the present invention refers to the conclusion of a tool and a specific method to improve salinity tolerance of living organisms eliminating sodium (Na+) from waters, soils, mud and any other mediums containing this element, based on the use of phytochelatine synthase gene from Nicotiana glauca.
Soil salination is one of the most important concerns regarding agricultural development.
Global climatic changes produce an increase in hydric stress, a factor associated to saline stress, since hydric deficit is also a cause of salt concentration increase in soils.
It is known that salt concentration within the cell of many organisms fluctuates around 50-10 mM, thus benefiting the proteic structure, for example, due to electrostatic forces. However, a 300-500 mM concentration inhibits metabolic reactions, altering balance between electrostatic and hydrophobic forces (Serrano R (1996) Int Rev of Cyt 165:1-52.).
Cells response to sodium chloride (NaCl) is determined by a group of diverse mechanisms:
Na+ may enter the cell through various types of channels, “voltage-dependent cation channels” and “voltage-independent cation channels” (VIC). The VICs are the principal path to Na+ entrance to plant cells (Xiong L. et al. (2002) in Salt Tolerance. The Arabidopsis Book (American Society of Plant Biologists, pp 1-22). Due to the similarity between Na+ and K+, voltage dependent K+ carriers might facilitate the entrance of Na+. For example, Arabidopsis AtKHT1 (sodium carrier with sequence homology to HKT family of potassium carriers), is involved in the conduction of Na+ from stems to roots. This circulation seems to play an important role in plants' tolerance to saline stress (Berthomieu P et al. (2003) EMBO journal 22:2004-2014).
Variation in membrane potential (values 130 mV) enables the entrance of Na+. Hyper-polarization in the plasmatic membrane of yeast is produced by the absence of the PMP3 (SNA1) gene. Homologues in A thaliana are RCI2A and RCI2B (BLT101 in wheat). The over-expression of RCI2A might ease the growth suppression and photo-oxidant damages reducing the entrance of Na+ in the roots (Mitsuya S. et al. (2006) Physiologia Plantarum 128:95-102).
The role played by Ca2+ in the intricate group of responses to NaCl has been recently clarified in various aspects. The over-expression of ACA4 (Ca2+ vacuolar ATPase of Arabidopsis thaliana) in yeast increases tolerance to salt (Geisler M. et al. (2000) Plant Physiol 124:1814-1827).
Na+ is evacuated out of the cells by means of Na+/H+ anti-carriers located in the plasmatic membrane. Accordingly, the over-expression of the SOS1 gene of A. thaliana, encoding for the anti-carrier of Na+/H+ of SOS1 plasmatic membrane, improves the tolerance to salinity (Shi H. et al. (2003) Nat biotechnol 21:81-85). Besides the extrusion of Na+ ions, the compartment effect in the vacuole is one of the most important causes of tolerance to salinity. The protons gradient enabling the anti-carrying is produced by H+-ATPases and H+-vacuolar pyrophosphatases (PPases). Transgenic plants over-expressing AVP1, vacuolar H+-PPase, show a saline tolerance correlated to increase in ionic content inside the plants (Yamaguchi T. et al. (2005) Trends Plant Sci 10:615-620). Likewise, plants over-expressing AtNHK1 (Na+/H+ vacuolar anti-carrier of Arabidopsis thaliana), were capable of growing, blossoming and producing seeds in presence of 200 mM NaCl in transgenic plants of Brassica napus (Zhang H-X. et al. (2001) Proc Natl Acad Sci USA 98:12832-12836) and transgenic tomato (Zhang H-X. et al. (2001) Nat biotechnol 19:765-768). The over-expression of AgNHX1 (proceeding from the halophyte plant Atriplex gmelini), BnNHX1 (Brassica napus), HbNHX1 (Hordeum brevisubculatum) and GhNHX1 (Gossipyum hirsutum) also play the same role (Yamaguchi T. et al. (2005) Trends Plant Sci 10:615-620). The heterologous expression of TsVP (H+-PPase cloned from Thellungiella halophila) in enal mutant yeast (pump eliminating Na+ out of the cell) suppresses hypersensitivity to Na+. The tobacco transgenic plant over-expressing TsVP attains 60% more of dry weight than the wild type when exposed to 300 mN NaCl (Gao F. et al. (2006) J Exp Bot 57:3259-3270).
Osmolytes also play a relevant role in tolerance to salinity. They protect against water loss and changes in the plasmatic membrane structure as a result of the elimination of ROS (“Reactive Oxygen Species”) toxic effects generated by saline stress. Proline, glycine, bethaine, trehalose, manitol and sorbitol, abundantly produced and accumulated in cells treated with salt, represent an important component in responses to saline stress (Sahia C. et al. (2006) Physiol Plant 127:1-9). Over-expression of enzymes involved in the detoxification path of ROS(SOD, CAT, GST, APX, GPX) result in an increase in tolerance to saline stress (Xiong L, Zhu J-K (2002)). Transgenic tobacco seeds over-expressing a cDNA encoding for an enzyme with glutation S-transferase activity (GST) and glutation peroxidase (GPX), grow faster than control seeds when exposed to low temperatures and saline stress (Roxas V-P, Smith R-K, Jr, Allen E-R, Allen R-D (1997) Nat biotechnol 15:988-991). Glyoxalase I (gly I) and glyoxalase II (gly II) enzymes are necessary for detoxification form methylglyoxal and confer tolerance to salinity in tobacco transgenic plants (Singla-Pareek S-L, Reddy M-K, Sopory S-K (2003) Proc Natl Acad Sci USA 100:14672-14677).
There is a clear connection between oxidizing and osmotic stress through the so called “Mitogen-Activated Protein Kinase” (MAPK). The EhHOG gene, encoding an MAPK having an essential role in the path of yeast and other eukaryote osmo-regulation, was isolated from Eurotium herbariorum of the Dead Sea. When EhHOG was over-expressed in the hog1 mutant of S. cerervisiae, the growth and aberrant morphology of hog1 were restored in high osmotic stress conditions (Jin Y, Weining S, Nevo E (2005) Proc Natl Acad Sci USA 102:18992-18997).
Various genes induced by saline stress belong to the LEA family. Diverse types of Arabidopsis LEA are known; RD (“Responsive to Dehydration”), COR (“COld-Regulated”), LTI (“Low Temperature-Induced”), KIN (“cold induced”). All these types are induced by saline and hydric stress, low temperatures and ABA. Transgenic rice over-expressing SNAC1 (“STRESS-RESPONSIVE NAC 1”) is more sensitive to ABA and looses water slower due to closure of the stomas; SNAC1 might improve tolerance to drought and to salinity in rice. (Hu H. et al. (2006) Proc Natl Acad Sci USA 103:12987-12992).
Thus, sensibility of plants to sodium presence is not a feature inherent to them, since many adaptations are known in soil as well as in seawater, at high saline concentrations, as is the case of halophyte plants. This indicates that tolerance might be resolved through gene transference. To date, genes described in defense against saline stress were involved in transport, extrusion, osmo-protection, vacuolar anti-carriers, transcription factors, etc.
Plants containing diverse groups of molecules used to ease effects of salinity have been developed in the last decades. For example, osmo-protectants, which are solutes compatible with proline (amino acids), glycine-bethaine, dehydrine and sugars (manitol, trehalose, etc) that work as osmolytes and protect cells from dehydration and thus loss of bulge, improve maintenance of roots and trigger in response to water deficit.
In August 1999 Eduardo Blumwald, an Argentinean scientist working in Toronto, publishes the use of a vacuolar anti-carrier of Arabidopsis thaliana that ejects H+ to cytoplasm while accepting Na+ ions (patent no. Ca 2,323,756 and afterwards patent U.S. Pat. No. 6,936,750). This enables plants over-expressing it to be able to live in highly saline environments.
In August 2001 the same researcher, now in Davis, University of California, brings to light the obtention of a tomato plant genetically modified growing and developing in salty water irrigation. It is important to outline that even though all along the past century a good number of researchers have been trying to develop crop varieties tolerant to salt using classical improvement techniques, none of the efforts rendered the expected results.
In 2001 the demonstration that atHKT1 is a Na+ carrier to the interior of the Arabidopsis thaliana root (Rus et al., Plant Physiology 136:2500-2511 (2001) is made public. Thus, theoretically, the over-expression of this gene in a plant would allow a greater income of Na+ through the roots of individuals modified with said gene.
The Spanish Patent Application No. 2,173,019, published in 2002, defines the use of the sodium ATPasa gene of Neurospora crassa in the improvement of tolerance to salinity.
Now, the authors of the present invention have developed a method to improve the tolerance to salinity based, for the first time, on the use of molecules capable of linking directly to sodium in order to block its toxic action inside the cell.
These molecules are the phytochelatines (PCs). PCs are peptides rich in cysteine that are not genetically codified. Its synthesis starts or is induced due to the presence of heavy metals, as cadmium, with the concourse of the phytochelatine synthase enzyme (PCS) using GSH as substrate to form the peptide [γ-Glu-Cys]n=2-11-Gly (PCs) (Steffens, J. C. (1990) The heavy metal-binding peptides of plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 41, 533-575).
The PCS gene of different species has been used to improve tolerance and accumulation of heavy metals in diverse vegetable species as a solution to the problem of soils contamination by these contaminants, that is, selected plants have been endowed with a higher capacity to tolerate and, what is more interesting, accumulate heavy metals through phyto-extraction. Thus the PCs seem to play an essential role in the regulation of cellular equilibrium in ions of “free” and complexed heavy metals through a simple and efficient mechanism (Erwin G. et al. (1989) Proc Natl Acad Sci USA 86:6838-6842. Moreover, many authors previously suggested that PCs might play a role in detoxification of heavy metals (Cobbett C (2002) Annu Rev Plant Biol 53: 159-182). This has resulted in patents of genes encoding for phytochelatines synthases (U.S. Pat. No. 6,489,537 and U.S. Pat. No. 6,844,485), but in no case have they been used to stand salinity, so the proposal herewith presented generates a new method to confront contamination by salts or salination.
On the other hand, the state of the art accounts for non biological methods used for the restoration of saline-sodic soils that generally consist in the addition of calcium sulfate (gypsum) to facilitate the cationic exchange and wait for diverse rainfall washes of the substituted cations. However, the migration of salts to deeper horizons is not the solution to the problem since it could ascend again by capillarity or contaminate water reservoirs.
To this end, the present invention also provides a procedure to reduce or eliminate sodium from any medium (solid, liquid or gaseous) containing said alkaline metal, consisting in the use of PCs in a living organism whose capacity to resist the effects of salinity is limited and subtract it from a concrete means through its accumulation in it.
Besides the use in this method of already known sequences encoding for PCSs of different species, the authors of the present invention have sequenced for the first time the gene of Nicotiana glauca PCS, which can be expressed in constitutive or induced form in N. glauca or in any other living organism with limited tolerance to salinity.
The method object of the present invention presents significant advantages with respect to procedures developed in the state of the art to stand salinity. On one hand, it is a method of easy application and great usefulness, since it enables the direct application to the contaminated medium, which means, in the case of salinized soils, avoiding the loss of soil due to the superficial wash of sluice waters from those soils that do not allow the growth of wild plants. Besides, it presents economic benefits since it takes advantage of the capacity of living organisms to decrease erosion and/or accumulate the salts.
AhPCS1 (Arabidopsis halleri, AAS45236.1), AtPCS1 (Arabidopsis thaliana, AAD16046.1), AtPCS2 (Arabidopsis thaliana, AAK94671.1), AsPCS (Allium sativum, AA013809.1), AyPCS (Athyrium yokoscense, BAB64932.1), BjPCS1 (Brassica juncea, BAB85602.1), BnPCS (Brassica napus, CAK24968.1), CdPCS (Cyonodon dactylon, AAO13810.2), CePCS (Caenorhabditis elegans, NP 4964575.3), GmhPCS (homo-phytochelatine synthase, Glycin max, AAL78384.1), NgPCS1 (Nicotiana glauca), NtPCS1 (Nicotiana tabacum, AAO74500.1), LsPCS1 (Lactuca sativa, AAU93349.1), LjPCS1 (Lotus japonicus, AAQ01752.1), LjPCS2 (Lotus japonicus AAT80341.1), LjPCS3 (Lotus japonicus, AAY81940), OsPCS (Oriza sativa, AAO13349.2), PvPCS (Pteris vittata, AAT11885.1), SpPCS (Schizosaccharomyces pombe, 010075), SrPCS (Sesbania rostrata, AAY83876.1), StPCS (Solanum tuberosum, CAD68109.1), TcPCS1 (Thlaspi caerulescens, AAT07467.1), TjPCS (Thlaspi japonicum, BAB93119.1), TaPCS (Triticum aestivum, AAD50592.1) and TIPCS (Thypha latifolia, AAG22095.3).
The object of the invention is the NgPCS1 gene encoding the phytochelatine synthase of Nicotiana glauca with the SEQ ID NO 1 sequence.
It is also object of the invention the use of the NgPCS1 gene in a method to improve the tolerance to salinity and accumulation of sodium in any living organism.
Another object of the invention is the use of the NgPCS1 gene in a method to recuperate a salinized medium through modified organisms expressing a phytochelatine synthase codified by SEQ ID NO 1 or sequences having at least a 35% similarity with SEQ ID No. 1.
Finally, it is an object of the invention the use of phytochelatines obtained in vivo or in Vitro through enzymatic reaction mediated by phytochelatine synthase codified by the SEQ ID NO 1, or sequences with at least a 35% similarity, as sodium chelators.
In a main aspect, the invention refers to an isolated sequence of a nucleic acid encoding for phytochelatine synthase of Nicotiana glauca (NgPCS1), characterized by the SEQ ID NO 1.
The term “encoding” refers to a property inherent of specific nucleotide sequences in a polynucleotide such as gene, cDNA or mRNA serving as mould for the synthesis of polymers and macromolecules in biological processes or in processes carried out in vitro.
Another main embodiment of the invention contemplates a vector comprising the SEQ ID NO 1. In a preferred form, said vector is a plasmid.
Another main embodiment of the invention, refers to a stable transgenic organism (cell or genetically modified organism) comprising the sequence SEQ ID NO 1.
Phytochelatine synthase of Nicotiana glauca (NgPCS1) located in the cytoplasm, is the enzyme that mediates the production of phytochelatines (PCs), using glutathione (GSH) as substrate. The over-expression of NgPCS1 results in an increased tolerance to Na+, as well as tolerance to heavy metals already observed in other PCSs. The mechanism through which an improvement of tolerance is produced is the chelation, through the PCs, of Na+ ions and the further seclusion in vacuoles of the PC-Na+ complexes.
Thus, another main aspect of the invention relates to the use of the SEQ ID No 1 sequence to improve the tolerance to salinity and/or accumulation of sodium (Na+) in a living organism (animal, vegetal or microorganism).
This has enabled the authors of the present invention to develop a method to improve the tolerance to salinity and/or accumulation of Na+ in a living organism comprising the following steps:
transforming the living organism with the SEQ ID NO 1, or a sequence with at least a 35% similarity with the SEQ ID NO 1; and
expressing the sequence (SEQ ID NO 1, or a sequence with at least a 35% similarity with the SEQ ID NO 1), controlled by functional regulatory sequences in the living organism.
Sequences with at least a 35% similarity with the SEQ ID NO 1 encompass those genes having phytochelatine synthase function, that is, from eukaryotes as the Caenorhabditis elegans worm and bacteria to plants (having a higher similarity).
The transformation step is carried out by any of the known state of the art methods. In a particular embodiment, in a first step the construction of a vector comprising the SEQ ID NO 1, or a sequence with at least a 35% similarity with the SEQ ID NO 1 is carried out and, afterwards, said vector is introduced in the living organism.
To improve tolerance to sodium in a living organism it must express the gene of the sequence introduced under the transcriptional control of a regulatory sequence that may be constitutive (always facilitating the expression) or induced (facilitating the expression only if there is sodium).
In the present invention, the term “functional” refers to the regulatory sequences having effect on the functionality of the gene as to the transcription (start and ending) and translation (start and ending) of messenger RNA and others not described.
Among the regulatory sequences of the present invention are the promoters and others less common as certain introns, the sequences of transcription terminus and sequences of start and ending for the posterior translation of messenger RNA.
Phytochelatine synthase of the species Nicotiana glauca may be thus expressed in constitutive or induced form in N. glauca or in any other living organism whose capacity to stand the effects of salinity is limited.
The constitutive expression of PCs produces an improvement in the growth of yeasts and plants so as to be used to solve various problems: (a) cultivation of numerous plants in salinized soils or in waters with saline contamination, that might generate two direct benefits, the revaluation of abandoned lands on account of salination thanks to biomass production, as well as the restoration of the same to be cultivated again and (b) the cultivation of modified microorganisms in saline contamination media to reduce the content of salts in such media.
In a particular embodiment, the living organism used in the method of the present invention is a yeast, preferrably Saccharomyces cerevisiae.
In another particular embodiment, the living organism is a plant, preferably Nicotiana glauca.
In another main aspect of the invention, the use of the SEQ ID No 1 sequence is used to reduce or eliminate sodium from a liquid, solid or gaseous medium.
This application enables the development of a method to reduce or eliminate Na+ from a liquid, solid or gaseous medium based on the following steps:
In a preferred form, the genetic transformation process is carried out through electroporation. This process succeeds in the production of host cells of Escherichia coli or Agrobacterium tumefaciens carrying a vector with the desired insert (alter being selected with the corresponding antibiotic). As previously clarified, these cells are also considered in the present invention as transgenic organisms, though they are not directly employed in the sodium link in a salinized medium but serve as amplifier means in the case of E. coli and as instrument of infection in the case of A. tumefaciens.
To carry out the sodium elimination process it is crucial to be sure that the organisms used contain the transgene inserted in its genome or otherwise express it through vectors. Thus, to obtain organisms expressing the sequence SEQ ID NO 1, or a similar sequence in at least a 35%, it is necessary to carry out a selection step. The selection is carried out with antibiotics, since the transgenic organism incorporates through the vector a gene resistant to antibiotics (GRA). It is located next to the gene of SEQ ID NO 1, between the left border (LB) and the right border (RB) defining the flanking regions of the transference DNA (tDNA) (
In case microorganisms expressing the gene to eliminate the salts from a salinized medium are used, the collection of organisms shall be carried out through floculation, precipitacion, centrifugation or any other method enabling the separation of microorganisms that have accumulated the salts of the medium.
In case plants expressing the gene to eliminate the salts from a salinized medium are used, the plant is to be collected, triturated before or after being dried and the remains are to be dumped in a residues' dumping place according to the legislation.
Finally another main embodiment of the invention comprises the use of phytochelatines (PCs), obtained in vivo or in vitro by enzymatic reaction mediated by phytochelatine synthase codified by the SEQ ID NO 1, or sequences with at least a 35% similarity, as sodium chelants.
The design of primers in conserved zones of the encoding region of the PCS gene of N. tabacum led to the amplification of a PCR fragment of an expected size (1.5 Kb). The open reading frame codified a protein (NgPCS1) with a molecular mass of 55.14 kD; 501 residues of amino acids and a pH of 6.32. The hydropathy profile was correlated to a cytoplasmatic protein (
Cloning and research of different PCSs led to the conclusion that these genes can confer accumulation and tolerance to Cd2+ (Clemens S. et al. (1999) EMBO J. 18: 3325-3333. 27). As expected, NgPCS1 can also confer tolerance to Cd2+. This is especially evident in this work from the experiments carried out at a 100 μM concentration (
NgPCS1 conferred tolerance to Na+ when it over-expressed in yeasts in concentrations of NaCl oscilating from 0.6 to 1 M (
To examine the capacity of PCS to improve the tolerance to Na+ in plants, N. glauca specimens over expressing wheat PCS (TaPCS1) previously tested to determine the accumulation of heavy metals, were grown. Gisbert C. et al. (2003) Biochem Biophys Res Commun 303:440-445) (Martinez M. et al. J (2006) Chemosphere 64:478-48524).
The binary vector pBI121 (Clontech) was used for the transformation. The GUS gene of the binary vector was substituted by the encoding DNA of wheat phytochelatine synthase TaPCS1 through the BamHI and ECL13611 restriction sites. Next, the introduction of the obtained plasmid, containing the TaPCS1 cDNA in Agrobacterium tumefaciens C58C1 RifR was carried out, and afterwards the transference by infection of N. glauca plants was carried out. The new construction was electropored in Agrobacterium tumefaciens cells. The transformants were selected in LB plates with kanamicine and contacted with 1 cm diameter disks during 10 minutes with a suspension of A. tumefaciens containing the desired construction. After generating adult plants through the regenerating program in vitro of N. glauca explants resistant to kanamicine, the seeds of the different transgenic lines obtained were recollected, selecting those containing one or various integrations in the plant genome, recovering those capable of growing in presence of the antibiotic kanamicine. The last step consisted in verifying if the integration of TaPCS1 cDNA to the Nicotiana glauca genome sensibly improved the tolerance of the plant to Na+. The tolerance to Na+ study was carried out germinating the transgenic seeds in a substrate with vermiculite and dolomite as well as in hydroponic conditions, applying NaCl at 7 and 2 weeks respectively, to a final concentration of 200, 300 and 500 mM.
Two aspects were evident. In the first place, both types of specimens, the wild type and those that over expressed PCS, followed exactly the same growth pattern in relation to the tested NaCl concentrations, indicating a net effect of PCS in the tolerance to NaCl (
The strain of Saccharomyces cerevisiae, YPH499 (MATa ura3-52 leu2Δ1 lys2-801 Ade2-101 trp1Δ63, his3-D200 was used in this study. For growth assays in solid and liquid media, S. cerevisiae cells were grown in minimum synthetical medium (SD) with or without 2% agar bacto, respectively, containing 1% sucrose, 1% galactose, 0.7% nitrogenous base for yeasts without amino acids and with ammonic sulfate (Pronadisa) and MES-Tris 50 mM (pH 6,0). The SD medium was complemented with adenine (30 μg/ml), histidine (30 μg/ml), leucine (100 μg/ml), lysine (100 μg/ml) and tryptophan (80 g/ml). The transformation through the procedure with litium acetate and the selection of the transformants in yeasts was carried out as described (Ito H, Fukuda Y, Murata K, Kimura A (1983) J Bacteriol 153:163-168) and using the URA3 marker for the selection in yeasts. The yeasts cells carrying the empty vector pYES2 were used as negative control. To investigate the kinetics of saline stress, cells were grown up to a DO at 600 nm of 1, approximately, and were inoculated at a concentration of 106 célls per ml in liquid medium without NaCl or containing 0.6 M, 0.7 M and 1.4 M of NaCl. For dripping assays, cells were grown to saturation in SD diluted with water (1/2, 1/5, 1/10, 1/20, 1/100, 1/1.000 y 1/10.000), and distributed through replica plater 8×6 array (Sigma-Aldrich) in plates containing 0.6 M, 0.7 M y 1 M de NaCl y CdCl2 100 μM.
For the planthouse experiment, seeds of N. glauca (wild type) and three F3 different transgenic lines (TaP12, TaP17 and TaP18, lines L1, L2 y L3 respectively) were sterilized as follows: the seeds were submerged in 30% commercial lye, plus 0.01% Triton X-100 detergent during 7 minutes to avoid fungal and bacterial growth, a second washing was carried out afterwards using a dissolution of 70% ethanol in water with 0.01% Triton X-100. Finally, the seeds were 5-folded consecutive washed with deionized water each lasting 5 minutes to eliminate any remainder of disinfectant dissolution. The submerged seeds were placed in Petric plates with a medium prepared with agar 6 g/liter, MS salts (Murashige T, Skoog F (1962) Physiol Plant 15:473-497) and sucrose 10 g/liter at pH 5.7 tamponed with MES (2-[N-morfoline acid]ethanosulphonic) 0.25 g/liter. At ten days (when the first leaves had developed), three plantules per line and pot treatment containing vermiculite and dolomite in the same proportions were transplanted and covered with film during some days to obtain better acclimate conditions. The six weeks plants were placed in a different tray for each treatment and were watered once a week with or without NaCl 200, 350 or 500 mM during two weeks. For the in vitro experiment, three plantules were placed for WT (wild type) and each line of N. glauca, growing in sterile conditions as described in (Gisbert C. et al. (2003) Biochem Biophys Res Commun 303:440-445), in 50 ml Falcon tubes containing MilliQ water with or without NaCl 200, 350 and 500 mM at room temperature in a soft shaker (25 rpm in an ELMI S4 shaker) during 7 days.
The encoding DNA was synthesized from 2 μg of total isolated RNA of N. glauca leaves through reverse transcriptase of M-MuLV (virus of Moloney murine leukemia) with an oligo(dT)18 primer (kit synthesis Ferment of the first strand encoding DNA) according to recommended procedures in the kit. A microliter of produced encoding DNA was used as mould in a reaction of conventional PCR 50 μL. Design of the primers for polymerase chain reaction (PCR): the conserved domain in the N-terminal termini for NtPCS1, AtPCS1, TaPCS1, BjPCS1 y OsPCS1 was observed. Two different primers in the 5′ termini, FW1 (SEQ ID NO 2) and FW2 (SEQ ID NO 3) were designed. The encoding sequences in the C-terminal region analyzed for this gene are less conserved than those of the N-terminal extreme. Thus, the sequence of NtPCS1 RNA messenger was used, designing three different primers (SEQ ID NO 4), RV2 (SEQ ID NO 5) y RV3 (SEQ ID NO 6).
After carrying out the experiments of PCR using different combinations, only two bands with the expected size of 1.5 KB were obtained, corresponding in both cases to the primers FW1/RV1 and FW2/RV2. Using an agarose gel with 1% TAE buffer, the reactions of PCR were carried out and extracted through pressure-freezing of the cut band and DNA precipitation through 1/50 volumes of NaCl 5M and 2 volumes of absolute EtOH. After measuring the DNA concentration in a Nanoprop ND-100 spectrophotometer, the amplified fragments were cloned in the pGEM-T Easy vector (Promega, Southampton, UK).
E. coli DHSa was used as host. After selecting the right transformants and isolating the plasmidic DNA (Marligen Bioscience, quick plasmids' isolation system) the cloned fragments were sequenced in a DNA ABI Prism (Perkin-Elmer) sequencer using the sites T7 and SPC6 located in the vector. Both complete sequences of the 1.5 Kb fragments were aligned with NtPCS1 using the William Pearson LALIGN program, observing a 93% identity in the sequence of the FW1/RV1 fragment. The sequence of the second fragment, FW2/RV2, revealed a 91% identity between the nucleotides 592 and 1501 of the NtPCS1 encoding sequence. Alignment of both sequence fragments resulted in the same nucleotides composition between the 592 position and the terminal codon. The correct C-terminal sequence corresponding to the RV1 primer was assayed using the second NgPCS1 sequenced fragment. NgPCS1 was directionally subcloned in the KpnI/BamHIH sites of the pYES2 expression vector through a new amplification by PCR with the primers FW2 and RV2 and with the additional KpnI y BamHI sequences, respectively. pYES2 includes the Amp of E. coli gene, the selectionable URA3 yeasts marker and the inducible promoter GAL1 for expression in yeasts cells. E. coli was transformed by electroporation selecting the transformants for Ampr. The plasmidic DNA of the correct clone containing NgPCS1 in pYES2 was transformed in the yeast strain YPH499 and was selected as previously described. The sequences of NgPCS1, NtPCS1, AtPCS1 y TaPCS1 were aligned with the CLUSTAL W (Thompson J-D, Higgins D-G, Gibson T-J (1994) Nucleic Acids Res 22: 4673-4680) program. A hydropathy profile of Kyte-Doolittle was built to learn the hydrophatic character of NgPCS1 (Kyte J, Doolittle R (1982) J Mol Biol 157:105-132).
Yeast cells were grown in 20 ml of SD with the appropriate amino acids measuring the absorbance at 600 nm up to 0.6. Then, NaCl was added up to a final NaCl concentration of 1.4 M and incubated at 28° C. shaking (150 rpm) during 3 hours, centrifugating during 5 minutes at 7,000 rpm (Beckman JA-20 rotor) and washed four times with 10 ml of a dissolution containing MgCl2 20 mM and sorbitol 1.5 M. Finally the intracellular content was extracted through incubation with 0.5 ml MgCl2 20 mM solution during 12 minutes at 95° C. After centrifugation during 2 minutes at maximum speed, the aliquots of the supernatant were analyzed with an atomic absorption spectrometer (Varian) in the flame emission mode.
The O2− superoxide anion was detected, as described by Yamamoto Y. et al. (2002) Plant Physiol 128:63-72, using dihydroetide (DHE), a reduced form of etide bromide that is not fluorescent and can passively pierce the membrane of living cells. Once in the cell, it oxidizes to yield a fluorescent colorant that links to nearby DNA. Production of O2− in the roots of N. glauca was observed after tinging the roots with DHE 10 μM in CaCl2 100 μM, at pH 4.75 during 13:30 h. Fluorescence images were obtained with a reverse Leica TCS SL confocal microscope. For the detection of the DHE, samples were excited at 488 nm using an argon laser and the emission was measured between 550 and 620 nm. The measurements by confocal microscopy and fluorescence were repeated at least five times with similar results. N. glauca seeds were grown during six weeks in MS medium as described for vegetable materials. The DHE was administered after nine days of accommodation to hydroponic conditions.
| Number | Date | Country | Kind |
|---|---|---|---|
| P200702926 | Oct 2007 | ES | national |
This application is a national stage entry of PCT/ES2008/000662 filed Oct. 24, 2008, under the International Convention claiming priority over Spanish Application No. P200702926 filed Oct. 24, 2007.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/ES08/00662 | 10/24/2008 | WO | 00 | 4/20/2010 |
