Biological contamination-removal method

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is of application in the metal elimination that contaminates soils and aquatic zones.

2. State of the Art

At present, the state of the art provides alternative ways of solving the problem. The most habitual are also the costliest and they base on, fundamentally chemical and physical methods, as they are: the committal of the contaminated soils with inert materials, the cleanliness "in situ" with the machines and chemical suitable material, the storage as poisonous residues in the places regulated for it (with correspondent license), etc.

In the United States there have been carried out experiences of recovery of the contaminated soils, using natural plants, of the type of the corn, tomato, sunflower, Brassica juncea, etc., and trees principally willow and black poplar. Most of these pioneering experiences has been realized by the American army moved by the desire to recover soils been contaminated with the metallic garbage that provoke the bullets that are in use in the tests of shot. These metals contain fundamentally lead.

Also another type of experiences have been performed in New Jersey. It was carried out a selection of cultures of Brassica juncea (Brassica juncea (L) Czem) with aptitude to accumulate the elected metals; it was used enmendants of the soil or chelants substances. Later they allowed them to glow, and to be harvest, and the contain in metals have been analyzed. In these two cases, the used techniques have been fundamentally: treatment of contaminated soils, and regeneration by metal accumulation in the cultivated plants. The "U.S. Environmental Protection Agency", also acquaintance as EPA, has been the pioneering institution in the study of these processes of phytoremediation. Together with the company "Phytotech" was the manager of realizing the mentioned process of recovery of the contaminated soils in New Jersey. In the experiments in which the objective has been the recovering of munition, as the one mentioned above, it has participated actively the engineers' body of the American army (Army Corps of Engineers). The company Phytotech is one of pioneers in the whole world. In Spain there exist references of ELECNOR, which it applies technologies of bioremediation patented by the North American company ECS.

Some examples of vegetable used material: hybrid trees of black poplar have been in use for extracting nickel, cadmium and zinc of contaminated soils (University of Georgia, USA, "Savannah River Ecology Laboratory"). In the department of energy of Ashtabula, Ohio, plants of sunflower have been in use for remediation of soils and waters contaminated by uranium.

Some examples of important patents are the following ones: U.S. Patent. Phytorecovery of metals using seedlings. U.S. Pat. No. 911,655, Aug. 15, 1997, Phytotech, Inc. (Monmouth Junction, N.J.). U.S. Patent. Method of using pelargonium sp. as hyperaccumulators for remediating contaminated soil. U.S. Pat. No. 185,797, Nov. 4, 1998, University of Guelph (Guelph, Calif.). U.S. Patent. Methods for removing pollutants from contaminated soil materials with a fern plant. U.S. Pat. No. 546,941, Apr. 11, 2000, University of Florida (Gainesville, Fla.). The invention describes the use of a variety that is less extensive geographically that N glauca. It is a question of a fern that basically accumulates enormous quantities of arsenic. Methods are described to carry out the decontamination using one or different varieties of ferns. The use of this type of plants has fundamentally the disadvantage of which there is located in very humid habitats which limits his use. Besides the need to receive very much water contribution can have negative effects for the soil where they grow since, on having contributed big quantities of water, this one can displease the metals at deeper levels or spread them.

Since it parts with the information contributed in the exposed patents, the work it has centred on processes of bioremediation, especially using hyperaccumulators (those plants that possess an extraordinary capacity of absorption of metals, and of accumulation in his biomass).

The phytoremediation consists basically of the use of plants or of vegetable material, to decontaminate soils with high concentrations of harmful elements. Recently a review has been realized, about the topic, by two Spanish investigators, in the publication: "Phytoextraction: to cost-effective plant-based technology for the removal of metals from the environment". Carlos Garbisu, Itziar Alkorta. Bioresource Technology 77 (2001) 229-236. Where the state of the investigation is described in phytoremediation, topcoat being based on the concrete aspect of the phytoextraction.

One key article (review), for the importance of the works realized by the authors are the followings: Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P., Angle, J. S., Baker, A. J. M., 1997. Phytoremediation of soil metals. Current Opin. Biotechnol. 8, 279-284. Salt, D. E., Smith, R. D., Raskin, I., 1997. Phytoremediation of soil metals. Current Opin. Biotechnol. 8, 279-284. This is an article of reference for the punctual contribution and Corn and buckthorn are two species that accumulate lead. (Huang, J. W., Cunningham, S. D., 1996. Lead phytoextraction: species variation in lead uptake). As for the cadmium, Salix viminalis, Thlaspi caerulescens and Alyssum murale seem to be the best species (SALIX EXPERT PHYTOEXTRACTOR, Maria Greger, Dept of Botany, Stockholm University, S-106 91 Stockholm, Sweden).

Concerning to the molecular biology of metal absorption, fundamentally researchers have worked with plants of the genre of Brassica, and among them especially Brassica juncea. In these type of plants genetic transformations have been realized by means of skills of biotechnology. Also they have been carried out in another type of vegetable varieties as black poplar and certainly Arabidopsis. Regarding the genes that have been in use for decontaminating heavy metals, it is necessary to distinguish basically three families: metallothioneins, phytochelatins, and genes that regulate the processes of oxidation-reduction. It is necessary to emphasize the pioneering work realized with bacterial genes that are specialized in the absorption of mercury, which marked a milestone in this field: Rugh, C. L., Wilde, H. D., Stack N. M., Thompson, D. M., Summers, A. O., Meagher, R. B., 1996. "Mercuric ion reduction in transgenic Arabidopsis thaliana plants expressing to modified bacterial mere gene". Proc. Natl. Acad. Sci. USA 93, 3182-3187.

The most important works in this field, apart the mentioned one, have been carried out by Schroeder, Rea, and Therry. During the same year, Schroeder studied for the first time the enzyme produced by the gene TaPCS1, which is a phytochelatin of wheat. (The EMBO Journal Vol. 18 Not 12 pp. 3325-3333, 1999. "Tolerance to toxic metals by to gene family of phytochelatin synthases from plants and yeast". Stephan Clemens, Eugene J. Kim, Dieter Neumann and Julian I. Schroeder). The second one cloned, the homolog phytochelatin of Arabidopsis (Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 7110-7115, June 1999). "AtPCS1, to phytochelatin synthase from Arabidopsis: Isolation and in vitro reconstitution". Olena K. Vatamaniuk, Stephane Mari, Yu-Ping Lu, and Philip A. Rea). And the third one transformed in several occasions, Brassica juncea with genes that increase the metal absorption ("Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing .gamma.-Glutamylcysteine Synthetase". Yong Liang Zhu, Elizabeth A. H. Pilon-Smits, Alice S. Tarun, Stefan U. Weber, Lise Jouanin, and Norman Terry. Plant Physiology, December 1999, Vol. 121, pp. 1169-1177).

SUMMARY OF THE INVENTION

When a place is contaminated with heavy metals, it can be the origin of a serious problem of environmental health and even of human health. The metals can happen to be dissolved in the water of rivers or source of water nearby, and also they can happen to be absorbed by plants or trees, giving place at its entry in the nourishing chain. Even, not happening any of these two processes, the metal can be accessible to any alive being that accidentally manipulates the waters or contaminated soils. Sometimes, there takes place such a serious problem of pollution (as it happened with the company Boliden, in the Aznalcollar region in the year 99), that the metals devastate the flora and fauna that they find to its step. It emerges therefore, a technical problem that consists of the manipulation of these contaminated places, either in order that they are not a danger for the environment, or in order that they are returned to his natural state before being contaminated.

The present invention provides a procedure to decontaminate a media of growth contaminated with metals, using individuals of the plant species Nicotiana glauca, and Nicotiana glauca modified genetically (for inclusion of the gene TaPCS1), during a period of sufficient time in order that the metals are absorbed by the roots and accumulated in the different tissues of the plant (root, stem, leaves). Being the metals of a group consisting of lead, arsenic, cadmium, copper, mercury, iron, chromium, uranium, nickel and zinc.

The proposed procedure uses the plant species Nicotiana glauca, chosen of a group of vegetable surviving species in a contaminated area, located in Valencia city.

At present, the state of the art provides alternative ways of solving the problem. The most habitual are also the costliest and they base on fundamentally chemical and physical methods, since they are: the committal of the soils contaminated with inert materials, the cleanliness "in situ" with the machines and chemical suitable material, the storage as poisonous residues in the places adapted for it (with license), etc. All these skills have the big disadvantage, of their high cost, in addition to which in any cases it is not possible to carry out the finished cleanliness. The committal, it does not look like a real solution to the problem, but rather a postponement of the same one in the time. The same might be said, of the storage of the soils contaminated in the places conditioned for it.

The techniques that use hyperaccumulator plants have the great disadvantage of which these plants possess some characteristics that dissuade them as solution, namely: low biomass, adjustment to a very concrete habitat, normally only accumulate a specific metal while they do not resist a set of diverse metals, and have a short cycle of life. What is looked actually, is that the plants absorb the metal pollutant (or the set of metals pollutants), and that they transport it to the parts able to be collected. Later this material would be harvested and it would arrange suitably or would burn itself (also it might study other alternatives).

In the current state of the art, since it has been mentioned previously, it has been elected another type of plants different from the hyperaccumulators, among them it is necessary to stand out on every Brassica juncea. Later, on these plants there have introduced genes that they qualify the plant species to a better response. In general, the use of the biotechnology to solve this type of problems of pollution, has the clear disadvantage of the enormous consumption of time that is needed to solve the problem, and that besides the process of decontamination has to be carried out almost always "in situ". Nevertheless, the phytoremediation (disciplines that use vegetable material to decontaminate contaminated places), it is always a much less costly procedure in terms of money.

In the present invention, one manages to improve the state of the art previously developed. In this way: 1.--Wild chosen flora: it is adapted to specific climatology and edaphology 2.--It is very competitive ("weeds") in "normal" situations and of metal excess. 3.--Adapted to soils of low nutritional and water content (they resist water and nutritional stress). 4.--Improved natural capacity. It means that it is probable that they are individuals modified genetically with regard to those who grow in not contaminated zones. 5.--Improved capacity by means of genetic modification. There has been introduced a gene of wheat, which level of expression is permanently increased. The result is a major capacity of absorption of heavy metals, as Pb, Cd, etc.

The point one, it does not mean that these plants are not a transportable technology to other points of the world geography. Probably the opposite will happen, since one the advantages of the used plant species is that it survives habitually in extensive geographical areas of the world. This way for example, although it is original of the south and central part of the American continent, it spreads also over all United States and Canada, the whole Australian continent, and part of Europe and Japan. Besides, this plant species reproduces by cuttings, it replicates with facility in the field, provides abundant seeds, has aptitude to banish animals (which is a guarantee of his difficulty to enter the feed chain), is in use as ornamental plant in the United States and Canada, and all that together with the familiar beneficial characteristics for phytoremediation as they are: deep root system, high biomass, and adjustment to soils nutritionally low and of high pollution for metals. Another additional, not despicable advantage exists still, and the fact is that the technology of transformation of genes to vegetable species is limited to a limited group of plant species, among them the plant species that the investigators more have worked is Nicotiana tabaccum. Therefore, Nicotiana glauca, might benefit from the whole scientific technological undivided assets that has developed for Nicotiana tabaccum.

In addition to the importance that has the vegetable species that has been chosen to be biotechnologically modified, it is necessary to emphasize the importance of the gene that has been transferred. The gene TaPCS1, it was studied in depth demonstrating a great specialization and yield in the absorption of lead and cadmium in yeast (used like model system). It was the first phytochelatin synthase that was studied, and it belongs to one vegetable species that is cultivated habitually in the exterior of the laboratories (it does not happen equally with Arabidopsis), what it could mean a better capacity of adjustment to the external environment. This gene had never been transformed into plant previously, hence the innovation and importance of this fact.

The importance of the role played by the phytochelatins in the detoxification and homeostasis of heavy metals, is still today an object of discussion. Recent works have shed some light in the molecular base of this process and also in the role of the phytochelatins and the phytochelatin synthases in the process of metal accumulation. Nevertheless part from the described investigation very recently it is incoherent with previous works and it needs an urgent clarification.

Phytoremediation and Metal-Binding Peptides

In the last decades the concern for the global Earth environment has impulse a major research focus in decontamination. The phytoremediation is an emerging and low cost technology that utilizes plants to remove, transform or stabilize toxic chemicals located in water, sediments or soils. Physiological studies indicate that heavy metal tolerance is one of the prerequisites of heavy metal hyperaccumulation in plants (Kramer et al., 1997; Raskin et al., 1997). Phytoremediation of heavy metals more likely require genetic engineering metal tolerance in selected wild plant species, which are fast-growing, of high biomass, and tolerant to a wide range of environmental conditions.

Living organisms have developed molecular processes that allow cells the capability to detoxify (in some particular concentration ranges) heavy metals. In eukaryotic cells, these mechanisms include mainly sequestration and chelation by specific ligands. Among these kind of molecules most of researchers interest has been focused in the last years in two of them: metallothioneins and phytochelatins. Metallothioneins are ubiquitous low molecular weight proteins and polypeptides of extremely high metal and sulfur content (Kagi J. H. and Schaffer A., 1988). Phytochelatins (PCs) are thiolate peptides with the primary structure (.gamma.-Glu-Cys).sub.n-Gly, (where n=2-11) non-translationally synthesized from glutathione (Grill, E. et al. (1989). PCs have been identified in a wide variety of plant species including monocots, di-cots, gymnosperms and algae (Rauser, 1999).

GSH is Substrate of Phytochelatin Synthase.

PCs are synthesized from Glutathione (GSH). GSH is synthesised in a two-step pathway (FIG. 1) catalysed by .gamma.-glutamylcysteine synthetase (.gamma.-ECs) and glutathione synthetase (GS); requires energy since both are ATP-dependent enzymes. The level of GSH decreases on the induction of PCs in plant cell cultures and the exposure of whole plants or plant cell cultures to an inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO), also inhibits the induction of PCs and/or confers hypersensitivity to metal ions. Furthermore, in cell cultures the effects of BSO can be reversed by the addition of GSH evidencing a marked interaction between GSH and phytochelatin biosynthesis regulation.

The Key Role of PCs in Heavy Metal Tolerance is Discussed

What is the role played by PCs in heavy metal (HM) tolerance and accumulation? Do they are essential instruments in engineering phytoremediation? Do they play a basic role in plants that emerge more tolerant by natural selection under HM pressure? Most significant recent advances in the understanding of the general role played by PCs comes from molecular genetic studies using different plant and fungi model systems. Specially for the differential metal tolerance observed among naturally occurring selected plants, resistant and sensitive ecotypes.

Negative Evidence

Some researchers think that previous studies of phytochelatin production in response to Cd and Cu demonstrated that PCs are not responsible for metal tolerance although they are indeed involved in the detoxification process. This affirmation relays upon the fact that root tips of Cd-tolerant plants of Silene vulgaris exhibit a lower rate of phytochelatin production accompanied by a lower rate of longer chain phytochelatin synthesis than those of Cd-sensitive plants Silene vulgaris (De Knecht, et al. 1994). Regarding their role in naturally selected plants that have increased HM accumulation, some researchers believe that the investigation of tolerant Silene vulgaris plants from the copper mining dump shows that PCs are not responsible for the development of the heavy metal tolerant phenotypes. This is based in the fact that Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Although the binding of HM ions to PCs exists it seems to play only a transient role in the heavy metal detoxification mechanism of this plant species (Leopold, et al. 1999). Similar findings have been reported for the hyperaccumulator Thlaspi caerulescens and the related non-accumulator T. arvense. Total PC levels found in the hyperaccumulator were generally lower respect to the non-accumulator, despite correspondingly higher metal concentrations. However again, similarly to Silene vulgaris, PCs were produced by both species in response to Cd, and phytochelatin levels showed a similar positive correlation with Cd-concentration in leaf and root tissues (Ebbs, et al. 2002). What are the molecular bases of PCs enzymatic regulation for this apparent absence of relevance in HM tolerance? Although Cd-tolerant Silene vulgaris plants produce three times less PCs than Cd-sensitive ones, when are exposed to external supply of Cd, neither the PCS activity nor PC degradation accounts by for the difference in PCs concentration (De Knecht, et al. 1995). Therefore, all these evidences may suggest a lack of importance for the PCs role in Silene vulgaris and Thlaspi caerulescens respect to Cd tolerance. Consequently, from all this information some researchers have concluded that differential synthesis of PCs is not involved in producing differential metal tolerance. However it also can be argued that PCs concentration is three times higher in Cd-sensitive Silene vulgaris plants after external Cd supply respect to the non sensitive, therefore whether they really play a key role or not in these sensitive plants should be questioned.

Very recently a set of evidences against the possible central role of PCs in HM detoxification have been presented. When Arabidopsis thaliana PCS gene (AtPCS1) was overexpressed with the goal of increasing PC synthesis and therefore metal accumulation, transgenic lines showed paradoxically hypersensitivity to Cd. However, this hypersensitivity disappeared when GSH was supplemented in the medium. Consequently, it is argued that Cd hypersensitivity seems due to the toxicity of PCs, as they existed at supraoptimal levels when compared with GSH levels (Lee, S. et al. 2003).

Positive Evidence

The first evidence comes from two basic facts: plant cells when exposed to HM rapidly accumulate PCs and PCs production is activated by HM ions (Grill et al., 1989). When the activity of the partially purified enzyme was determined in vitro, the enzyme was active only in the presence of HM ions (being Cd the best activator). When the activity was determined in vivo (in both intact plants and plant cell cultures), HM also induced PC biosynthesis (Rauser, 1995). Lack and restoration of function has been also an important source of evidences. For example, it was reported that phytochelatin-deficient Saccharomyces pombe and Arabidopsis thaliana mutants are hypersensitive to Cd. Furthermore, AtPCS1 was able to suppress the Cd-sensitive phenotype in Brewer's yeast (Vatamaniuk, et al., 1999). Stronger evidence however, has come from a wheat cDNA, TaPCS1, whose expression in S. cerevisiae results in a dramatic increase in Cd tolerance (Clemens, et al., 1999). This work demonstrated that TaPCS1 expression led to an augment in Cd.sup.2- accumulation inside the yeast cells even at concentrations that do not affect the growth. More important, this group showed that heterologous expression of PCS genes is sufficient to confer enhanced metal tolerance. Performing growth assays with S. cerevisiae cells expressing AtPCS1 and SpPCS (S. pombe homolog), they have demonstrated that phytochelatin synthesis alone can significantly increase cellular Cd tolerance. Besides, when wheat roots were investigated for TaPCS1 expression, RT-PCR experiments indicated that this PCS expression is constitutive and enhanced by Cd, consistent with the reported constitutive activity of PCS (Grill, et al., 1989) in roots and stems (Chen, et al., 1997) and coherent with the suggested requirement for organisms to express metal tolerance genes constitutively (Zenk, 1996). Therefore PCS activity is regulated transcriptionally and post-transcriptionally by HM. Are these evidences strong enough to answer the question of whether PCs are essential for HM tolerance? There are now more observations supporting the idea of a key role of PCs in HM tolerance. Very recently the overexpression of gene TaPCS1 in Nicotiana glauca selected in a contaminated environment increases Cd tolerance and furthermore lead accumulation. Roots growth was improved drastically (near 160%) and leaves were bigger and greener in the transformed plants respect to wild type, in the presence of 50 .mu.M Cd and even 0.8 mM lead. TaPCS1 transformed plants are able to growth in mining soils having as high as 1572 ppm of lead (2602 ppm of Zn), and accumulating twice lead level than wild type plants. TaPCS1 enhanced expression triggered a higher lead transport (around 200% accumulation) to the root tissue and to the aerial parts (near 150%). Furthermore this improved metal tolerance might be a first step towards engineering hyperaccumulation in this fast-growing, high biomass plant species (Gisbert, et al., 2003). All these positive evidences together strongly suggest that indeed PCs play a role of physiological and ecological relevance in this plant species and many other organisms beyond the manifest role of PCs in HM detoxification.

PCs are Basic Instruments in HM Tolerance, at Least in Some Organisms

A set of evidences has questioned the role played by PCs in HM detoxification. Differential Cd tolerance in S. vulgaris in tolerant and sensitive ecotypes is not affected by differential PC production per se. Increased Cd tolerance does not result from an increased accumulation of PCs, from a faster synthesis of longer PCs (forming more stable complexes than shorter forms), or from an increased incorporation of sulfide into PC-Cd complexes in the roots (which likely increases the stability and the potential amount of metal bound per unit of PC-SH). However, tolerant plants reach the same PC concentration as sensitive plants when exposed to higher Cd concentrations. Since the difference between the amounts of PCs induced neither is caused by a difference in the specific PCS activity, nor the rate of PC breakdown, it might then result from a lower Cd concentration in the cytosol caused by a faster transport of PC-Cd complexes, across the tonoplast into the vacuole (Salt, and Rauser, 1995). It also is possible, that tolerant plants contains a higher proportion of acid-soluble Cd, which is not bound to PCs or another complex in addition to PC-Cd is into the vacuole, or that the Cd ion itself is transported via the tonoplast antiporter activity (Salt, and Wagner 1993). Same arguments might likely explain the similar PC concentrations for T. caerulescens and T arvensis. Nevertheless it is interesting to note that the accumulator T. caerulescens produces a positive Cd gradient across the plant from roots to leaves (1000-3000 ppm) while T arvensis develops a higher but negative Cd gradient (7500-1000 ppm). Consequently, and following the same accumulation pattern in both Thlaspi species, shoot Cd and PC levels are higher in absolute values for T. caerulescens, and accordingly PCS showed a less rate of saturation for this ecotype in leaves. Consistently, in this tissue a higher ratio of Cd to .gamma.-GluCys subunits has been observed for T. caerulescens as if Cd were preferentially sequestered as high molecular weight complexes (Ebbs, S. et al., 2002). Although these arguments might also contribute to understand the hypersensitivity reported by AtPCS1 overexpression in Arabidopsis, evidences are clearly contradictories with previously reported works (Vatamaniuk, et al. 1999; Clemens, et al., 1999). Perhaps, nonspecific protein-protein or others unknown interactions caused by the modification of the AtPCS1 C-terminal region by the FLAG epitope (DYKDDDL) used for protein recognition, may contribute to the observed Cd hypersensitivity in lines overexpressing AtPCS1 (Lee, et al. 2003). The C-terminal domain clearly has some role in PCS activity since the cad1-5 mutant synthesizes a truncated polypeptide predicted to lack nine of the 10 Cys residues in this C-terminal domain, producing an altered phenotype. It is likely that this domain acts as a local sensor by binding heavy metal ions (presumably via the multiple Cys residues, but possibly also others) and bringing them into contact with the activation site in the catalytic domain (Cobbett, C. S., 2000). Therefore, a possible negative interference of AtPCS1 C-terminal FLAG region with PC homeostasis should not be discarded. Beyond of what be the possible explanation for this Cd hypersensitivity, the facts are that after addition of a plant-specific phytochelatin chemically synthesized and, more important, after AtPCS1 expression in mammalian cells, the transfected cells exhibited production of plant-specific PC and higher resistance to Cd.sup.2+ (Takagi, et al., 2003). It is known that mammalian cells can not synthesize PC because of their lack of PCS.

The work reported specially by Clemens et al., 1999, Tagaki et al., 2003 and, very recently, by Gisbert et al., 2003 have presented solid and consistent evidences for a primary role of PCs in HM tolerance and detoxification in a wide range of organisms. Perhaps negative evidences accumulated might possibly indicate that PCs are essential but not the solitary essential component of the more complex mechanisms developed by organisms to detoxify HM. Consequently other basic elements, when increased or more effective, might reduce the need of a significant augment of PCs and PCS concentrations in such specific cases.

REFERENCES FOR THIS PARAGRAPH

Kramer, U. et al. (1997) The role of metal transport and tolerance in nickel hyperaccumulation by Thlaspi goesingense Halacsy. Plant Physiol. 115, 1641-1650 Raskin, I., et al. (1997) Phytoremediation of metals: using plants to remove pollutants from the environment. Curr. Opin. Biotechnol. 8, 221-226 Kagi J. H. and Schaffer A. (1988) Biochemistry of metallothionein. Biochemistry 27, 8509-8515 Grill, E. et al. (1989) Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific .gamma.-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc. Natl Acad. Sci. USA 86, 6838-6842 Rauser, W. E. (1999) Structure and function of metal chelators produced by plants; The case for organic acids, amino acids, phytin and metallothioneins. Cell. Biochem. Biophys. 31, 19-48 De Knecht, J. A. et al. (1994) Phytochelatins in Cadmium-Sensitive and Cadmium-Tolerant Silene vulgaris. Plant Physiol. 104, 255-261 Leopold, I. et al. (1999) Phytochelatins and heavy metal tolerance. Phytochemistry 50, 1323-1328 Ebbs, S. et al. (2002) Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Planta 214, 635-640 De Knecht, J. A. et al. (1995) Synthesis and degradation of phytochelatins in cadmium-sensitive and cadmium-tolerant Silene vulgaris. Plant. Sci. 106, 9-18 Lee, S. et al. (2003) Overexpression of Arabidopsis thaliana phytochelatin synthase (AtPCS1) paradoxically leads to hypersensitivity to cadmium stress. Plant Physiol. 131, 656-663 Rauser, W. E. (1995) Phytochelatins and related peptides: structure, biosynthesis, and function. Plant Physiol. 109, 1141-1149 Vatamaniuk O. K. et al. (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc Natl Acad Sci USA 96, 7110-7115 Clemens, S. et al., (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J. 18, 3325-3333 Chen, J. et al. (1997) Characterization of phytochelatin synthase from tomato. Physiol. Plant. 101, 165-172 Zenk, M. H. (1996) Heavy metal detoxification in higher plants--a review. Gene, 179, 21-30 Gisbert, C. et al. (2003). A plant genetically modified that accumulates Pb is specially promising for phytoremediation. Biochem. Biophys. Res. Commun. 303, 440-445 Salt, D. E. and Rauser, W. E. (1995) MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol 107, 1293-1301 Salt D. E., Wagner G. J. (1993) Transport of Cd in tonoplast vesicles from oat roots. Evidence for a Cd/H antiport activity. J Biol Chem 268, 12297-12302 Cobbett, C. S. (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiol. 123, 825-832 Takagi, M. et al. (2003) Cellular Toxicity of Cadmium Ions and Their Detoxification by Heavy Metal-Specific Plant Peptides, Phytochelatins, Expressed in Mammalian Cells. J. Biochem. 131, 233-239.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. is a chart showing PC biosynthesis in plants and yeast. Glutathione (GSH) is a substrate for phytochelatin synthase (PCS). Cd is a positive effector of .gamma.-ECS, Glutathione synthase (GS) and PCS. .gamma.-ECS is also feedback regulated by GSH. Both .gamma.-ECS and CS are ATP dependent. Cd is a positive effector of CS. The phytochelatin is bound to Cd and forms a complex PC-Cd. The complex is transported inside the vacuole by a transporter type YCF1.

DETAILED DESCRIPTION

The binary Ti vector pBI121 (Clontech) was used for transformation. The GUS gene of the binary vector was replaced with the wheat phytochelatin synthase 1 cDNA (TaPCS1, Accession No. AF093752) to gain the new construct pBITaPCS1. The TaPCS1 cDNA (donation of Professor Julian Schroeder, University of California, San Diego) was originally cloned in pYES2 (Invitrogen) and designated pYESTaPCS1. The plasmid was digested with XhoI and converted to blunt ends with the DNA polymerase I (Klenow fragment). Afterwards, pYESTaPCS1 was digested with BamHI to produce a 2-kb insert containing the TaPCS1 cDNA. PBI121 was digested with BamHI and ECL136II. The 2-kb TaPCS1 insert was ligated to the BamHI-Ecl136II sites of plasmid pBI121. The new construct (pBITaPCS1) was electroporated into Agrobacterium tumefaciens strain C58C1RifR Rif (Van Larebeke et al. 1974).

Nicotiana glauca leaf explants were infected with A. tumefaciens after two days of preculture on organogenic medium NB2510 [MS salts (Murashige y Skoog, 1962) including Gamborg B5 vitamins (DUCHEFA), 3% sucrose, 2.5 .mu.g mL.sup.-1 naphthalene acetic acid (NAA), 1 .mu.g mL.sup.-1 of 6 benzyl aminopurine (BA), and 0.8% agar (bacteriologic agar "Europeo" PRONADISA) in the dark. Explants from adult and young leaves were infected by immersion on Agrobacterium culture for 10 min. After 1 day of cocultivation, explants were transferred to selection medium NB2510 containing 100 .mu.g mL.sup.-1 kanamycin and 350 .mu.g mL.sup.-1 carbenicillin. Two months after infection, shoots were individually removed from the call using explants and transferred to bottles containing 30 ml of B1 medium (MS salts including Gamborg B5 vitamins, 0.3 .mu.g mL.sup.-1 indol acetic acid or 0.2 .mu.g mL.sup.-1 NAA, 1% sucrose, 100 .mu.g mL.sup.-1, and 0.7% plant agar).

SEQUENCE LISTINGS

1

2711044DNAArabidopsis thaliana 1atggcttcaa attcagcact tctcatgaaa acaatcttcc tcgtactcat ttttgtctct 60tttgcaatct ctccagcaac ttcaactgcg ccggaagaat gtggaagcga gtcagcgaac 120ccgtgcgtca acaaagctaa agctttgcct ctcaaagtca tagcaatctt cgtaatcctc 180attgcaagca tgattggtgt tggagctcct ctctttagcc gtaacgtttc gttcctccaa 240ccagacggaa acatcttcac tatcattaag tgtttcgcct ccgggatcat ccttggaacc 300ggttttatgc acgttttacc tgattctttc gaaatgttgt catctatatg tcttgaagag 360aacccgtggc ataaatttcc tttctccgga tttctcgcta tgttatcggg tctaatcact 420ctagccattg actccatggc cacgagccta tacaccagca agaacgcagt tggtatcatg 480ccccatggtc atggtcatgg tcacggcccc gcaaatgatg ttaccttacc aataaaagaa 540gatgattcgt caaatgcaca gctcttgcga taccgagtca ttgccatggt cttggaactt 600gggatcatag ttcactcggt ggtcattgga ttatctctag gagcaactag tgacacttgc 660accattaaag gacttatagc agctctttgc ttccatcaaa tgttcgaagg catgggtctt 720ggcggttgta tcctccaggc tgagtataca aatatgaaga aatttgttat ggcgttcttt 780ttcgcggtaa caacaccatt cggaatagcg ttagggatcg ctctatcaac tgtttaccaa 840gataatagcc caaaagcttt gatcacggtt ggacttctaa atgcatgctc cgctggattg 900ctcatttaca tggcactcgt ggatcttcta gctgcggagt tcatgggacc taagcttcaa 960ggtagcatca aaatgcagtt caagtgttta atcgcggctc ttctcgggtg cggtggaatg 1020tcgattatcg ccaaatgggc ttaa 104421053DNAArabidopsis thaliana 2atggctacta ccaagctcgt ctacattctc ctcatcctat tcaccttcac cgtatctccg 60gcgatctcaa cggccccgga acattgtgat agcggctttg ataacccgtg catcaacaaa 120gctaaggctt taccactcaa aatcgtagcc attgttgcca tacttacaac aagcttgata 180ggcgtgacct ctcctctttt cagccgttac atttcgttcc tccgtcccga tggcaatggt 240ttcatgatcg tcaaatgttt ttcttctgga atcatccttg gaaccggttt catgcacgtc 300ttgcctgact ctttcgagat gttgtcatcg aaatgtctta gtgataatcc gtggcataag 360ttcccttttg cgggttttgt cgctatgatg tccggtctag tcactctagc cattgactcc 420attaccacca gcctttatac cggtaagaac tcagtcggac cagtgcctga tgaagagtat 480ggcattgatc aagagaaagc gattcacatg gtaggccaca atcatagtca cggtcatggt 540gtagtgctag caactaaaga tgatggacag cttttgcgct accaagtcat tgccatggta 600ttggaggttg gcattttatt tcattctgtg gtcattggac tatctctagg agcaactaat 660gattcatgta ccattaaagg actcatcata gctctttgct tccatcactt gttcgaaggc 720ataggtcttg gtggctgcat cctccaggca gattttacaa atgtgaagaa gttcttgatg 780gcattctttt tcactggaac aacaccttgt ggtatctttc ttggaatcgc attgtcgagt 840atctatagag ataacagtcc aaccgcgttg attacgattg gactgttaaa tgcttgctcg 900gccggaatgc tcatctacat ggccctcgtc gaccttctag ctaccgagtt catggggtca 960atgctccaag gtagcatcaa acttcagatc aagtgcttca cggcggcttt gcttggctgc 1020gccgtaatgt cggtcgtcgc cgtgtgggct taa 105331068DNAArabidopsis thaliana 3atgtctgaat gtggatgttt ttcggcaaca actatgttga gaatttgtgt agtattgata 60atatgtttgc atatgtgttg tgcctcgagt gattgtacaa gtcacgatga tcctgtgtct 120caagacgaag cagagaaagc gacgaagcta aagcttggtt cgatagcttt acttcttgta 180gccggaggag tcggcgtgag tctaccgttg atcgggaaaa ggattccggc gttacaaccg 240gaaaatgata tcttcttcat ggtgaaagct tttgctgcag gagtgatcct ctgcacaggt 300ttcgttcata tcttaccaga cgcgttcgag agattgagct ctccatgtct tgaggacact 360acagctggga agttcccgtt tgctggtttt gtagcgatgc tgtcggcgat ggggactctt 420atgatcgaca cattcgcgac agggtattac aagaggcaac attttagcaa taaccatggg 480agcaagcaag tgaacgtagt agtagatgaa gaagagcatg cgggtcatgt tcacattcac 540acgcacgcta gtcacggaca cacacatggt tcgaccgagt tgatcagaag acgtatagtg 600tcgcaggtgc ttgagattgg gatagttgtg cattcggtta ttatagggat atcacttgga 660gcttcacaga gcatagacac cataaagcca ctcatggctg cactatcttt ccatcagttc 720tttgaaggtc ttggcctcgg tggatgcatc tccctggcgg atatgaagtc gaaatcgaca 780gtgctaatgg cgacattttt ctcggtgacg gcgccacttg ggataggaat agggttgggg 840atgtcaagtg gtttaggcta caggaaagag agcaaagagg caataatggt ggaaggaatg 900ttgaatgctg catctgctgg gatactcata tacatgtcac ttgttgatct tcttgctact 960gattttatga atccaagatt gcaatccaat ctctggcttc acttggctgc ttatctctct 1020ctcgtcctag gcgcaggttc catgtctctc ctcgccatct gggcctga 106841062DNAArabidopsis thaliana 4atggctttgt cttccaaaac cctaaagtca actctcgtct tcctctctat tattttcctc 60tgtttctcct tgatcctagc tcacggcggc atagacgacg gcgacgaaga agaggagacc 120aaccagccac ctccggccac cggaacaacc accgtcgtga atctccgatc caaaggcttg 180gtgcttgtga agatctactg tattataata ctcttcttta gcacattctt agccggaatt 240tcaccttact tttaccgatg gaacgagtcg tttctcctcc taggaactca attctccggt 300ggtatattcc tcgcgaccgc tctaatccat ttcctcagcg acgctaacga gactttccga 360gggttaaaac acaaagagta tccttacgct ttcatgttag cagccgctgg atattgcctt 420acaatgctgg cagatgtggc ggttgcgttt gtagcggctg ggagtaataa caaccacgtc 480ggagctagcg tcggagagtc gagggaggat gatgacgtgg cagtgaaaga ggaaggacgt 540cgtgagataa agagtggtgt tgatgtgagt caagcgctta tacgaactag tggatttgga 600gacacagctt tgctgatttt tgctctttgt tttcactcca tctttgaggg aatcgccatt 660ggtctctcag acactaaaag cgacgcttgg agaaacctat ggacaatatc gttgcacaag 720gtctttgcgg ccgtagcaat gggaatagct cttctcaagc taatccctaa acgtccattc 780ttcctcactg tcgtctactc cttcgccttt gggatatcga gtcccatagg tgtcgggatt 840ggcattggaa tcaatgccac tagccaagga gctggtggtg actggaccta cgcgatctct 900atggggcttg cgtgtggagt ttttgtgtac gttgcggtta accatctcat ctcaaaaggg 960tataagcctc ttgaggaatg ttacttcgac aagccaatct acaagtttat tgccgtcttc 1020ctcggtgttg ctttgctctc tgttgtaatg atttgggatt ga 106251020DNAArabidopsis thaliana 5atgaagacta agagcgtgaa actcttattc ttcttcttct ccgtctccct ccttctcatc 60gccgtcgtca acgccgccga aggccattca catggtggac caaaatgtga atgctcacac 120gaagacgacc atgaaaacaa agccggagct cggaaataca agatcgccgc aattcctaca 180gttctaatag ccggcataat cggagttctt ttccctttgt taggcaaagt cttcccttct 240ttgcgtccag aaacatgttt cttcttcgtc acgaaagctt tcgcagccgg agttatcttg 300gctaccggat ttatgcatgt cttgcctgag gcttacgaga tgcttaactc tccatgtttg 360atatctgaag catgggaatt tccgttcacc ggatttattg cgatgattgc tgcgatcttg 420acgttatccg ttgatacatt tgccacttcg agtttctata aatcgcattg caaagcgtct 480aagagggtca gtgatggaga aaccggcgag tcctccgttg actccgagaa ggtccaaatt 540ctccggacta gagttattgc acaggtattg gagttgggaa taatagtaca ctcagtggta 600ataggaatat cactaggagc ttcacagagc ccagatgctg caaaagctct gtttattgcc 660ttaatgtttc atcaatgctt cgaaggtcta ggccttggtg gttgtattgc tcagggaaaa 720ttcaagtgtt tgtcagtaac aatcatgtcg acgttcttcg caataacgac accgatagga 780atcgttgtgg gaatgggaat agcaaattct tacgatgagt cttcaccaac ggctctgatc 840gttcaaggag ttttgaacgc tgcatccgca ggcattctca tctacatgtc tttggttgac 900cttctcgcag cagatttcac gcaccctaaa atgcaatcca atactgggct tcaaattatg 960gcccatattg ctctccttct tggtgctggc ctcatgtctc tattggctaa atgggcttga 102061131DNASaccharomyces cerevisiae 6atgagcaacg ttactacgcc gtggtggaaa caatgggacc cttctgaagt tacacttgcc 60gataaaaccc ctgatgatgt gtggaagacc tgtgttttgc aaggtgttta ctttggtgga 120aacgagtaca atggtaactt aggtgccaga atatcttccg tctttgttat tcttttcgtg 180agtacttttt tcaccatgtt cccattaatc tcaacaaaag tgaaaagatt gagaattcct 240ctatatgttt accttttcgc aaagtatttt ggttccggtg ttattgttgc aaccgcattt 300atccacttaa tggaccctgc ttatggtgcg attggtggta ccacttgtgt aggacaaacc 360ggtaactggg gtctttattc atggtgtcct gccattatgc taacgagttt gaccttcact 420ttccttactg atctattcag tagcgtctgg gttgaaagaa agtatggtct ttcccatgac 480catacccacg atgaaattaa agacactgtt gtgagaaaca ctgcagctgt ttcaagtgag 540aatgacaatg agaatggtac tgcaaatgga tctcatgaca ccaagaacgg agtagagtat 600tatgaagatt cagacgctac atccatggat gttgttcaat catttcaagc acaattttat 660gcctttttaa ttttagaatt cggtgtgatt ttccactccg ttatgatcgg tctaaacctg 720ggaagtgttg gtgatgagtt ctcctcccta taccctgtct tagtgttcca tcaatcattt 780gaaggtttag gtattggtgc aagattgtca gccattgaat tccctagatc aaagagatgg 840tggccatggg ccctatgtgt tgcgtatggg ttaaccacac caatctgtgt ggccatcggt 900ttgggtgttc gtaccagata cgtcagcggt tcttacactg cgcttgttat ctctggtgtt 960ttggatgcca tttctgctgg tatcttattg tacactggtt tggttgaact actagcaaga 1020gactttatat tcaatcctca aagaacaaag gatctaagag aattgtcctt caacgttata 1080tgcactcttt tcggtgctgg tatcatggct ttgatcggta agtgggctta a 113171269DNASaccharomyces cerevisiae 7atggttgatc ttatagcgag ggatgactcc gtagatactt gccaagcttc taacggctac 60aatgggcacg caggtcttag aattctggca gtattcatta tactgatatc gtcaggattg 120ggagtttatt tcccaatttt gtcatcacgg tattcgttta taaggctacc aaattggtgc 180tttttcatag cgaagttctt cggttctggt gtcattgttg ccacagcgtt cgttcatctt 240ctacagcccg cagccgaagc tctgggagat gaatgtcttg gtggcacatt tgccgaatat 300ccatgggctt ttgggatctg tttaatgtcg cttttcttac ttttcttcac tgaaatcatc 360acgcattatt ttgtagcgaa aacgctggga cacgatcatg gggaccatgg ggaagttacc 420agtattgatg ttgatgctcc cagttcggga tttgtcatca gaaatatgga ctcggatcct 480gtatctttca ataacgaagc tgcctactcc atccataatg acaaaactcc gtacactact 540agaaatgaag agattgtcgc tactcctata aaggaaaaag aacccggctc aaatgttact 600aattatgatc tggaaccggg aaaaacagag tcactagcta atgaactagt tccaaccagt 660tcccatgcga caaatctcgc ttctgtacct ggaaaagatc attattctca cgaaaatgac 720catcaagatg tctcccagtt ggccacacgt atcgaggagg aagataaaga gcagtatctc 780aatcagatac tagctgtttt tattctagaa tttggcatca tctttcactc tgtatttgtg 840ggtctttcgc tatctgtcgc gggtgaagaa ttcgaaacct tatttatcgt tttaactttc 900caccaaatgt tcgaaggttt gggtctaggc acaagagttg ccgaaacgaa ttggccagaa 960agtaagaagt acatgccttg gttaatggga ttagccttca ctttaacgtc acccatagca 1020gtcgcggtag gtattggtgt cagacactct tggatacctg gctctagaag agcattaatt 1080gctaatggtg tttttgactc gatatcatca ggaattctta tttatactgg actagtcgaa 1140ttaatggctc atgaattctt atactctaat caattcaaag gacctgatgg cctcaaaaaa 1200atgcttagtg catatctcat catgtgttgt ggagctgctt taatggctct tctagggaaa 1260tgggcatag 12698948DNAArabidopsis thaliana 8atggctacga ctactcaaca catgaatcaa atcttcctcg tactcctcct aatctccttc 60gcaatctctc cggcgatctc aacggttcca aaagaatgcg agaccgattc aacagactct 120tgcatcgata aaaccaaagc cttacctctc aaaattgtag caatcgttgc catcctcgtg 180acaagcatga tcggagtcgc agctcctctc tttagccgat atgtcacatt cctgcatcca 240gatggtaaaa tctttatgat cattaagtgt tttgcatccg ggatcatcct aggaaccggc 300ttcatgcatg ttttgcccga ttctttcgag atgttgtcct ctccatgtct tgaagacaat 360ccatggcaca agtttccctt cactggcttt gtcgctatgt tgtccggtct tgtaactctt 420gctattgact ctatcgctac aagtctctac accaagaaag ctgtcgctga tgacagcgaa 480gaaaggacta ctcctatgat aattcaaatc gatcatttgc ctctaacaac taaagaacgt 540agctctacat gctcaaaaca actattgcgg taccgagtta tcgccacggt cttggagctt 600gggataatag ttcactccgt ggtcattgga ctatctctag gtgcaaccaa cgacacatgc 660actattaaag gccttattgc agctctttgc ttccatcaaa tgttcgaagg gatgggtctc 720ggcgccttgt cgagcgttta caaagacaac agtccaacag cattaatcac ggtaggactg 780ctcaatgctt gttctgcagg attgctcatt tatatggcac ttgtagatct tctagccgca 840gagtttatgg gatctatgct ccaaagaagc gtcaagcttc agcttaactg cttcggggca 900gctttgcttg ggtgtggcgg aatgtcagtc ctggccaagt gggcataa 94891428DNAArabidopsis thaliana 9atggcgggaa tcgtgacaga gccgtggtca gtagctgaga acggaaaccc aagcataacg 60gcgaaaggat cgagcagaga actaagactt ggaagaaccg ctcacaacat gtcttcttct 120tctttgagga aaaaatcaga cctccgagtg attcagaagg ttccatacaa aggtcttaaa 180gattttctct ctaatctcca agaagtcatt ctcggaacaa agcttgccat tctttttccg 240gccattcctg ccgccattat ttgcacctat tgtggcgtca gtcagccttg gatatttgga 300cttagcttgc taggactgac acctttggct gagcgagtca gctttttgac agagcaacta 360gctttctaca ccggtcctac attgggtggt ctattgaacg caacgtgtgg aaacgcgact 420gaattgataa tcgcgattct tgctttgacc aataacaaag tcgcagtggt gaaatattcg 480ctgctaggtt cgattttgtc gaacctttta ttggttctag ggacttcact cttctgtgga 540ggaatcgcta atatccgaag ggaacagcgg ttcgaccgga aacaagccga tgtgaacttc 600ttcttacttc tactgggttt cttgtgtcac ttgctgccat tgttggtggg atacttgaaa 660aacggagagg cttcggctgc tgttttgtcc gacatgcaac tgagtatatc gcgaggcttc 720agtattgtta tgttgatcag ctacattgca tatcttgttt tccaactgtg gactcaccgc 780caattgttcg atgcacaaga acaggaagat gagtatgatg acgatgtgga gcaagaaacc 840gcggtgatta gtttttggag cggttttgca tggttggttg ggatgacact cgttatcgca 900ttgctatcgg agtatgttgt agccacgatt gaggaagcat cggataaatg gaacttatca 960gtgagtttca taagcatcat attgcttcct attgttggaa atgcagctga acatgctgga 1020gccgttattt ttgcctttaa gaacaagctt gacatatctt tgggagttgc gttaggctct 1080gcgactcaga ttggcttatt cgttgtcccc ttgaccatca tcgtggcgtg gattttggga 1140attaatatgg atctcaattt tggtcccctc gaaactggct gtcttgctgt ttccataatc 1200atcacagcgt tcacattaca ggatgggagt tctcactaca tgaagggact ggtcctcttg 1260ctttgctatt tcattattgc catctgtttc ttcgtcgaca aacttcccca gagtgagtta 1320gttttcaaat gtatatgcat gctattatta gggaaaacaa taattgaggc atacaacacc 1380catatatcaa atggaaatgc ttcatcaaat aaagttaaaa cgggttaa 1428101380DNAArabidopsis thaliana 10atgggaagta tcgtggagcc atgggcagca atcgccgaga acggaaacgc aaacgtgacc 60gcgaaaggct cgagcaggga gctgcgacat gggagaacag cacacaacat gtcttcatcg 120tcgctaagga agaaatcaga cctgagattg gttcagaaag ttccatgcaa aactctcaag 180aacattctct ctaatcttca agaagtcatt cttggtacta agcttactct cttatttctc 240gccatccctc tcgccattct tgccaattct tacaactacg gtcgtccgtt gatatttgga 300ctgagcttga taggactgac acctctagct gagcgagtta gctttttgac agagcaacta 360gctttctaca ctggtccaac agtgggcggt ttgttgaacg cgacttgtgg aaacgcgaca 420gagctgataa tcgcgatact agcgttggcc aataacaaag tggcagtggt gaaatactct 480ctattgggtt caattctctc aaaccttctc ttggttcttg gcacttccct cttctttggt 540ggtatcgcca atatccgccg cgagcagcgg ttcgaccgga aacaagccga tgtgaacttc 600ttcttgctgc ttatgggcct gttgtgtcat ttgctgccat tattgttaaa atatgcagca 660accggcgaag tatcgacctc tatgattaac aaaatgtcgc tcactctgtc gcggacaagc 720agcatagtta tgcttattgc ttacattgct tatctcatct tccagctctg gactcaccgc 780caattgtttg aggcacaaca ggatgatgat gatgcatatg atgatgaggt tagtgttgaa 840gaaactccag tgataggatt ctggagcgga tttgcttggc tcgttgggat gacaatagtc 900atcgcattgc tatcagagta tgttgtggac acgatcgagg atgcatcgga ctcatgggga 960ctatcagtga gtttcataag catcatattg cttcccattg ttgggaatgc ggctgagcat 1020gctggagcca tcattttcgc attcaagaac aagctcgaca tatctctagg ggttgcgttg 1080ggctctgcaa ctcagatttc tttgttcgtg gtcccattga gtgttatcgt tgcgtggatc 1140ctgggaataa aaatggatct caactttaac atccttgaaa ctagctctct agctttggcc 1200attatcatca cagccttcac tttacaggat ggaacttctc attacatgaa gggactggtt 1260ctattgttat gctatgtcat catcgcggcg tgtttcttcg tcgaccaaat tccccaacca 1320aatgatttgg acgtgggact tcaacccatg aacaatttgg gagaagtttt ctcagcttaa 1380111530DNAArabidopsis thaliana 11atgccacaac tcgagaacaa cgagccactt ctaatcaacg aggaagaaga agaagaaaca 60gcgtacgatg aaacagagaa ggtacatatc gtaagaaacg aagaggagga cgatctagaa 120cacggcgtcg gatgcggcgg cgcaccaccg ttctcatgga agaagctatg gttattcacc 180ggacctgggt ttttaatgag cattgcgttt ttagatccag ggaatctcga aggagatctt 240caagccggtg cggttgctgg gtactctttg ttatggcttc tcatgtgggc aacagcaatg 300ggtcttttgg ttcagctttt gtcggctagg cttggtgttg cgacaggtcg tcacttagct 360gagctttgtc gtgatgagta tcctacttgg gcaagaatgg ttttgtgggt tatggctgaa 420ttggctttga ttggatctga tattcaagaa gttattggta gtgctattgc tatcaagatt 480ttgagtaatg ggattttgcc tctttgggct ggtgttgtta ttactgctct tgattgtttc 540gtcttcttgt ttcttgagaa ctacggaata aggaagctcg aggctgtgtt tgcagttctt 600atcgctacaa tgggagtctc attcgcttgg atgtttggtc aagctaagcc aagtggctct 660gagcttctca ttgggatttt ggtaccgaaa ctgagttcaa gaacgataca gaaagcagtt 720ggagttgtgg gttgcattat aatgccacac aatgtgtttc ttcactcagc tcttgttcaa 780tctagagaag tcgataaacg acagaaatac cgagtccaag aagcgctaaa ctactacaca 840atagaatcca caattgctct tttcatctcc tttttgatca atctgtttgt cacaactgtt 900ttcgccaaag ggttttataa tactgacctc gccaatagca tcggtttggt taacgcggga 960cagtatcttc aggagaaata tggaggcggt gtgttcccga tactatacat ttgggcgatc 1020gggctattag ctgctggcca aagcagcact attaccggta catatgcggg acagttcata 1080atgggcgggt ttcttaattt caaaatgaag aaatggttga gagctttgat cacacgaagc 1140tgcgctatca ttccaactat tatcgttgcg ctagtgtttg attcatcgga agctacactc 1200gatgtcttaa acgagtggct taacgtgctt cagtccattc aaatcccctt tgcactcatt 1260cccttacttt gtttggtctc caaggaacaa atcatgggta gcttcaagat tggtcctttg 1320tacaagacaa tcgcgtggct tgttgctgcg ctcgtgataa tgatcaacgg ttatcttttg 1380ttggagttct tctccaatga ggttagtggc atagtctata ccggttttgt gacactgttc 1440acagcttctt atggtgcatt catcctctac ctcattgctc gtggcatcac tttcactcct 1500tggccgttca aagcggagtc tagtcattga 1530121044DNAArabidopsis thaliana 12atggctacga ctactcaaca catgaatcaa atcttcctcg tactcctcct aatctccttc 60gcaatctctc cggcgatctc aacggttcca aaagaatgcg agaccgattc aacagactct 120tgcatcgata aaaccaaagc cttacctctc aaaattgtag caatcgttgc catcctcgtg 180acaagcatga tcggagtcgc agctcctctc tttagccgat atgtcacatt cctgcatcca 240gatggtaaaa tctttatgat cattaagtgt tttgcatccg ggatcatcct aggaaccggc 300ttcatgcatg ttttgcccga ttctttcgag atgttgtcct ctccatgtct tgaagacaat 360ccatggcaca agtttccctt cactggcttt gtcgctatgt tgtccggtct tgtaactctt 420gctattgact ctatcgctac aagtctctac accaagaaag ctgtcgctga tgacagcgaa 480gaaaggacta ctcctatgat aattcaaatc gatcatttgc ctctaacaac taaagaacgt 540agctctacat gctcaaaaca actattgcgg taccgagtta tcgccacggt cttggagctt 600gggataatag ttcactccgt ggtcattgga ctatctctag gtgcaaccaa cgacacatgc 660actattaaag gccttattgc agctctttgc ttccatcaaa tgttcgaagg gatgggtctc 720ggcggttgca tccttcaggc agaatataca aatgtgaaga aatttgtgat ggccttcttc 780tttgctgtta caacaccatc aggaatcgct cttggtatag ccttgtcgag cgtttacaaa 840gacaacagtc caacagcatt aatcacggta ggactgctca atgcttgttc tgcaggattg 900ctcatttata tggcacttgt agatcttcta gccgcagagt ttatgggatc tatgctccaa 960agaagcgtca agcttcagct taactgcttc ggggcagctt tgcttgggtg tggcggaatg 1020tcagtcctgg ccaagtgggc ataa 1044131095DNAArabidopsis thaliana 13atgactaaat ctcatgtcat tttctctgcc tccatcgctc tctttctcct actctccatc 60tcgcatttcc cgggagctct ctctcaatcc aataaagatt gccaatcaaa atccaactac 120tcatgtatag acaaaaacaa agcattagat ctcaaactct tatcaatctt ctcaattctc 180atcactagtc taatcggtgt ttgcctcccg ttctttgccc gatcaattcc cgctttccaa 240cccgagaaat ctcacttcct catcgtaaaa tctttcgcct ccggaatcat cctttccact 300ggtttcatgc atgtcttgcc tgattctttc gagatgcttt catctccttg tcttaacgat 360aacccctggc acaagtttcc tttcgccggc tttgtagcca tgatgtctgc cgtgttcacg 420ctcatggttg actctattac caccagcgtc

ttcaccaagt caggaaggaa agatctacgt 480gctgacgtag catccgttga gactcctgac caagagatag ggcacgtaca ggttcatggc 540catgttcata gccatactct tcctcacaat cttcatggag agaatgataa agagcttggt 600tcttatttac agcttctgcg gtatcgtatt cttgcaatcg tattggagct aggaatagtg 660gtgcagtcga tagtgatagg actatcggta ggagacacta acaatacttg caccatcaaa 720ggactcgtcg ctgcgctttg cttccatcaa atgttcgaag gcatgggtct cggcggttgc 780atccttcagg cggagtacgg gtgggttaaa aaggcggtga tggctttctt ttttgcggtg 840acgacgcctt ttggagtggt tctagggatg gcactatcta aaacatacaa agagaatagc 900cctgaatcgc ttataacagt tgggttgctc aacgcttcct cggcaggact actcatctac 960atggctttag ttgaccttct agctgccgat tttatgggtc aaaaaatgca aaggagcatc 1020aagcttcaat taaagtcata tgctgccgtt ttgcttggtg ctggtggcat gtccgtcatg 1080gccaagtggg cttga 1095141401DNACapsela bursa-pastoris 14atggctggaa tcgtaacaga accgtgggca tcggcggaga acggaaacgc aagcatgaca 60gcgaaaggat cgagcagaga actgagacat ggaagaacag ctcacaacat gtcatcttct 120tctctaagga agaagtcaga cctccgagtg attcagaagg ttccatacaa aggtctcaaa 180gattttctca ccaatctcca agaagtcatc ctcggcacta agctcgccat tcttttcccg 240gccattcctg ccgccattat ctgcacctat tgtggcgtca gtcagccgtg gatatttgga 300ctgagcatgc taggactgac acctttggct gaacgagtca gctttctgac agagcaacta 360gctttctaca ccggtccaac attgggtggt ctactgaacg caacgtgtgg aaacgcgact 420gaattgataa ttgcgattct ggcattgacc aataataagg tcgcagtggt gaaatattcg 480ctgttaggtt cgattttgtc aaatcttctt ttagttctag ggacttcact tctctgcggc 540ggaatcgcta atatcaggag ggagcggcgg ttcgaccgga aacaagcgga tgtgaacttc 600ttcttactcc taatgggttt gttgtgtcac ttgctcccat tgatgttcgt atacgtggca 660accgcagaga ctccggctgc tcttgtttct gacatgacac tgactctgtc gcggggcagc 720agtattttta tgttgatcgg ttacattgca tatctcgttt tccagctttg gtctcaccgc 780caattgttcg acgcacaaga tcaggaagat gagtatgatg acgatgtaga ggaagaaacc 840gcggtgatta gtttttggag cggttttgct tggttggttg ggatgaccct tgtcatcgca 900ttgctatccg agtatgttgt tgccaccatt gagaccgcat cggaatcatg gaacctatca 960gtaagtttca taagcatcat attgcttccc attgttggaa atgcggctga acacgctgga 1020gccatcattt ttgcctttaa gaacaagctc gacatatcat tgggagttgc attaggctct 1080gcgactcaga ttggcttatt cgtcgtaccc ttgaccataa tcgtggcgtg gattctagga 1140attaatatgg atcttaactt caatctcctc gaaaccggtt ctcttgctct ttccattatc 1200atcactgcct tcacattaca ggatgggact tcacactaca tgaagggact ggtcctcttg 1260ctttgctatt tcattattgc cttctgtttc ttcgtcgaca aacttcctca gaaacaacca 1320aatggttttc acatgggact tcaacagata aacaatgttg tcactggaat cactggaaca 1380ggaggagctt cttcaactta a 1401151686DNACaenorhabditis elegans 15atggaagtaa caatggaaga tcgctctgtc aaagctgata aggctgatag agatgataac 60aatactacct ccaccgaact gcttggtaaa atgcgtcaaa caaaagtgat cagtacaaac 120caaaacaata atcatttgat tgatcgtatg gtgtcaatat atgagatgga aatgatcaaa 180tcccaatcca cagaaactat cagcgatgta tcagacgttt tagaatttac ggttatcgat 240aattgttcac acggtacaca tactcttgaa catgatctca ggcttaaagg agagcctatt 300ggcaagtcgg aatctgttaa aggtgttagc agaagcctta ttattcaaat tgggatgact 360gtgatattct gcgcacttga attcatcacg ggagtcgtct gttcatctat tgcaatgctt 420gctgacagtt accatatggc agccgacgtg atggcgctca ttgtggcatt cacgtgcatt 480aaaatcgcta ctcgcccttc cacgcgcctc ggatacggct gggtccgtgc tgaaacactt 540ggaggattct tcaacggaat ctttatgtgt acggtatgtg ttctcgtgtt tcaagaggca 600gttggacgta tcatcaatgt acacatgatt acacaccctc tacaagtact tgtcatcgga 660ttcatcggat tgctaatcaa cttattcggt atgttcaact tgagtggtca cggtcacagt 720catggaggcg gaagtcatgg tcacagtcac ggaggaagtc atggtcacag tcacaataac 780aagaagacta agaaaaatga tgggcatggt cacagtcatg caaatggcca tggacactct 840catgatggga aaagtgattg caatggtgaa gaagagccgg atcacactag attgaatgga 900aaatttcgaa gtgcttctgc gatggcaaac tccgacgcca atgtgcgact actggataat 960gatgacaact ccaatgacat tattgaacgt cgtctttctg gagttaatag tcagaatacc 1020atcatcgcga cggtcgatcg tcaaatgaca ccatatggaa cacacatggc cagtgaagtt 1080ctcaatgtgt cttctaacaa tctcgataaa agtgctcaaa agacagaaca gaaaaaagac 1140aaaaatgtga acattcacgg agtttggctt catttattgt cggatgcctt cggatctgtg 1200atcgtcatga tttctgccgg atttgtatac tttttgccaa catggaaaat tgccgcttat 1260ttggatccaa ttttaagtat ttccctggcc tccatcatgg gtttcactgc agtggttctt 1320gtcaaaacat cgggagaaaa gctgctcaag caaaccccag aagggctcga cctcgaaaag 1380gtgaaaaaag atttgtgcag cattgttggt gtatccaaag tggaaaagct gtctgtgtgg 1440acgttgtgtg ggcaacgaat cattgctgcc gcccacgtga acatatgtca cccagcagtt 1500tttcctgaag ctgcttataa aattaaaaat tatttccacg acttgggagt acactcaacg 1560actattgaac ccacctttga ggatacctgc atgcagtcga tgagaataat ggttaaaaaa 1620gttgttgatg gcaaaagcat cgaggagcca gtcagtgtgt caactgaaaa tgaaatcacc 1680gaataa 1686161701DNAPhaseolus vulgaris 16attttccaat ttcgtactgg attcattgct ccctcttctc gtgtatttgg aagaggtgtt 60ttggttgaag attcttgggc aatagcaagg aggtatctat catcatattt cttagttgac 120attcttgctg ttcttcccct cccacaggtg gtgattctag ttatcattcc aaagatgagt 180ggttttaaat cacttaatac caagaacttg ctgaaatttg ttgtcttctt ccaatatgtg 240cctcgtttat tgcgggtcat tccattatat agagaagtta caagagcctc tggcattctc 300actgaaactg cttgggctgg agctgcattt aatctatttc tttacatgct tgcaagtcat 360gttgttggtg ccttttggta cttgttttct atagaacgag aaaccacatg ttggcaagaa 420gcctgtcgaa gtaatacaac agtgtgtaac aaggcagata tgtattgtaa tgattattgg 480ggtgggctga gcaaaatttc gacattcctg aatacttctt gcccaataca gaaggaagat 540aaaaatctct ttgattttgg aatgttcctt gatgctcttc aatccggtgt tgtggagtca 600agagattttc cacaaaaatt cttttactgc ttttggtggg gcttaaaaaa tttgagttct 660cttggccaga acctgggaac aagtcctatg tttgggaaat atgctttgca gttttcattt 720ctgtatctgg tttgggtatt ttcattcctc attggaaata tgcagacata tttgcagtca 780acaaccacaa gattggagga gatgagagtg aagaggaggg atgcagaaca gtggatgtct 840cacccgattg cttctgatgg cctgagagtg cgaatcagac gatatgagca gtacaaatgg 900caagaaacca gaggcgtaga tgaagacaat ttggttcgta atcttcccaa ggatttaaga 960agagacatta agcgacatct ttgtttggct ttgctaatga gagtgccaat gtttgagaaa 1020atggatgaac aacttctgga tgcaatgtgt gaccgtctga agccggtgct gttcactgaa 1080gaaagctaca ttgtgaggga aggagaccca gttgatgaga tgctgttcat aatgcgtggg 1140aagttactga ccataacaac taacggtgga agaaccggtt tcttcaactc ggagtatctg 1200aaagctggtg acttctgtgg agaggagctt ctgacgtggg ccttggatcc ccattcctca 1260tccaaccttc ccacctcaac cagaacagtc caaactcttt cagaagtgga agccttcgcc 1320ctcaaagccg atgacttgaa gttcgtggca tcacagtttc ggcgccttca cagtaagcag 1380ctacgccaca ctttccggtt ctactcgcaa caatggcggt cgtgggctgc gtgcttcatc 1440caagctgcat ggcggcgata cagtaagagg aagcttgaag aatccctggt tgaagacgag 1500aatagactgc aaaatgtgtt ggctaaatca ggtggaagct cacctagcct tggtgctaca 1560atatatgctt caaggtttgc tgcaaatgca cttacattgc tacgccgtaa tggtgcaaag 1620aagggtaggg tggcagagag attacccccc atgctttttc agaagcctgc agaacctgat 1680tttactgcgg atgaagaata a 1701171551DNAGlycine max 17atgtctggga gccaccaaga gcagccactg ttagagaact cgttcataga agaagacgag 60ccgcaagaaa cagcttatga ttcgtcggag aagatagtgg tggtcggagt cgacgagttc 120gatgacgagg agaattgggg gagagtgccc cgattctcgt ggaagaagct atggctgttc 180accgggccgg gctttctgat gagcatagcg tttctggacc ctggaaactt agagggggac 240cttcaggcgg gtgccattgc agggtactca ttgttgtggc ttctgatgtg ggccacagca 300atgggcctcc tgatccagct cctctcggca cggctcggcg tggccacagg gaagcacctc 360gccgagctct gccgagagga gtatcctccg tgggcccgga tagtgctctg gatcatggcg 420gaactcgctc tcattggctc cgatattcag gaggttattg ggagcgctat tgcaatcagg 480attcttagtc atggggttgt gcccctttgg gctggggttg tcattactgc tcttgattgt 540tttatttttc tctttcttga gaactatggt gtgaggactt tggaagcttt ttttgctatt 600ctcattggtg tgatggcaat ctcgttcgca tggatgtttg gtgaagccaa gcccagtggc 660aaggaacttc ttcttggagt tttgattcca aaactcagct ccaaaactat acagcaggct 720gttggagttg tggggtgcct tattatgcct cacaatgtgt tcttgcactc tgctcttgtt 780cagtcaaggc aggttgaccg cagcaagaaa ggccgagttc aagaagctct taattattac 840tcgatagagt ccacccttgc ccttgtagtt tcctttatta taaatatttt tgtaacaaca 900gtgtttgcta agggatttta tggctctgaa cttgcaaaca gcataggtct tgtaaatgca 960ggacagtatc tagaggagac atatgggggt ggactatttc caattttata catatggggt 1020attggattat tagcagcagg ccaaagtagc actattactg ggacttatgc aggacaattc 1080atcatgggag gttttctaaa tttaaggtta aagaagtgga tgagggcgtt gattacccga 1140agttgtgcaa taattccaac tatgatagtt gctcttttat tcgatacctc ggaggaatcg 1200ttagatgttt tgaatgagtg gcttaatgtt cttcagtcag tccagatccc ctttgcactt 1260attcccttgc tttgtctggt gtcaaaggag cagataatgg gcactttcag aattggtgct 1320gtcctcaaga ctacttcatg gctcgtggct gctctggtga tagtgattaa tggctatctt 1380ttgacggaat tcttttcctc tgaagtgaat ggaccaatga ttggcactgt agtgggtgta 1440ataactgctg catatgttgc cttcgtagta taccttattt ggcaagccat cacctattta 1500ccttggcaaa gtgtaacaca accaaagaca attgctcatt cagagggttg a 1551181053DNALycopersicon esculentum 18atggcaaatt ataatttcaa gtacatcgcc attttcctcc ttctcatctc aattttggcc 60cctcgagtac tatcagtagt agaagattgt ggagcagaag aagacaactc atgtgtcaat 120aaatccaaag cgttaccctt aaaaatcata gccatagtct ccatccttat cactagtatg 180atcggagtat gtcttccact agtcacacgt tctattccgg ccctaagccc ggaaagaaac 240ctttttgtga tagttaaggc atttgctgct ggaattatcc tggctacggg gtttatgcac 300gtgctaccgg actcgtttga catgttgtca tcgagttgcc ttaaggagca cccgtggcac 360aaattcccct ttactggatt tgtggcaatg ttgtccgcta tagtaacgat ggctattgac 420tctatagcta ctagtttata cagcaaaaag cataatggtg gtgtggttaa tccagaaggt 480gatcaagaaa tggctgtggc tggaaatcat gttcattccc atcatcatca tggatccctt 540tcgactaaag atggacttga tggcaaaaaa ttactaagat acagagtaat tgccatggtg 600ttagagcttg gaattattgt tcactccata gtgattgggc tatcactagg tgcgtcaagc 660aatacatgta cgattaaagg actcgtagct gcactttgct ttcatcaaat gtttgaagga 720atgggccttg gtggttgcat cctacaggcg gagtataagt tcatgaagaa ggctataatg 780gcgtttttct tcgcagtaac aacaccattt ggtatagcac ttgggatagc attgtcaact 840acttatgagg aaaatagtcc acgggcgtta ataactgttg gattactgaa tgcatcatct 900gctggacttt tgatatatat ggctttggtt gatcttcttg ctgctgattt tatgggtgac 960aaattacaag gcagtgtcaa actacaaatt aagtcttaca tggctgttct tcttggtgct 1020ggtggaatgt cagtcatggc catttgggct taa 1053191059DNALycopersicon esculentum 19atgagtgatt ataatttcaa gcacatcgcc atcatcttta ttctcatatc aattttcatc 60cctcgagttt tatcagtagt agaagattgt ggagcgcaag aagataactc atgtgtcaac 120aaatccaaag cgttaccctt aaaaatcata gccatagtct ccatccttat cactagtatg 180atcggagttt gtcttccact agtcacacgt tctatcccgg ccctaagccc ggaaagaaac 240ctttttgtga tagtcaaggc atttgctgcc ggaattatat tggctacggg gtttatgcac 300gtgcttcctg actcattcga catgttgtca tcgagttgcc ttaaggagaa tccatggcac 360aaattcccct tcactggatt tgttgctatg ttgtccgcta tagttacaat ggctattgac 420tctatagcaa ctagtatgta tagcaaaaaa catagagctg gtttggttaa tccagaaact 480ggtggtgctg atcaagaaat gggtgcagta aatggtggac attcacatca tcatcatgga 540tcactttcca ctaaagacgg agttgaaggc actaaattac tacgatatag agtcatcgct 600atggtgttag agctgggaat catagttcac tcaatagtaa taggaatttc acttggagct 660tcaaacaata catgtacaat taaaggatta gttgctgcac tttgctttca tcaaatgttt 720gaaggaatgg gacttggtgg ttgcattctc caggctgagt acaagttttt gaagaagaca 780ctaatggcat ttttcttcgc agtaacaact ccatttggta tagcacttgg tatggcattg 840tcaactactt atgaggaaac tagcccacgg gcgttaataa ctgttggatt actgaatgca 900tcatctgctg ggcttttgat ttatatggct ttggttgatc ttcttgctgc tgattttatg 960ggtgacaaat tacaaggcag tgtcaaacta caaattaagt cttacatggc tgttctttta 1020ggtgctggtg gaatgtctct catggccaaa tgggcctag 1059201707DNAMus musculus 20atggtgttgg atcctaaaga aaagatgcca gacgatggcg cttctgggga ccatggagac 60tctgccagcc ttggcgccat caaccctgcc tacagcaact catccctccc acattccact 120ggagactctg aggagccctt caccacctac tttgatgaga aaatccccat tcctgaggag 180gagtactctt gttttagctt tcgtaaactc tgggcgttca cggggcctgg ctttcttatg 240agcattgcct acctagaccc aggaaacatc gaatctgatt tgcagtctgg agcagtggct 300ggatttaagc tgctctgggt gctcctcttg gccaccattg tggggctgct gctccagcgc 360cttgcagcga gacttggagt ggtcaccggc ttgcatcttg ctgaagtatg tcaccgtcag 420tatcccaagg tcccacggat catcctgtgg ctgatggtgg agttggcaat cattggttct 480gacatgcagg aagtcattgg ctcagccatc gccatcaatc tcctgtctgc aggaagggtc 540cctgtgtggg gcggagtcct catcaccatc gcagacactt ttgtgtttct ttttttggac 600aaatatggct tgcggaagct ggaagcgttt tttggctttc tcatcactat catggccctc 660acgtttggat atgagtacat tacagtgaag cccagccaga gccaagtact caggggcatg 720ttcgtgccgt cctgtccagg gtgccgcacc cctcaggtgg agcaggcggt gggcatcgtg 780ggagctgtga tcatgccgca caacatgtac ctgcattctg ccttagtcaa gtctagacag 840gtgaatcggg ccaataagca ggaagtgcgg gaagccaata agtacttctt catcgagtcc 900tgcatcgcgc tctttgtttc cttcatcatc aatgtctttg tcgtgtccgt ctttgctgaa 960gcattttttg agaaaaccaa caagcaggtg gttgaagtct gcaaaaataa cagcagcccc 1020catgctgacc tctttcccag tgacaactct actctggctg tggacatcta caaagggggt 1080gttgtgcttg gatgttactt cgggcctgca gctctctaca tctgggcagt ggggatcctg 1140gctgccggtc agagctccac catgactgga acctattctg gccagtttgt catggaggga 1200ttcctgaacc taaaatggtc gcgctttgcc cgagtgatcc tgacccggtc tatcgccatc 1260atccccaccc tgctcgtcgc tgtcttccag gatgtggagc acctaacggg gatgaatgac 1320ttcctgaatg tcctgcagag cttacagctc ccctttgctc tcatacccat cctcacgttc 1380acaagcctgc ggccagtgat gagtgagttt tccaatggaa taggctggag gattgccggt 1440ggcatcctgg tcctgatcgt ctgctccatc aacatgtact ttgtagtggt ttatgtccag 1500gagctagggc atgtggcact ctatgtggtg gctgcagtgg ttagcgtggc ttatttgacc 1560tttgtgttct acttgggttg gcagtgtttg attgcattgg gtctgtcttt cctggactgt 1620ggacgctcgt accgcctggg actgaccgct cagcctgaac tctatcttct gaacaccgtg 1680gatgctgact cagtggtgtc cagatga 1707211083DNAMedicago trunculata 21atggaagtgg gggtgtcctt caaatgcacg gcttattcag taactataca caaagcaatc 60ttcattgttt tcatcttaat cactttttta acatcacaag ccctagctga ttgtgaaagt 120gaatccacaa acagttgcaa caacaaagaa aaggctcagc ctctaaaact catagccata 180ttctcaatct tagcaactag tgtgattggt gtgtgtttac ccttggcgac acgttcaatc 240ccggctttaa gcccagaagg agatcttttc ataatcgtga aatgtttcgc ggctggtatt 300attcttggga ccgggttcat gcatgtactc cctgattcgt acgagatgtt gtggagtgat 360tgtttagatg agaaaccatg gcacgagttt cccttttcgg gacttgtggc tatgttctcc 420gcggtggtca caatgatggt cgattctata gctactagtt attatagtaa gaagggtaag 480agcggagttg tgattccaga gagccatggt ggagatgatc aagagattgg acattcacat 540ggtggtcacc atcatattca taatgggttc aagacagaag aaagtgacga gccacaactt 600ctacgttatc gcgtagtggt catggtatta gaacttggaa ttgtagttca ttcagtggtg 660ataggacttg gaatgggagc ctccaataat acgtgctcaa taaaaggtat cttatcagca 720gccttgtgct tccatcaaat gtttgaaggc atgggtcttg gtggctgcat tctccaggca 780aagtacaagt tcttaaagaa tgcaatgttg gtattcttct tctcaattac aacaccactt 840ggaattgcaa taggacttgc catgtcaaca agttacaaag agaatagtcc agtagcacta 900atcaccgttg gattgcttaa tgcatcatct gctggtcttt taatctacat ggctttggtt 960gacttattag ctgctgattt catgagtaag aggatgcaga gtagtattaa gcttcaatta 1020aaatcttatg tagcagtatt ccttggtgct ggtggaatgt ctctcatggc taaatgggct 1080taa 1083221395DNAOriza sativa 22atgcgcccgg ccttctcgtg gcgcaagctg tggcggttca cggggcccgg gttcctcatg 60tgcatcgcgt tcctcgaccc ggggaacctg gagggcgacc tgcaggccgg cgccgcggcg 120gggtaccagc tgctgtggct gctgctgtgg gcgacggtca tgggcgccct ggtgcagctg 180ctctccgcgc ggctcggggt cgccacgggg aagcacctcg ccgagctctg cagggaggag 240tacccgccct gggccacggc cgcgctctgg gccatgaccg agctcgcgct cgtcggcgcg 300gacatccagg aggtgattgg cagcgcgatt gccatcaaga tcctctccgc tggcaccgtc 360ccgctctggg gcggcgtcgt catcaccgcg ttcgattgct tcatcttttt attcctggag 420aactatggag tgagaaaatt ggaagcattt ttcggagtcc tgattgcagt catggcagta 480tcatttgcaa ttatgtttgg tgaaacaaag ccaagtggca aggaccttct gattggtttg 540gtggttccaa agttgagttc aaggacaatc aaacaagcag ttggaattgt gggctgcata 600atcatgcccc acaatgtctt cttgcactca gcactagtcg agtcaaggaa gattgacaca 660aacaagaaat cccgtgttca agaagcagtg ttctattaca acattgagtc cattcttgcc 720ctcgttgttt cgttctttat taacatctgt gtcacaacag tttttgcgaa aggattttat 780ggatctgaac aagctgatgg tataggtctt gagaatgctg gacagtactt acagcagaaa 840tatgggactg cattctttcc tatactgtat atctgggcta ttgggctgtt agcatctgga 900cagagtagca ctattactgg cacatatgca ggccaatttg ttatgggagg cttccttaat 960cttcggttga agaagtggtt aagagcaatg attactcgaa gctttgcaat tattccaact 1020atgattgtgg ctttattttt tgacacggag gatcctacaa tggacattct gaatgaggca 1080ctcaatgttc ttcaatccat acagatacca tttgcactga ttcctctcat cacactcgtc 1140tcaaaggagc aagtcatggg atcatttgtg gttggtccta tcacaaaagt gattagctgg 1200attgttacag tattcttgat gctcatcaat gggtatctta tactgtcctt ctatgccact 1260gaagtccggg gagcattggt tcggtcaagc ttgtgcgttg tattggcagt ttaccttgca 1320ttcatcgtct atcttatcat gcaaaatacc tcactgtatt ctcgcctccg ctcagcaatg 1380acaaagagca catga 1395231047DNApisum sativum 23atggctaatc cagtaactaa acaaaaatta atctccattg tgtttatctt aatcactctt 60ttcacatcac aagccctagc tgattgtgaa accgaaagca caaacagttg taacaacaaa 120gaaaaggctc tgtctcttaa aatcatagca atattctcaa tattagtaac tagcatgatt 180ggagtgtgtc tacccttggt gtcacgttcc gtcccggctt taagcccgga cggaaatctg 240ttcgtgatcg tgaagtgttt cgcggccggt atcattcttg gaactgggtt catgcatgta 300cttcctgatt cgttcgacat gttgtggtcg gattgtttgc aggagaaacc gtggcacgag 360tttccgtttt cgggatttgc ggctatgatc tctgcggtgg ttacaatgat ggtggattct 420ctggctacta gctattatac tcagaagggt aagaaaggtg ttataattcc agctgaaggt 480gaagttggag atcaagagat gggtgctgtc catgctggtc accatcacca ttaccaggtg 540aagacggaag gcgaggagtc acagcttctc cgttatcgtg taatcgccat ggtattagaa 600ctcggaatag tagttcattc gatcgtgata ggacttgcca tgggatcctc caataacaca 660tgctcgataa aaggtctagt tgcggcactt tgcttccatc aaatgttcga aggcatgggt 720cttggtggtt gcatcctcca ggcggagtac aagtttgtaa agaaggctat aatggtgttt 780ttcttctcaa taacaacacc acttggaatt gcaataggga ttgcaatgtc tagtaattac 840aaagagaaca gtccaaaagc attaatcact gttggattgc ttaatggatc atctgctggt 900cttttaatct acatggcttt ggttgatctt cttgctgctg atttcatgag taggaggatg 960cagggtagta ttaaacttca attaaaatct tatgttgctg tgtttcttgg tgctggtggc 1020atgtctctca tggctaaatg ggcttga 1047241044DNAThlaspi caerulescens 24atggcttcaa

cttcaacact tctcatgaaa acaatcttcc tcgtactcat ctttgtctct 60tttgcaatct ctcctgcaac ttcaacggcg ccggatgatt gtgcaagcga gtcagcgaac 120ccgtgcgtca acaaagctaa agctttgcct ctcaaaatca tagcaatcgc cgcaatccta 180gttgcaagca tgattggtgt tggagctcct ctttttagcc gctccgtgcc gttccttcaa 240cccgacggga acattttcac catcgttaag tgtttcgcct cagggattat ccttggaacc 300gggtttatgc acgttttgcc tgattcgttc gatatgttgt catctaaatg tcttggagag 360aacccgtggc acaaatttcc cttctccgga tttctcgcta tgttggcctg tctagtgact 420ctagtcatcg attccatggc tacaaccctc tatactagca agaacgtagt ggggatcgta 480ccccatggtc atggtcatgg tcatggcccc gaaaatgatg ttgccttacc aataaaagaa 540gatgattccg cgaatgcaca actcttgcga tatcgagtca ttgctatggt attggaactt 600ggaattatag ttcactctgt ggtcattgga ctatctctag gagcaacaag tgacacttgt 660accattaaag gactcatcgc agctctttgt ttccatcaaa tgttcgaagg catgggtctt 720ggcggttgca tcctccaggc tgagtatacg aacatgaaaa agtttgttgt ggccttcttt 780tttgcggtaa caacgccttc cggaatagca ttagggattg ctctttcgac cgtttacaga 840gaaaatagtc cctctgcatt gatcactgtt gggttactca atgcatgctc tgcgggattg 900ctcatctaca tggcgcttgt cgaccttcta gcggccgagt tcatgggacc aaagcttaaa 960ggtagcatca aaatgcaggc caagtgtttc cttgcagctc ttctcgggtg cggtggcatg 1020tcgatcatcg ccaaatgggc ttaa 104425399DNAThlaspi caerulescens 25gtgtcattgc catggttagt gtccayatgg aaataatttg agacggccgt taaattcgac 60gtagcatttt aattaaaatc atttaccaaa gtaaaccatt tttttgaatt tcttgaatag 120gtattggagc ttgggatttt atttcattct gtggtcattg gactatctct aggagcaact 180aatgatgcat gtaccattaa aggactcatc atagctcttt gcttccatca cttgttcgaa 240ggcataggtc tcggtggctg catcctccag gtaccaatat acattcatac tatatatata 300gtttatgtct cttctatcaa ttagaaatat acgctcattt gtatatataa tttatgtgaa 360acaggcagat tttacaaatg tgaagaagtt ctcgatggc 399261539DNAThlaspi caerulescens 26atgtcacggc tcgagaacga tcgtccgctt ctcatcgaca gaatcgacga ggaagaagaa 60gagacggcgt acgacgaaac agagaaagtt cacatcgtga gagacgaaga cgacaacgag 120agggatctcg agtacggcgt cggatgcggc ggcgcgccgc cgttctcgtg gaggaagtta 180tggctattca ccggacctgg attcctgatg agcattgcgt ttctcgaccc agggaacctc 240gaaggagatc tccaatccgg tgctgtcgcc ggatactcgc ttctctggtt gctcatgtgg 300gcaacggcga tgggacttct ggttcagctt ctctcggcga ggctcggcgt cgcgacgggt 360cgtcacttgg cggagctttg ccgggaagag tatccgagtt gggcgggtat ggttttgtgg 420gttatggcgg aattggcttt gatcggatct gatattcagg aagtgatcgg aagtgcgatt 480gctattaaga ttttgactaa tgggattttg cctctctggg ctggtgtcat catcactgct 540cttgactgtt tcttcttctt gttctttgag aactacggaa taaggaagct cgaggcagtg 600tttgcggttt tgatcgctac aatgggagtc tcattcgctt ggatgtttgg tcaagccaag 660ccaagtggct ctgagcttct cgttggcata ttggtaccga aactgagctc aagaacgata 720caaaaagcag taggagttgt gggttgtatt ataatgccgc acaacgtgtt tcttcactca 780gctcttgttc agtctcgaga agtcgacaaa cggcagaaat acagagtcca agaagctata 840aactactaca caatcgaatc cacgctcgct ctcttcgtct cctttctgat caatctcttt 900gtcacgacgg ttttcgctaa agggttttac aacactgact tagccgatag cattggcctt 960gtaaacgccg gacagtatct tcaggacaag tatggaggcg gcttgttccc gatactgtac 1020atttggggaa tcggtttatt agctgcggga cagagcagta ccattacagg tacttacgca 1080ggacaattca tcatgggagg gtttcttaat ttcagaatga agaaatggat gagagctttg 1140atcacacgaa gctgcgcgat cattccaacg atcatcgttg cgctagtgtt tgattcatcg 1200gaagctacac tcgatgtctt gaacgagtgg cttaacgtgc ttcaatcaat tcaaatccct 1260tttgctctca tcccattgct ctgtttggtt tccaaggaac gaatcatggg tagtttcaaa 1320atcggtcctt tgtgtcagac aatcgcgtgg ctagtcgctg cgctcgtgat catgatcaac 1380ggctatcttt tggttgagtt tttctcatcg gaggttagtg gcatcgtcta caccggtttt 1440gtgattgtgt tcacggcttc gtatggtgca ttcatagtct acctcattgc tcgtggcatc 1500aatttcactc cgtggcgtcc taaagcagag tctagttga 1539271533DNAThlaspi japonicum 27atgtcggagt cggagagagc gcgtccattt ttagaatcgg aggagaaagc ttttgaagaa 60actgacaagg ttcacgttgt cggagtcgac gaagatgatg acgtcggtta cgacgagctc 120ggaaatgcgc ctccgttctc atggaagaag ctttggctgt tcaccggacc tggctttctg 180atgagcatcg ccttcctcga tcccggtaac ctcgagagcg atctccaagc cggagccatc 240gctggttact ctctgatttg gctcttgatg tgggcgacgg cgatcgggct tctgattcag 300cttctctctg ctcgtctcgg cgtcgccacc ggccgacacc tggcggagct gtgtcgggaa 360gagtatccga cttgggccag gatggtgctt tggatcatgg cggagattgc tttgatcggt 420gccgatattc aggaagtcat cggaagtgcc atagctatca agatcttgtc taatggattg 480atccctctct gggctggtgt tgtaatcact gctctcgatt gtttcatatt tctaattctg 540gagaattacg gagtaaggaa actagaagct gtgtttgctg ttttgattgc aacaatggcg 600ctttcatttg cttggatgtt tggccagaca aagcctagtg gaaccgaact tcttgttgga 660gctttggtcc caaaactaag ctccaggact ataaaacaag ctgttggaat tgtgggatgc 720atcatcatgc ctcacaatgt gtacttgcac tcagcgcttg tgcaatcgag agaaatcgat 780ccgaaaaaga gattccgtgt caaagaagcc ctcaggtact attccattga atccaccgga 840gctctcgtgg tttccttcat aatcaacgtc tgtgtgacca ccgtgtttgc taaatctttc 900tataagacag acatagcgga tactatcggt cttgcaaacg caggagatta cttacaggaa 960aaatacggcg gcggatattt tccggtgtac tatatatggg ccgtcggact tttagctgct 1020ggtcagagta gtaccatcac tggtacatac gccggacagt ttataatggg agggttcttg 1080aatctcaaga tgaagaaatg gattagagcg acaatcacaa gaagctgcgc gatcatccct 1140acgatgatcg tggcgattgt ctttaattct tcggctacat tgctcgacga gctcaacgaa 1200tggctaaacg ttcttcagtc tgttcagatc cctttcgctg tgatccctct cctttgcttg 1260gtctccaacg agaggatcat gggcagcttc aaaatcaaac ctttaatgca ggcaatctcg 1320tggcttgtag ctgctcttgt gatagccatt aacgcgtatc tgatggtaaa tttcttctcg 1380ggagctgcga agagtgtggt catgctcgtg cttgtgatca tattcgttgt tgcgtatgtt 1440ttttttgtgc tttaccttat ctcaagaggc ttcacgtaca ctccctggca gttagtggct 1500tcggagaaag taaaagagag ggatgatgag taa 1533