Patent Application
20080288174
The present invention relates to a method for determining the function of a gene. This method involves determining the amount of transcript for each of a set of candidate genes in samples taken from different phenotypic and/or genotypic states of an organism and determining the amount of each of a plurality of metabolites in different samples taken from the same states as those mentioned above. Subsequently, the data obtained is analyzed by suitable mathematical methods in order to identify a transcript and one or more metabolites which correlate in the different states, thereby identifying a transcript corresponding to a gene which influences the amount of these one or more metabolites in the organism. The invention furthermore relates to a method for identifying a gene which is capable of modifying the amount of a metabolite in an organism and to a method for identifying a metabolite which is capable of modifying the amount of a transcript in an organism. Likewise, the present invention relates to uses of the genes and metabolites identified in the aforementioned methods.
Since the advent of genetic engineering, many techniques have become available that allow the purposeful and specific modification of the transcript level of a given gene. This may lead to either an increase or to a decrease of the transcript level in a so-manipulated cell. Corresponding techniques have been described for many biological systems comprising various classes of prokaryptes and eukaryotes. The change in the transcript level may be effected transiently or, such as in the case of corresponding genetically modified organisms, constitutively.
However, until today, it cannot be reliably foreseen what effect the modified transcript level of a given gene has on the phenotype of the organism. The term “phenotype” refers herein to any possible detectable property of an organism, in particular including the amount of a polypeptide or, further downstream in the reaction path of gene expression, the amount of a metabolite in a cell. The uncertainty about the effect of a modification of the transcript level is mainly due to the complexity of the events that contribute to the expression of a certain phenotype which often involves a redundancy of the various gene expression and regulatory mechanisms, in particular when eukaryotes, and there especially higher eukaryotes such as mammals, plants or insects, are considered. For instance, up to now, one cannot reliably predict whether a significant reduction of a transcript, e.g. by antisense or RNA interference approaches, will effectively lead to a decrease of the corresponding protein activity in the cell. However, if already at the protein level the effect of a transcript reduction is uncertain, the more unpredictable is then the outcome of such a measure at the level of metabolites. Thus, when it is for instance the task to increase or decrease the amount of a certain metabolite, such as a nutritibonally relevant compound in a plant, the scientists often fail to reach this goal or only achieve a minor success (see for instance DellaPenna, Science 285 (1999), 375-379). Here, the effect of a modified transcription of a gene that is involved in a biochemical pathway is often compensated by feedback control mechanisms or alternative pathways, as it beforehand could not have been foreseen. Thus, those who attempt to reach such goals are normally faced with an enormous extent of trial and error. Such experimental approaches are usually costly and time-consuming since they often involve the production of genetically modified organisms. Thus, there is a need for methods that allow it to more effectively apply the well-established techniques of modifying transcript levels in order to modify the metabolite composition of an organism.
Taking the inverse view, there is likewise a need for effector molecules that are capable of specifically modifying the transcription rate of a gene. This applies particularly to pathogenic conditions and diseases where, for instance due to a mutation, a certain gene is aberrantly over- or underexpressed.
In the post-genomic era, scientists working in medical and biological disciplines are confronted with the need of assigning functions to genes which have been identified in the course of genome sequencing. Based on this and following the aim to more comprehensively understand the regulatory processes in a cell, systems biology has evolved in the recent years (Kitano, 2002; Ideker, 2001; Oltvai, 2002). This field refers to multi-parallel analyses of a multitude of parameters of a given biological system at a range of different molecular levels following a systematic perturbation of the biological system.
The starting point of systems biology can be seen in the genomic research making use of the advanced technologies allowing the sequencing of enkaryotic genomes (see e.g. Arabidopsis and Human Genome Sequencing, 2000 & 2001) and the analysis of the expression level of complete genomes or large proportions thereof (i.e. transcriptomic analyses) (Lockhart, 1996). However, it is becoming increasingly clear that a wide range of post-translational factors bear functional importance in the cell as well. Thus, the development of strategies to allow similar comprehensive studies of the protein and metabolite complements (proteomics (Shevchenko, 1996) and metabolomics (Fiehn, 2000; Roessner, 2001)) of the cell have begun, but still at a relatively early stage. Despite the well-known connectivity between the molecules described by the transcriptomic, proteomic and metabolomic approaches, only a few studies have been described where it was tried to correlate parameters across the various levels (Ideker, 2001; Gygi, 1999; Futcher, 1999). In particular, approaches where transcriptomic data is directly correlated with metabolomic data has to date not yet been described.
In view of the above explanations, it is clear that there is a need for techniques allowing an improved deployment of transcription modification to achieve a deliberate change of metabolite composition in a biological system.
Thus, the technical problem underlying the present invention is the provision of means and methods that render it possible to modify the level of one or more metabolites in a given organism in a more reliable and predictable manner.
This technical problem is solved by the provision of the embodiments as characterized in the claims.
Accordingly, the present invention relates to a method for determining the function of a gene comprising
The method of the invention is based on experiments by which it was surprisingly possible to find significant correlations between the amount of a transcript and the amount of a metabolite when different developmental stages of wild-type potato tubers and transgenic potato tubers are compared (see Example 2). Interestingly, it has been observed that the amount of one transcript may correlate significantly with the amount of more than one metabolite. Likewise, it has been seen that the amount of one metabolite may correlate significantly with the amount of more than one transcript. The observed correlations provide the information that the gene corresponding to the identified transcript may have a function that influences the amount of the one or more metabolites in the organism under investigation. Correspondingly, it can be expected that this gene is a promising target for an intervention that leads to a modified transcript amount of this gene in the corresponding organism if it is intended to modify the amount of said one or more metabolites. Furthermore, on the other hand, it is likewise conceivable that a metabolite the amount of which significantly correlates with the amount of one or more transcripts, or a structurally related compound exercising essentially similar effects, can be an effector molecule useful for modifying the amount of said transcript in the corresponding organism.
Specifically, in the experiments described in Example 2, 518 out of the 23715 transcript-metabolite pairs analysed exhibited significant correlations, whereby 329 correlations were positive and 189 negative. Some of the observed correlations confirmed interrelationships between gene expression and metabolite level that were already known. This provides the proof that the method of the invention works and gives reasonable results. This refers for instance to the strong negative correlation observed between sucrose and sucrose transporter expression (
By the method of the invention, it is possible to determine the function of a gene. The method involves the above-mentioned steps (a) to (c) by which a significant correlation between the amount of one or more transcripts with the amount of one or more metabolites in different states of an organism is determined. Such a correlation provides the information that, in the organism under investigation, a gene corresponding to the (or a) transcript of this correlation may have the function that it influences the amount of the one or more metabolites to which it correlates.
In connection with the method of the invention, the term “gene” refers to the conventional meaning of this term in the field of molecular genetics. In particular, a gene whose function is determined by applying the method of the invention is a nucleic acid molecule which, under suitable conditions, is transcribed and, if the gene encodes a polypeptide, translated. A “coding sequence” is that part of a gene which encodes an amino acid sequence.
The term “function of a gene” means any possible function that a gene may have as long as this function influences the amount of one or more metabolites in the organism under investigation. Typically the function of the gene will be exerted by the gene product, in most cases a polypeptide, that it encodes. The term “function of a gene” embraces the possibility that, for the gene in question, already a function is known, and that, by applying the method of the invention, a novel function will be revealed. Therefore, the term “determining the function of a gene” may in particular mean determining one of the functions of a gene, preferably an additional function of a gene of which one or more functions are already known. If for example, a gene is known to encode a transcription factor, the novel function may be to influence the amount of a nutritionally relevant or essential metabolite. It may be possible that the known function is directly or indirectly involved in the novel function, but that this causality was hitherto not known. In the given example, this would mean that, for instance, the transcription factor is responsible for the synthesis of an enzyme that participates in the biosynthesis of the metabolite.
The term “influences the amount of said metabolite(s) in said organism” means any kind of causal relationship between the activity of a gene or its gene product and the amount of a metabolite in the organism. Such an influence may for example be a more or less direct influence in that the gene encodes an enzyme which participates in the biosynthesis or the metabolization or degradation of the metabolite. On the other hand, the influence may be indirect such as that the gene regulates the activity or the amount of an enzyme which participates in the biosynthesis or the metabolization or degradation of the metabolite.
It is preferred that the gene of which the function is determined by the method of the invention encodes an enzyme, a regulatory protein, a transport protein or a transcription factor.
The term “set of candidate genes” refers to a plurality of genes the amount of transcript of which is determined in step (a). The method of the invention requires that a selection is made of the genes to be analyzed, thereby taking into account a number of factors. These factors include the availability of sequence information of the genes rendering it feasible to obtain specific and significant data on the transcript amount. It is certainly favorable if it is known that the candidate gene is transcribed in the organism of which the samples are taken. Furthermore, experimental restrictions as to the feasibility to produce suitable probes for the respective gene may play a role. Also, the genes to be analyzed in step (a) may be pre-selected by the individual user according to certain predictions on the gene function or requirements concerning for example the intended use of the gene of which a novel function is sought. For instance, in the experiments underlying the present invention, the candidate genes were selected among genes encoding enzymes involved in the primary metabolism and transcription factors. Among these in turn, genes were selected from available tomato EST libraries. In particular, each candidate gene corresponds to one so-called Tentative Consensus (TC) sequence, each being created by assembling ESTs into virtual transcripts. TCs contain full or partial cDNA sequences (ESTs) obtained by classical methods. TCs contain information on the source library and the abundance of ESTs and in many cases represent full-length transcripts. Alternative splice forms are built into separate TCs. To create TCs, CAP3, a DNA sequence assembly program, was used (Huang, X. and Madan, A. (1999) CAP3: A DNA Sequence Assembly Program. Genome Research, 9: 868-877).
The tomato genes used are annotated as described by Van der Hoeven (Plant Cell 14 (2002), 1441-1456). At least two EST clones were selected for microarray construction for each analyzed candidate gene. This example for selecting candidate genes may be adapted by the individual user of the present invention according to his needs. In particular, it may be recommendable to select more than one, if not more than two or even more than three EST clones or corresponding probe molecules being specific for one candidate gene for constructing a microarray or an equivalent device for analyzing the amount of transcripts in step (a) of the method of the invention. Here the methodologies according to the state of the art, such as described in Aharoni (Plant Mol. Biol. 48 (2002), 99-118), may be applied.
In general the number of genes which are analyzed in step (a) should be as big as possible in order to be able to obtain as many as possible correlations between transcripts and metabolites.
In a preferred embodiment, a set of at least 20, preferably of at least 50, more preferably at least 100, and even more preferred of at least 200 genes is used in step (a).
The term “transcript” refers to the RNA that is produced upon transcription of each candidate gene which may be in particular mRNA or also pre-mRNA, i.e. the primary transcript of a gene or a premature processed form thereof. The “amount of transcript” determined in step (a) is the quantity of the transcript in the sample and may for example be expressed in the form amount per fresh or dry weight of the sample. The amount of transcript depends on several factors, however mainly on the transcription rate of the corresponding gene and on the RNA degradation rate.
In step (a), the transcript amount may be determined by applying any suitable technique available to the person skilled in the art. Preferred are techniques that allow the parallel quantification of a plurality of transcripts, especially if data retrieval can be carried out partially or fully automatically. In the field of transcriptome analysis corresponding suitable techniques have been described which mainly focus on the use of DNA chips or microarrays (see for example Aharoni (loc. cit.), Colebatch, 2002 and Thimm, 2001).
In a preferred embodiment, the determination of the amount of transcript is performed as described in the Examples.
In a preferred embodiment of the method of the invention, for determining the amount of transcripts, probes are used that are homologous with respect to the organism of which the samples are taken.
This means that each probe, at least in the region where it is aimed that hybridization takes place, is essentially complementary to the sequence of the transcript of the respective candidate genes. Preferably, the complementary sequence of the probe is identical with the complement of said transcript over the corresponding stretch. However, the use of homologous probes is not a mandatory requirement. It is also possible to use heterologous probes, i.e. for instance derived from a different species than that of the organism under investigation. In this case, however, one should take care that each probe reliably hybridizes with the respective transcript.
The amount of transcript of the candidate genes and the amount of metabolites is determined from two or more samples from an organism, wherein the samples correspond to different phenotypic and/or genotypic states of said organism. The term “organism” refers to any living matter that is capable of gene expression. In particular, “living matter” may be one or more cells, a tissue, an organ or a complete organism such as a plant or an animal. The living matter may be in a naturally occurring form or in a man-made form such as in a cultured form, e.g. cell culture, protoplast culture, tissue culture or the like or in the form of a genetically modified organism. In connection with metabolite determination, the term “organism” also includes the direct environment of the living matter, wherein the “direct environment” is characterized by the presence of a metabolite or a gene product produced by said living matter. This gene product may for example influence the metabolite content in the environment of the cell. The direct environment may for example be the extracellular space around a cell, the apoplast, the cell wall, the interstitial space or a culture medium. Furthermore, the metabolite sample may be taken from a certain part of the organism as for example from certain cellular compartments such as plastids, mitochondria, the nucleus, vacuole etc.
The samples analyzed in steps (a) and (b) are taken from different phenotypic and/or genotypic states of said organism. This is explained by the fact that correlations within the transcript and metabolite composition can only be found if the organism is in different states, whereby these states must be connected with differences in the transcript content and in the metabolite content of the organism. It is the idea behind the present invention that a correlation between a transcript and a metabolite may indicate a causal interrelatedness between the two compounds. Therefore, it is envisaged that, in accordance with the correlation observed, the artificial modification of the amount of one compound may lead to a modification of the amount of the other compound. Thereby, the first compound may be either the transcript or the metabolite.
The term “phenotypic state” refers to differences in the phenotype of the organism under investigation. “Phenotype” means any kind of feature that can be detected and which is not a feature of the genome. Such phenotypic states may for example be visually identified such as a morphological or anatomical difference like they can be observed at different developmental stages. Phenotypic states may likewise manifest themselves by the composition of chemical compounds or the occurrence of a disease. Thus, the phenotypic states may be a healthy state in comparison to one or more pathogenic states, different stages of a pathogenicity or an uninfected versus one or more infected organisms.
The term “genotypic state” reflects differences in the genome of the organism. Thus, if the samples are taken from different genotypic states of an organism, the term “organism” specifically refers to organisms according to the definition given above which belong to the same taxonomic unit, but which differ in at least one genetic trait. Specifically, the “taxonomic unit” is a genus, preferably a species, and more preferably an even lower taxonomic rank such as a race, variety, cultivar, strain, isolate, population or the like. Most preferably, the taxonomic rank is an isogenic line with variance in only a limited number, preferably three, more preferably two genetic traits and most preferably one genetic trait, whereby “genetic tirait” refers to a chromosomal region, a gene locus or, as it is preferred, to a gene. Typically, differences in the genotypic state can be differences between a wild-type organism and one or more corresponding mutant or transgenic organisms or between different mutant or transgenic organisms. A certain genotypic state may be stable or transient as is the case with transduced or transfected cells for instance containing a plasmid, phage or viral vector. Advantageously, organisms of different genotypic state are analyzed when they are in the same developmental stage.
It is immediately clear that the terms “phenotypic” and “genotypic” states may overlap. In particular, normally a genotypic state, if the differing genetic trait(s) is/are expressed in the organism, lead(s) to a difference in the phenotype.
According to the above explanations, in a preferred embodiment of the method of the invention, the different phenotypic and/or genotypic states are different developmental stages, taxonomic units, wild-type and mutant or transgenic organisms, infected and uninfected states, diseased and healthy states or different stages of a pathogenicity.
For each phenotypic and/or genotypic state of the organism, samples are taken in order to determine the amount of the transcripts and the metabolites in these samples.
The term “sample” encompasses any amount of material taken from the organism that is susceptible to the method of the invention. For instance, a sample can be fresh material such as a tissue explant, a body fluid or an aliquot from a bacterial or cell culture, preferably deprived of the culture medium, that may be directly subjected to extraction. On the other hand, samples may also be stored for a certain time period, preferably in a form that prevents degradation of the transcripts and metabolites in the sample. For this purpose, the sample may be frozen, for instance in liquid nitrogen, or lyophilized.
The samples may be prepared according to methods known to the person skilled in the art and as described in the literature. In particular, the preparation should be carried out in a way that the respective compounds to be analyzed are not degraded during the extraction in order to prevent a falsification of the determination in steps (a) and (b). The samples for transcription analysis may for example be prepared according to procedures described in Logemann (1987). The samples for metabolite analysis may for example be prepared according to procedures described in Roessner (2000).
Advantageously, the sample preparation involves the employment of suitable methods in order to remove detection-disturbing compounds from the transcripts (RNA) and/or the metabolites prior to determining the amounts of said transcripts and/or metabolites in the samples. This refers in particular to detection-disturbing compounds which are carbohydrates or other compounds that may disturb identification and quantification of RNA. Routinely, compounds that may disturb the detection of RNA or metabolites are removed by suitable techniques known to the skilled practitioner if such a removal improves the quality and significance of the detection (i.e. the determination of the amounts of said compounds in the sample). For example, it has been shown that the presence of carbohydrates disturbs the detection of RNA by microarrays and that the removal of the carbohydrates from the sample may significantly improve the quality of the detected signals.
In a preferred embodiment of the method of the invention, the amount of transcripts and the amount of metabolites is each determined from the same sample.
This preferred embodiment is based on a technology described in PCT/EP03/00196 and in Fiehn (Eur. J. Biochem. 270 (2003), 579-588). The method described therein provides data useful for quantitatively analyzing metabolites, proteins and/or RNA in a biological source material, whereby said analysis involves suitable statistical evaluation and correlation analysis on the data obtained. In this method, extracting, identifying and quantifying of at least two compound classes of the group consisting of metabolites, proteins and RNA are each determined from one sample. Accordingly, in the preferred embodiment of the method of the present invention, steps (a) and (b) are carried out by applying the corresponding teachings of PCT/EP03/06196. In a particularly preferred embodiment, steps (a) and (b) are performed by (i) extracting the metabolites from the respective sample with at least one solvent or mixture of solvents; and (ii) extracting the RNA from the remainder of the sample after step (i). Thereby, it is a further option that metabolites may additionally be extracted from the yet undissolved remaining cellular material contained in the sample after (ii). Preferably, extraction is carried out by using a mixture of solvents that comprises at least one highly polar solvent, at least one less polar solvent and at least one lipophilic solvent. Thereby, the use of a mixture of solvents comprising water, methanol and chloroform is particularly preferred. More preferably, this mixture of solvents contains water, methanol and chloroform in the approximate proportion by volume of 1:2.5:1. Advantageously, the extraction in step (i) is carried out at a temperature between −60° C. and +4° C.
The term “metabolite” refers to any substance within an organism of which a sample useful for applying the method of the invention can be taken and for which techniques for determining the amount are available. According to the invention, nucleic acid molecules are not within the meaning of “metabolite”. Preferably, the metabolites addressed by the present invention have a low molecular weight, i.e. for instance not more than 4000 Da, preferably not more than 2000 Da, more preferably not more than 1000 Da. Typically, the metabolites to be analyzed may belong to the following, however non-limiting list of compounds: carbohydrates (e.g. sugars, oligo- and polysaccharides such as polyglucans as for example starch or polyfructans), sugar alcohols, amines, polyamines, amino alcohols, aliphatics, aliphatic alcohols, amino acids, lipids, fatty acids, fatty alcohols, organic acids, organic phosphates, organic or anorganic ions, nucleotides, sugar nucleotides, sterols, terpenes, terpenoids, flavons and flavonoids, glucosides, carotenes, carotenoids, cofactors, ascorbate, tocopherol and vitamins.
In a particularly preferred embodiment, the metabolites to be analyzed comprise sugars, sugars alcohols, organic acids, amino acids, ascorbate, tocopherol, fatty acids, vitamins and/or polyamines.
The number and selection of metabolites analysed in step (b) depends on the question for which kind of metabolites a correlation with transcripts is aimed to be determined. Moreover, this depends on the availability of suitable techniques for determining the amount of the respective metabolite, wherein “determining” means identifying and quantifying.
Accordingly, in a preferred embodiment of the invention, the amount of at least 20, more preferably at least 50, still more preferably at least 100, even more preferably at least 150 and most preferably at least 200 or even at least 300 metabolites is determined in step (b).
The determination of the amount of metabolites of interest can be done according to well-known techniques known in the prior art and familiar to the person skilled in the art. Preferably, techniques are applied that allow the identification and quantification in one step and, advantageously, are suited to record the respective metabolites contained in the sample in a comprehensive manner.
For example, the metabolites may be identified and quantified using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), NMR or FT-IR or combinations thereof. Further useful methods include LC/UV, refractory index determination, the use of radioactivity in connection with suitable methods known to the skilled person, thin layer chromatography (TLC), capillary electrophoresis (CE), CE/UV, CE/laser induced fluorescence (LIF), fluorescence detection, electrochemical detection (i.e. colorimetry), direct injection MS, flow injection MS, MS/MS, MS/MS/MS, and further combinations of MS steps (MSn), fourier transform ion mass spectrometry (FT/MS), and gel permeation chromatography (GPC). If appropriate, any of the above methods may be combined.
An exemplary non-biased analysis is described in Fiehn (2000). In this study, of different plant mutants, 326 distinct compounds (ranging from primary polar metabolites to sterols) were detected and relatively quantified, including both identified and non-identified compound's by applying a GC/MS analysis. Another example of a GC/MS analysis that can be applied in the method of the invention has been described by Roessner (2001), who used it for comprehensively studying the metabolism in potato tubers. Alternatively, metabolite data can be obtained by extended chromatographic analysis such as described by Tweeddale (1998) where, after growing wild type and mutant E. coli strains in minimal media and 14C-labelled glucose, 70 metabolites could be separated using two dimensional thin layer chromatography. The relative quantification of metabolites was carried out by radioactive detection.
In step (c), the data obtained in steps (a) and (b) are analyzed by applying suitable mathematical methods in order to identify a transcript and at least one metabolite the amounts of which significantly correlate in the different phenotypic and/or genotypic states.
The term “analyzed by applying suitable mathematical methods” refers to any mathematical analysis method that is suited to further process the quantitative data obtained in steps (a) and (b) in a way that significant correlations between transcripts and metabolites can be ascertained. These data represent the amount of the analyzed compounds present in each sample either in absolute terms (e.g. weight or moles per weight sample) or in relative terms (i.e. normalized to a certain reference quantity). Usually normalized data is used for performing correlation analyses. Normalization may involve the representation of the amount of an examined compound at each state by setting the figure in relation to one reference value determined at one specific state. Normalization may furthermore involve the correction of background levels and the combination of the transcript and the metabolite data sets into a single data sheet. Corresponding mathematical methods and computer programs are known to the skilled person. Examples include SAS, SPSS, systatR, R and Matlab. As the next step, the statistically pre-processed data may be subjected to a pairwise correlation analysis. Here series of pairs of data points from the analyzed compounds are looked at for correlation, whether positive or negative, for instance by using the Pearson's correlation coefficient.
In a preferred embodiment, the mathematical analysis of the method of the invention furthermore involves network analysis. Network analysis aims at finding out higher order interplays of multiple factors on the basis of pairwise correlation data. By taking several data sets each obtained from one sample, correlations between metabolites and transcripts as well as among these classes of compounds can be analysed in order to derive information about the network regulation of biological systems, e.g. upon genetic or environmental perturbation. The analysis of pairwise correlations in particular between metabolites and transcripts allows to establish links between regulatory and metabolic networks and the computation of general properties such as connectivity for both types of networks.
A comprehensive overview of methods for quantitatively analyzing data obtained according to the method of the invention including principle component analysis, “snapshot analysis”, Pearson correlation analysis, mutual information and network analyses can be found in Fiehn (2001).
According to the present invention, a significant correlation between the amount of a transcript and the amount of a metabolite in the different states, whereby the candidate genes may be pre-selected or non-selected, can be determined by a non-parametric Spearman's rank order correlation analyzis. Rank order correlation appears to be preferable to other methods because transcript and metabolite levels may be correlated in a non-linear manner. In particular, in a first step, for all possible transcript and metabolite pairs Spearman correlation coefficients may be calculated. Subsequently, the correlation coefficients may be compared with a Spearman Rank significance table for specific parameters (P=0.01). This approach has also been taken in the experiments described in Example 2. The value rs is the result of the Spearman correlation coefficient calculation. The formula for the Rank (Spearman) Correlation Coefficient is
In a preferred embodiment, step (c) of the method of the invention comprises the steps
In this embodiment, first those transcripts and metabolites are determined the amount of which shows significant differences between the analyzed states of the organism. Corresponding statistical analysis methods and applicable software are known to the person skilled in the art and described in the literature. This may for example be done as it is described in Example 1. On the so-pretreated data, the analysis for finding pairwise correlations between transcripts and metabolites may be performed as described above. Preferably, only the transcripts and metabolites showing significant differences as determined in steps (i) and (ii), respectively, may be used for the correlation analysis. This may have the advantage of saving computational and other capacities.
It is the result of the method of the invention to identify a transcript in step (c) that significantly correlates with at least one metabolite in the different states. This finding gives an indication that the gene corresponding to this transcript has a function that influences the amount of said metabolite(s) in said organism. The term “gene corresponding to a transcript” refers to the gene from which the transcript is transcribed. The gene can be identified according to conventional techniques common to the molecular biologist. The term “function that influences the amount of a metabolite” has already been defined further above.
In a further aspect, the present invention relates to a method for identifying a gene which is capable of modifying the amount of a metabolite in an organism comprising steps (a) to (c) of the aforementioned method for determining the function of a gene, wherein said transcript identified in step (c) corresponds to a gene being capable of modifying the amount of said metabolite(s) identified in step (c).
The term “gene capable of modifying the amount of a metabolite in an organism” refers to a gene having the function to influence the amount of a metabolite in the organism as it can be determined by the corresponding method described above. As a consequence, it is contemplated that an exogenously induced alteration of the expression of this gene as compared to the normal wild-type gene expression in the organism under the same developmental and environmental conditions will lead to a significant modification of the amount of said metabolite in the organism as compared to the amount of the metabolite in the corresponding organism with the expression of said gene not being altered.
Thus, when a correlation between a transcript and a metabolite has been revealed by the method of the invention, a gene corresponding to this transcript can be made the target of a specific alteration of its gene expression if it is intended to deliberately modify the amount of the metabolite. In this context, the term “altered gene expression” refers to any measures that lead to an altered amount of the transcript of said gene in the organism. Primarily encompassed are measures that modify the transcription rate or the stability of the transcript RNA. However, if the gene encodes a polypeptide, measures are also encompassed that modify the translation rate, the activity of the encoded gene product or post-translational modifications of the polypeptide resulting in a modified activity of the gene product. Depending on whether the correlation is positive or negative, an increased gene expression may lead to an increase or a decrease of the amount of the metabolite in the organism. Accordingly, a decreased gene expression may lead to a decrease or an increase of the metabolite in the organism. This effect can be employed, for example, in order to produce useful plants having an increased content in a nutritionally valuable metabolite such as a vitamin or having a reduced content in a undesirable metabolite such as a compound being responsible for allergic reactions. Likewise, the effect can be used in gene therapeutical approaches in order to increase or decrease a certain metabolite in a specific tissue or organ.
Often it will be necessary that the modification of the particular metabolite is not accompanied by the modification of other metabolites which might evoke undesirable side effects. In such a case, a gene can be selected for modification which does not show significant correlations with other metabolites.
Various methods for exogenously inducing an alteration of the expression of a specific gene are known and described in the literature that can be applied when a gene has been identified that is capable of modifying the amount of a metabolite in an organism.
An increase of gene expression may for example be achieved by over-expressing the corresponding gene product from a gene construct introduced into said organism by applying conventional methods such as those described in Sambrook. (2001) and Gassen (1999). However, the state of the art provides further methods for achieving an increased gene expression. For example, the corresponding endogenous gene may be modified at its natural location, e.g. by homologous recombination, for example by positively affecting the promoter activity. Applicable homologous recombination techniques (also known as “in vivo mutagenesis”) are known to the person skilled in the art and are described in the literature. One such technique involves the use of a hybrid RNA-DNA oligonucleotide (“chimeroplast”) which is introduced into cells by transformation (TIBTECH 15 (1997), 441-447; WO95/15972; Kren, Hepatology 25 (1997), 1462-1468; Cole-Strauss, Science 273 (1996), 1386-1389).
A reduction of gene expression may be achieved by different techniques described in the prior art. These include but are not limited to antisense, ribozyme, co-suppression, RNA interference, expression of dominant negative mutants, antibody expression and in vitro mutagenesis approaches. All of them include the introduction of a suitable nucleic acid molecule into a cell. Such a foreign nucleic acid molecule is present in cells of a correspondingly treated organism, but absent from the cells of the corresponding source organism. Thereby encompassed are nucleic acid molecules, e.g. gene sequences, which differ from the corresponding nucleic acid molecule in the source organism by at least one mutation (substitution, insertion, deletion, etc. of at least one nucleotide), wherein such a mutation inhibits the expression of the affected gene or reduces the activity of the gene product. Furthermore encompassed by the term “foreign” are nucleic acid molecules which are homologous with respect to the source organism but are situated in a different chromosomal location or differ, e.g., by way of a reversed orientation for instance with respect to the promoter.
In principle, the nucleic acid molecule to be introduced in accordance with the present embodiment may be of any conceivable origin, e.g. eukaryotic or prokaryotic. It may be from any organism which comprises such molecules. Furthermore, it may be synthetic or derived from naturally occurring molecules by, e.g., modification of its sequence, i.e. it may be a variant or derivative of a naturally occurring molecule. Such variants and derivatives include but are not limited to molecules derived from naturally occurring molecules by addition, deletion, mutation of one or more nucleotides or by recombination. It is, e.g., possible to change the sequence of a naturally occurring molecule so as to match the preferred codon usage of the target organism. It is preferred that the nucleic acid molecule introduced into the organism has to be expressed in order to exert its reducing effect upon gene expression of the target gene. The term “expressed” means that such a nucleic acid molecule is at least transcribed and, for some embodiments, also translated into a protein. Preferred examples of such nucleic acid molecules relate to those embodiments wherein a reduced gene expression is achieved by an antisense, co-suppression, ribozyme or RNA interference effect or by the expression of antibodies or other suitable polypeptides capable of specifically reducing the activity of the encoded gene product or by the expression of a dominant-negative mutant. These methods are further explained in the following.
In particular, gene expression may be reduced by using nucleic acid molecules encoding an antisense RNA or directly by using antisense RNA, said antisense RNA being complementary to transcripts of the gene the expression of which is to be reduced. Thereby, complementarity does not signify that the RNA has to be 100% complementary. A low degree of complementarity may be sufficient as long as it is high enough to inhibit the gene expression. The transcribed RNA is preferably at least 90% and most preferably at least 95% complementary to the transcript of the gene. In order to cause an antisense effect during the transcription the antisense RNA molecules have a length of at least 15 bp, preferably a length of more than 100 bp and most preferably a length or more than 500 bp, however, usually less than 2000 bp, preferably shorter than 1500 bp. For example, for plants, exemplary methods for achieving an antisense effect are described by Muller-Röber (EMBO J. 11 (1992), 1229-1238), Landschütze (EMBO J. 14 (1995), 660-666), D'Aoust (Plant Cell 11 (1999), 2407-2418) and Keller (Plant J. 19 (1999), 131-141). Likewise, an antisense effect may also be achieved by applying a triple-helix approach, whereby a nucleic acid molecule complementary to a region of the respective gene is designed according to the principles for instance laid down in Lee (Nucl. Acids Res. 6 (1979), 3073); Cooney (Science 241 (1998), 456) or Dervan (Science 251 (1991), 1360).
A similar effect as with antisense techniques can be achieved by applying RNA interference (RNAi). Thereby, the formation of double-stranded RNA leads to an inhibition of gene expression in a sequence-specific fashion. More specifically, in RNAi constructs, a sense portion comprising the coding region of the gene to be inactivated (or a part thereof, with or without non-translated region) is followed by a corresponding antisense sequence portion. Between both portions, an intron not necessarily originating from the same gene may be inserted. After transcription, RNAi constructs form typical hairpin structures. The RNAi technique may be carried out as described by Smith (Nature 407 (2000), 319-320), Marx (Science 288 (2000), 1370-1372) or Elbashir (Nature 411 (2001), 428-429).
Furthermore, DNA molecules can also be employed which, during their expression lead to the synthesis of an RNA which reduces the expression of the gene to be inactivated due to a co-suppression effect. The principle of co-suppression as well as the production of corresponding DNA sequences is precisely described, for example, in WO 90/12084. Such DNA molecules preferably encode an RNA having a high degree of homology to transcripts of the target gene. It is, however, not absolutely necessary that the coding RNA is translatable into a protein. The principle of the co-suppression effect is known to the person skilled in the art and is, for example, described in Jorgensen, Trends Biotechnol. 8 (1990), 340-344; Niebel, Curr. Top. Microbiol. Immunol. 197 (1995), 91-103; Flavell, Curr. Top. Microbiol. Immunol. 197 (1995), 43-36; Palaqui and Vaucheret, Plant. Mol. Biol. 29 (1995), 149-159; Vaucheret, Mol. Gen. Genet. 248 (1995), 311-317; de Bome, Mol. Gen. Genet. 243 (1994), 613-621 and in other sources.
Likewise, ribozymes which specifically cleave transcripts of the gene to be inactivated can be used. Ribozymes are catalytically active RNA molecules capable of cleaving RNA molecules at a specific target sequence. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of the group I intron ribozyme type or of ribozymes exhibiting the so-called “hammerhead” motif as a characteristic feature. By means of recombinant DNA techniques, the specific recognition of the target RNA molecule may be modified by altering the sequences flanking the hammerhead motif. By base pairing with sequences in the target molecule, the flanking sequences determine the position at which cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule. In order to produce nucleic acid molecules encoding a ribozyme which specifically cleaves the transcript of the gene to be inactivated, for example, a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA sequences which are complementary to sequences of the transcript. Sequences encoding the catalytic domain may for example be the catalytic domain of the satellite DNA of the SCMo virus (Davies, Virology 177 (1990), 216-224 and Steinecke, EMBO J. 11 (1992), 1525-1530) or that of the satellite DNA of the TobR virus (Haseloff and Gerlach, Nature 334 (1988), 585-591); The expression of ribozymes in order to decrease the activity of certain proteins in cells is known to the person skilled in the art and is, for example, described in EP-B1 0 321 201. The expression of ribozymes in plant cells is for example described in Feyter (Mol. Gen. Genet. 250 (1996), 329-338).
Furthermore, nucleic acid molecules encoding antibodies specifically recognizing the polypeptide encoded by the gene to be inactivated or specific fragments or epitopes of such a polypeptide can be used for inhibiting the gene expression of said gene. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Köhler and Milstein (Nature 256 (1975), 495) and Galfré (Meth. Enzymol. 73 (1981) 3), which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies or fragments thereof to the aforementioned polypeptide can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. In plants, expression of antibodies or antibody-like molecules can be achieved by methods well known in the art. These include the expression of, for example, full-size antibodies (Düring, Plant. Mol. Biol. 15 (1990), 281-293; Hiatt, Nature 342 (1989), 469-470; Voss, Mol. Breeding 1 (1995), 39-50), Fab-fragments (De Neve, Transgenic Res. 2 (1993), 227-237), scFvs (Owen, Bio/Technology 10 (1992), 790-794; Zimmermann, Mol. Breeding 4 (1998), 369-379; Taviadoraki, Nature 366 (1993), 469-472; Artsaenko, Plant J. 8 (1995), 745-750) and variable heavy chain domains (Benvenuto, Plant Mol. Biol. 17 (1991), 865-874) have been successfully expressed in tobacco, potato (Schouten, FEBS Lett. 415 (1997), 235-241) or Arabidopsis, reaching expression levels as high as 6.8% of the total protein (Fiedler, Immunotechnology 3 (1997), 205-216).
Moreover, also nucleic acid molecules encoding peptides or polypeptides capable of reducing the activity of the polypeptide encoded by the gene to be inactivated other than antibodies can be used in the present context. Examples of suitable peptides or polypeptides that can be constructed in order to achieve the intended purpose can be taken from the prior art and include, for instance, binding proteins such as lectins.
In addition, nucleic acid molecules encoding a mutant form of the polypeptide encoded by the gene to be inactivated can be used to interfere with the activity of the wild-type protein. Such a mutant form preferably has lost its biological activity and may be derived from the corresponding wild-type protein by way of amino acid deletion(s), substitution(s), and/or additions in the amino acid sequence of the protein. Mutant forms of such proteins may show, in addition to the loss of the hydrolytic activity, an increased substrate affinity and/or an elevated stability in the cell, for instance, due to the incorporation of amino acids that stabilize proteins in the cellular environment. These mutant forms may be naturally occurring or, as is preferred, genetically engineered mutants.
It is also possible that the nucleic acid molecule, the presence of which in the genome of an organism leads to a reduction of gene expression, does not require its expression to exert its reducing effect on gene expression. Correspondingly, preferred examples relate to methods wherein this effect is achieved by in vivo mutagenesis or by the insertion of a heterologous DNA sequence in the target gene. The term “in vivo mutagenesis”, relates to methods where the sequence of the gene to be inactivated is modified at its natural chromosomal location such as for instance by techniques applying homologous recombination. This may be achieved by using a hybrid RNA-DNA oligonucleotide (“chimeroplast”), as described above.
The term “insertion of a heterologous DNA sequence” refers to DNA sequences which can be inserted into the target gene via appropriate techniques other than in vivo mutagenesis. The insertion of such a heterologous DNA sequence may be accompanied by other mutations in the target gene such as the deletion, inversion or rearrangement of the sequence located at the insertion site. In connection with preparing transgenic plants, this embodiment includes randomly introducing a heterologous DNA sequence into the respective plant genome, thereby generating a pool, i.e. a plurality, of transgenic plants having a genome into which the heterologous DNA sequence is randomly spread over various chromosomal locations. This generation of transgenic plants is followed by selecting those transgenic plants out of the pool which show the desired genotype, i.e. an inactivating insertion in the target gene and/or the desired phenotype, i.e. a reduced activity of the polypeptide encoded by the target gene and/or a modified amount of the metabolite which correlates with the transcript of said gene.
Suitable heterologous DNA sequences that can be taken for such an approach are described in the literature and include for instance vector sequences capable of self-integration into the host genome or mobile genetic elements. Particularly preferred in this regard are T-DNA or transposons which are well-known, to the person skilled in the art from so-called tagging experiments used for randomly knocking out genes in plants. The production of such pools of transgenic plants can for example be carried out as described in Jeon (Plant J. 22 (2000), 561-570) or Parinov (Curr. Op. Biotechnol. 11 (2000), 157-161).
Another example of insertional mutations that may result in gene silencing includes the duplication of promoter sequences which may lead to a methylation and thereby an inactivation of the promoter (Morel, Current Biology 10 (2000), 1591-1594).
Furthermore, it is immediately evident to the person skilled in the art that the above-described approaches, such as antisense, ribozyme, co-suppression, in-vivo mutagenesis, RNAi, expression of antibodies, other suitable peptides or polypeptides or dominant-negative mutants and the insertion of heterologous DNA sequences, can also be used for reducing the expression of a gene that encodes a regulatory protein such as a transcription factor that controls the expression of the gene to be inactivated. It is also evident from the above descriptions any of the above-mentioned approaches can be combined in order an effective reduction of gene expression of the target gene.
In yet another aspect, the present invention refers to a method for identifying a metabolite which is capable of modifying the amount of a transcript in an organism comprising steps (a) to (c) of the aforementioned method for determining the function of a gene, wherein a metabolite identified in step (c) is a candidate for a metabolite being capable of modifying the amount of said transcript identified in step (c).
The term “metabolite capable of modifying the amount of a transcript in an organism” refers to a metabolite the amount of which significantly correlates with said transcript in the different states of the organism as it can be determined by performing steps (a) to (c) of the above-described method for determining the function of a gene. It is thus contemplated that an exogenously induced alteration of the amount of the metabolite as compared to the normal amount of the metabolite in the organism under the same developmental and environmental conditions may lead to a significant modification of the amount of the transcript in the organism as compared to the amount of the transcript in the corresponding organism with the amount of said metabolite not being altered. Thus, when a correlation between a metabolite and a transcript has been revealed by the method of the invention, a metabolite or a thereto structurally related compound can be regarded as a candidate for a compound that may modify the expression of the gene corresponding to this transcript.
The term “candidate” reflects that the metabolite identified in step (c) may not necessarily be causative for the observed differences of the correlated transcript in the different states of the organism under investigation. Thus, it may be required that the identified metabolite be further tested for the property of being capable of modifying the amount of the transcript in an organism. For this purpose, gene expression assays may be conducted according to methods known in the prior art as for instance described in Sambrook (2001) and Gassen (1999). For example, the amount of the respective transcript may be detected for the organism to which a certain amount of the candidate metabolite is added in comparison with a corresponding organism at the same conditions to which no metabolite is added. As already mentioned above, such a candidate may also be a compound which is structurally related to the specific metabolite and which has a similar effect on the transcript level as the metabolite.
It is envisaged that the method of the present embodiment may for example provide novel starting points for developing therapeutically useful agents that may be used for ameliorating an aberrant over- or under-expression of a gene in a patient who suffers from a genetic disease.
Generally, most of the metabolites that occur in nature may be synthesized chemically or by microorganisms or by a combination of these two possibilities and often are commercially available. Correspondingly, modifications to a metabolite compound may be introduced according to methods of organic chemistry and biochemistry known in the art. Compounds being structurally related with a metabolite thus produced may be tested for their activity of modifying the amount of a transcript with which said metabolite correlates according gene expression assays known in the art, as mentioned above.
In this context, an “alteration of the amount of a metabolite” may on the one hand mean an increase of this amount in the organism. This may be achieved by the addition of this metabolite or of a structurally related compound having a similar effect on the transcript level to be modified to the organism. Alternatively, other methods for increasing the amount of a certain metabolite in an organism known in the prior art may be used as well such as measures that indirectly lead to an increase of the metabolite in the organism, e.g. by influencing the biosynthesis or degradation of the metabolite. On the other hand, the “alteration of the amount of a metabolite” may be a reduction of this amount in the organism.
For administering a metabolite or a structurally related compound having a corresponding activity, said metabolite or structurally related compound may be formulated in a composition.
If for example administration is meant for plants the composition may be composed in the form of a plant protection composition, wherein the metabolite or the structurally related compound may be formulated by conventional means commonly used for the application of, for example, herbicides or pesticides. For example, certain additives known to those skilled in the art such as stabilizers or substances which facilitate the uptake by the plant cell, plant tissue or plant may be used as for example harpins, elicitins, salicylic acid (SA), benzol(1,2,3)thiadiazole-7-carbothioic acid (BTH), 2,6-dichloro isonicotinic acid (INA), jasmonic acid (JA) or methyljasmonate.
If for example administration is meant for mammals or corresponding mammalian cells or tissues, the composition may be composed in the form of a pharmaceutical composition and may further comprise a pharmaceutically acceptable carrier and/or diluent. The composition may furthermore contain substances that stabilize or facilitate the uptake of the metabolite or compound by the cells. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the subject's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment.
Compositions may be administered locally or systemically. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Often it will be necessary that the modification of the particular transcript level induced by the correlated metabolite is not accompanied by the modification of the amount of other transcripts which might evoke undesired side effects. In such a case, a metabolite can be selected which does not'show significant correlations with other transcripts.
Furthermore, the present invention relates to the use of a gene the function of which has been determined by the above-described method for determining the function of a gene, of a nucleic acid molecule comprising the coding sequence of said gene or a fragment or derivative of said coding sequence showing said function or of a polypeptide encoded by said gene or nucleic acid molecule for applying said function. With the method of the invention, it is possible to elucidate gene functions that have not been thought of before. Therefore, the present invention also refers to the use of the genes for applying the function that has been identified by the method of the invention.
In the experiments underlying this invention, several new correlations between transcript level and metabolite level have been revealed. Accordingly, the present invention refers in particularly preferred embodiments to the uses of the genes corresponding to these transcripts for applying the newly revealed functions each of which involves influencing the amount of the respective metabolite, preferably in a plant, especially in potato, more specifically in potato tuber.
In particular, these uses are evident for the person skilled in the art from the following transcript-metabolite correlations (see also Example 2):
Since most molecular biological applications do not use a gene sequence (in particular if it contains one or more introns), but the coding sequence or fragments or derivatives thereof, the uses of the present inventions are not limited to the genes identified according to the methods of the invention, but also refer to nucleic acid molecules comprising the coding sequence of said gene or a fragment or derivative of said coding sequence showing said function.
The terms “nucleic acid molecule” and “coding sequence of a gene” refer to the conventional meaning of these terms as they are familiar to the skilled person. The term “nucleic acid molecule” furthermore encompasses molecules which, in addition to said coding sequence, comprise additional sequences useful for the intended uses. Such additional nucleotide sequences may in particular be additional coding sequences fused to the aforementioned coding sequence, said additional coding sequence for example encoding a tag facilitating easy purification of the encoded fusion protein, a stabilizing moiety inhibiting degradation of the fusion protein or targeting signal sequences. Furthermore, such additional nucleotide sequences may be expression control sequences operably linked to the coding sequence or vector sequences allowing the propagation of the nucleic acid molecule in a suitable host. Corresponding construction manuals for nucleic acid molecules are known to the skilled person and described in the literature (see e.g. Sambrook (2001) and Gassen (1999)).
The term “fragment of said coding sequence showing said function” refers to fragments of the coding sequence of the gene that has the function of influencing the amount of a metabolite to which the transcript of said gene shows a correlation, whereby the fragment when expressed in a corresponding organism shows a similar, preferably the same effect on the amount of the metabolite as the gene or its entire coding sequence. This effect can be determined by well-known methods known in the art, for example involving the expression of the fragment in the organism or a biological test system derived from this organism (e.g. a cell culture or the like) and measuring the amount of the metabolite therein. This measurement may be compared with one obtained from a corresponding experiment undertaken with the gene or the entire coding sequence, and preferably with a measurement obtained from a control setting, wherein no heterologous nucleic acid molecule is expressed. If the amount of the metabolite upon fragment expression does not significantly deviate from that measured upon expression of the gene or the entire coding sequence, the fragment shows a similar or even the same function as the gene or the entire coding sequence.
The term “derivative of said coding sequence showing said function” refers to nucleotide sequences the complementary strand of which hybridizes with the coding sequence and wherein the derivative when expressed in a corresponding organism shows a similar, preferably the same effect on the amount of the metabolite with which the transcript of the coding sequence correlates as the gene or the coding sequence. This effect can be determined as described above in connection with fragments of the coding sequence.
If the coding sequence encodes a polypeptide, such a derivative may encode a polypeptide which has a homology, that is to say a sequence identity, of at least 30%, preferably of at least 40%, more preferably of at least 50%, even more preferably of at least 60% and particularly preferred of at least 70%, especially preferred of at least 80% and even more preferred of at least 90% to the entire amino acid sequence encoded by the coding sequence.
Moreover, such a derivative may have a homology, that is to say a sequence identity, of at least 40%, preferably of at least 50%, more preferably of at least 60%, even more preferably of more than 65%, in particular of at least 70%, especially preferred of at least 80%, in particular of at least 90% and evern more preferred of at least 95% when compared to the coding sequence.
The use of the present embodiment also relates to derivatives the sequence of which deviates from above-described hybridizing or homologous nucleotide sequences due to the degeneracy of the genetic code and which have a similar, preferably the same function as the coding sequence.
In the context of the present invention the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA. In an especially preferred embodiment, the term “hybridization” means that hybridization occurs under the following conditions:
Derivatives which hybridize with the coding sequence of a gene the function of which is identified by applying the method of the invention can for instance be isolated from genomic libraries or cDNA libraries of bacteria, fungi, plants or animals. According to one aspect of the invention, it is preferred that such polynucleotides are from plant origin, particularly preferred from a plant belonging to the dicotyledons, more preferably from the family of Solanaceae. Preferably, the derivative is a variant, preferably an ortholog of said coding sequence. Alternatively, such derivatives can be prepared by genetic engineering or chemical synthesis.
Such hybridizing polynucleotides may be identified and isolated by using a nucleic acid molecule comprising the coding sequence or parts or reverse complements thereof, for instance by hybridization according to standard methods (see for instance Sambrook (2001). Fragments used as hybridization probes can also be synthetic fragments which are prepared by usual synthesis techniques, and the sequence of which is substantially identical with the coding sequence of the gene, a function of which has been determined by the method of the invention.
The molecules hybridizing with said coding sequence comprise fragments, derivatives and allelic variants of the specific coding sequence corresponding to the gene a function of which has been determined by applying the method of the invention.
Preferably, the degree of homology is determined by comparing the respective nucleotide sequence with the coding sequence of the gene a function of which has been identified by applying the method of the invention. When the sequences which are compared do not have the same length, the degree of homology preferably refers to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence. The degree of homology can be determined conventionally using known computer programs such as the DNAstar program with the ClustalW analysis. This program can be obtained from DNASTAR, Inc., 1228 South Park Street, Madison, Wis. 53715 or from DNASTAR, Ltd., Abacus House, West Ealing, London W13 0AS UK (support@dnastar.com) and is accessible at the server of the EMBL outstation.
When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.
Preferably, the degree of homology of the hybridizing polynucleotide is calculated over the complete length of its coding sequence. It is furthermore preferred that such a hybridizing polynucleotide, and in particular the coding sequence comprised therein, has a length of at least 300 nucleotides, preferably at least 500 nucleotides, more preferably of at least 750 nucleotides, even more preferably of at least 1000 mucleotides, particularly preferred of at least 1500 nucleotides and most preferably of at least 2000 nucleotides.
Preferably, sequences hybridizing to the coding sequence of the gene a function of which has been identified by applying the method of the invention comprise a region of homology of at least 90%, preferably of at least 93%, more preferably of at least 95%, still more preferably of at least 98% and particularly preferred of at least 99% identity to an above-described polynucleotide, wherein this region of homology has a length of at least 500 nucleotides, more preferably of at least 750 nucleotides, even more preferably of at least 1000 nucleotides, particularly preferred of at least 1500 nucleotides and most preferably of at least 2000 nucleotides.
Homology, moreover, means that there is a functional and/or structural equivalence between the corresponding polynucleotides or polypeptides encoded thereby. Polynucleotides which are homologous to the above-described molecules and represent derivatives of these molecules are normally variations of these molecules which represent modifications having the same biological function. They may be either naturally occurring variations, for instance sequences from other ecotypes, varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The allelic variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques. Deviations from the above-described polynucleotides may have been produced, e.g., by deletion, substitution, insertion and/or recombination.
The polypeptides encoded by the different variants of the coding sequence of the gene, a function of which has been determined by applying the method of the invention, possess certain characteristics they have in common. These include for instance biological activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behavior in gel electrophoreses, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc.
In connection with the present use of the invention, the term “polypeptide” refers to any polypeptide encoded by the above-mentioned gene, nucleic acid molecule, coding sequence, fragment or derivative. This polypeptide may, e.g., be a naturally purified product or a product of chemical synthetic procedures or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptide may be glycosylated or non-glycosylated. The polypeptide may also include an initial methionine amino acid residue. It may be further modified to contain additional chemical moieties not normally part of the polypeptide. Those derivatized moieties may, e.g., improve the stability, solubility, the biological half life or absorption of the polypeptide. The moieties may also reduce or eliminate any undesirable side effects of the polypeptide and the like. An overview for these moieties can be found, e.g., in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa. (1990)). Polyethylene glycol (PEG) is an example for such a chemical moiety which has been used for the preparation of therapeutic polypeptides. The attachment of PEG to polypeptides has been shown to protect them against proteolysis (Sada et al., J. Fermentation Bioengineering 71 (1991), 137-139). Various methods are available for the attachment of certain PEG moieties to polypeptides (for review see: Abuchowski et al., in “Enzymes as Drugs”; Holcerberg and Roberts, eds. (1981), 367-383). Generally, PEG molecules are connected to the polypeptide via a reactive group found on the polypeptide. Amino groups, e.g. on lysines or the amino terminus of the polypeptide are convenient for this attachment among others.
In addition, the present invention relates in a further embodiment to the use of a gene identified by the above-described method for identifying a gene which is capable of modifying the amount of a metabolite in an organism, of a nucleic acid molecule comprising the coding sequence of said gene or a fragment or derivative of said coding sequence which is capable of modifying the amount of a metabolite in an organism or of a polypeptide encoded by said gene or nucleic acid molecule for modifying the amount of a metabolite in an organism.
This use is in principle a preferred embodiment of the aforementioned use of a gene, the function of which has been determined by the method for determining the function of a gene, in that the “function” can be seen as the capacity of modifying the amount of a metabolite in an organism. Accordingly, the definitions for “coding sequence”, “nucleic acid molecule”, “fragment”, “derivative” and “polypeptide” given above likewise apply to the present embodiment.
Accordingly, also the preferred embodiments enumerated under (1) to (14), above, as to the uses of the genes corresponding to transcripts for applying the newly revealed functions also apply to the modification of the amount of the respective metabolite, preferably in a plant, especially in potato, more specifically in potato tuber.
Moreover, the present invention relates in a further embodiment to the use of a metabolite identified by the above-described method for identifying a metabolite which is capable of modifying the amount of a transcript in an organism for modifying the amount of a transcript in an organism.
This use is in principle a preferred embodiment of the above-outlined use of a gene, the function of which has been determined by the method for determining the function of a gene, in that the “function” can be seen as the capacity that the amount of the transcript of the gene can be modified by the metabolite.
Accordingly, the correlations enumerated under (1) to (14), above, give rise to preferred embodiments of the present use. In particular, it is conceivable that each of the metabolites mentioned there can be used for modifying the amount of the respective transcript for which the metabolite shows a correlation. These uses will preferably be applicable to plants, especially to potato, more specifically to potato tuber.
These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database “Medline” may be utilized which is available on the Internet, for example under http://www.ncbi.nim.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.ch/biology/research_tools.html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.google.de. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
Furthermore, the term “and/or” when occurring herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.
The present invention is further described by reference to the following non-limiting figures, tables and examples.
The Figures and the Tables show:
The filter is organized in 16×24 block (
Description A1 to P24 corresponding to the position on the 384 well-plates.
The orientation is left-bottom since the origin of each plate A1 is located in the left bottom position of each block on the filter.
The description of R1-R24 and C1-C24 corresponding to the primary rows (R) and columns (C) in AIS program.
The secondary grit contain 4×4 spots (
The control DNA (human gene) is spotted at the position r4-c1.
Each secondary grid contains different clones from 6 different plates (T1-T6) (
The whole filter consists of 384 secondary grids which adds up to 2112 individual clones represented by the spots.
For the: developing tubers the results from potato tuber after 8 weeks growth were chosen as the reference for calculating the relative amounts of transcripts in the subsequent stages of development. For the transgenic tubers, the results from wild-type tubers after 10 weeks growth were chosen as the reference.
a) 9 weeks
b) 10 weeks
c) 13 weeks
d) 14 weeks
e) SP29 line (SP)
f) INV30 line (INV2-30)
The data obtained from the transgenic lines are normalized to the mean response calculated for the wild-type tubes after 10 weeks of growing. For the developing wild-type tubers, 8 weeks grown tubers were used as reference. The values are presented as the mean ±SE of all independently determined replicates. Those metabolites that significantly differ from the reference are shown in boldface.
a) Developing wild-type tubers
b) Transgenic lines
Correlations between selected transcripts (identified by reference to the EST clone to which it hybridises, see “EST ID” and “Annotation”) and metabolites (see “MB”) were calculated using the method of Spearman, taking correlations with significant value P=0.01 as the threshold of significance. Under “sig 0.001”, it is indicated whether a calculated correlation is also significant for p=0.001, the number 1 meaning significant and 0 non-significant. “NOP” stands for the number of correlated pairs.
Solanum tubersosum L. cv Desiree was obtained from Saatzucht Lange AG (Bad Schwartau, Germany). The generation of the transgenic plant lines SP29 and INV2-30 used in this study has been detailed previously (Sonnewald, 1997; Tretheway, 2001; Roessner, 2001). Plants were handled as described in the literature (Tauberger, 2000; Regierer, 2002). Plants were maintained in tissue culture with a 16-hr.light/8-hr-dark regime on Murashige and Skoog (1962) medium that contained 2% sucrose. In the greenhouse, plants from all lines and wild-type were grown under the same light regime with a minimum of 250 μmol photons m−2 sec−1 at 22° C. Wild-type tubers were harvested after 8, 9 10, 13 and 14 weeks growing. Transgenic lines were harvested after 10 weeks of development.
Total RNA was isolated from 2 g fresh weight of tuber tissue using REB isolation buffer (25 mM TrisHCl pH.8, 75 mM EDTA, 75 mM NaCl, 1% w/v SDS, 1 M β-MercaptoEtOH) and 10M LiCl2 as described by Logemann et al. (1987).
Microarrays were constructed on nylon filters as described previously (Thimm, 2001; Colebatch, 2002). More than 2000 tomato clones were collected from the cDNA library constructed in the laboratories of Dr. Steve Tanksley, Cornell. University Solanaceae Genome Network, Dr. Greg Martin, Boyce Thompson Institute and Dr. Jim Giovannoni, Boyce Thompson Institute and provided by TIGR—Institute for Genomic Research (Van der Hoeven, Plant Cell 14 (2002), 1441-1456). These ESTs correspond to approximately 1000 tomato genes which are highly homologous to those from potato (Fulton, 2002).
In particular, using the web site annotation http://www.tigr.org/tdb/tgi/lgi/ form LeGI Release 7.0—May 22, 2001, about one thousand interesting TCs (Tentative Consensus) were selected. For each TC, at least two ESTs were picked to the Solanum library, i.e. of each bacterial colony containing a specific EST, an alignot was selected and transferred to a new 96 well plate. (for the complete list of selected clones see Table 4). 96 additional potato clones were added, but the obtained results were not used for this work. The cDNA was amplified by PCR using LacZ-specific primers (forward LacZ1 5′ GCTTCCGGCT CGTATGTTGT GTG 3′ (SEQ ID NO:1) and reverse LacZ2 5′ AAAGGGGGATGTGCT GCAAGGCG 3′ (SEQ ID NO:2)) and Taq polymerase. The PCR products were selectively checked on an agarose gel before spotting. The PCR products were spotted automatically onto the nylon membranes (Biogrid, Biorobotics, Cambridge, UK; Nytran Supercharg, 22.2×22.2. cm, Schleicher and Schüll, Dassel, Germany). The Solanum library was spotted six times onto one membrane (for details of the spotting scheme see
To normalize the amount of spotted cDNA, a reference hybridization for each filter was carried but using a [33P]-labeled PCR product-specific primer (T4 polynucleotide kinase, New England Biolabs, Beverly, Mass.; [33P]ATP, Hartmann Analytic, Braunschweig, Germany; 5′ TTCCCAGTCACGA (SEQ ID NO:3)). The filters were hybridized at 5° C. overnight and washed for 40 min at 5° C. in Ssarc (4×SSC, 7% [v/v] Sarcosyl NL30, and 4 μM EDTA). Filters were exposed o/n on imaging plates and detected with phosphorimager (BAS-1800, Fuji, Tokyo). Radioactivity Was removed from the filters by washing two times in SSarc at 65° C. for 30 minutes. After every stripping, removing quality was checked by o/n exposition with imaging plates.
For reverse transcription, 10 μg total RNA was used (SuperScript II, GibcoBRL, Karlsruhe, Germany; [33P]CTP, Hartmann Analytic, Germany). After reverse transcription, RNA was hydrolyzed with NaOH (0.25N) and neutralized with HCl (0.2 N) and sodium phosphorylate buffer (40 mM, pH 7.2). Labelling efficiency was controlled by scintillation counting (LS6500, Beckman Munich) after removal of unincorporated oligonucleotides by Sephadex G-50 chromatography (NICK Columns, Amersham Pharmacia).
After pre-hybridisation for 2 h at 65° C. in Church buffer (7% [w/w] SDS, 1 mM EDTA, pH 8.0, and 0.5 M sodium phosphate, pH 7.2) containing salmon sperm DNA (100 ng ml−1, Roth, Carl GmbH&Co, Karlsruhe, Germany), filters were hybridised with the labelled cDNA probe at 65° C. for 24 h. Washing steps were carried out at 65° C. for 20 min each with 1×SSC, 0.1% (w/v) SDS, 4 mM Na2PO4 (pH 1.2); 0.2×SSC, 0.1% (w/v) SDS, 4 mM Na2PO4 (pH 7.2); 0.1 SSC, 0.1% (w/v) SDS, 4 mM Na2PO4 (pH 7.2). The filters were exposed on imaging plates for 16 h and signals were detected using a phosphorimager (BAS-1800 II, Fuiji) followed by stripping for two times for 30 minutes at 80° C. (0.1% [w/v] SDS, 5 mM Na2PO4, pH 7.2). After every stripping, removing quality was checked by the o/n exposition with imaging plates. For transgenic lines and 10 weeks tubers, three repetitions of hybridisation with a newly synthesized and labelled cDNA probe of corresponding RNA were performed. For 8, 9, 13 and 14 weeks old tubers four repetitions were done.
For data analysis, the signal intensities of the reference and complex hybridisation were quantified using the Array vision 5.1 software (Imaging Research Inc., Haverhill, UK). A predefined grid, determining the area of signal quantification, was manually optimised to ensure correct signal recording. The quantified signals, defined as photo-stimulated luminescence mm−2, were assigned to the corresponding cDNA clones stored in a suitable database (“Haruspex”) (http://www.mpimp-golm.mpg.de/haruspex/index-e.html). The cDNAs on the filter were arranged as 4×4 arrays, each containing six doubly spotted clones, a human gene (desmin) (not used in this study), and an empty field to determine specific local background (LB) (for details see
For each clone, the average value for every repetition was calculated. Clones were described as differentially expressed in the tested situation while differences between average tested values and average reference values were two fold and additionally test showed significant change at the level of P 0.05.
The preparation and derivatisation of samples for metabolite analysis, and the subsequent operation of the GC-MS and evaluation of the resultant chromatograms and normalization of the results were carried out exactly as described (Roessner, 2001).
For GC-TOF analysis, the organic phase was dried and dissolved in 50 μl of methoxamine hydrochloride (20 mg/mL pyridine) and incubated at 30° C. for 90 min with continuous shaking. Then 80 μL of N-Methyl-N-trimethylsilyltrifluoroacetamid (MSTFA) was added to derivatize polar functional groups at 37° C. for 30 min. The derivatized samples were stored at room temperature for 120 min before injection. GC-TOF analysis was performed on a HP 5890 gas chromatograph with standard liners and splitless injection at 230° C. injector temperature. The GC was operated at constant flow of 1 mL/min Helium and a 40 m 0.25 mm ID 0.25 μm RTX-5 column with 10 m integrated pre-column. The temperature gradient started at 80° C., was held isocratic for 2 min, and subsequently ramped at 15° C./min to a final temperature of 330° C. which was held for 6 min. 20 spectra per second were recorded between m/z 85 to 500. After data acquisition was finished, reference chromatograms were defined that had a maximum of detected peaks over a signal/noise threshold of 20 and used for automated peak identification based on mass spectral comparison to a standard NIST 98. Automated assignments of unique ions for each individual metabolite were taken as default as quantifiers, and manually corrected where necessary. All artifactual peaks caused by column bleeding or phtalates and polysiloxanes derived from MSTFA hydrolysation were removed from the results table. All data were normalized to plant mg FW and to the internal references and log-transformed. t-test, correlation analysis, and variance analysis were performed in Excel 5.
The dried protein pellet was dissolved in freshly prepared 1M Urea in 0.05 M Tris buffer pH 7.6. The complex protein mixture was digested with modified trypsin (Böhringer Mannheim) according to the manufacturer's instructions. The tryptic digest was dried down and dissolved in 300 μwater (1% formic acid). Unsoluble material was removed by centrifugaton. An aliquot of the digest (˜100 μg protein) was injected onto two-dimensional chromatography on a thermofinnigan proteomeX system coupled to an LCQDecaXp ion trap (Thermofinnigan). The chromatographic separation was done according to manufacturer's instructions. After a 12 cycle run the MS/MS spectra were searched against an Arabidopsis thaliana database (downloaded from the TAIR homepage www.arabidopsis.org) using Turbosequest implemented in Bioworks 3.0 (Thermofinnigan). Matches were filtered according to Wolters et al. (2001). Additionally, we used the multiple scoring filter of Bioworks 3.0 with 50 percent ion coverage. For the quantification approach aliquots of the complex tryptic digest of Arabidopsis leaf protein (50 μL) were analysed on reversed phase chromatography. Quantification was achieved by integrating peak areas of target peptides representative for proteins. These peak areas were normalised to the sum of internal standard peptides that had been added to the mixture (Chelius et al. (2002), Bondarenko et al. (2002)).
Several different data transformations, distance measure and clustering methods were tested on the data set, before using principal component analysis (PCA). PCA was performed independently on the data sets obtained from transcript and metabolite profiling with the software package S-Plus 2000 (Insightfull, Berlin, Germany) using the default weighted covariance estimation function. The data was log10 transformed prior to further analysis and metabolites or ESTs that were not common to all samples were removed.
Correlation analysis was used to select ESTs which gave highly reproducible results with respect to other ESTs of the consensus sequence from which they were derived. For this purpose the significance threshold was set to P>0.001. Only ESTs that were above this threshold according to the Spearman method and which appeared reliable during every experiment were selected for subsequent analysis. Table 1 shows the EST clones for which corresponding differentially expressed transcripts were observed. Correlations between selected transcripts and metabolites were calculated using the method of Spearman, taking correlations with significant value P=0.01.
The parallel analysis of a given biological system is described using two phenotyping technologies, the current technology of metabolic profiling based on GC-MS analysis and gene expression analysis using classical array technology. In attempting these experiments, it was first tried to answer the question what the relative power of the two phenotyping technologies is to discriminate biological systems which either differ in the developmental state or exhibit well characterized transgenic changes.
When these experiments were designed, it was decided to use a plant system, namely the potato tuber system, to evaluate the above questions. The reasons for this were two-fold: first, the potato tuber displays well-defined but nevertheless highly related developmental stages and allows the assessment of a number of well-characterized transgenic situations. Second, the laboratory of the inventors is very well acquainted with the analysis of the potato tubers on both the biochemical and the molecular levels (Fernie, 2002). As a first step, the expression of 1000 genes represented by at least two expressed sequence tags (ESTs) comprising the genes of many transcription factors and a wide variety of biosynthetic genes were analysed on a custom array. After analysing the data sets obtained, 279 ESTs were selected which gave highly reproducible results with respect to the consensus sequence from which they were derived. In order to see whether various developmental stages could be discriminated from one another and from the transcript profiles of transgenic potato tubers ectopically expressing a more efficient pathway of sucrose mobilization (Roessner, 2001), a principal component analysis (PCA) was performed. Table 1 is a presentation of those clones of which the corresponding transcripts are differentially expressed. As can be seen in
Metabolic profiling was then carried out on samples corresponding to those in the above-described transcript analysis in order to determine the levels of the major metabolites of primary metabolism including sugars, sugar alcohols, organic acids, amino acids as well as the nutritionally important compounds ascorbate and tocopherol. The corresponding metabolite profiles are depicted in Table 2. When a principal component analysis was carried out on the data set obtained from the metabolic profiling studies (
In conclusion, the discriminatory power of transcript and metabolite profiling approaches are different with metabolite profiling allowing a greater resolution of the different systems studied here. Whether or not this reflects the fact that changes on the transcript level are less pronounced as compared to changes on the metabolite level or merely highlights limitations of the profiling technologies used remains an open question. However, whatever the reason, these results imply that the discrimination of biological systems should be performed at more than one level.
As a further question, it was examined whether the combined analysis of transcript and metabolite profiling data presents a useful approach for the identification of candidate genes that may change the metabolic composition of a given biological system. For this purpose, all the transcript and metabolite data points obtained in the analysis described in Example 1 were run through pairwise correlation analysis in order to determine for each transcript whether it is correlated with any of the metabolites. Out of the 23715 analysed pairs 329 positive and 189 negative correlation's were identified. The used significance threshold of P=0.01 in the non-parametric Spearman's rank correlation analysis is a rather conservative estimation of the number of chance correlations. Therefore, the identification of 518 correlations of high statistical significance was a surprising result. A couple of representative correlations is shown in
First, as with any new approach it is important to see whether the data obtained is in agreement with observations made following different, more established experimental strategies. This is clearly the case if one contemplates for instance the strong negative correlation between sucrose and sucrose transporter expression (
As a conclusion from the afore-described experiments, one has to note that, although, as might have been expected from previous experimental work aimed at comparing the transcript and protein levels (Ideker, 2001: Gygi, 1999; Futcher, 1999) and mathematical studies (TerKuile, 2001), the number of strong, metabolite-transcript correlations observed is relatively small, it is conceivable that they allow the generation of clearly verifiable hypotheses. Of particular interest in this regard are the observation of the correlation of genes with the essential amino acid lysine and with the vitamins ascorbate and tocopherol. These linkages define strong candidate genes for the manipulation of the content of these nutritionally important compounds in plants.
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tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
Homo sapiens. ESTs gb|AA712990,
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
thaliana}GP|12325076|gb|AAG52485.1|AC018364_3|AC018364 transc
thaliana}GP|5051786|emb|CAB45079.1||AL078637 tr
annuum}
thaliana}PIR|B84813|B84813 probable RING
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tuberosum}SP|Q43844|NUKM_SOLTU NAD
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar transp
thaliana}GP|12325076|gb|AAG52485.1|AC018364_3|AC018384 transc
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
thaliana}PIR|F84565|F84565 probable homeodom
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar transp
thaliana}PIR|T49898|T49898 CCAAT
thaliana}GP|13878053|gb|AAK44104.1|AF370289_1|AF370289 putative fructok
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|9280230|dbj|BAB01720.1||AB023045 Dof zin{umlaut over (c)} finger p
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
tuberosum}SP|Q43844|NUKM_SOLTU NAD
annuum}
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|B84813|B84813 probable RING
thaliana}PIR|F84565|F84565 probable homeodom
thaliana}PIR|B84813|B84813 probable RING
thaliana}GP|4240118|dbj|BAA74838.1||AB007800 NADH-cytochr
tuberosum = potatoes, root, Peptid
thaliana}PIR|T48250|T48250 serine/threoni
thaliana}PIR|C84588|C84588 probable NADH-ubiquin
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
thaliana}PIR|H86356|H86356 probable UDP-gluco
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
thaliana}PIR|T49898|T49898 CCAAT
thaliana}PIR|T51527|T51527 UDP-glucose dehydrog
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
sapiens. ESTs gb|AA712990,
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}PIR|T07016|T07016 6-ph
thaliana}GP|9280230|dbj|BAB01720.1||AB023045 Dof zinc finger p
esculentum}SP|Q42884|ARC1
thaliana}GP|4240118|dbj|BAA74838.1||AB007800 NADH-cytochr
thaliana}PIR|B84813|B84813 probable RING
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|5051786|emb|CAB45079.1||AL078637 tr
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
annuum}
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
thaliana}GP|9280230|dbj|BAB01720.1||AB023045 Dof zinc finger p
thaliana}PIR|H86356|H86356 probable UDP-gluco
0.24
±0.31
0.27
±0.53
0.69
±0.07
2.43
±0.42
0.66
±0.12
0.76
±0.04
0.35
±0.07
3.28
±0.17
0.56
±0.14
0.48
±0.20
1.69
±0.09
0.80
±0.06
0.59
±0.21
0.61
±0.12
0.62
±0.19
0.74
±0.07
0.64
±0.10
0.21
±0.16
0.29
±0.11
0.33
±0.83
0.09
±0.26
0.12
±0.26
0.13
±0.56
0.46
±0.34
0.55
±0.11
0.54
±0.09
±0.07
0.44
±0.10
±0.21
0.24
±0.13
0.34
±0.49
0.53
±0.13
0.37
±0.25
0.32
±0.46
0.30
±0.30
0.22
±0.18
0.18
±0.15
0.23
±0.38
0.28
±0.15
0.26
±0.12
0.45
±0.15
0.32
±0.11
0.44
±0.35
0.24
±0.20
0.27
±0.19
0.21
±0.50
0.50
±0.13
0.36
±0.16
0.44
±0.55
0.46
±0.05
0.39
±0.22
0.15
±0.18
0.17
±0.23
1.73
±0.09
1.78
±0.09
0.59
±0.08
0.24
±0.16
0.19
±0.14
0.22
±0.62
0.21
±0.36
0.10
±1.21
0.31
±0.30
0.58
±0.15
0.67
±0.18
0.60
±0.10
0.27
±0.32
0.35
±0.43
0.16
±0.36
0.09
±0.23
0.08
±0.84
3.63
±0.08
0.10
±0.02
1.20
±0.02
1.03
±0.06
1.90
±0.08
1.00
±0.00
2.96
±0.16
1.00
±0.00
15.33
±0.15
0.73
±0.33
0.68
±0.07
1.21
±0.15
8043.33
±0.02
1.00
±0.00
0.84
±0.10
1.32
±0.14
2.29
±0.16
1776.67
±0.08
1.47
±0.02
0.82
±0.09
thaliana}PIR|T49898|T49898 CCAAT
thaliana}PIR|T49898|T49898 CCAAT
thaliana}PIR|T49898|T49898 CCAAT
thaliana}PIR|T49898|T49898 CCAAT
thaliana}PIR|T49898|T49898 CCAAT
tabacum}PIR|T01985|T01985 zinc-finger protein
tabacum}PIR|T01985|T01985 zinc-finger protein
tabacum}PIR|T01985|T01985 zinc-finger protein
tabacum}PIR|T01985|T01985 zinc-finger protein
tabacum}PIR|T01985|T01985 zinc-finger protein
tabacum}PIR|T01985|T01985 zinc-finger protein
tabacum}PIR|T01985|T01985 zinc-finger protein
thaliana}PIR|B84813|B84813 probable RING
thaliana}PIR|B84813|B84813 probable RING
annuum}
annuum}
annuum}
annuum}
annuum}
annuum}
annuum}
annuum}
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
thaliana}GP|9280230|dbj|BAB01720.1||AB023045 Dof zinc finger p
thaliana}GP|9280230|dbj|BAB01720.1||AB023045 Dof zinc finger p
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}GP|12325076|gb|AAG52485.1|AC018364_3|AC018364 transc
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20”
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|8843734|dbj|BAA97282.1|
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
tuberosum}SP|Q43844|NUKM_SOLTU NAD
thaliana}PIR|F84565|F84565 probable homeodom
thaliana}PIR|F84565|F84565 probable homeodom
thaliana}PIR|F84565|F84565 probable homeodom
thaliana}PIR|F84565|F84565 5 probable homeodom
thaliana}PIR|F84565|F84565 probable homeodom
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|T49898|T49898 CCAAT
thaliana}PIR|T49898|T49898 CCAAT
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
glutinosa}SP|O04866|ARGD_ALNGL ACETYLORNITHINE AM
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}PIR|T07016|T07016 6-ph
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713 tran
tuberosum}SP|P37830|G6PD_SOLTU GLUCOSE-6-PHOSPHATE 1-D
thaliana}PIR|T48250|T48250 serine/threoni
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
tuberosum}SP|Q43644|NUAM_SOLTU NADH-UBIQUINONE OXID
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|B84813|B84813 probable RING
thaliana}PIR|B84813|B84813 probable RING
thaliana}PIR|B84813|B84813 probable RING
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}GP|12325273|gb|AAG52580.1|AC016529_11|AC016529 putat
thaliana}GP|12325273|gb|AAG52580.1|AC016529_11|AC016529 putat
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
Homo sapiens. ESTs gb|AA712990,
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum|GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
tuberosum = potatoes, root, Peptid
tuberosum = potatoes, root, Peptid
tuberosum = potatoes, root, Peptid
tuberosum = potatoes, root, Peptid
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}PIR|T45701|T45701 dTDP-glucose 4-6
thaliana}
thaliana}
thaliana}
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
tabacum}GP|1679853|emb|CAB05369.1||Z82982 caffeoyl-Co
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|8843734|dbj|BAA97282.1|
thaliana}GP|7270932|emb|CAB80611.1||AL161595 cytochrome P
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar transp
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar transp
thaliana}GP|13878053|gb|AAK44104.1|AF370289_1|AF370289 putative fructok
thaliana}GP|13878053|gb|AAK44104.1|AF370289_1|AF370289 putative fructok
thaliana}GP|13878053|gb|AAK44104.1|AF370289_1|AF370289 putative fructok
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
thaliana}GP|5051786|emb|CAB45079.1||AL078637 tr
thaliana}GP|5051786|emb|CAB45079.1||AL078637 tr
thaliana}GP|5051786|emb|CAB45079.1||AL078637 tr
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
batatas}SP|Q40089|ATP4_IPOBA ATP SYNT
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-BOUND GLYCOGEN
thaliana}GP|6065740|emb|CAB58230.1||AJ012758 nucleoti
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
tabacum}GP|1545805|dbj|BAA10929.1||D64052 cytochrome P450 like—
tabacum}GP|1545805|dbj|BAA10929.1||D64052
hybrida}SP|Q07346|DCE_PETHY GLUTAMATE
tabacum}
thaliana}GP|4512664|gb|AAD21718.1||AC006931
stramonium}SP|Q96556|SPE1_DATST SPERMIDINE
tabacum}
esculentum = tomatoes, cv. Super First, fruits, Peptide,
esculentum}SP|P29000|INVA_LYCES ACID BETA-
esculentum}SP|P29000|INVA_LYCES ACID BETA-
esculentum = tomatoes, cv Tiny Tim LA154, flowers,
melongena}SP|P37122|C762_SOLME CYTOCHROME
scutellarioides}
scutellarioides}
aestivum}SP|P93596|CP51_WHEAT CYTOCHROME
thaliana}GP|13878053|gb|AAK44104.1|AF370289_1|AF370289
thaliana}PIR|T47754|T47754 leucine zipper-cont
thaliana}GP|6598933|gb|AAF18728.1|AC018721—
tuberosum}PIR|T07685|T07685 omega-3 fatty acid
tabacum}GP|3721540|dbj|BAA33531.1||D83583 Sulfite
faba}GP|2104681|emb|CAA66481.1||X97907
tuberosum}SP|P37830|G6PD_SOLTU GLUCOSE-6-
tuberosum}SP|P37830|G6PD_SOLTU GLUCOSE-6-
thaliana}PIR|T47556|T47556 pyruvate kinase-like
tabacum}
mays}
tabacum}
thaliana}
tuberosum}SP|Q43848|TKTC_SOLTU
thaliana}PIR|T52043|T52043 probable glutamate-
versicolor}GP|2429282|gb|AAD05034.1||AF014056
thaliana}
thaliana}GP|6513940|gb|AAF14844.1|AC011664_26|AC011664
tabacum}
mays}
tabacum}PIR|T01934|T01934 adenosylmethionine
thaliana}PIR|H84870|H84870 probable
thaliana}PIR|T49899|T49899 zinc finger t
thaliana}
max}SP|P48513|TF2B_SOYBN TRANSCRIPTION
thaliana}GP|7269850|emb|CAB79709.1||AL161575
batatas}
hybrida}
tuberosum}GP|668987|emb|CAA59063.1||X84320
caryophyllus.
Populus deltoides vegetative storage protein. (L
hybrida}
melongena}SP|P37117|C714_SOLME CYTOCHROME
melongena}SP|P37117|C714_SOLME CYTOCHROME
oleracea}
thaliana}GP|5731763|emb|CAB52582.1||X92419
thaliana}GP|2244754|emb|CAB1077.1||Z9733
esculenta}SP|Q40288|UFO6_MANES FLAVONOL 3-
thaliana}GP|5006473|gb|AAD37511.1|AF139098_1|AF139098
tabacum}GP|1103487|emb|CAA91228.1||Z56282
thaliana}PIR|C84588|C84588 probable NADH-ubiquin
tabacum}SP|P93394|UPP_TOBAC URACIL
peruvianum}
thaliana}GP|7270437|emb|CAB80203.1||AL161586
glutinosa}SP|O04866|ARGD_ALNGL
oleracea}PIR|T09153|T09153 glucose-6-phosphate
thaliana}PIR|D84614|D84614 hyp
thaliana}GP|7378610|emb|CAB83286.1||AL162751
thaliana}PIR|A84437|A84437 probable PHD-type zin
tabacum}
thaliana}GP|9280230|dbj|BAB01720.1||AB023045 Dof
thaliana}PIR|T07719|T07719 aldose 1-epimerase
tabacum}GP|1805359|dbj|BAA19155.1||AB000623
tabacum}SP|P00823|ATPA_TOBAC ATP SYNTHASE
tuberosum}SP|P52578|IFRH_SOLTU ISOFLAVONE
tuberosum}GP|1488652|emb|CAA62817.1||X91615
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20
domestica}PIR|T16995|T16995 probable cinnamyl-alco
thaliana}PIR|D84614|D84614 hyp
sativa}
thaliana}GP|7270098|emb|CAB79912.1||AL161580
thaliana}
tabacum}GP|1419094|emb|CAA65173.1||X95932
tabacum}GP|1805359|dbj|BAA19155.1||AB000623
thaliana}GP|12323975|gb|AAG51946.1|AC01
thaliana}
sativa}
thaliana}
tuberosum}SP|P52903|ODPA_SOLT
tabacum}GP|2204236|emb|CAA74176.1||Y13861
esculentum}PIR|T07393|T07393 myb-related transcripti
thaliana}GP|12321383|gb|AAG50761.1|AC0791
thaliana}PIR|H86450|H86450 probable zinc-fing
thaliana}GP|7269478|emb|CAB79482.1||AL1
thaliana}PIR|T47628|T47628 sugar-phosphate isom
thaliana}GP|13374859|emb|CAC34493.1||AL589883
tuberosum}
thaliana}PIR|E84680|E84680 probable
thaliana}
thaliana}PIR|C84630|C84630 probable cinnamoyl CoA
tabacum}GP|1805359|dbj|BAA19155.1||AB000623
mays}
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}□GP|1621012|emb|CAA70038.1||Y08786
tuberosum}SP|Q43644|NUAM_SOLTU NADH-
thaliana}PIR|F96633|F96633 hypothetical protein
thaliana}PIR|D84614|D84614 hyp
thaliana}PIR|B96699|B96699 probable lipoxygenase
thaliana}GP|5302810|emb|CAB46051.1||Z97342
tuberosum}
thaliana}GP|2244749|emb|CAB10172.1||Z97335
tuberosum}
thaliana}PIR|T47605|T47605 RING finger-like protein-
Arabido
Streptomyces coelicolor A3(2) gb|AL163641. EST gb
thaliana}PIR|G84831|G84831 probable bZIP transcript
tabacum}SP|O24160|TG21_TOBAC TGACG-
tuberosum}SP|Q
thaliana}PIR|H86450|H86450 probable zinc-fing
thaliana}GP|7269544|emb|CAB79546.1||AL16
thaliana}PIR|H96690|H96690 probable formyl
tuberosum}
hybrida}
tabacum}
melongena}SP|P37123|C771_SOLME CYTOCHROME
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar
thaliana}PIR|E84509|E84509 probable vanadate res
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}GP|13374859|emb|CAC34493.1||AL589883
thaliana}GP|7268879|emb|CAB79083.1||AL161553
thaliana}
thaliana}GP|7270404|emb|CAB80171.1||AL161585
thaliana}GP|2244749|emb|CAB10172.1||Z97335
thaliana}PIR|F86441|F86441 probable cytochrome P450
thaliana}GP|3132474|gb|AAC16263.1||AC003096
thaliana}PIR|G84831|G84831 probable bZIP transcript
thaliana}PIR|T48253|T48253 myb-like protein-
Arabidopsis thaliana
thaliana}PIR|H84786|H84786 probable
somniferum}SP|P54771|TYD5_PAPSO
Arabidopsis thaliana and contains two PF|00847 AP
hirsutum}GP|2266947|gb|AAB80714.1||AF008939
oleracea}PIR|T09153|T09153 glucose-6-phosphate
thaliana}
thaliana}PIR|F86307|F86307 hypothetical protein
thaliana}PIR|T48643|T48643 cinnamoyl CoA reductas
thaliana}GP|6065740|emb|CAB58230.1||AJ012758
thaliana}GP|6562306|emb|CAB62604.1||AL133421 diac
thaliana}GP|7378610|emb|CAB83286.1||AL162751
thaliana}GP|2244999|emb|CAB10419.1||Z97341
thaliana}GP|6682260|gb|AAF23312.1|AC016661_37|AC0166
tuberosum}GP|575418|emb|CAA57894.1||X82544
sativa}GP|13489165|gb|AAK27799.1|AC022457_2|AC022457
tuberosum}SP|P93568|UGS2_SOLTU SOLUBLE
tuberosum}GP|1495802|emb|CAA65268.1||X96405 13-
thaliana}
tuberosum}PIR|JX0128|XNPOU UTP-glucose-1-
tabacum}
tabacum}
hybrida}
tabacum}SP|Q42962|PGKY_TOBAC
esculentum}SP|Q42884|ARC1
esculentum}SP|Q42884|ARC1
hybrida}GP|439493|dbj|BAA05079.1||D26086 zinc-
ananassa}GP|10121330|gb|AAG13131.1|AF193791_1|AF
tuberosum}GP|1486472|emb|CAA68164.1||X99853
chinense}
pleracea}SP|P31853|ATPX_SPIOL ATP SYNTHASE
thaliana}GP|2864617|emb|CAA16964.1||AL0
max}SP|Q96558|UGDH_SOYBN UDP-GLUCOSE 6-
thaliana}PIR|T51527|T51527 UDP-glucose dehydrog
tuberosum}GP|1407705|gb|AAB67865.1||U60202
thaliana}GP|2981618|dbj|BAA25249.1||AB008855 3-
thaliana}GP|12325273|gb|AAG52580.1|AC016529_11|
sativus}SP|P08216|MASY_CUCSA MALATE
thaliana}
thaliana}
domestica}PIR|T16995|T16995 probable cinnamyl-alco
thaliana}GP|6598664|gb|AAD25643.2|AC007109_1|AC007109
tabacum}GP|1679853|emb|CAB05369.1||Z82982
thaliana}GP|12324405|gb|AAG52172.1|AC020665_17|
thaliana}PIR|H84807|H84807 probable phospho
tuberosum}
thaliana}PIR|D86257|D86257 hypothetical protein
pennellii}SP|O04973|LU1A_LYCPN 2-
thaliana}PIR|D84747|D84747 probable cinnamoyl-CoA
thaliana}PIR|T00419|T00419 dTDP-glucose 4-6-dehy
thaliana}
sativa}GP|9967277|dbj|BAB12338.1||AB047975
thaliana}GP|4240118|dbj|BAA74838.1||AB007800
thaliana}GP|13877669|gb|AAK43912.1|AF370593_1|AF370593
napus}GP|1416514|dbj|BAA09644.1||D63168
tabacum}EGAD|126596|143593 citrate synthase
oleracea}SP|Q01402|NDK2_SPIOL NUCLEOSIDE
unguiculata}PIR|T08124|T08124 ferritin 2 precursor-
hybrida}SP|P11650|CFIA_PETHY CHALCONE--
thaliana}GP|2981618|dbj|BAA25249.1||AB008855 3-
thaliana}
sativa}□GP|777387|gb|AAB46611.1||L25335 aspartate
Arabidopsis thal
tabacum}
tabacum}
thaliana}PIR|H84784|H84784 probable glucosyl
thaliana}PIR|H96622|H96622 probable ABC transporter
thaliana}GP|4325341|gb|AAD17340.1||AF128393
thaliana}GP|13194812|gb|AAK15568.1|AF
sativa}GP|12328532|dbj|BAB21190.1||AP002909
hybrida}PIR|S36655|S36655 U
thaliana}PIR|E84610|E84610 probable aspartate amin
thaliana}GP|7269379|emb|CAB81339.1||AL161563
esculentum}SP|Q00497|AROK_LYCES SHIKIMATE
thaliana}PIR|T02486|T02486 hyp
tuberosum}SP|P80269|NUIM_SOLTU NADH-
thaliana}
thaliana}PIR|D96806|D96806 probable aminotrans
thaliana}GP|3281856|emb|CAA19751.1||AL031004
tabacum}SP|Q42967|DCUP_TOBAC
thaliana}PIR|C84784|C84784 probable glucosyl
tuberosum}SP|P37830|G6PD_SOLTU GLUCOSE-6-
thaliana}PIR|C96702|C96702 probable ABC transpor
thaliana}
sativa}GP|495725|gb|AAB42144.1||L25042 acetyl-CoA
hybrida}
tuberosum}GP|639834|emb|CAA58823.1||X83999
thaliana}PIR|T51457|T51457 phosphoglucomutase-like
chacoense}SP|P93530|C7D6_SOLCH CYTOCHROME
sativa}GP|9967162|dbj|BAB12275.1||AB047923
sativus}SP|Q39639|PLSB_CUCSA GLYCEROL-3-
tremuloides}
Arabidopsis thaliana and is a member of the PF|0
thaliana}
tuberosum}GP|639834|emb|CAA58823.1||X83999
thaliana}GP|7378610|emb|CAB83286.1||AL162751
annuum}PIR|T09541|T09541 transketolase (EC 2.2.1.1)
thaliana}PIR|F84618|F84618 probable flavonol
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20
sativa}
thaliana}PIR|F86441|F86441 probable cytochrome P450
miliaceum}GP|20597|emb|CAA45022.1||X63428
tabacum}SP|P27154|CAPP_TOBAC
Arabidopsis thaliana
thaliana}GP|7269334|emb|CAB79393.1||AL161562 a
tuberosum}
thaliana}GP|7269448|emb|CAB79452.1||AL161564
roseus}PIR|T09999|T09999 cytochrome P450-
reflexa}GP|458456|gb|AAA16513.1||U06754 alpha
thaliana}
tabacum}
faba}GP|2104679|emb|CAA66480.1||X97906
tuberosum}
tabacum}GP|1360086|emb|CAA66605.1||X97946 Zn
hirsutum}PIR|T09745|T09745 myb-related protein-upl
tuberosum}PIR|T07016|T07016 6-ph
tabacum}GP|1171579|emb|CAA64635.1||X95342
hybrida}
tuberosum}SP|Q43644|NUAM_SOLTU NADH-
thaliana}GP|12006420|gb|AAG44850.1|AF283757
thaliana}
thaliana}PIR|T48643|T48643 cinnamoyl CoA reductas
thaliana}
thaliana}PIR|T51333|T51333 transcription factor
thaliana}PIR|E96720|E96720 probable alpha-amylase
esculentum]
tabacum}SP|Q42954|KPYC_TOBAC PYRUVATE
tuberosum}PIR|T07391|T07391 probable alpha-
thaliana}GP|13374859|emb|CAC34493.1||AL589883
tabacum}PIR|T01933|T01933 probable aldose 1-
thaliana}GP|4115931|gb|AAD03441.1||AF118223
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-
thaliana}GP|12324537|gb|AAG52223.1|AC021665_6|AC021665
thaliana}PIR|E96720|E96720 probable alpha-amylase
tabacum}PIR|T03396|T03396 invertase inhibitor
thaliana}GP|13877661|gb|AAK43908.1|AF370589_1|AF370589
japonica}SP|P21820|TPIS_COPJA
thaliana}PIR|E96742|E96742 probable ABC transpor
thaliana}
tuberosum}PIR|T07016|T07016 6-ph
roseus}
thaliana}GP|4689366|gb|AAD27870.1|AF134155_1|AF134155
thaliana}
thaliana}GP|2245095|emb|CAB10517.1||Z97343
thaliana}PIR|T02609|T02609 probable heat shoc
tabacum}
thaliana}PIR|T45654|T45654 zinc-finger-like protein-
Arabido
vulgaris}GP|1142619|gb|AAB00686.1||U18348
esculenta}SP|Q40288|UFO6_MANES FLAVONOL 3-
tuberosum}SP|P93568|UGS2_SOLTU SOLUBLE
esculentum}GP|1654138|gb|AAB65766.1||U37839
tuberosum}PIR|T07391|T07391 probable alpha-
thaliana}PIR|A84588|A84588 probable tyrosine
tuberosum}GP|1117793|gb|AAD09202.1||U24232
thaliana}
melongena}SP|P37124|C772_SOLME CYTOCHROME
tuberosum}SP|Q43644|NUAM_SOLTU NADH-
thaliana}GP|2245025|emb|CAB10445.1||Z97341
tabacum}PIR|T01933|T01933 probable aldose 1-
plumbaginifolia}SP|P05495|ATP0_NICPL ATP
melongena}SP|P37118|C712_SOLME CYTOCHROME
esculentum}
thaliana}GP|3513735|gb|AAC33951.1||AF080118 cont
thaliana}GP|2244749|emb|CAB10172.1||Z97335
thaliana}GP|2244749|emb|CAB10172.1||Z97335
esculentum}SP|P43282|METM_LYCES S-
tuberosum}
sativus}GP|393707|emb|CAA47926.1||X67696 acetyl-
vulgare}SP|Q40082|XYLA_HORVU XYLOSE
tabacum}GP|1574946|gb|AAC49913.1||U38612
thaliana}
hybrida}SP|P48495|TPIS_PETHY
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}
tuberosum}PIR|T07014|T07014 phosphoglycerate
thaliana}GP|7269604|emb|CAB81400.1||AL161571
tuberosum}SP|Q43844|NUKM_SOLTU NAD
hybrida}
tuberosum}SP|P50433|GLYM_SOLTU SE
tuberosum}SP|P50433|GLYM_SOLTU SE
thaliana}GP|12325190|gb|AAG52541.1|AC013289
thaliana}PIR|B84606|B84606 probable ATP synthase
esculentum}SP|P54767|DCE_LYCES GLUTAMATE
tabacum}PIR|T01985|T01985 zinc-finger protein
unguiculata}PIR|T08124|T08124 ferritin 2 precursor-
tuberosum}SP|P2
maxima}SP|P49299|CYSZ_CUCMA CITRATE
tuberosum}
thaliana}GP|9392679|gb|AAF87256.1|AC068562_3|AC068562
tuberosum}PIR|T07402|T07402 probable isocitrate
thaliana}PIR|H84687|H84687 legumin-like protein
niger}SP|P24397|HY6H_HYONI HYOSCYAMINE 6-
thaliana}GP|13374859|emb|CAC34493.1||AL589883
thaliana}GP|7268088|emb|CAB78426.1||AL161
thaliana}PIR|T51543|T51543 TOM (target of myb1)-
thaliana}
thaliana}PIR|F86227|F86227 hypothetical protein
thaliana}PIR|C84588|C84588 probable NADH-ubiquin
thaliana}PIR|T47595|T47595 RING finger protein
thaliana}GP|3250736|emb|CAA76803.1||Y17593
sativa}PIR|JC5125|JC5125 aspartate transaminase (EC
tabacum}SP|P06288|ATPI_TOBAC ATP SYNTHASE
napus}SP|P2910
thaliana}
hybrida}
thaliana}PIR|H84774|H84774 probable homeodom
hirsutum}GP|2266947|gb|AAB80714.1||AF008939
thaliana}GP|12325076|gb|AAG52485.1|AC018364_3|AC018364
tabacum}GP|1237250|emb|CAA65580.1||X96784
thaliana}GP|3892048|gb|AAC78257.1|AAC78257|AC
carota}SP|P28734|AATC_DAUCA ASPARTATE
thaliana}
thaliana}GP|7270157|emb|CAB79970.1||AL161581 H+-
thaliana}GP|4309698|gb|AAD15482.1||AC006266
papaya}PIR|T08168|T08168 glutaminyl-peptide
thaliana}
thaliana}PIR|T49033|T49033 zinc finger-like protein-
thaliana}GP|4835225|emb|CAB42903.1||AL049
tuberosum}SP|P50433|GLYM_SOLTU SE
tuberosum}SP|P50433|GLYM_SOLTU SE
tabacum}GP|870726|gb|AAC41659.1||L38260 biotin
sativum}SP|Q43814|OTC_PEA ORNITHINE
esculentum}
tuberosum}GP|1857447|gb|AAB48444.1||U82367 UDP-
thaliana}
annuum}
thaliana}
tabacum}SP|Q42948|DAPA_TOBAC
thaliana}GP|7267861|emb|CAB78204.1||AL
thaliana}GP|7270932|emb|CAB80611.1||AL161595
thaliana}PIR|T46196|T46196 cytochrome P450-like
thaliana}GP|3132474|gb|AAC16263.1||AC003096
peruvianum}
thaliana}PIR|H84784|H84784 probable glucosyl
thaliana}
thaliana}GP|7576196|emb|CAB87947.1||AL163912
thaliana}PIR|F84832|F84832 glycerol-3-phosphate deh
sativa}GP|11761117|dbj|BAB19107.1||AP002839
thaliana}GP|5051786|emb|CAB45079.1||AL078637 tr
thaliana}GP|7340661|emb|CAB82941.1||AL162506
thaliana}GP|7270436|emb|CAB80202.1||AL161586
thaliana}GP|13877669|gb|AAK43912.1|AF370593_1|AF370593
thaliana}GP|7270045|emb|CAB79860.1||AL161579
oleracea}SP|Q41364|SOT1_SPIOL 2-
thaliana}
hybrida}
thaliana}GP|7270098|emb|CAB79912.1||AL161580
annuum}PIR|T09541|T09541 transketolase (EC 2.2.1.1)
Populus deltoides vegetative storage protein. (L
thaliana}PIR|D84581|D84581 probable CCCH-type z
thaliana}
thaliana}GP|7270932|emb|CAB80611.1||AL161595
tuberosum}SP|P52578|IFRH_SOLTU ISOFLAVONE
thaliana}
hybrida}PIR|S32110|S32110 cytochrome P450 PET-1-
tabacum}GP|19893|emb|CAA46940.1||X66145
tuberosum}
thaliana}GP|11908040|gb|AAG41449.1|AF3268
thaliana}GP|10092278|gb|AAG12691.1|AC025814_15|
thaliana}GP|7268946|emb|CAB81256.1||AL161555
thaliana}GP|4929803|gb|AAD34162.1|AF152555_1|AF152555
tuberosum}
thaliana}GP|13899119|gb|AAK48981.1|AF370554_1|AF370554
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar
thaliana}PIR|T47554|T47554 cytochrome P450
caryophyllus.
thaliana}PIR|B96768|B96768 protein enolase F2P9.10
thaliana}PIR|T07717|T07717 probable ABC-type
thaliana}GP|7270045|emb|CAB79860.1||AL161579
thaliana}PIR|E96612|E96612 probable transcription
thaliana}GP|9964121|gb|AAG09829.1|AF287699_1|AF287699
thaliana}PIR|F84832|F84832 glycerol-3-phosphate deh
melongena}SP|P37119|C713_SOLME CYTOCHROME
thaliana}
tuberosum}SP|P93568|UGS2_SOLTU SOLUBLE
thaliana}GP|7268718|emb|CAB78925.1||AL161550
esculentum}GP|2102691|gb|AAB57733.1||U64817
thaliana}PIR|C84858|C84858 probable citrate synthase
thaliana}PIR|T01151|T01151 probable acetone-cyanohy
tabacum}GP|1360078|emb|CAA66601.1||X97942 Zn
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713
thaliana}GP|2245029|emb|CAB10449.1||Z97341
thaliana}PIR|E96661|E96661 GMP synthase, 61700-64653
max}GP|902938|dbj|BAA09462.1||D50866 beta-
thaliana}GP|7270718|emb|CAB80401.1||AL161591
thaliana}PIR|H86356|H86356 probable UDP-gluco
caryophyllus.
thaliana}GP|7270370|emb|CAB80137.1||AL1615
glaucum}
thaliana}PIR|T46159|T46159 cytochrome P450-like
tabacum}PIR|T02203|T02203 finger protein Dof-
thaliana}GP|4115931|gb|AAD03441.1||AF118223
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20
tabacum}GP|861067|emb|CAA46942.1||X66147
thaliana}PIR|H84687|H84687 legumin-like protein
tuberosum}PIR|T06995|T06995 probable MADS box
annuum}PIR|T09541|T09541 transketolase (EC 2.2.1.1)
thaliana}PIR|T00527|T00527
hybrida}
thaliana}PIR|T47556|T47556 pyruvate kinase-like
thaliana}
thaliana}GP|7268827|emb|CAB79032.1||AL161552
thaliana}PIR|D96550|D965
thaliana}PIR|C84544|C84544 probable gluconokinase
tabacum}GP|1545805|dbj|BAA10929.1||D64052
hybrida}SP|Q07346|DCE_PETHY GLUTAMATE
tabacum}
thaliana}GP|4512664|gb|AAD21718.1||AC006931
stramonium}SP|Q96556|SPE1_DATST SPERMIDINE
tabacum}
esculentum = tomatoes, cv. Super First, fruits, Peptide,
esculentum}SP|P29000|INVA_LYCES ACID BETA-
esculentum}SP|P29000|INVA_LYCES ACID BETA-
esculentum = tomatoes, cv Tiny Tim LA154, flowers,
melongena}SP|P37122|C762_SOLME CYTOCHROME
scutellarioides}
scutellarioides}
aestivum}SP|P93596|CP51_WHEAT CYTOCHROME
thaliana}GP|13878053|gb|AAK44104.1|AF370289_1|AF370289
thaliana}PIR|T47754|T47754 leucine zipper-cont
thaliana}GP|6598933|gb|AAF18728.1|AC018721—
tuberosum}PIR|T07685|T07685 omega-3 fatty acid
tabacum}GP|3721540|dbj|BAA33531.1||D83583 Sulfite
faba}GP|2104681|emb|CAA66481.1||X97907
tuberosum}SP|P37830|G6PD_SOLTU GLUCOSE-6-
tuberosum}SP|P37830|G6PD_SOLTU GLUCOSE-6-
thaliana}PIR|T47556|T47556 pyruvate kinase-like
tabacum}
mays}
tabacum}
thaliana}
tuberosum}SP|Q43848|TKTC_SOLTU
thaliana}PIR|T52043|T52043 probable glutamate--
versicolor}GP|2429282|gb|AAD05034.1||AF014056
thaliana}
thaliana}GP|6513940|gb|AAF14844.1|AC011664_26|AC011664
thaliana}PIR|F84565|F84565 probable homeodom
tabacum}
mays}
tabacum}PIR|T01934|T01934 adenosylmethionine
thaliana}PIR|H84870|H84870 probable
thaliana}PIR|T49899|T49899 zinc finger t
thaliana}
max}SP|P48513|TF2B_SOYBN TRANSCRIPTION
thaliana}GP|7269850|emb|CAB79709.1||AL161575
batatas}
hybrida}
tuberosum}GP|668987|emb|CAA59063.1||X84320
caryophyllus.
Populus deltoides vegetative storage protein.
hybrida}
melongena}SP|P37117|C714_SOLME CYTOCHROME
melongena}SP|P37117|C714_SOLME CYTOCHROME
oleracea}
thaliana}GP|5731763|emb|CAB52582.1||X92419
thaliana}GP|2244754|emb|CAB10177.1||Z9733
esculenta}SP|Q40288|UFO6_MANES FLAVONOL 3-
thaliana}GP|5006473|gb|AAD37511.1|AF139098_1|AF139098
tabacum}GP|1103487|emb|CAA91228.1||Z56282
thaliana}PIR|C84588|C84588 probable NADH-ubiquin
tabacum}SP|P93394|UPP_TOBAC URACIL
peruvianum}
thaliana}GP|7270437|emb|CAB80203.1||AL161586
glutinosa}SP|O04866|ARGD_ALNGL
oleracea}PIR|T09153|T09153 glucose-6-phosphate
thaliana}PIR|D84614|D84614 hyp
thaliana}GP|7378610|emb|CAB83286.1||AL162751
thaliana}PIR|A84437|A84437 probable PHD-type zin
tabacum}
thaliana}GP|9280230|dbj|BAB01720.1||AB023045 Dof
thaliana}PIR|T07719|T07719 aldose 1-epimerase
tabacum}GP|1805359|dbj|BAA19155.1||AB000623
tabacum}SP|P00823|ATPA_TOBAC ATP SYNTHASE
tuberosum}SP|P52578|IFRH_SOLTU ISOFLAVONE
tuberosum}GP|1488652|emb|CAA62817.1||X91615
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20
domestica}PIR|T16995|T16995 probable cinnamyl-alco
thaliana}PIR|D84614|D84614 hyp
sativa}
thaliana}GP|7270098|emb|CAB79912.1||AL161580
thaliana}
tabacum}GP|1419094|emb|CAA65173.1||X95932
tabacum}GP|1805359|dbj|BAA19155.1||AB000623
thaliana}GP|12323975|gb|AAG51946.1|AC01
thaliana}
sativa}
thaliana}
tuberosum}SP|P52903|ODPA_SOLT
tabacum}GP|2204236|emb|CAA74176.1||Y13861
esculentum}PIR|T07393|T07393 myb-related transcripti
thaliana}GP|12321383|gb|AAG50761.1|AC0791
thaliana}PIR|H86450|H86450 probable zinc-fing
thaliana}GP|7269478|emb|CAB79482.1||AL1
thaliana}GP|13374859|emb|CAC34493.1||AL589883
tuberosum}
thaliana}PIR|E84680|E84680 probable
thaliana}
thaliana}PIR|C84630|C84630 probable cinnamoyl CoA
tabacum}GP|1805359|dbj|BAA19155.1||AB000623
mays}
tuberosum}PIR|T07016|T07016 6-ph
tuberosum}□GP|1621012|emb|CAA70038.1||Y08786
tuberosum}SP|Q43644|NUAM_SOLTU NADH-
thaliana}PIR|F96633|F96633 hypothetical protein
thaliana}PIR|D84614|D84614 hyp
hybrida}SP|Q07474|MAD2_PETHY FLORAL
thaliana}PIR|B96699|B96699 probable lipoxygenase
thaliana}GP|5302810|emb|CAB46051.1||Z97342
tuberosum}
thaliana}GP|2244749|emb|CAB10172.1||Z97335
tuberosum}
domestica}SP|O04136|HKL3_MALDO HOMEOBOX
thaliana}PIR|T47605|T47605 RING finger-like protein-
Streptomyces coelicolor A3(2) gb|AL163641. EST gb
thaliana}PIR|G84831|G84831 probable bZIP transcript
tabacum}SP|O24160|TG21_TOBAC TGACG-
tuberosum}SP|Q
plumbaginifolia}SP|O04937|DHEA_NICPL
thaliana}PIR|H86450|H86450 probable zinc-fing
thaliana}GP|7269544|emb|CAB79546.1||AL16
thaliana}PIR|H96690|H96690 probable formyl
tuberosum}
hybrida}
tabacum}
melongena}SP|P37123|C771_SOLME CYTOCHROME
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar
thaliana}PIR|E84509|E84509 probable vanadate res
thaliana}PIR|H86356|H86356 probable UDP-gluco
thaliana}GP|13374859|emb|CAC34493.1||AL589883
thaliana}GP|7268879|emb|CAB79083.1||AL161553
sativa}GP|11761120|dbj|BAB19110.1||AP002839
thaliana}
thaliana}GP|7270404|emb|CAB80171.1||AL161585
chacoense}SP|P93531|C7D7_SOLCH CYTOCHROME
thaliana}GP|2244749|emb|CAB10172.1||Z97335
thaliana}GP|3132474|gb|AAC16263.1||AC003096
thaliana}PIR|G84831|G84831 probable bZIP transcript
thaliana}PIR|T48253|T48253 myb-like protein-
Arabidopsis thaliana
thaliana}PIR|H84786|H84786 probable
somniferum}SP|P54771|TYD5_PAPSO
Arabidopsis thaliana and contains two PF|00847 AP
hirsutum}GP|2266947|gb|AAB80714.1||AF008939
oleracea}PIR|T09153|T09153 glucose-6-phosphate
thaliana}
thaliana}PIR|F86307|F86307 hypothetical protein
thaliana}PIR|T48643|T48643 cinnamoyl CoA reductas
thaliana}GP|6065740|emb|CAB58230.1||AJ012758
thaliana}GP|6562306|emb|CAB62604.1||AL133421 diac
thaliana}GP|7378610|emb|CAB83286.1||AL162751
thaliana}GP|2244999|emb|CAB10419.1||Z97341
thaliana}GP|6682260|gb|AAF23312.1|AC016661_37|AC0166
tuberosum}GP|575418|emb|CAA57894.1||X82544
sativa}GP|13489165|gb|AAK27799.1|AC022457_2|AC022457
tuberosum}SP|P93568|UGS2_SOLTU SOLUBLE
tuberosum}GP|1495802|emb|CAA65268.1||X96405 13-
thaliana}
tuberosum}PIR|JX0128|XNPOU UTP-glucose-1-
tabacum}
tabacum}
hybrida}
tabacum}SP|Q42962|PGKY_TOBAC
esculentum}SP|Q42884|ARC1
esculentum}SP|Q42884|ARC1
hybrida}GP|439493|dbj|BAA05079.1||D26086 zinc-
ananassa}GP|10121330|gb|AAG13131.1|AF193791_1|AF
tuberosum}GP|1486472|emb|CAA68164.1||X99853
chinense}
oleracea}SP|P31853|ATPX_SPIOL ATP SYNTHASE
thaliana}GP|2864617|emb|CAA16964.1||AL0
max}SP|Q96558|UGDH_SOYBN UDP-GLUCOSE 6-
thaliana}PIR|T51527|T51527 UDP-glucose dehydrog
tuberosum}GP|1407705|gb|AAB67865.1||U60202
thaliana}GP|2981618|dbj|BAA25249.1||AB008855 3-
thaliana}GP|12325273|gb|AAG52580.1|AC016529_11|
sativus}SP|P08216|MASY_CUCSA MALATE
thaliana}
thaliana}
domestica}PIR|T16995|T16995 probable cinnamyl-alco
thaliana}GP|6598664|gb|AAD25643.2|AC007109_1|AC007109
tabacum}GP|1679853|emb|CAB05369.1||Z82982
thaliana}GP|12324405|gb|AAG52172.1|AC020665_17|
thaliana}PIR|H84807|H84807 probable phospho
tuberosum}
thaliana}PIR|D86257|D86257 hypothetical protein
pennellii}SP|O04973|LU1A_LYCPN 2-
thaliana}PIR|D84747|D84747 probable cinnamoyl-CoA
thaliana}PIR|T00419|T00419 dTDP-glucose 4-6-dehy
thaliana}
sativa}GP|9967277|dbj|BAB12338.1||AB047975
thaliana}GP|4240118|dbj|BAA74838.1||AB007800
thaliana}GP|13877669|gb|AAK43912.1|AF370593_1|AF370593
hapus}GP|1416514|dbj|BAA09644.1||D63168
tabacum}EGAD|126596|143593 citrate synthase
oleracea}SP|Q01402|NDK2_SPIOL NUCLEOSIDE
unguiculata}PIR|T08124|T08124 ferritin 2 precursor-
hybrida}SP|P11650|CFIA_PETHY CHALCONE--
thaliana}GP|2981618|dbj|BAA25249.1||AB008855 3-
thaliana}
sativa}□GP|777387|gb|AAB46611.1||L25335 aspartate
Arabidopsis thal
tabacum}
tabacum}
thaliana}PIR|H84784|H84784 probable glucosyl
thaliana}PIR|H96622|H96622 probable ABC transporter
thaliana}GP|4325341|gb|AAD17340.1||AF128393
thaliana}GP|13194812|gb|AAK15568.1|AF
sativa}GP|12328532|dbj|BAB21190.1||AP002909
hybrida}PIR|S36655|S36655 U
thaliana}PIR|E84610|E84610 probable aspartate amin
thaliana}GP|7269379|emb|CAB81339.1||AL161563
esculentum}SP|Q00497|AROK_LYCES SHIKIMATE
thaliana}PIR|T02486|T02486 hyp
tuberosum}SP|P80269|NUIM_SOLTU NADH-
thaliana}
thaliana}PIR|D96806|D96806 probable aminotrans
thaliana}GP|3281856|emb|CAA19751.1||AL031004
tabacum}SP|Q42967|DCUP_TOBAC
thaliana}PIR|C84784|C84784 probable glucosyl
tuberosum}SP|P37830|G6PD_SOLTU GLUCOSE-6-
thaliana}PIR|C96702|C96702 probable ABC transpor
thaliana}
sativa}GP|495725|gb|AAB42144.1||L25042 acetyl-CoA
hybrida}
tuberosum}GP|639834|emb|CAA58823.1||X83999
thaliana}PIR|T51457|T51457 phosphoglucomutase-like
chacoense}SP|P93530|C7D6_SOLCH CYTOCHROME
sativa}GP|9967162|dbj|BAB12275.1||AB047923
sativus}SP|Q39639|PLSB_CUCSA GLYCEROL-3-
tremuloides}
Arabidopsis thaliana and is a member of the PF|0
thaliana}
tuberosum}GP|639834|emb|CAA58823.1||X83999
thaliana}GP|7378610|emb|CAB83286.1||AL162751
annuum}PIR|T09541|T09541 transketolase (EC 2.2.1.1)
thaliana}PIR|F84618|F84618 probable flavonol
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20
sativa}
thaliana}PIR|F86441|F86441 probable cytochrome P450
miliaceum}GP|20597|emb|CAA45022.1||X63428
tabacum}SP|P27154|CAPP_TOBAC
Arabidopsis thaliana
thaliana}GP|7269334|emb|CAB79393.1||AL161562 a
tuberosum}
thaliana}GP|7269448|emb|CAB79452.1||AL161564
roseus}PIR|T09999|T09999 cytochrome P450-
reflexa}GP|458456|gb|AAA16513.1||U06754 alpha
thaliana}
tabacum}
faba}GP|2104679|emb|CAA66480.1||X97906
tuberosum}
tabacum}GP|1360086|emb|CAA66605.1||X97946 Zn
hirsutum}PIR|T09745|T09745 myb-related protein-upl
tuberosum}PIR|T07016|T07016 6-ph
tabacum}GP|1171579|emb|CAA64635.1||X95342
hybrida}
tuberosum}SP|Q43644|NUAM_SOLTU NADH-
thaliana}GP|12006420|gb|AAG44850.1|AF283757—
thaliana}
thaliana}PIR|T48643|T48643 cinnamoyl CoA reductas
thaliana}
thaliana}PIR|T51333|T51333 transcription factor
thaliana}PIR|E96720|E96720 probable alpha-amylase
esculentum]
tabacum}SP|Q42954|KPYC_TOBAC PYRUVATE
tuberosum}PIR|T07391|T07391 probable alpha-
thaliana}GP|13374859|emb|CAC34493.1||AL589883
tabacum}PIR|T01933|T01933 probable aldose 1-
thaliana}GP|4115931|gb|AAD03441.1||AF118223
tuberosum}SP|Q00775|UGST_SOLTU GRANULE-
thaliana}GP|12324537|gb|AAG52223.1|AC021665_6|AC021665
thaliana}PIR|E96720|E96720 probable alpha-amylase
tabacum}PIR|T03396|T03396 invertase inhibitor
thaliana}GP|13877661|gb|AAK43908.1|AF370589_1|AF370589
japonica}SP|P21820|TPIS_COPJA
thaliana}PIR|E96742|E96742 probable ABC transpor
thaliana}
tuberosum}PIR|T07016|T07016 6-ph
roseus}
thaliana}GP|4689366|gb|AAD27870.1|AF134155_1|AF134155
thaliana}
thaliana}GP|2245095|emb|CAB10517.1||Z97343
thaliana}PIR|T02609|T02609 probable heat shoc
tabacum}
thaliana}PIR|T45654|T45654 zinc-finger-like protein-
Arabido
vulgaris}GP|1142619|gb|AAB00686.1||U18348
esculenta}SP|Q40288|UFO6_MANES FLAVONOL 3-
tuberosum}SP|P93568|UGS2_SOLTU SOLUBLE
esculentum}GP|1654138|gb|AAB65766.1||U37839
tuberosum}PIR|T07391|T07391 probable alpha-
thaliana}PIR|A84588|A84588 probable tyrosine
tuberosum}GP|1117793|gb|AAD09202.1||U24232
thaliana}
melongena}SP|P37124|C772_SOLME CYTOCHROME
tuberosum}SP|Q43644|NUAM_SOLTU NADH-
thaliana}GP|2245025|emb|CAB10445.1||Z97341
tabacum}PIR|T01933|T01933 probable aldose 1-
plumbaginifolia}SP|P05495|ATP0_NICPL ATP
melongena}SP|P37118|C712_SOLME CYTOCHROME
esculentum}
thaliana}GP|3513735|gb|AAC33951.1||AF080118 cont
thaliana}GP|2244749|emb|CAB10172.1||Z97335
thaliana}GP|2244749|emb|CAB10172.1||Z97335
esculentum}SP|P43282|METM_LYCES S-
tuberosum}
sativus}GP|393707|emb|CAA47926.1||X67696 acetyl-
vulgare}SP|Q40082|XYLA_HORVU XYLOSE
tabacum}GP|1574946|gb|AAC49913.1||U38612
thaliana}
hybrida}SP|P48495|TPIS_PETHY
thaliana}PIR|H84774|H84774 probable homeodom
thaliana}
tuberosum}PIR|T07014|T07014 phosphoglycerate
thaliana}GP|7269604|emb|CAB81400.1||AL161571
tuberosum}SP|Q43844|NUKM_SOLTU NAD
hybrida}
tuberosum}SP|P50433|GLYM_SOLTU SE
tuberosum}SP|P50433|GLYM_SOLTU SE
thaliana}GP|12325190|gb|AAG52541.1|AC013289—
thaliana}PIR|B84606|B84606 probable ATP synthase
esculentum}SP|P54767|DCE_LYCES GLUTAMATE
tabacum}PIR|T01985|T01985 zinc-finger protein
unguiculata}PIR|T08124|T08124 ferritin 2 precursor-
tuberosum}SP|P2
maxima}SP|P49299|CYSZ_CUCMA CITRATE
tuberosum}
thaliana}GP|9392679|gb|AAF87256.1|AC068562_3|AC068562
tuberosum}PIR|T07402|T07402 probable isocitrate
thaliana}PIR|H84687|H84687 legumin-like protein
niger}SP|P24397|HY6H_HYONI HYOSCYAMINE 6-
thaliana}GP|13374859|emb|CAC34493.1||AL589883
thaliana}GP|7268088|emb|CAB78426.1||AL161
thaliana}PIR|T51543|T51543 TOM (target of myb1)-
thaliana}
thaliana}PIR|F86227|F86227 hypothetical protein
thaliana}PIR|C84588|C84588 probable NADH-ubiquin
thaliana}PIR|T47595|T47595 RING finger protein
thaliana}GP|3250736|emb|CAA76803.1||Y17593
sativa}PIR|JC5125|JC5125 aspartate transaminase (EC
tabacum}SP|P06288|ATPI_TOBAC ATP SYNTHASE
napus}SP|P2910
thaliana}
hybrida}
thaliana}PIR|H84774|H84774 probable homeodom
hirsutum}GP|2266947|gb|AAB80714.1||AF008939
thaliana}GP|12325076|gb|AAG52485.1|AC018364_3|AC018364
tabacum}GP|1237250|emb|CAA65580.1||X96784
thaliana}GP|3892048|gb|AAC78257.1|AAC78257|AC
carota}SP|P28734|AATC_DAUCA ASPARTATE
thaliana}
thaliana}GP|7270157|emb|CAB79970.1||AL161581 H+-
thaliana}GP|4309698|gb|AAD15482.1||AC006266
papaya}PIR|T08168|T08168 glutaminyl-peptide
thaliana}
thaliana}PIR|T49033|T49033 zinc finger-like protein-
thaliana}GP|4835225|emb|CAB42903.1||AL049
tuberosum}SP|P50433|GLYM_SOLTU SE
tuberosum}SP|P50433|GLYM_SOLTU SE
tabacum}GP|870726|gb|AAC41659.1||L38260 biotin
sativum}SP|Q43814|OTC_PEA ORNITHINE
esculentum}
tuberosum}GP|1857447|gb|AAB48444.1||U82367 UDP-
thaliana}
annuum}
thaliana}
tabacum}SP|Q42948|DAPA_TOBAC
thaliana}GP|7267861|emb|CAB78204.1||AL
thaliana}GP|7270932|emb|CAB80611.1||AL161595
thaliana}PIR|T46196|T46196 cytochrome P450-like
thaliana}GP|3132474|gb|AAC16263.1||AC003096
peruvianum}
thaliana}PIR|H84784|H84784 probable glucosyl
thaliana}
thaliana}GP|7576196|emb|CAB87947.1||AL163912
thaliana}PIR|F84832|F84832 glycerol-3-phosphate deh
sativa}GP|11761117|dbj|BAB19107.1||AP002839
thaliana}GP|5051786|emb|CAB45079.1||AL078637 tr
thaliana}GP|7340661|emb|CAB82941.1||AL162506
thaliana}GP|7270436|emb|CAB80202.1||AL161586
thaliana}GP|13877669|gb|AAK43912.1|AF370593_1|AF370593
thaliana}GP|7270045|emb|CAB79860.1||AL161579
oleracea}SP|Q41364|SOT1_SPIOL 2-
thaliana}
hybrida}
thaliana}GP|7270098|emb|CAB79912.1||AL161580
annuum}PIR|T09541|T09541 transketolase (EC 2.2.1.1)
Populus deltoides vegetative storage protein. (L
thaliana}PIR|D84581|D84581 probable CCCH-type z
thaliana}
thaliana}GP|7270932|emb|CAB80611.1||AL161595
tuberosum}SP|P52578|IFRH_SOLTU ISOFLAVONE
thaliana}
hybrida}PIR|S32110|S32110 cytochrome P450 PET-1-
tabacum}GP|19893|emp|CAA46940.1||X66145
tuberosum}
thaliana}GP|11908040|gb|AAG41449.1|AF3268
thaliana}GP|10092278|gb|AAG12691.1|AC025814_15|
thaliana}GP|7268946|emb|CAB81256.1||AL161555
thaliana}GP|4929803|gb|AAD34162.1|AF152555_1|AF152555
tuberosum}
thaliana}GP|13899119|gb|AAK48981.1|AF370554_1|AF370554
thaliana}GP|2464913|emb|CAB16808.1||Z99708 sugar
thaliana}PIR|T47554|T47554 cytochrome P450
caryophyllus.
thaliana}PIR|B96768|B96768 protein enolase F2P9.10
thaliana}PIR|T07717|T07717 probable ABC-type
thaliana}GP|7270045|emb|CAB79860.1||AL161579
thaliana}PIR|E96612|E96612 probable transcription
thaliana}GP|9964121|gb|AAG09829.1|AF287699_1|AF287699
thaliana}PIR|F84832|F84832 glycerol-3-phosphate deh
melongena}SP|P37119|C713_SOLME CYTOCHROME
thaliana}
tuberosum}SP|P93568|UGS2_SOLTU SOLUBLE
thaliana}GP|7268718|emb|CAB78925.1||AL161550
esculentum}GP|2102691|gb|AAB57733.1||U64817
thaliana}PIR|C84858|C84858 probable citrate synthase
thaliana}PIR|T01151|T01151 probable acetone-cyanohy
tabacum}GP|1360078|emb|CAA66601.1||X97942 Zn
thaliana}GP|12658412|gb|AAK01128.1|AF331713_1|AF331713
thaliana}GP|2245029|emb|CAB10449.1||Z97341
thaliana}PIR|E96661|E96661 GMP synthase, 61700-64653
max}GP|902938|dbj|BAA09462.1||D50866 beta-
thaliana}GP|7270718|emb|CAB80401.1||AL161591
thaliana}PIR|H86356|H86356 probable UDP-gluco
caryophyllus.
thaliana}GP|7270370|emb|CAB80137.1||AL1615
glaucum}
thaliana}PIR|T46159|T46159 cytochrome P450-like
tabacum}PIR|T02203|T02203 finger protein Dof-
thaliana}GP|4115931|gb|AAD03441.1||AF118223
lineata}GP|12003188|gb|AAG43481.1|AF203688_1|AF20
tabacum}GP|861067|emb|CAA46942.1||X66147
thaliana}PIR|H84687|H84687 legumin-like protein
tuberosum}PIR|T06995|T06995 probable MADS box
annuum}PIR|T09541|T09541 transketolase (EC 2.2.1.1)
thaliana}PIR|T00527|T00527
hybrida}
thaliana}PIR|T47556|T47556 pyruvate kinase-like
thaliana}
thaliana}GP|7268827|emb|CAB79032.1||AL161552
thaliana}PIR|D96550|D965
thaliana}PIR|C84544|C84544 probable gluconokinase
| Number | Date | Country | Kind |
|---|---|---|---|
| 03004838.3 | Mar 2003 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2004/002201 | 3/4/2004 | WO | 00 | 5/7/2007 |
