Patent Application
20120198587
1. Field of the Invention
The present invention relates to methods and materials for identifying genes and the regulatory networks that control gene expression in an organism. More particularly, the present invention relates to soybean genes encoding transcription factors or other functional proteins that are expressed in a tissue specific, developmental stage specific, or biotic and abiotic stress specific manner.
2. Description of the Related Art
Gene expression is controlled at the transcriptional level by a very diverse group of proteins called transcription factors (TF or TFs). These proteins identify specific promoters of the genes regulated by them, and through protein-DNA and/or protein-protein interactions, these TFs help to assemble the basal transcription machinery in the cell. Transcription factors are master controllers in many living cells. They control or influence many biological processes, including cell cycle progression, metabolism, growth, development, reproduction, and responses to the environment. (Czechowski et al. 2004).
TFs play critical roles in all aspects of a higher plant's life cycle. Although several studies have analyzed the function of individual TFs, collectively these studies have provided information on only a few TFs. Therefore, it is important to identify and to understand the functions of more TFs in order to dissect their specific role in plant development, stress tolerance and plant-microbe interaction.c
Molecular tailoring of novel TFs, for example, has the potential to overcome a number of limitations in creating transgenic soybean plants with stress tolerance and better yield. A number of published reports show that genetic engineering of plants, both monocot and dicot, to modify gene expression can lead to enhanced stress tolerance. For example, over-expression of different types of TFs, such as DREB1A, ANAC, MYB, MYC and ZFHD in Arabidopsis strongly improved the drought and salt tolerance of transgenic plants (Liu et al. 1998; Abe et al. 2003; Tran et al. 2007).
Recently, introduction of SNAC 1 and ZmNF-YB2 TFs into rice and maize, respectively, enhanced the drought tolerance of transgenic plants, as demonstrated by field studies. Transgenic rice over-expressing the SNAC1 gene had 22-34% higher seed set than a negative control in the field under severe drought stress conditions at the reproductive stage, whereas transgenic maize over-expressing the ZmNF-YB2 gene (from Monsanto) produced a ˜50% increase in yield, relative to the controls, when water was withheld from the planted field area during the late vegetative stage (Hu et al. 2006; Nelson et al. 2007). The regulations forcing the listing or banning of trans-fats have spurred the development of low-linolenic soybeans. Recently, some modified zinc finger TFs (ZFP-TFs) that can specifically down-regulate the expression of the endogenous soybean FAD2-1 gene, which catalyzes the conversion of oleic acid to linoleic acid, were introduced into soybean. Seed-specific expression of these ZFP-TFs in transgenic soybean somatic embryos repressed FAD2-1 transcription and increased significantly the levels of oleic acid, indicating that engineering of TFs is capable of regulating fatty acid metabolism and modulating the expression of endogenous genes in plants (Wu et al. 2004).
Other studies have demonstrated the role of TFs during legume nodulation by characterizing mutant plant phenotypes. For example, The Medicago truncatula MtNSP1 and MtNSP2 genes encode two GRAS family TFs (Catoira et al., 2000; Oldroyd and Long, 2003; Kalo et al., 2005; Smit et al., 2005) that are essential for nodule development. MtERN, a member of the ETHYLENE RESPONSIVE FACTOR (ERF) family (Middleton et al., 2007), was shown to play a key role in the initiation and the maintenance of rhizobial infection. The Lotus japonicus NIN gene encodes a putative TF gene (Schauser et al., 1999). Mutants in the L. japonicus nm gene or the Pisum sativum ortholog (i.e. Sym35) failed to support rhizobial infection and did not show cortical cell division upon inoculation (Schauser et al., 1999; Borisov et al., 2003). In contrast, the L. japonicus astray mutant exhibited hypernodulation. The ASTRAY gene encodes for a bZIP TF (Nishimura et al., 2002).
DNA microarray analysis allows fast and simultaneous measurement of the expression levels of thousands of genes in a single experiment. However, current DNA microarray technology fails to accurately measure the expression levels of genes expressed at very low levels. For example, TFs are often missed in DNA microarray analysis due to the very low levels they are usually expressed in cells.
Drought is one of the major abiotic stress factors limiting crop productivity worldwide. Global climate changes may further exacerbate the drought situation in major crop-producing countries. Although irrigation may in theory solve the drought problem, it is usually not a viable option because of the cost associated with building and maintaining an effective irrigation system, as well as other non-economical issues, such as the general availability of water (Boyer, 1983). Thus, alternative means for alleviating plant water stress are needed.
In soybean, drought stress during flowering and early pod development significantly increases the rate of flower and pod abortion, thus decreasing final yield (Boyer 1983; Westgate and Peterson 1993). Soybean yield reduction of 40% because of drought is common experience among soybean producers in the United States (Muchow & Sinclair, 1986; Specht et al. 1999).
Mechanisms for selecting drought tolerant plants fall into three general categories. The first is called drought escape, in which selection is aimed at those developmental and maturation traits that match seasonal water availability with crop needs. The second is dehydration avoidance, in which selection is focused on traits that: lessen evaporatory water loss from plant surfaces or maintain water uptake during drought via a deeper and more extensive root system. The last mechanism is dehydration tolerance, in which selection is directed at maintaining cell turgor or enhancing cellular constituents that protect cytoplasmic proteins and membranes from drying.
The molecular mechanisms of abiotic stress responses and the genetic regulatory networks of drought stress tolerance have been reviewed recently (Wang et at 2003; Vinocur and Altman 2005; Chaves and Oliveira 2004; Shinozaki et al. 2003). Plant modification for enhanced drought tolerance is mostly based on the manipulation of either transcription and/or signaling factors or genes that directly protect plant cells against water deficit. Despite much progress in the field, understanding the basic biochemical and molecular mechanisms for drought stress perception, transduction, response and tolerance remains a major challenge in the field. Utilization of the knowledge on drought tolerance to generate plants that can tolerate extreme water deficit condition is even a bigger challenge.
Analysis of changes in gene expression within a target plant is important for revealing the transcriptional regulatory networks. Elucidation of these complex regulatory networks may contribute to our understanding of the responses mounted by a plant to various stresses and developmental changes, which may ultimately lead to crop improvement. DNA microarray assays (Schena et al 1995; Shalon et al. 1996) have provided an unprecedented opportunity for the generation of gene expression data on a whole-genome scale.
Gene expression profiling using cDNAs or oligonucleotides microarray technology has advanced our understanding of gene regulatory network when a plant is subject to various stresses (Bray 2004; Denby and Gehring 2005). For example, numerous genes that respond to dehydration stress have been identified in Arabidopsis and have been categorized as “rd” (responsive to dehydration) or “erd” (early response to dehydration) (Shinozaki and Yamaguchi-Shinozaki 1999).
There are at least four independent regulatory pathways for gene expression in response to water stress. Out of the four pathways, two are abscisic acid (ABA) dependent and the other two are ABA independent (Shinozaki and Yamaguchi-Shinozaki 2000). In the ABA independent regulatory pathways, a cis-acting element is involved and the Dehydration-responsive element/C-repeat (DRE/CRT) has been identified. DRE/CRT also functions in cold response and high-salt-responsive gene expression. When the DRE/CRT binding protein DREB1/ICBF is overexpressed in a transgenic Arabidopsis plant, changes in expression of more than 40 stress-inducible genes can be observed, which lead to enhanced tolerance to freeze, high salt, and drought (Seki et al, 2001; Fowler and Thomashow 2002; Murayama et al. 2004).
The production of microarrays and the global transcript profiling of plants have revolutionized the study of gene expression which provides a unique snapshot of how these plants are responding to a particular stress. However, no transcriptional profiling or transcriptome changes have been reported for soybean plants under various stress conditions, such as drought, flooding, disease infections, etc. There is also a lack of knowledge with respect to tissue specific expression of soybean genes and regulation of gene expression during different stage of soybean growth or reproduction. Moreover, no studies have systematically classified soybean TFs based on the structure of these proteins.
The instrumentalities described herein overcome the problems outlined above and advance the art by providing genes and DNA regulatory elements which may play an important role in regulating the growth and reproduction of a plant under normal or distress such as drought conditions, among others. Methodology is also provided whereby these genes responsive to various distress conditions may be introduced into a host plant to enhance its capability to grow and reproduce under such conditions. The regulatory elements may also be employed to control expression of heterologous genes which may be beneficial for enhancing a plant's capability to grow under such conditions.
Expression of many plant proteins are regulated by a group of proteins termed transcription factors (TFs). The expression of TFs may themselves be regulated. TF genes are generally expressed at relatively low levels which makes the detection and quantitation of their expression difficult. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is the most sensitive technology currently available to quantify gene expression. High-throughput qRT-PCR has been used in several other plant species (e.g. A. thaliana, O. sativa and M. truncatula) to quantitate the expression of TF genes. See Czechowski T, Bari R P, Stitt M, Scheible W R, Udvardi M K (2004) Plant J 38: 366-379; Caldana C, Scheible W R, Mueller-Roeber B, Ruzicic S (2007). Plant Methods 3: 7; and Kakar K, Wandrey M, Czechowski T, Gaertner T, Scheible W R, Stitt M, Torres-Jerez I, Xiao Y, Redman J C, Wu H C, Cheung F, Town C D, Udvardi M K (2008) Plant Methods 4: 18.
It is also disclosed here a library of primers specifically designed for transcription factors (TF) In one embodiment, qRT-PCR may be used to profile gene expression in various soybean tissues using the primers specific for these genes. In another embodiment, the same primers may be used to identified genes whose expression levels change during various developmental or reproductive stages, such as during nodulation by rhizobia in roots, under drought stress, under flooding, or in developing seeds. Among the variety of results obtained was the identification of a number of transcription factors that are specifically expressed in soybean tissues, such as leaves, seeds, roots, etc.
In addition to qRT-PCR, high-through-put sequencing technologies (Illumina-Solexa) may be used to profile gene expression. Compared to more conventional high-through-put technologies (e.g. DNA microarray hybridization), Illumina-Solexa sequencing is more sensitive and allows full coverage of all genes expressed. qRT-PCR and high-through-put sequencing may also be combined to quantify low expressed genes such as TF genes. Using the most sensitive technologies available (i.e. qRT-PCR and high-through-put sequencing technologies (Illumina-Solexa)), a large number of TF genes have been identified and disclosed herein which may prove important in response to various environmental stresses, or to control plant development.
In one embodiment, microarray experiments may be conducted to analyze the gene expression pattern in soybean root and leaf tissues in response to drought stress. Tissue specific transcriptomes may be compared to help elucidate the transcriptional regulatory network and facilitate the identification of stress specific genes and promoters.
In another embodiment, a number of soybean TFs are shown to be expressed only in certain soybean tissues but not in others. These TFs may play an important role in regulating gene expression within the specific tissues. The DNA elements, responsible for tissue specific expression of these genes may be used to control the expression of other genes. Such DNA elements may include but are not limited to a promoter, an enhancer, etc. For instance, sometimes it may be desirable to express a plant transgene only in certain tissues, but not in others. To accomplish this goal, a transgene from the same or different plant may be placed under control of a tissue-specific promoter in order to drive the expression of the gene only in the certain tissues.
In another embodiment, certain soybean TF genes are expressed during seeding, or only at specific stage during seeding (termed “TFIS” for “TF implicated in seeding”). These TFs may play a role in seed filling and may function to control seed compositions. In one aspect, manipulation of these TFs through gene overexpression, gene silencing, or transgenic expression may prove useful in controlling the number, size or composition of the seeds.
In one embodiment, a method is disclosed for generating a transgenic plant from a host plant to create a transgenic plant that is more tolerant to an adverse condition when compared to the host plant. The method may include a step of altering the expression levels of a transcription factor or fragment thereof, and the adverse condition may be selected from one or more of an environmental conditions, such as, by way of example, too high or too low of water, salt, acidity, temperature or combination thereof. Preferably, the transcription factor has been shown to be upregulated or downregulated in an organism in response to the adverse condition, more preferably, by at least two fold. In another aspect, the organism is a second plant that is different from the host plant.
In one aspect, the transcription factor may be endogenous or exogenous to the host plant. “Exogenous” means the transcription factor is from a plant that is genetically different from the host plant. “Endogenous” means that the transcription factor is from the host plant.
In one embodiment, the transcription factor is encoded by a coding sequence such as polynucleotide sequence of SEQ ID. No. 2299, SEQ ID. No. 2300, SEQ ID. No. 2301, SEQ ID. No. 2302, or other transcription factors that are inducible by the adverse condition or those that may regulate expression of proteins that play a role in plant response to the adverse condition.
In another embodiment, the regulatory sequence in the genes encoding the transcription factors of this disclosure may be operably linked to a coding sequence to promote the expression of such coding sequence. Preferably, such coding sequence encode a protein that play a role in plant response to the adverse condition.
In another embodiment, some plant TF genes are induced by drought (these genes are termed DRG or TFIRD) or flooding stress (termed TFIRF). These TFs may help mobilize or activate proteins in plants in response to the drought or flooding conditions.
For purpose of this disclosure, genes whose expression are either up- or down-regulated in response to drought condition are referred to as Drought Response Genes (or DRGs). A DRG that is a transcription factor is also termed “Transcription factors in response to drought” (“TFIRD”). For purpose of this disclosure, a “DRG protein” refers to a protein encoded by a DRG. Some DRGs may show tissue specific expression patterns in response to drought condition. A transcription factor that is induced by flooding is termed “TFIRF” for “Transcription factors in response to Flooding.”
It is to be recognized that although the present disclosure primarily uses drought as an example of environmental distress, the methodology disclosed herein to identify plant genes that are upregulated or downregulated in response to various environmental stimuli and the methodology to manipulate such genes to enhance a plant's capability to growth under stress are applicable to other situations such as flooding, infection, etc.
The microarray experiments described in this disclosure may not have uncovered all the DRGs in all plants, or even in soybean alone, due to the variations in experimental conditions, and more importantly, due to the different gene expressions among different plant species. It is also to be understood that certain DRGs or TFs disclosed here may have been identified and studied previously; however, regulation of their expression under drought condition or their role in drought response may not have been appreciated in previous studies. Alternatively, some DRGs or TFs may contain novel coding sequences. Thus, it is an object of the present disclosure to identify known or unknown genes whose expression levels are altered in response to drought condition.
In order to generate a transgenic plant that is more tolerant to drought condition when compared to a host plant, the expression levels of a protein encoded by an endogenous Drought Response Gene (DRG) or a fragment thereof may be altered to confer a drought resistant phenotype to the host plant. More particularly, the transcription, translation or protein stability of the protein encoded by the DRG or TF may be modified so that the levels of this protein are rendered significantly higher than the levels of this protein would otherwise be even under the same drought condition. To this end, either the coding or non-coding regions, or both, of the endogenous DRG or TF may be modified.
In another aspect, in order to generate a transgenic plant that is more tolerant to drought condition when compared to a host plant, the method may comprise the steps of: (a) introducing into a plant cell a construct comprising a Drought Response Gene (DRG) or a fragment thereof encoding a polypeptide; and (b) generating a transgenic plant expressing said polypeptide or a fragment thereof. In one embodiment, the Drought Response Gene or a fragment thereof is derived from a plant that is genetically different from the host plant. In another embodiment, the Drought Response Gene or a fragment thereof is derived from a plant that belongs to the same species as the host plant. For instance, a DRG identified in soybean may be introduced into soybean as a transgene to confer upon the host increased capability to grow and/or reproduced under mild to severe drought conditions.
The DRGs or TFs disclosed here include known genes as well as genes whose functions are not yet fully understood. Nevertheless, both known or unknown DRGs or TFs may be placed under control of a promoter and be transformed into a host plant in accodance with standard plant transformation protocols. The transgenic plants thus obtained may be tested for the expression of the DRGs or TFs and their capability to grow and/or reproduce under drought conditions as compared to the original host (or parental) plant.
Although the TFs or DRGs disclosed herein are identified in soybean, they may be introduced into other plants as transgenes. Examples of such other plants may include corn, wheat, rice, cotton, sugar cane, or Arabidopsis. In another aspect, homologs in other plant species may be identified by PCR, hybridization or by genome search which may share substantial sequence similarity with the DRGs or TFs disclosed herein. In a preferred embodiment, such a homolog shares at least 90%, more preferably 98%, or even more preferably 99% sequence identity with a protein encoded by a soybean DRG or TF.
In another embodiment, a portion of the DRGs disclosed herein are transcription factors, such as most of the DRGs or fragments thereof listed in Table 6. Conversely, a portion of the TFs disclosed herein are DRGs. It is desirable to introduce one or more of these DRGs or fragments thereof into a host plant so that the transcription factors may be expressed at a sufficiently high level to drive the expression of other downstream effector proteins that may result in increased drought resistance to the transgenic plant.
It is further an object to identify the non-coding sequences of the DRGs, termed Drought Response Regulatory Elements (DRREs) for purpose of this disclosure. These DRREs may be used to prepare DNA constructs for the expression of genes of interest in a host plant. The DREEs or the DRGs may also be used to screen for factors or chemicals that may affect the expression of certain DRGs by interacting with a DREE. Such factors or chemicals may be used to induce drought responses by activating expression of certain genes in a plant.
For purpose of this disclosure, the genes of interest may be genes from other plants or even non-plant organisms. The genes of interest may be those identified and listed in this disclosure, or they may be any other genes that have been found to enhance the capability of a host plant to grow under water deficit condition.
In a preferred embodiment, the genes of interest may be placed under control of the DRREs such that their expression may be upregulated under drought condition. This arrangement is particularly useful for those genes of interest that may not be desirable under normal conditions, because such genes may be placed under a tightly regulated DRRE which only drives the expression of the genes of interest when water deficit condition is sensed by the plant. Under control of such a DRRE, expression of the gene of interest may be only detected under drought condition.
It is an object of this disclosure to provide a system and a method for the genetic modification of a plant, to increase the resistance of the plant to adverse conditions such as drought and/or excessive temperatures, compared to an unmodified plant.
It is another object of the present invention to provide a transgenic plant that exhibits increased resistance to adverse conditions such as drought and/or excessive temperatures as compared to an unmodified plant.
It is another object of the present invention to provide a system and method of modifying a plant, to alter the metabolism or development of the plant.
In one embodiment, a gene of interest may be placed under control of a tissue specific promoter such that such gene of interest may be expressed in specific site, for example, the guard cells. The expression of the introduced genes may enhance the capacity of a plant to modulate guard cell activity in response to water stress. For instance, the transgene may help reduce stomatal water loss. In addition, other characteristics such as early maturation of plants may be introduced into plants to help cope with drought condition.
Preferably, the transgene is under control of a promoter, which may be a constitutive or inducible promoter. An inducible promoter is inactive under normal condition, and is activated under certain conditions to drive the expression of the gene under its control. Conditions that may activate a promoter include but are not limited to light, heat, certain nutrients or chemicals, and water conditions. A promoter that is activated under water deficit condition is preferred.
In another aspect, a tissue specific promoter, an organ specific promoter, or a cell-specific promoter may be employed to control the transgene. Despite their different names, these promoters are similar in that they are only activated in certain cell, tissue or organ types. It is to be understood that a gene under control of an inducible promoter, or a promoter specific for certain cells, tissues or organs may have low level of expression even under conditions that are not supposed to activate the promoter, a phenomenon known as “leaky expression” in the field. A promoter can be both inducible and tissue specific. By way of example, a transgene may be placed under control of a guard cell specific promoter such that the gene can be inducibly expressed in the guard cell of the transgenic plant.
In another aspect, the present disclosure provides a method of generating a transgenic plant having an altered stress response or an altered phenotype compared to an unmodified plant. The coding sequences of the genes that are disclosed to be upregulated may be placed under a promoter such that the genes can be expressed in the transgenic plant. The method may contain two steps: (a) introducing into a plant cell capable of being transformed and regenerated into a whole plant a construct comprising, in addition to the DNA sequences required for transformation and selection in plants, an expression construct including the coding sequence of a gene that a operatively linked to a promoter for expressing said DNA sequence; and (b) recovery of a plant which contains the expression construct.
The transgenic plant generated by the methods disclosed above may exhibit an altered trait or stress response. The altered traits may include increased tolerance to extreme temperature, such as heat or cold; or increased tolerance to extreme water condition such as drought or excessive water. The transgenic plant may exhibits one or more altered phenotype that may contribute to the resistance to drought condition. These phenotypes may include, by way of example, early maturation, increased growth rate, increased biomass, or increased lipid content.
In accordance with the disclosed methods, the coding sequence to be introduced in the transgenic plant preferably encodes a peptide having at least 70%, more preferably at least 90%, more preferably at least 98% identity, and even more preferably at least 99% identity to the polypeptide encoded by the DRGs disclosed in this application. In an alternative aspect, DNA sequence may be oriented in an antisense direction relative to said promoter within said construct.
In accordance with the methods of the present invention, the promoter is preferably selected from the group consisting of an constitutive promoter, an inducible promoter, a tissue specific promoter, and organ specific promoter, a cell-specific promoter. More preferably the promoter is an inducible promoter for expressing said DNA sequence under water deficit conditions.
In another aspect, the present invention provides a method of identifying whether a plant that has been successfully transformed with a construct, characterized in that the method comprises the steps of: (a) introducing into plant cells capable of being transformed and regenerated into whole plants a construct comprising, in addition to the DNA sequences required for transformation and selection in plants, an expression construct that includes a DNA sequence selected from at least one of the DRGs disclosed herein, said DNA sequence may be operatively linked to a promoter for expressing said DNA sequence; (b) regenerating the plant cells into whole plants; and (c) subjecting the plants to a screening process to differentiate between transformed plants and non-transformed plants.
The screening process may involve subjecting the plants to environmental conditions suitable to kill non-transformed plants, retain viability in transformed plants. For instance by growing the plants in a medium or soil that contains certain chemicals, such that only those plants expressing the transgenes can survive. In one particular embodiment, after obtaining a transgenic plant that appear to be expressing the transgene, a functional screening may be carried out by growing the plants under water deficit conditions to select for those that can tolerate such a condition.
In another aspect, the present disclosure provides a kit for generating a transgenic plant having an altered stress response or an altered phenotype compared to an unmodified plant, characterized in that the kit comprises: an expression construct including a DNA sequence selected from at least one of the DRGs disclosed herein, said DNA sequence may be operatively linked to an promoter suitable for expressing said DNA sequence in a plant cell.
Preferably the kit further includes targeting means for targeting the activity of the protein expressed from the construct to certain tissues or cells of the plant. Preferably the targeting means comprises an inducible, tissue-specific promoter for specific expression of the DNA sequence within certain tissues of the plant. Alternatively the targeting means may be a signal sequence encoded by said expression construct and may contain a series of amino acids covalently linked to the expressed protein.
In accordance with the kit of the present invention, the DNA sequence may encode a peptide having at least 70%, more preferably at least 90%, more preferably at least 98%, or even 99% identity to the peptide encoded by coding sequences selected from at least one of the DRGs disclosed herein. In one aspect, said DNA sequence may be oriented in an antisense direction relative to said promoter within said construct.
The methods and materials described herein relate to gene expression profiling using microarrays, quantitative RT-PCR, or high throughput sequencing methods, and follow-up analysis to decode the regulatory network that controls a plant's response to stress. More particularly, drought response is analyzed at the molecular level to identify genes and/or promoters which may be activated under water deficit conditions. The coding sequences of such genes may be introduced into a host plant to obtain transgenic plants that are more tolerant to drought than unmodified plants.
It is to be understood that the materials and methods are taught by way of example, and not by limitation. The disclosed instrumentalities may be broader than the particular methods and materials described herein, which may vary within the skill of the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the related art. The following terminology and grammatical variants are used in accordance with the definitions set out below.
The present disclosure provides genes whose expression levels are altered in response to stress conditions in soybean plants using genome-wide microarray (or gene chip) analysis of soybean plants grown under water deficit conditions. Those genes identified using microarray analysis may be subject to validation to confirm that their expression levels are altered under the stress conditions. Validation may be conducted using high throughput two-step qRT-PCR or by the delta delta CT method.
Sequences of those genes that have been validated may be subject to further sequence analysis by comparing their sequences to published sequences of various families of genes or proteins. For instance, some of these DRGs may encode proteins with substantial sequence similarity to known transcription factors. These transcription factors may play a role in the stress response by activating the transcription of other genes.
The present disclosure provides a system and a method for expressing a protein that may enhance a host's capability to grow or to survive in an adverse environment characterized by water deficit. Although plants are the most preferred host for purpose of this disclosure, the genetic constructs described herein may be introduced into other eukaryotic organisms, if the traits conferred upon these organisms by the constructs are desirable.
The term “transgenic plant” refers to a host plant into which a gene construct has been introduced. A gene construct, also referred to as a construct, an expression construct, or a DNA construct, generally contains as its components at least a coding sequence and a regulatory sequence. A gene construct typically contains at least on component that is foreign to the host plant. For purpose of this disclosure, all components of a gene construct may be from the host plant, but these components are not arranged in the host in the same manner as they are in the gene construct. A regulatory sequence is a non-coding sequence that typically contribute to the regulation of gene expression, at the transcription or translation levels. It is to be understood that certain segments in the coding sequence may be translated but may be later removed from the functional protein. An example of these segments is the so-called signal peptide, which may facilitate the maturation or localization of the translated protein, but is typically removed once the protein reaches its destination. Examples of a regulatory sequence include but are not limited to a promoter, an enhancer, and certain post-transcriptional regulatory elements.
After its introduction into a host plant, a gene construct may exist separately from the host chromosomes. Preferably, the entire gene construct, or at least part of it, is integrated onto a host chromosome. The integration may be mediated by a recombination event, which may be homologous, or non-homologous recombination. The term “express” or “expression” refers to production of RNAs using DNAs as template through transcription or translation of proteins from RNAs or the combination of both transcription and translation.
A “host cell,” as used herein, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA which has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like. A “host plant” is a plant into which a transgene is to be introduced.
A “vector” is a composition for facilitating introduction, replication and/or expression of a selected nucleic acid in a cell. Vectors include, for example, plasmids, cosmids, viruses, yeast artificial chromosomes (YACs), etc. A “vector nucleic acid” is a nucleic acid vector into which heterologous nucleic acid is optionally inserted and which can then be introduced into an appropriate host cell. Vectors preferably have one or more origins of replication, and one or more sites into which the recombinant DNA can be inserted. Vectors often have convenient markers by which cells with vectors can be selected from those without. By way of example, a vector may encode a drug resistance gene to facilitate selection of cells that are transformed with the vector. Common vectors include plasmids, phages and other viruses, and “artificial chromosomes.” “Expression vectors” are vectors that comprise elements that provide for or facilitate transcription of nucleic acids which are cloned into the vectors. Such elements may include, for example, promoters and/or enhancers operably coupled to a nucleic acid of interest.
“Plasmids” generally are designated herein by a lower case “p” preceded and/or followed by capital letters and/or numbers, in accordance with standard nomenclatures that are familiar to those of skill in the art. Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use as described below. The properties, construction and use of such plasmids, as well as other vectors, is readily apparent to those of ordinary skill upon reading the present disclosure.
When a molecule is identified in or can be isolated from a organism, it can be said that such a molecule is derived from said organism. When two organisms have significant difference in the genetic materials in their respective genomes, these two organisms can be said to be genetically different. For purpose of this disclosure, the term “plant” means a whole plant, a seed, or any organ or tissue of a plant that may potentially deveolop into a whole plant.
The term “isolated” means that the material is removed from its original environment, such as the native or natural environment if the material is naturally occurring. For example, a naturally-occurring nucleic acid, polypeptide, or cell present in a living animal is not isolated, but the same polynucleotide, polypeptide, or cell separated from some or all of the coexisting materials in the natural system, is isolated, even if subsequently reintroduced into the natural system. Such nucleic acids can be part of a vector and/or such nucleic acids or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
A “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA evolution or other procedures. A “recombinant polypeptide” is a polypeptide which is produced by expression of a recombinant nucleic acid. An “amino acid sequence” is a polymer of amino acid residues (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence.
The terms “nucleic acid,” or “polynucleotide” refer to a deoxyribonucleotide, in the case of DNA, or ribonucleotide in the case of RNA polymer in either single- or double-stranded form, and unless otherwise specified, encompasses known analogues of natural nucleotides that can be incorporated into nucleic acids in a manner similar to naturally occurring nucleotides. A “polynucleotide sequence” is a nucleic acid which is a polymer of nucleotides (A,C,T,U,G, etc. or naturally occurring or artificial nucleotide analogues) or a character string representing a nucleic acid, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence.
A “subsequence” or “fragment” is any portion of an entire sequence of a DNA, RNA or polypeptide molecule, up to and including the complete sequence. Typically a subsequence or fragment comprises less than the full-length sequence, and is sometimes referred to as the “truncated version.”
Nucleic acids and/or nucleic acid sequences are “homologous” when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and/or protein sequences are homologous when their encoding DNAs are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Similarly, nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. The homologous molecules can be termed homologs. For example, any naturally occurring DRGs, as described herein, can be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original DRGs. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. Methods for determining sequence identity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.
The terms “identical” or “sequence identity” in the context of two nucleic acid sequences or amino acid sequences of polypeptides refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A “comparison window”, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482; by the alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci. U.S.A. 85:2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-244 and Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-10890; Huang et al (1992) Computer Applications in the Biosciences 8:155-165; and Pearson et al. (1994) Methods in Molecular Biology 24:307-331. Alignment is also often performed by inspection and manual alignment.
In one class of embodiments, the polypeptides herein are at least 70%, generally at least 75%, optionally at least 80%, 85%, 90%, 98% or 99% or more identical to a reference polypeptide, e.g., those that are encoded by DNA sequences as set forth by any one of the DRGs disclosed herein or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or more identical to a reference nucleic acid, e.g., those that are set forth by any one of the DRGs disclosed herein or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, said percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned.
The term “substantially identical” as applied to nucleic acid or amino acid sequences means that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, preferably at least 95%, more preferably at least 98% and most preferably at least 99%, compared to a reference sequence using the programs described above (preferably BLAST) using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.
The term “polypeptide” is used interchangeably with the terms “polypeptides” and “protein(s)”, and refers to a polymer of amino acid residues. A ‘mature protein’ is a protein which is full-length and which, optionally, includes glycosylation or other modifications typical for the protein in a given cellular environment.
The term “variant” or “mutant” with respect to a polypeptide refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. Alternatively, a variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variation can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
A variety of additional terms are defined or otherwise characterized herein. In practicing the instrumentalities described herein, many conventional techniques in molecular biology, microbiology, and recombinant DNA are optionally used. These techniques are well known to those of ordinary skill in the art. For example, one skilled in the art would be familiar with techniques for in vitro amplification methods, including the polymerase chain reaction (PCR), for the production of the homologous nucleic acids described herein.
In addition, commercially available kits may facilitate the purification of plasmids or other relevant nucleic acids from cells. See, for example, EasyPrep™ and FlexiPrep™ kits, both from Pharmacia Biotech; StrataClean™ from Stratagene; and, QIAprep™ from Qiagen. Any isolated and/or purified nucleic acid can be further manipulated to produce other nucleic acids, used to transfect cells, incorporated into related vectors to infect organisms, or the like. Typical cloning vectors contain transcription terminators, transcription initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both.
Various types of mutagenesis are optionally used to modify DRGs and their encoded polypeptides, as described herein, to produce conservative or non-conservative variants. Any available mutagenesis procedure can be used. Such mutagenesis procedures optionally include selection of mutant nucleic acids and polypeptides for one or more activity of interest. Procedures that can be used include, but are not limited to: site-directed point mutagenesis, random point mutagenesis, in vitro or in vivo homologous recombination (DNA shuffling), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, mutagenesis by chimeric constructs, and many others known to persons of skill in the art.
In one embodiment, mutagenesis can be guided by known information about the naturally occurring molecule or altered or mutated naturally occurring molecule. By way of example, this known information may include sequence, sequence comparisons, physical properties, crystal structure and the like. In another class of mutagenesis, modification is essentially random, e.g., as in classical DNA shuffling.
Polypeptides may include variants, in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, typically 90% identity, preferably at least 95% identity, more preferably at least 98% identity and most preferably at least 99% identity, to the amino acid sequences as encoded by the DNA sequences set forth in any one of the DRGs disclosed herein.
The aforementioned polypeptides may be obtained by any of a variety of methods. Smaller peptides (less than 50 amino acids long) are conveniently synthesized by standard chemical techniques and can be chemically or enzymatically ligated to form larger polypeptides. Polypeptides can be purified from biological sources by methods well known in the art, for example, as described in Protein Purification, Principles and Practice, Second Edition Scopes, Springer Verlag, N.Y. (1987) Polypeptides are optionally but preferably produced in their naturally occurring, truncated, or fusion protein forms by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (2001) Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y.; and Ausubel et al., eds. (1997) Current Protocols in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., N.Y (supplemented through 2002). RNA encoding the proteins may also be chemically synthesized. See, for example, the techniques described in Oligonucleotide Synthesis, (1984) Gait ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.
The nucleic acid molecules described herein may be expressed in a suitable host cell or an organism to produce proteins. Expression may be achieved by placing a nucleotide sequence encoding these proteins into an appropriate expression vector and introducing the expression vector into a suitable host cell, culturing the transformed host cell under conditions suitable for expression of the proteins described or variants thereof, or a polypeptide that comprises one or more domains of such proteins. The recombinant proteins from the host cell may be purified to obtain purified and, preferably, active protein. Alternatively, the expressed protein may be allowed to function in the intact host cell or host organism.
Appropriate expression vectors are known in the art, and may be purchased or applied for use according to the manufacturer's instructions to incorporate suitable genetic modifications. For example, pET-14b, pcDNAlAmp, and pVL1392 are available from Novagen and Invitrogen, and are suitable vectors for expression in E. coli, mammalian cells and insect cells, respectively. These vectors are illustrative of those that are known in the art, and many other vectors can be used for the same purposes. Suitable host cells can be any cell capable of growth in a suitable media and allowing purification of the expressed protein. Examples of suitable host cells include bacterial cells, such as E. coli, Streptococci, Staphylococci, Streptomyces and Bacillus subtilis cells; fungal cells such as Saccharomyces and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells, mammalian cells such as CHO, COS, HeLa, 293 cells; and plant cells.
Culturing and growth of the transformed host cells can occur under conditions that are known in the art. The conditions will generally depend upon the host cell and the type of vector used. Suitable culturing conditions may be used such as temperature and chemicals and will depend on the type of promoter utilized.
Purification of the proteins or domains of such proteins, if desired, may be accomplished using known techniques without performing undue experimentation. Generally, the transformed cells expressing one of these proteins are broken, crude purification occurs to remove debris and some contaminating proteins, followed by chromatography to further purify the protein to the desired level of purity. Host cells may be broken by known techniques such as homogenization, sonication, detergent lysis and freeze-thaw techniques. Crude purification can occur using ammonium sulfate precipitation, centrifugation or other known techniques. Suitable chromatography includes anion exchange, cation exchange, high performance liquid chromatography (HPLC), gel filtration, affinity chromatography, hydrophobic interaction chromatography, etc. Well known techniques for refolding proteins can be used to obtain the active conformation of the protein when the protein is denatured during intracellular synthesis, isolation or purification.
In general, DRG proteins or domains, or antibodies to such proteins can be purified, either partially (e.g., achieving a 5×, 10×, 100×, 500×, or 1000× or greater purification), or even substantially to homogeneity (e.g., where the protein is the main component of a solution, typically excluding the solvent (e.g., water or DMSO) and buffer components (e.g., salts and stabilizers) that the protein is suspended in, e.g., if the protein is in a liquid phase), according to standard procedures known to and used by those of skill in the art. Accordingly, the polypeptides can be recovered and purified by any of a number of methods well known in the art, including, e.g., ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography, gel electrophoresis and the like. Protein refolding steps can be used, as desired, in making correctly folded mature proteins. High performance liquid chromatography (HPLC), affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired. In one embodiment, antibodies made against the proteins described herein are used as purification reagents, e.g., for affinity-based purification of proteins comprising one or more DRG protein domains or antibodies thereto. Once purified, partially or to homogeneity, as desired, the polypeptides are optionally used e.g., as assay components, therapeutic reagents or as immunogens for antibody production.
In addition to other references noted herein, a variety of purification methods are well known in the art, including, for example, those set forth in R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana, Bioseparation of Proteins, Academic Press, Inc. (1997); Bollag et al., Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ; Harris and Angal Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England (1990); Scopes, Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY (1993); Janson and Ryden, Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY (1998); and Walker, Protein Protocols on CD-ROM Humana Press, NJ (1998); and the references cited therein.
After synthesis, expression and/or purification, proteins may possess a confoimation different from the desired conformations of the relevant polypeptides. For example, polypeptides produced by prokaryotic systems often are optimized by exposure to chaotropic agents to achieve proper folding. During purification from, e.g., lysates derived from E. coli, the expressed protein is optionally denatured and then renatured. This is accomplished, e.g., by solubilizing the proteins in a chaotropic agent such as guanidine HCl. In general, it is occasionally desirable to denature and reduce expressed polypeptides and then to cause the polypeptides to re-fold into the preferred conformation. For example, guanidine, urea, DTT, DTE, and/or a chaperonin can be added to a translation product of interest. Methods of reducing, denaturing and renaturing proteins are well known to those of skill in the art. Debinski, et al., for example, describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. The proteins can be refolded in a redox buffer containing, e.g., oxidized glutathione and L-arginine. Refolding reagents can be flowed or otherwise moved into contact with the one or more polypeptide or other expression product, or vice-versa.
In another aspect, antibodies to the DRG proteins or fragments thereof may be generated using methods that are well known in the art. The antibodies may be utilized for detecting and/or purifying the DRG proteins, optionally discriminating the proteins from various homologues. As used herein, the term “antibody” includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional antibody fragments, which are those fragments sufficient for binding of the antibody fragment to the protein.
General protocols that may be adapted for detecting and measuring the expression of the described DRG proteins using the above mentioned antibodies are known. Such methods include, but are not limited to, dot blotting, western blotting, competitive and noncompetitive protein binding assays, enzyme-linked immunosorbant assays (ELISA), immunohistochemistry, fluorescence-activated cell sorting (FACS), and other protocols that are commonly used and widely described in scientific and patent literature.
Sequence of the DRG genes may also be used in genetic mapping of plants or in plant breeding. Polynucleotides derived from the DRG gene sequences may be used in in situ hybridization to determine the chromosomal locus of the DRG genes on the chromosomes. These polynucleotides may also be used to detect segregation of different alleles at certain DRG loci.
Sequence information of the DRG genes may also be used to design oligonucleotides for detecting DRG mRNA levels in the cells or in plant tissues. For example, the oligonucleotides can be used in a Northern blot analysis to quantify the levels of DRG mRNA. Moreover, full-length or fragment of the DRG genes may be used in preparing microarrays (or gene chips). Full-length or fragment of the DRG genes may also be used in microarray experiments to study expression profile of the DRG genes. High-throughput screening can be conducted to measure expression levels of the DRG genes in different cells or tissues. Various compounds or other external factors may be screened for their effects expression of the DRG gene expression.
Sequences of the DRG genes and proteins may also provide a tool for identification of other proteins that may be involved in plant drought response. For example, chimeric DRG proteins can be used as a “bait” to identify other proteins that interact with DRG proteins in a yeast two-hybrid screening. Recombinant DRG proteins can also be used in pull-down experiment to identify their interacting proteins. These other proteins may be cofactors that enhance the function of the DRG proteins, or they may be DRG proteins themselves which have not been identified in the experiments disclosed herein.
The DRG polypeptides may possess structural features which can be recognized, for example, by using immunological assays. The generation of antisera which specifically bind the DRG polypeptides, as well as the polypeptides which are bound by such antisera, are a feature of the disclosed embodiments.
In order to produce antisera for use in an immunoassay, one or more of the immunogenic DRG polypeptides or fragments thereof are produced and purified as described herein. For example, recombinant protein may be produced in a host cell such as a bacterial or an insect cell. The resultant proteins can be used to immunize a host organism in combination with a standard adjuvant, such as Freund's adjuvant. Commonly used host organisms include rabbits, mice, rats, donkeys, chickens, goats, horses, etc. An inbred strain of mice may also be used to obtain more reproducible results due to the virtual genetic identity of the mice. The mice are immunized with the immunogenic DRG polypeptides in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol. See, for example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), which provides comprehensive descriptions of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity. Alternatively, one or more synthetic or recombinant DRG polypeptides or fragments thereof derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
Antisera that specifically bind the DRG proteins may be used in a range of applications, including but not limited to immunofluorescence staining of cells for the expression level and localization of the DRG proteins, cytological staining for the expression of DRG proteins in tissues, as well as in Western blot analysis.
Another aspect of the disclosure includes screening for potential or candidate modulators of DRG protein activity. For example, potential modulators may include small molecules, organic molecules, inorganic molecules, proteins, hormones, transcription factors, or the like, which can be contacted to a cell or certain tissues that express the DRG proteins to assess the effects, if any, of the candidate modulator upon DRG protein activity.
Alternatively, candidate modulators may be screened to modulate expression of DRG proteins. For example, potential modulators may include small molecules, organic molecules, inorganic molecules, proteins, hormones, transcription factors, or the like, which can be contacted to a cell or certain tissues that express the DRG proteins, to assess the effects, if any, of the candidate modulator upon DRG protein expression. Expression of a DRG gene described herein may be detected, for example, via Northern blot analysis or quantitative (optionally real time) RT-PCR, before and after application of potential expression modulators. Alternatively, promoter regions of the various DRG genes may be coupled to reporter constructs including, without limitation, CAT, beta-galactosidase, luciferase or any other available reporter, and may similarly be tested for expression activity modulation by the candidate modulator. Promoter regions of the various genes are generally sequences in the proximity upstream of the start site of transcription, typically within 1 Kb or less of the start site, such as within 500 bp, 250 by or 100 by of the start site. In certain cases, a promoter region may be located between 1 and 5 Kb from the start site.
In either case, whether the assay is to detect modulated activity or expression, a plurality of assays may be performed in a high-throughput fashion, for example, using automated fluid handling and/or detection systems in serial or parallel fashion. Similarly, candidate modulators can be tested by contacting a potential modulator to an appropriate cell using any of the activity detection methods herein, regardless of whether the activity that is detected is the result of activity modulation, expression modulation or both.
A method of modifying a plant may include introducing into a host plant one or more DRG genes described above. The DRG genes may be placed in an expression construct, which may be designed such that the DRG protein(s) are expressed constitutively, or inducibly. The construct may also be designed such that the DRG protein(s) are expressed in certain tissue(s), but not in other tissue(s). The DRG protein(s) may enhance the ability of the host plant in drought tolerance, such as by reducing water loss or by other mechanisms that help a plant cope with water deficit growth conditions. The host plant may include any plants whose growth and/or yield may be enhanced by a modified drought response. Methods for generating such transgenic plants is well known in the field. See e.g., Leandro Peña (Editor), Transgenic Plants: Methods and Protocols (Methods in Molecular Biology), Humana Press, 2004.
The use of gene inhibition technologies such as antisense RNA or co-suppression or double stranded RNA interference is also within the scope of the present disclosure. In these approaches, the isolated gene sequence is operably linked to a suitable regulatory element. In one embodiment of the disclosure, the construct contains a DNA expression cassette that contains, in addition to the DNA sequences required for transformation and selection in said cells, a DNA sequence that encodes a DRG proteins or a DRG modulator protein, with at least a portion of said DNA sequence in an antisense orientation relative to the normal presentation to the transcriptional regulatory region, operably linked to a suitable transcriptional regulatory region such that said recombinant DNA construct expresses an antisense RNA or portion thereof of an antisense RNA in the resultant transgenic plant.
It is apparent to one of skill in the art that the polynucleotide encoding the DRG proteins or a DRG modulator proteins can be in the antisense (for inhibition by antisense RNA) or sense (for inhibition by co-suppression) orientation, relative to the transcriptional regulatory region. Alternatively a combination of sense and antisense RNA expression can be utilized to induce double stranded RNA interference. See, e.g., Chuang and Meyerowitz, PNAS 97: 4985-4990, 2000; also Smith et al., Nature 407: 319-320, 2000.
These methods for generation of transgenic plants generally entail the use of transformation techniques to introduce the gene or construct encoding the DRG proteins or a DRG modulator proteins, or a part or a homolog thereof, into plant cells. Transfoimation of a plant cell can be accomplished by a variety of different methodology. Methods that have general utility include, for example, Agrobacterium based systems, using either binary and/or cointegrate plasmids of both A. tumifaciens and A. rhyzogenies, (See e.g., U.S. Pat. No. 4,940,838, U.S. Pat. No. 5,464,763), the biolistic approach (See e.g, U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, U.S. Pat. No. 5,149,655), microinjection, (See e.g., U.S. Pat. No. 4,743,548), direct DNA uptake by protoplasts, (See e.g., U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,453,367) or needle-like whiskers (See e.g., U.S. Pat. No. 5,302,523). Any method for the introduction of foreign DNA into a plant cell and for expression therein may be used within the context of the present disclosure.
Plants that are capable of being transformed encompass a wide range of species, including but not limited to soybean, corn, potato, rice, wheat and many other crops, fruit plants, vegetables and tobacco. See generally, Vain, P., Thirty years of plant transformation technology development, Plant Biotechnol J. 2007 March; 5(2):221-9. Any plants that are capable of taking in foreign DNA and transcribing the DNA into RNA and/or further translating the RNA into a protein may be a suitable host.
The modulators described above that may alter the expression levels or the activity of the DRG proteins (collectively called DRG modulators) may also be introduced into a host plant in the same or similar manner as described above.
The DRG proteins or the DRG modulators may be used to modify a target plant by causing them to be assimilated by the plant. Alternatively, the DRG proteins or the DRG modulators may be applied to a target plant by causing them to be in contact with the plant, or with a specific organ or tissue of the plant. In one embodiment, organic or inorganic molecules that can function as DRG modulators may be caused to be in contact with a plant such that these chemicals may enhance the drought response of the target plant.
In addition to the DRG modulators, DRG polypeptides or DRG nucleic acids, a composition containing other ingredients may be introduced, administered or delivered to the plant to be modified. In one aspect, a composition containing an agriculturally acceptable ingredient may be used in conjunction with the DRG modulators to be administered or delivered to the plant.
Bioinformatic systems are widely used in the art, and can be utilized to identify homology or similarity between different character strings, or can be used to perform other desirable functions such as to control output files, provide the basis for making presentations of information including the sequences and the like. Examples include BLAST, discussed supra. For example, commercially available databases, computers, computer readable media and systems may contain character strings corresponding to the sequence information herein for the DRG polypeptides and nucleic acids described herein. These sequences may include specifically the DRG sequences listed herein and the various silent substitutions and conservative substitutions thereof.
The bioinformatic systems contain a wide variety of information that includes, for example, a complete sequence listings for the entire genome of an individual organism representing a species. Thus, for example, using the DRG sequences as a basis for comparison, the bioinformatic systems may be used to compare different types of homology and similarity of various stringency and length on the basis of reported data. These comparisons are useful to identify homologs or orthologs where, for example, the basic DRG gene ortholog is shown to be conserved across different organisms. Thus, the bioinformatic systems may be used to detect or recognize the homologs or orthologs, and to predict the function of recognized homologs or orthologs. By way of example, many homology determination methods have been designed for comparative analysis of sequences of biopolymers including nucleic acids, proteins, etc. With an understanding of hydrogen bonding between the principal bases in natural polynucleotides, models that simulate annealing of complementary homologous polynucleotide strings can also be used as a foundation of sequence alignment or other operations typically performed on the character strings corresponding to the sequences herein. One example of a software package for calculating sequence similarity is BLAST, which can be adapted to the present invention by inputting character strings corresponding to the sequences herein.
The software can also include output elements for controlling nucleic acid synthesis (e.g., based upon a sequence or an alignment of a sequences herein) or other operations which occur downstream from an alignment or other operation performed using a character string corresponding to a sequence herein.
In an additional aspect, kits may embody any of the methods, compositions, systems or apparatus described above. Kits may optionally comprise one or more of the following: (1) a composition, system, or system component as described herein; (2) instructions for practicing the methods described herein, and/or for using the compositions or operating the system or system components herein; (3) a container for holding components or compositions, and, (4) packaging materials.
The nonlimiting examples that follow report general procedures, reagents and characterization methods that teach by way of example, and should not be construed in a narrowing manner that limits the disclosure to what is specifically disclosed. Those skilled in the art will understand that numerous modifications may be made and still the result will fall within the spirit and scope of the present invention.
The soybean genome has been sequenced by the Department of Energy-Joint Genome Institute (DOE-JGI) and is publicly available. Mining of this sequence identified 5671 soybean genes as putative regulatory genes, including transcription factors. These genes were comprehensively annotated based on their domain structures. (
To provide easy access to all soybean TF genes, SoyDB—a central knowledge database has been developed for all the transcription factors in the soybean genome. The database contains protein sequences, predicted tertiary structures, DNA binding sites, domains, homologous templates in the Protein Data Bank (Berman 2000) (PDB), protein family classifications, multiple sequence alignments, consensus DNA binding motifs, web logo of each family, and web links to general protein databases including SwissProt (Boeckmann et al. 2003), Gene Ontology (Ashburner et al 2000), KEGG (Kanehisa et al. 2008), EMBL (Angiuoli et al. 2008), TAIR (Rhee et al. 2003), InterPro (Mulder et al. 2002), SMART (Letunic et al. 2006), PROSITE (Hulo et al. 2006), NCBI, and Pfam (Bateman et al. 2004). The database can be accessed through an interactive and convenient web server, which supports full-text search, PSI-BLAST sequence search, database browsing by protein family, and automatic classification of a new protein sequence into one of 64 annotated transcription factor families by hidden Markov model. Major groups of these families are shown in
The database schema were implemented in MySQL, together with web-based database access scripts. The scripts automatically execute bioinformatics tools, parse results, create a MySQL database, generated PHP web scripts, and search other protein databases. The fully automated approach can be easily used to create protein annotation databases for any species.
Several bioinformatics tools were used to generate annotations of the soybean transcription factors. An accurate protein structure prediction tool MULTICOM (Cheng 2008) was also used to predict the tertiary structure of each transcription factor when homologous template structures could be found in the PDB. According to the official evaluations during the 8th community-wide Critical Assessment of Techniques for Protein Structure Prediction (CASP8) (http://predictioncenter.org/casp8/), MULTICOM was able to predict with high accuracy three dimensional structures with an average GDT-TS score 0.87 if suitable templates can be found. GDT-TS score ranges from 0 to 1 measuring the similarities of the predicted and real structures, while 1 indicates completely the same and 0 completely different. In SoyDB, the predicted tertiary structure is visualized by Jmol Zemla 2003). Users can view the structures from various perspectives in a three dimensional way.
The predicted structure was parsed into domains by Protein Domain Parser (PDP) (Hughes and Krough 1995). Since a few transcription factors did not have homologous templates in the PDB, DOMAC (Cheng 2007), an accurate ab initio domain prediction tool, was also used to predict the domains for each protein. During the structure prediction process, MULTICOM also generates the sequence alignments between the transcription factor and its homologous templates using PSI-BLAST.
The protein sequences in the same family were aligned into a multiple sequence alignment by MUSCLE (Edgar 2004). A consensus sequence was derived from the multiple sequence alignment. The multiple alignments were also used to identify the conserved signatures (DNA binding sites) for each family. The conserved binding sites were visualized by WebLogo (Crooks et al. 2004).
In order to annotate the functions of soybean transcription factors, each protein sequence was searched against other protein databases by PSI-BLAST periodically. The other databases include Swiss-port, TAIR, RefSeq, SMART, Pfam, KEGG, SPRINTS, EMBL, InterPro, PROSITE, and Gene Ontology. Web links to other databases were created at SoyDB when the same transcription factor or its homologous protein was found in other databases. For almost every transcription factor, several links to the outsides databases were created, which greatly expanded the annotations. For example, the expanded annotations include: protein features in Swiss-Prot, protein function in Gene Ontology, pathways in KEGG, function sites in PROSITE, and so on.
The comprehensive collection and analyses in SoyDB allows us to perform comparison of TF family distribution across the plant kingdom. The large number of soybean TF genes (5671) described in this study is likely due to the two soybean whole genome duplication events that are known to have occurred, one estimated at 40-50 million years ago (mya) and the most recent approximately 10-15 million years ago (Schlueter, J., et al., Gene duplication and paleopolyploidy in soybean and the implications for whole genome sequencing. BMC genomics, 2007. 8(1): p. 330; and Schlueter, J., et al., Mining EST databases to resolve evolutionary events in major crop species. Genome, 2004. 47(5): p. 868-876.) By comparing the total number of genes in different organisms, it was found that the increase of plant gene number is related to multicellularity and ploidy. For example, compared to the unicellular eukaryote Chlamydomonas reinhardtii where 15,143 genes are predicted (Merchant, S., et al., The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Science, 2007. 318(5848): p. 245), larger numbers of protein-encoding genes are reported in multicellular plant organisms [e.g. Physcomitrella patens (35,938; See Rensing, S., et al., The Physcomitrella Genome Reveals Evolutionary Insights into the Conquest of Land by Plants. Science, 2008. 319(5859): p. 64), Arabidopsis thaliana (32,944; TAIR, http://www.arabidopsis.org/)] and the tetraploid Glycine max [(66,153, Phytozome, http://www.phytozome.net/soybean).
It is hypothesized that TF gene number also follows the same trend as land plants, which have a larger number of TF genes compared to algae. To perform the most complete and current comparisons of plant TF genes and their distributions across TF gene families, we mined the last updated DBD database [9] in eleven plant species (C. reinhardtii, P. patens, Oryza sativa, Zea mays, Sorghum bicolor, Lotus japonicum, Medicago truncatula, A. thaliana, Vinis vinifera, Ricinus communis, and Populus trichocarpa). These species were then compared with the soybean TF genes stored in our SoyDB database.
Our analysis shows that the unicellular C. reinhardtii has the lowest number of TF genes when compared to multicellular land plants (the exceptions are L. japonicus and M. truncatula where only a partial genome sequence is available). This trend also reflects the differences of total gene number in the organisms. For example, it is interesting to note that homeobox, MYB, NAC, and WRKY TF genes in C. reinhardtii lack or have very low representations compared to the eleven other plant models. Previous studies defined a role for homeobox and WRKY genes in plant organ and plant cell development. Therefore, the occurrence of these genes only in multicellular plants may reflect their special roles in development. In addition, a close relationship between TF gene number and total gene number is observed when comparing the TF gene numbers of G. max and A. thaliana with their total gene numbers (i.e. G. max encodes 66,153 protein-coding genes including 5,683 TF genes; A. thaliana encodes 32,944 protein-coding genes and 1,738 TF genes). Thus, the family distribution of soybean TF genes is similar to other land plant species, except for P. patens (e.g. AP2 represents 7% of total TF genes in soybean vs. 8-12% for other land plants; bZIP: 3% vs. 3-7%; bHLH: 7% vs. 8-11%; homeobox: 6% vs. 4-7%; MYB: 14% vs. 7-14%; NAC: 4% vs. 4-9%; WRKY: 3% vs. 4-7%; ZF-C2H2: 7% vs. 5-9%).
In order to quantitate the expression of TF genes in soybean, a library containing 1149 sets (or pairs) of PCR primer was designed and synthesized. The sequences of these primers and the Identifier of the corresponding gene are listed in Table 1. These primers allowed for sensitive measurement of the expression levels of 1034 different soybean transcription factors (20% of total TF soybean genes). The number and classification of these TF genes are shown in
The primers in the primer library described in Example 2 were used to quantitate TF gene expression in 10 tissues from soybean plants. Briefly, soybean strain Williams 82 was grown under normal conditions. RNA samples from 10 different tissues were prepared as described in Example 7 and in U.S. patent application Ser. No. 12/138,392. cDNA were prepared from these RNA samples by reverse transcription. The cDNA samples thus obtained were then used as templates for PCR using primer pairs specific for soybean TFs. The PCR products of each TF gene in different tissues were quantitated and the results are summarized in Table 2.
The tissue specific expression of some of these TFs was confirmed by creating a transcriptional fusion with GUS (i.e., β-glucosidase) or GFP (green fluorescent protein) reported genes. The coding regions of the reporter gene was cloned under control of the promoter of the tissue specific TF gene as described below.
Briefly, the Gateway system by Invitrogen Inc. (Carlsbad, Calif.) was used to clone promoter upstream to the GFP and GUS cDNAs. A 2 kb DNA fragment 5′ to the first codon of the bHLH gene was identified by mining genomic sequences available on Phytozome website (http://www.phytozome.net/soybean.php). Through two independent PCR reactions, AttB sites at the extremities of the promoter sequences were created. Genomic DNA from the soybean strain Williams 82 was used as template for PCR. Using the Gateway® BP Clonase® II enzyme mix, the promoter fragment was introduced first into the pDONR-Zeo vector (Invitrogen, Carlsbad, Calif.) then into pYXT1 or pYXT2 destination vectors using the Gateway® LR Clonase® II enzyme mix (Invitrogen, Carlsbad, Calif.). pYXT1 and pYXT2 were destination vectors carrying the GUS and GFP reporter genes respectively (Xiao et al., 2005).
A. rhizogenes (strain K599) was transformed by electroporation with bHLHpromoter-pYXT1 and bHLHpromoter-pYXT2 vectors. Soybean hairy root transformation was carried out essentially as described by Taylor et al. (2006). Briefly, two-week old soybean shoots were cut between the first true leaves and the first trifoliate and placed into rock-wall cubes (Fibrgro, Sarnia, Canada). Each shoot was inoculated with 4 ml of A. rhizogenes (OD600=0.3) and then allowed to dry for approximately 3 days (23° C., 50% humidity, long day conditions) before watering with deionized water. After one week, the plants were transferred to pots with vermiculite:perlite mix (3:1) wetted with nitrogen-free plant nutrient solution (Lullien et al., 1987). One week later, the shoots were transferred to the green house (27° C., 20% humidity, long day conditions). Two weeks after vermiculite-perlite transfer, the shoots were inoculated with B. japonicum (10 ml, OD600=0.08).
In order to identify soybean TF genes whose expression levels are regulated at different seed developmental stages, soybean tissues including roots, leaves, stems and seeds were harvested and RNA extracted. qRT-PCR was performed as described in Examples 7-9 and in U.S. patent application Ser. No. 12/138, 392 to determine the expression levels of each TF at different seed developmental stages, ER5 (early R5 stage-R5 starting of seed filling), LR5 (late R5 stage-seed filing ongoing), R6 (seed filling stage), and R7 (maturation stage) and R8 matures seed stage. TF Genes that showed stage specific expression during seed development are termed “Transcription Factors Implicated in Seed Development” (TFISD). Examples of TFISD include, for example, Myb, C2C2, bZip, CCAAT binding, DOF, etc.
Further functional investigation of these TFISDs will help to understand the mechanisms regulating seed filling and seed composition. These soybean TFISDs, such as bZip and CCAAT, are overexpressed in Arabidopsis thaliana under the control of inducible or constitutive promoters. The expression levels of various genes implicated in seed development are determined to help elucidate which downstream genes are regulated by a TFISD. The filling or composition of the seeds and other characteristics of the seeds are also examined to establish the relationship between the expression of a TFISD and seed development.
In another aspect, the DNA elements responsible for the stage specific expression of a TFISD during seed development are determined using various reporter genes as described above. These DNA elements include but are not limited to promoters, enhancers, attenuators, methylation sites etc. Structural or functional genes are placed under control of the DNA elements of the soybean TFISDs such that they are expressed at specific stage during seed development. The structural or functional genes may be from soybean or other plants that have been identified to control seed composition, such as protein and/or oil content.
Some soybean strains are naturally more resistant to flooding than others. To identify soybean genes that may confer upon a plant flood resistant phenotype, the gene expression of two soybean strains are profiled. One strain, PI 408105A (PI—Plant introduction), is flooding stress tolerant; the other strain, S99-2281 (Breeding line), is flooding stress sensitive.
The two soybean strains were grown under normal conditions and water was introduced to flood the plants. Tissues samples were collected at Day 1, Day 3, Day 7 and Day 10 post flooding. Microarray profiling was used to determine the expression levels of all genes across the entire genome as described above.
The expression patterns of soybean regulatory genes regulated during nodule development were studied using qRT-PCR. Expression of 126 soybean TF genes were profiled to identify soybean TFs that are upregulated or downregulated during root nodule development. Table 3 lists the changes of expression levels for these 126 genes recorded at 4 days, 8 days and 24 days after inoculation. These genes are candidate genes that control nodule development, plant-symbiont interaction or nitrogen fixation and assimilation.
The expression pattern of 13 of these TF genes through different stages of nodule development after inoculation of B. japonicum are shown in
Using a RNAi gene-silencing strategy, the functions of some TFs implicated in nodule development were further characterized. When one of these TFs, MYB, was silenced, lower number but bigger nodules were observed. This result suggests that this MYB gene plays a role in the nodulation process (
Panel A of
Next, the localization of the TF genes during nodulation was determined by using the GUS or GFP reporter genes system described above. Transcriptional fusions containing promoter sequences of the TF genes and coding sequence of the reporter gene were constructed and introduced into soybean plants. Briefly, Gateway system (Invitrogen, Carlsbad, Calif.) was used to clone the promoter of the Glyma03g31980 gene upstream of the GFP and GUS cDNAs. By mining genomic sequences available on Phytozome website (http://www.phytozome.net/soybean.php), a 1967 by DNA fragment 5′ to the first codon of the Glyma03g31980 gene was identified. By two independent PCR reactions, the AttB sites were created at the extremities of the promoter sequences. Soybean Williams 82 genomic DNA was used as template and the following primers were used for these two PCRs:
Using the Gateway® BP Clonase® II enzyme mix, the Glyma03g31980 promoter fragment was introduced first into the pDONR-Zeo vector (Invitrogen, Carlsbad, Calif.), then into pYXT1 or pYXT2 destination vectors using the Gateway® LR Clonase® II enzyme mix (Invitrogen, Carlsbad, Calif.). pYXT1 or pYXT2 destination vectors carry the GUS or GFP reporter genes, respectively (Xiao et al., 2005). A. rhizogenes (strain K599) was transformed by electroporation with Glyma03g31980promoter-pYXT1 and Glyma03g31980promoter-pYXT2 vectors.
The expression of the reporter genes was monitored by following the GUS (blue) or GFP (green) signals.
Genetic material and the growing system: cv Williams 82 was used for the green house experiments. Plants were grown in Turface-sand medium in 3 gallon pots. One-month old soybean plants were subjected to gradual stress by withholding water and the samples were collected in three biological replicates. To quantitate the stress level we monitored relative water content (RWC), leaf water potential, and turface-soil mixture water potential and moisture content. Leaf RWC, leaf water potential, and soil water content were 95%.-0.3 MPa, and 20% (v/v), respectively, for well-watered samples. These values were 65%, −1.6 MPa, 9.6% for the water-stressed samples.
RNA isolation and the microarray: Flash-frozen plant tissue samples were ground under liquid nitrogen with a mortar and pestle. Total RNA is extracted using a modified Trizol (Invitrogen Corp., Carlsbad, Calif.) protocol followed by additional purification using RNEasy columns (Qiagen, Valencia, Calif.). RNA quality is assayed using an Agilent 2100Bioanalyzer to determine integrity and purity; RNA purity is further assayed by measuring absorbance at 200 nm and 280 nm using a Nanoprop spectrophotometer.
Microarray hybridization, data acquisition, and image processing: We used the pair wise comparison experimental plan for the microarray experiments. A total number of 12 hybridizations were conducted as: 2 biological conditions×3 biological replicates×2 tissue types. First strand GDNA were synthesized with 30 pg total RNA and T7-Oligo(dT) primer. The total RNA were processed to use on Affymetrix Soybean GeneChip arrays, according to the manufacturer's protocol (Affymetrix, Santa Clara, Calif.). The GeneChip soybean genome array consists of 35,611 soybean transcripts (details as in the results description). Microarray hybridization, washing and scanning with Affymetrix high density scanner were performed according to the standard protocols. The scanned images were processed and the data acquired using GCOS. Having selected genes that are significantly correlated with phenotype or treatment, data mining is conducted using a variety of tools focusing on class discovery and class comparison in order to identify and prioritize candidates.
Confirmation of gene expression by qRT-PCR: Validation of the microarray profiling and the expression of significant genes at significant time points in the experiments were determined by a high-throughput two-step quantitative RT-PCR (qRT-PCR) assay using SYBR Green on the ABI 7900 HT and by the delta delta CT method (Applied Biosystems) developed in course of these studies.
One-month old soybean plants were subjected to gradual stress by withholding water and the samples were collected in three biological replicates. To quantitate the stress level we monitored relative water content (RWC), leaf water potential, and surface-soil mixture water potential and moisture content. Total RNA isolation and microarray hybridizations were conducted using standard protocols. We used 60K soybean Affymetrix GeneChips for the transcriptome profiling. The GeneChip® Soybean Genome Array is a 49-format, 11-micron array design, and it contains 11 probe pairs per probe set. Sequence Information for this array includes public content from GenBank® and dbEST. Sequence clusters were created from UniGene Build 13 (Nov. 5, 2003). The GeneChip® Soybean Genome Array contains ˜60,000 transcripts and 37,500 transcripts are specific for soybean. In addition to extensive soybean coverage, the GeneChip® Soybean Genome Array includes probe sets to detect approximately 15,800 transcripts for Phytophthora sojae (a water mold that commonly attacks soybean crops) as well as 7,500 Heterodera glycines (cyst nematode pathogen) transcripts. (www.affymetrix.com) The affymetrix chip hybridization data of the soybean root under stress were processed. The statistical analysis of the data was performed using the mixed linear model ANOVA (log2 (pm)˜probe+trt+array (trt)). The response variable “log2 (pm)” is the log base 2 transformed perfect match intensity after RMA background correction and quantile normalization; the covarlate “probe” indicates the probe levels since for each gene there are usually 11 probes; “trt” is the treatment/condition effect and it specifies if the array considered is treatment or control; “array(trt)” is the array nested within trt effect, as there are replicate arrays for each treatment.
FDR adjusted p-value is less than 0.01 cutoff point where fdrp is less than 0.01.
The statistically analyzed data were sorted and the functional classifications (KOG and G0) were performed. Significantly differentially expressed transcripts in root and leaf tissues between well-watered and water stressed condition are:
p value adjusted FDR 5%
The functional classification of the differentially expressed genes in soybean leaf under drought condition is summarized in Table 4, which shows the numbers of genes that are either up- or down-regulated in each category as defined by protein function.
Sequences for the genes and proteins disclosed in this disclosure can be found in GenBank, a nucleotide and protein sequence database maintained by the National Center for Biotechnology Information (NCBI), or in the Soybean genome database maintained by the University of Missouri at Columbia, Mo. Both databases are freely available to the general public.
The functional classification of the differentially expressed genes in soybean root under drought condition is summarized in Table 5, which shows the numbers of genes that are either up- or down-regulated in each category as defined by protein function.
Based on database mining of transcription factors, domain homology analysis, and the soybean microarray data obtained in Example 1 using drought-treated root tissues from greenhouse-grown plants, 199 candidate transcription factor genes or ESTs derived from these genes with putative function for drought tolerance were identified. 64 of the candidates showed high sequence similarity to known transcription factor domains and might possess high potential for drought tolerant gene identification. The remaining 135 of the candidates showed relatively low sequence similarity to known transcription factors domains and thus might represent a valuable resource for the identification of novel genes of drought tolerance. The candidates generally belonged to the NAM, zinc finger, bHLH, MYB, AP2, CCAAT-binding, bZIP and WRKY families.
On the basis of family novelty and the magnitude of drought-inducibility, three transcripts were chosen for a pilot experiment to characterize and isolate promoters for drought tolerance studies. The three candidates were BG156308, BI970909, and BI893889, which belonged to the bHLH, CCAAT-binding, and NAM families, respectively. Under drought condition, the expression levels of these three genes were increased from 2.5 to 252-fold. Moreover, no transcription factor from those families has been reported to control drought tolerance in soybean and other crops. Therefore, these candidate genes may represent novel members of these families that may also play a role in plant drought response. Functional characterization of these transcription factors may help elucidate pathways that are involved in plant drought response.
A set of 62 candidate drought response genes (or DRGs) identified in the microarray experiment were further confirmed by quantitative reverse transcription-PCR (qRT-RCR). Briefly, RNA samples from root or leaf tissues obtained from soybean plants grown under normal or drought conditions were prepared as described in Example 1. cDNA were prepared from these RNA samples by reverse transcription. The cDNA samples thus obtained were then used as template for PCR using primer pairs specific for 64 candidate genes. The PCR products of each gene under either drought or normal conditions were quantified and the results are summarized in Table 6. The Column with the heading “qRT-PCR Root log ratio of expression level” shows the base 2 logarithm of the ratio between the root expression level of the particular gene under drought condition and the expression level of the same gene under normal condition. Similarly, the Column with the heading “qRT-PCR Leaf log ratio of expression level” shows a similar set of data obtained from leaf tissues. The qRT-PCR results are generally consistent with the microarray data, suggesting that the genes whose expression levels are up-regulated or down-regulated are likely to be true Drought Response Genes (DRGs).
Table 7 lists additional soybean root related, drought related transcription factors that are up- or down-regulated in response to drought condition.
Glycine max DREB3
Glycine max NAC4
Glycine max C2H2
Glycine max HSTF5
Glycine max ACT1
Glycine max NAC1
Glycine max NAC5
Glycine max NAC3
Glycine max NAC2
Glycine max NAC6
Glycine max DREB2 gene
Soybean transcription factors belonging to different families are shown in
http://casp.rnet.missouri.edu/soydb
http://www.phytozome.net/soybean.php and
http://www.phytozome.net/cgi-bin/gbrowse/soybean/?start=5935000; stop=6024999; ref=Gm01; width=800; version=100;
cache=on; drag and drop=on; show_tooltips=on; grid=on; label=Transcripts-Glycine_max_est-Gmax_PASA_assembly
The sequences of all genes or proteins listed in this disclosure or those referenced by PublicID, GenBank ID, or soybean gene ID are hereby incorporated by reference into this disclosure as if fully reproduced herein.
The amino acid sequences of the TFs in each 64 Arabidopsis TF families were downloaded from DATF (Guo, et al., 2005) and the sequences were aligned by a multiple sequence alignment tool MUSCLE (Edgar, 2004). A hidden Markov model was trained for each Arabidopsis family by SAM (Hughey and Krogh, 1995) using the multiple sequence alignment. Each of the 6,690 soybean TFs was aligned individually to each of the 64 hidden Markov models and then was assigned to the TF family whose hidden Markov model generated the lowest e-value. This e-value indicates the fitness between the query TF sequence and the hidden Markov model, with smaller e-value indicating better fitness between them. Out of the entire soybean TFs, the highest e-value was 0.305 on one soybean TF, and a total of 166 soybean TFs had an e-value between 0.1-0.4, which indicates most of the soybean TFs had a confident classification to one of the 64 TF families from Arabidopsis.
Comparisons of TF numbers in each TF family between soybean and Arabidopsis: The numbers of transcription factors in each of the 64 families for soybean and Arabidopsis were compared (Table 1). For each family, the TF number of soybean was divided by the one in Arabidopsis. A higher ratio shows the families have an enriched number of soybean transcriptions as compared to Arabidopsis. Based on TAIR version 8 (Rhee, et al., 2003), Arabidopsis has 32,825 proteins, while soybean has 75,778 proteins based on the soybean genome sequencing completed in early 2008 by the Department of Energy-Joint Genome Institute (Schmutz, et al., 2009). Therefore, the soybean gene number is about two times bigger than Arabidopsis, and the >2.3 ratio (75,778/32,825) in Table 1 shows enrichment in soybean after considering the genome size difference between these two species.
Arabidopsis
The functions of the top 5 and bottom 5 TF families ranked by the TF number ratio between soybean and Arabidopsis are listed in Table 9. The functions are cited from the database DATF (Guo, et al., 2005). As shown in Table 9, soybean TFs are mostly enriched in those families that are involved in reproductions, such as pollen and flower development.
qRT-PCR provides one of the most accurate methods to quantify gene expression. Using this technology, the expression of 1034 out of the 5671 transcription factor genes (TF) identified in soybean (18%) was quantified during soybean root nodulation and in different tissues. See Example 2. The entire soybean genome has been published. See e.g., Schmutz et al., 2010. To better understand the regulation of soybean TF gene expression, it is important to note that two duplication events occurred in the soybean genome about 59 and 13 million years ago, respectively. These duplications have led to multiple copies of the same gene in the soybean genome which is also called homeologous genes.
The expression levels of homeologous soybean genes during soybean root nodulation and in response to KCl and KNO3 were compared using the qRT-PCR data (
This analysis unveiled numerous examples of homeologous soybean TF genes showing differential expression (
Despite the value of such analysis, it was frustrating to limit our analysis to a small fraction of the soybean TF genes. The restricted number of soybean TF genes analyzed by qRT-PCR is mainly limited by the design of specific primers for each gene analyzed. Consequently, the use of technologies such as Illumina-Solexa technology allowing the accurate quantification of the transcriptome of the entire set of soybean TF genes is required. Illumina-Solexa technology allows quantifying very accurately the expression of transcripts including low abundant transcripts such as TF gene transcripts and is not restricted to a subset of the soybean genes
Despite the value of such analysis, the number of soybean TF genes that can be analyzed by qRT-PCR is limited by the design and synthesis of specific primers for each gene analyzed. The use of technologies such as Illumina-Solexa technology may allow the accurate quantification of the transcriptome of the entire set of soybean TF genes. Illumina-Solexa technology may enable very accurate quantification of the expression of genes including low-abundance transcripts such as TF gene transcripts and is not restricted to a subset of the soybean genes.
With the help of the Illumina-Solexa technology, a soybean transcriptome atlas has been developed which shows, among others, the expression of the 5671 soybean TF genes across 14 different conditions and/or location, namely, Bradyrhizobium japonicum-inoculated and mock-inoculated root hairs isolated 12, 24 and 48 hours after inoculation, Bradyrhizobium japonicum-inoculated stripped root isolated 48 hours after inoculation (i.e. root devoid of root hair cells), mature nodule, root, root tip, shoot apical meristem, leaf, flower, green pod (Table 10). The upper half of Table 10 shows expression of these genes in 7 conditions/tissues, while the lower half of Table 10 shows expression of the same genes in the remaining 7 conditions/tissues. No transcripts were detected across the 14 conditions tested for 787 soybean TF genes (Table 10). Although this set of conditions is not exhaustive; this result suggests that these 787 genes might be pseudogenes (i.e. genes silenced during their evolution). Such a result confirmed previous reports based on qRT-PCR as described above.
This large scale analysis also enables the identification of soybean TF genes showing a repetitive induction of their expression during root hair cell infection by B. japonicum (Table 11). It is worth noting that some of these soybean TF genes were orthologs to Lotus japonicus and Pisum sativum TF genes that have been previously identified as key-regulators of the root hair infection by rhizobia (Table 11).
120 soybean TF genes were identified which were expressed at least 10 times more in one soybean tissues when compared to the remaining 9 tissues (i.e. mock-inoculated root hairs isolated 12 and 48 hours after treatment, mature nodule, root, root tip, shoot apical meristem, leaf, flower, green pod. See
NAC transcription factors (TFs) are plant specific transcription factors that have been reported to enhance stress tolerance in number of plant species. The NAC TFs regulate a number of biochemical processes which protect the plants under water-deficit conditions. A comprehensive study of the NAC TF family in Arabidopsis reported that there are 105 putative NAC TFs in this model plant. More than 140 putative NAC or NAC-like TFs have been identified in Rice. The NAC TFs are multi-functional proteins and are involved in a wide range of processes such as abiotic and biotic stress responses, lateral root and plant development, flowering, secondary wall thickening, anther dehiscence, senescence and seed quality, among others.
170 potential NACs were identified through the soybean genome sequence analysis. Full length sequence information of 41 GmNACs are available at present and 31 of them are cloned. Quantitative real time PCR experiments were conducted to identify tissue specific and stress specific NAC transcription factors in soybean and the results are shown in
The drought response of these genes was studied, and the results are shown in
A number of NAC TFs were cloned and expressed in the Arabidopsis plants to study the biological functions in-planta. Transgenic Arabidopsis plants were developed and assayed for various physiological, developmental and stress related characteristics. Two of the major gene constructs (following gene cassettes) were utilized for the transgene expression in Arabidopsis plants. One is CaMV35S Promoter-GmNAC3gene-NOS terminator, the other construct is CaMV35S Promoter-GmNAC4gene-NOS terminator. The coding sequence of the GmNAC3 gene is listed as SEQ ID No. 2299, while the coding sequence of the GmNAC4 gene is listed as SEQ ID No. 2300. For the transgenic experiments, the Arabidopsis ecotype Columbia was transformed with the above gene constructs using floral dip method and the transgenic plants were developed. Independent transgenic plants were assayed for the transgene expression levels using qRT-PCR methods (
Examination of the transgenic plants revealed that the transgenic plants showed improved root growth and branching as compared to controls (
A trend towards the enhanced root branching (more lateral roots) was observed under simulated drought stress conditions using the poly ethylene glycol (PEG) containing growth medium. Major observations during these studies include, for example, GmNACC3 and GmNACC4 are differentially expressed in soybean root, and both seemed to be expressed at a higher level in the root. It is likely that the proteins encoded by the transgenes in GmNACQ1 and GmNACQ2 help regulate lateral root development in transgenic Arabidopsis plants.
To identify other proteins that may be beneficial to a host plant, Arabidopsis transgenic plants with the following gene constructs were generated: (a) CaMV35S Promoter-GmC2H2 gene-NOS terminator; and (b) CaMV35S Promoter-GmDOF27 gene-NOS terminator. The coding sequence of the GmC2H2 gene is listed as SEQ ID No. 2301, while the coding sequence of the GmDOF27 gene is listed as SEQ ID No. 2302. The homozygous transgenic lines (T3 generation) were developed and the physiological assays were conducted, including, for example, examination of root and shoot growth, stress tolerance, and yield characteristics.
While the foregoing instrumentalities have been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.
In addition to those references that are cited in full in the text, additional information for those abbreviated citations is listed below:
This application claims priority to U.S. Provisional Application No. 61/270,204 filed Jun. 30, 2009, the contents of which are hereby incorporated into this application by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/40687 | 6/30/2010 | WO | 00 | 4/17/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61270204 | Jun 2009 | US |
