Patent Application
20150128305
The present invention relates to the field of plant molecular biology, genetic engineering, and transgenic plant, in particular to stress responsive protein, CaTLP1, from Cicer arietinum L., and its uses thereof. The invention further relates to a method for producing modified plants that exhibit enhanced tolerance to abiotic stress, and the modified plants produced by the process.
Plant environmental (abiotic) stress has been defined as “any change in environmental conditions that might reduce or adversely change a plant's growth or development” (J. Levitt, 1972, Responses of Plants to Environmental Stresses, Academic Press Inc., New York and London). Adverse environmental factors such as high winds, cold (chilling and freezing), heat, desiccation (drought), flood, salinity, soil mineral deficiency, soil mineral toxicity, and other unfavourable growing conditions are common stresses that affect environments constantly. Prolonged exposure to abiotic stresses can result in severe crop damage and impacts the growth and productivity of crops worldwide. Continuous exposure to abiotic stresses results in alterations in the plant metabolism which ultimately leads to cell death and consequently yield losses. Crop losses and crop yield losses of major crops such as rice, wheat, and maize caused by environmental stresses represent a significant economic and political factor that contribute to food shortage in many underdeveloped countries.
Environmental stress limits growth, development, and productivity of the crops, thereby playing a major role in determining the geographic distribution of many plant species. Major environmental stresses are united by the fact that at least a part of their detrimental effect on plant performance is caused by disruption of water status.
Water-deficit or dehydration is the most common environmental stress to which plants are often exposed, and in many regions forms the bottleneck to agricultural development. Dehydration is the most crucial environmental constraint on plant growth and development and agricultural productivity. Transitory or constant dehydration adversely affects the morpho-anatomical, physiological, and biochemical processes in plants, though the extent of damage varies considerably at different developmental stages. The perception and dehydration-induced signal transduction to switch on adaptive responses are critical steps in determining the survival of plants exposed to dehydration, and elucidation of the nature of these mechanisms has been an interesting area of research. The plant cell wall or extracellular matrix (ECM) is a dynamic organization essential not only for cell division, enlargement, and differentiation; but also acting in diverse environmental stress. Though the protein part accounts for only 10% of the cell wall mass, the proteins contribute substantially to the cellular dynamics as they comprise several hundreds of different molecules with diverse functions.
Plants are typically exposed to conditions of reduced environmental water content in different stages of their life cycle. Most plants have evolved strategies and specific patterns of stress mediated metabolism to protect themselves against the conditions of low water or desiccation. Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of the problems arising due to stress response in the plant. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to stresses are relatively slow and require specific resistant lines for crossing with the desired line. Most of the efforts to mitigate the effects of plant stress have included complex methodologies that are both time consuming and expensive. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent the significant problems encountered in conventional breeding. Additionally, the cellular processes leading to drought, cold, and salt tolerance are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways which makes the development of mitigation strategies increasingly difficult. The development of resistance to abiotic stress has previously been demonstrated by the use of mitigating compounds and isolated polynucleotide sequences.
U.S. Pat. No. 6,534,446 discloses methods for mitigating the effects of plant stress by use of plant stress mitigating compounds and compositions comprising gamma immunobutyric acid and glutamic acid.
U.S. Pat. No. 8,071,843 discloses a method for increasing stress resistance in plants by introducing an isolated polynucleotide encoding a raffinose synthase into the body of a plant, and selecting transformed plants having raffinose synthetic activity in the plant body greater than the wild type plant grown under the same conditions.
U.S. Pat. No. 7,897,843 discloses plant transcription factor polypeptides, polynucleotides that encode them, homologs from a variety of plant species, and methods of using the polynucleotides and polypeptides to produce transgenic plants having advantageous properties, including increased biomass or improved cold or other osmotic stress tolerance, as compared to wild-type or reference plants.
U.S. Pat. No. 7,368,632 discloses transgenic plants comprising a plant gene (ROBS), demonstrating increased plant growth, plant vigor, and improvement in the capacity to tolerate a variety of stress conditions.
Plants harbour a large number of tubby-like proteins (TLPs); for instance, 11 members in Arabidopsis (Lai C-P, Lee C-L, Chen P-H, Wu S-H, Yang C-C, Shaw J-F; 2004, Molecular analysis of the Arabidopsis TUBBY-like protein gene family; Plant Physiology; 134: 1586-1597), and 14 members in rice (Liu Q, 2008, Identification of rice TUBBY-like genes and their evolution. FEBS J 275: 163-171; Kou Y, Qiu D, Wang L, Li X, Wang S, 2009, Molecular analyses of the rice tubby-like protein gene family and their response to bacterial infection, Plant Cell Reports, 28: 113-121). In addition to the typical 270 amino acids conserved C-terminal Tub domain, plant TLPs have evolved with an F-box conserved at N-terminal sequence, which is otherwise highly divergent in animal. It has also been shown that these proteins may function as bipartite transcription regulators by binding to double-stranded DNA and activating transcription (Boggon T J, Shan W S, Santagata S, Myers S C, Shapiro L, 1999, Implication of tubby proteins as transcription factors by structure-based functional analysis, Science, 286: 2119-2125).
While a wide array of cellular functions of TLPs have been established in animals, their role in plants is still elusive. Nevertheless, the highly conserved evolution of tubby proteins and the existence of redundancy suggest their indispensable role in plants. In recent years; this protein family has been shown to be involved in abscisic acid (ABA)-dependent signalling in Arabidopsis and pathostress response in rice. The mode of action of tubby family proteins in plants is believed to be regulated by ABA, which is characteristically implicated in different stress responses.
The multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerance plants using biotechnological methods. Thus, there is a need for novel stress resistant or tolerant genes, such that better adaptation may be sought in plants leading to a healthy and robust agronomy.
Accordingly it is an aspect of the present invention to provide a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5.
It is another aspect of the present invention to provide a polypeptide having amino acid sequence as set forth in SEQ ID NO: 5, wherein expression of the polypeptide is responsible for conferring enhanced tolerance to abiotic stress in a transformed plant as compared to an untransformed plant.
It is yet another aspect of the present invention to provide a method for producing a transformed plant cell, plant, or a part thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the method comprises: transforming the plant cell, plant, or part thereof with a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, and selecting the transformed plant cell, plant or part thereof, wherein the transformed plant cell, plant, or part thereof is more tolerant to abiotic stress than an untransformed plant.
It is still another aspect of the present invention to provide a transgenic plant, plant cell or progeny thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the transgenic plant, plant cell or progeny thereof is more tolerant to abiotic stress than an untransformed plant.
The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
a) shows the results of electrofocusing of cell wall proteins on IPG strip.
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.
As used in the specification and the claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
The terms “comprise” “comprising” “including” “containing” “characterized by” and grammatical equivalents thereof are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only.”
The term “polypeptide” is defined as an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) a DNA-binding domain, or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.
The term “Protein” refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic. “Proteins” as used herein, includes a wide variety of peptide-containing molecules, including monomeric, dimeric, multimeric, heterodimeric, heterotrimeric, and heterotetrameric proteins; disulfide bonded protein; glycosylated proteins; helical proteins; and α and β sheet-containing proteins.
The term “recombinant” as used herein, means that a molecule (e.g., a nucleic acid or a polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the molecule within, or removed from, its natural environment or state.
The term “recombinant protein” or “recombinant polypeptide” as used herein, refers to a protein molecule which is expressed using a recombinant DNA molecule.
The term “recombinant polynucleotide” is defined as a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.
The term “expressing a protein” or “protein expression” means the function of a cell to transcribe recombinant DNA to mRNA and translate the mRNA to a protein. For expression the recombinant DNA usually includes regulatory elements including 5′ regulatory elements such as promoters, enhancers, and introns; other elements can include polyadenylation sites, transit peptide DNA, markers and other elements commonly used by those skilled in the art. Promoters can be modulated by proteins such as transcription factors and by intron or enhancer elements linked to the promoter. Promoters in recombinant polynucleotides can also be modulated by nearby promoters.
The term “host cell” refers to a microbial cell such as bacteria and yeast or other suitable cell including animal or a plant cell which has been transformed to express the homologous and heterologous proteins of interest. Host cells which are contemplated by the present invention include those in which the over-expressed proteins by the cell are sequestered in refractile bodies. An exemplary host cell is E. coli BL21 (DE3), which has been transformed to effect expression of the desired recombinant protein.
The term “wild type plant” refers to the form of the plant that occurs most frequently in nature. It is distinguished from mutant forms that may result from selective breeding.
A transgenic “plant cell” means a plant cell that is transformed with stably-integrated, non-natural, recombinant polynucleotides, e.g. by Agrobacterium-mediated transformation or by bombardment using micro particles coated with recombinant polynucleotides. A plant cell of this invention can be an originally-transformed plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, e.g. into a transgenic plant with stably-integrated, non-natural recombinant polynucleotides in its chromosomal DNA, or seed or pollen derived from a progeny transgenic plant.
A “transgenic” plant or seed means one whose genome has been altered by the stable incorporation of recombinant polynucleotides in its chromosomal DNA, e.g. by transformation, by regeneration from a transformed plant from seed or propagule or by breeding with a transformed plant. Thus, transgenic plants include progeny plants of an original plant derived from a transformation process including progeny of breeding transgenic plants with wild type plants or other transgenic plants.
The term “alignment” refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences.
The term “identity” or “similarity” refers to sequence similarity between two or more polynucleotide sequences, or two or more polypeptide sequences, with identity being a more strict comparison. “Sequence similarity” refers to the percentage of bases that are similar in the corresponding positions of two or more polynucleotide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of similar amino acid residues at positions shared by the polypeptide sequences. Two or more sequences can be anywhere from 0-1.00% similar, or any integer value there between. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison.
The present invention provides a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5. The present invention also provides a recombinant DNA construct comprising the polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5 operably linked to a promoter, a recombinant vector comprising the recombinant DNA construct, a host cell comprising the polynucleotide of the present invention that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 5, a process for producing a transformed plant cell, plant or a part thereof over-expressing abiotic stress responsive protein having amino acid sequence as set forth in SEQ ID NO: 5, a transgenic plant, plant cell or progeny thereof over-expressing abiotic stress responsive protein having amino acid sequence as set forth in SEQ ID NO: 5, wherein the transgenic plant, plant cell or progeny thereof is more tolerant to abiotic stress than a wild type plant and a transgenic seed or progeny obtained from the transgenic plant, plant cell or progeny thereof, wherein said seed or the progeny over-expresses abiotic stress responsive protein having amino acid sequence as set forth in SEQ ID NO: 5 and is more tolerant to abiotic stress than a wild type plant.
The invention disclosed in the present specification investigated the temporal changes in the proteome of C. arietinum that led to the identification of a tubby-like protein (TLP), designated CaTLP1, putatively involved in dehydration. A critical analysis of the differential proteome of extracellular matrix of chickpea under dehydration revealed a spot representing tubby-like protein (TLP) that was found to be highly regulated under dehydration (
The 3′-rapid amplification of cDNA using degenerate primers having sequences as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, designed from peptide sequence tags, yielded a 1.0 kb partial clone homologous to a chickpea EST sequence, gi-7635492. The coding region of the gene as set forth in SEQ ID NO: 4 was isolated, cloned in pGEM-T vector and named as pCaTLP1. Further, the full-length ORF of CaTLP1 was cloned in-frame with GST cDNA in pGEX4T-2 and expressed in Escherichia coli, BL21 (DE3) cells, which encode the fusion protein, GST-CaTLP1 with an estimated molecular mass of 72 kDa (
The CaTLP1 genomic DNA is 2,299 bp (SEQ ID NO: 6), indicating the presence of introns. The comparison of the full-length cDNA sequence with the corresponding genomic DNA sequence revealed that the coding region of the CaTLP1 is interrupted by three introns (
To characterize the CaTLP1 gene in more detail, Southern blotting was performed, which could be used to obtain preliminary information on the size and organization of the corresponding genomic sequence and the number of gene copies. Genomic DNA of chickpea was digested with different restriction enzymes and blotted to nylon membrane. The blot was hybridized with [32P]-labelled 314-bp CaTLP1 fragment under high stringency conditions. The restriction enzymes SspI and SacI, having single site in, the probe region, yielded two bands, whereas HindIII, EcoRI, and BamHI having no restriction sites yielded a single band, indicating the presence of single copy of the CaTLP1 (
The CaTLP1 binds avidly to double-stranded DNA, but poorly to single-stranded DNA. Gel shift assay revealed band shift with GST-CaTLP1 fusion protein in case of both the double-stranded DNA molecules, while no shift was observed with the purified GST (
It is known that there is a close relation between physiological function of a protein and its intracellular location. In mammalian cells, tubby protein is reported to be localized in the nucleus as well as in plasma membrane, and shown to have a direct link in G-protein signalling that regulate the gene expression. Mutation studies in the tubby-like protein family gene showed significant abnormalities and phenotypes that do not always overlap despite overlapping expression patterns. Therefore, the TLPs are multifunctional proteins, and may play multiple independent functional roles.
This is the first report demonstrating the localization of tubby protein in plants. CaTLP1 is localized predominantly in the cell wall and nucleus (
The characteristic properties of CaTLP1, its nuclear translocation and DNA-binding activity support, at least partly, a putative transcription factor. However, its inability to induce the transcription of reporter gene may account for the absence of transactivation domain in CaTLP1. Unlike highly diverse N-terminal sequence of animal TLPs, CaTLP1 contains conserved F-box domain. The role of F-box is established as cell surface receptor and transcription modulator. These proteins use broad and different mechanisms for target recognition, the most common mechanism being the formation of a SkpI/Cullin/F-box (SCF) complex. The presence of the conserved F-box domain in CaTLP1 suggests a key role in stress tolerance, possibly by protein-protein interaction. CaTLP1 presumably serves as a molecular sensor. Following the possible signal transduction initiating events, it translocates to the nucleus to carry out targeted biological function. Probably CaTLP1 binds to DNA and interacts with the regulator with the help of Tub and F-box domains, respectively. F-box domain may either regulate the activity or recruit other regulators at the promoter that can effectively switch on and off the downstream gene expression.
The organ specificity of CaTLP1 expression revealed low but detectable expression in roots, flowers and pods but substantially higher expression in vegetative organs such as stems and leaves, suggesting a possible synergistic quantitative relationship of CaTLP1 to growth and development of the plant (
The dehydration response of CaTLP1, evaluated using the Northern analysis revealed that the CaTLP1 transcripts were induced within 5 hours of dehydration with maximum accumulation at 12 hours that persisted up to 16 hours. However, the transcripts showed a drastic decrease after 24 hours and reached the background level at 48 hours (
Transient expression analysis of CaTLP1-YFP fusion protein in transfected tobacco cells showed fluorescence in both extracellular matrix and nucleus (
To investigate the potential of CaTLP1 to activate a large number of stress-responsive genes, the ORF of CaTLP1 was introduced in-frame with the GAL4 DNA-binding domain in yeast expression vector pGBKT7. The pGBKT7-CaTLP1 construct was then transformed into yeast strain AH109 harbouring MEL1 reporter gene. The colonies were observed for time-dependent colour development; but strangely, they remained white even after three days of incubation whereas the vector colonies turned blue (
Transgenic tobacco plants constitutively expressing CaTLP1 under the control of CaMV35S promoter were evaluated for their response to stress. Over-expression of CaTLP1 in transgenic tobacco plants rendered improved tolerance to dehydration, salinity and oxidative stresses. There were significant increases in root and leaf development, net photosynthesis, and plant biomass in CaTLP1-overexpressing transgenic lines (
The expression of CaTLP1 is associated with dehydration tolerance by maintaining turgidity and relative water content. Under progressive dehydration, the photosynthetic rate in transgenic plants remained unaltered in initial 24 hours, decreased linearly up to 96 hours, and sustained thereafter. In contrast, the decrease in photosynthetic rate in wild-type plants was found to be proportional to the duration of stress treatment. In wild-type plants, photosynthetic rate reduced to 12% after 120 hours of dehydration, while the rates were 60% and 50% in transgenic T1 and T4, respectively (
The present invention discloses the role of a tubby-like protein, CaTLP1, in plant growth and development and in stress tolerance. The sequence of CaTLP1 displayed high similarity with the tubby genes earlier reported in Arabidopsis and rice. Over-expression of CaTLP1 in transgenic tobacco plants showed enhanced growth and development compared to wild-type plants. The CaTLP1 binds to double-stranded DNA but is incapable of transcriptional activation. The gene structure and organization of CaTLP1 revealed that CaTLP1 is involved in osmotic stress-responsive pathway in plants. The transcripts are strongly expressed in vegetative tissues but weakly in reproductive tissues. CaTLP1-overexpressing plants showed improved tolerance to dehydration, high salinity, and oxidative stress indicating its possible role in multivariate stress-responsive pathways. CaTLP1 is up regulated by dehydration and high salinity, and also by abscisic acid (ABA) treatments suggesting that its stress-responsive function might be associated with ABA-dependent network. The present invention provides new insights into the underlying mechanism of action of plant TLPs and also facilitates the targeted genetic manipulation in crop plants to improve stress tolerance. The polynucleotide and polypeptide sequence disclosed in the present specification also facilitates growth and development in the plant.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the invention, as described herein.
In an embodiment of the present invention, there is provided a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5.
An embodiment of the present invention provides a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the nucleotide sequence of the polynucleotide has at least 90% sequence identity with the nucleotide sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7.
Another embodiment of the present invention provides a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the nucleotide sequence of the polynucleotide is as set in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7.
In yet another embodiment of the present invention, there is provided a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is responsible for conferring enhanced tolerance to abiotic stress in a transformed plant as compared to an untransformed plant.
In still another embodiment of the present invention, there is provided a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is responsible for conferring enhanced tolerance to abiotic stress in a transformed plant as compared to an untransformed plant, wherein the abiotic stress is selected from the group consisting of dehydration stress, salinity stress, oxidative stress, osmotic stress, drought stress, and phytohormone stress.
In an embodiment of the present invention, there is provided a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the polynucleotide is responsible for enhanced plant growth and development in a transformed plant as compared to an untransformed plant.
Another embodiment of the present invention provides a recombinant DNA construct comprising the polynucleotide as claimed in claim 1; or the polynucleotide having the nucleotide sequence encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5; or the polynucleotide having the nucleotide sequence asset forth in SEQ ID NO: 6; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 7, wherein the polynucleotide is operably linked to a promoter.
An embodiment of the present invention provides a recombinant vector comprising a recombinant DNA construct comprising the polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 6; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 7, wherein the polynucleotide is operably linked to a promoter.
In yet another embodiment of the present invention, there is provided a recombinant host cell comprising a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5 or the recombinant vector comprising a recombinant DNA construct comprising the polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5; or the polynucleotide having the nucleotide sequence, as set forth in SEQ ID NO: 4; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 6; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 7, wherein the polynucleotide is operably linked to a promoter.
In still another embodiment of the present invention, there is provided a recombinant host cell comprising a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5 or the recombinant vector comprising a recombinant DNA construct comprising the polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 4; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 6; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 7, wherein the polynucleotide is operably linked to a promoter, wherein the host cell is selected from the group consisting of E. coli, Agrobacterium, yeast and, plant cell.
In an embodiment of the present invention, there is provided a polypeptide having amino acid sequence as set forth in SEQ ID NO: 5, wherein expression of the polypeptide is responsible for conferring enhanced tolerance to abiotic stress in a transformed plant as compared to an untransformed plant.
An embodiment of the present invention provides a method for producing a transformed plant cell, plant, or a part thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the method comprises: transforming the plant cell, plant, or part thereof with a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, and selecting the transformed plant cell, plant or part thereof, wherein the transformed plant cell, plant, or part thereof is more tolerant to abiotic stress than an untransformed plant.
Another embodiment of the present invention provides a method for producing a transformed plant cell, plant, or a part thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the method comprises: transforming the plant cell, plant, or part thereof with a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, and selecting the transformed plant cell, plant or part thereof, wherein the transformed plant cell, plant, or part thereof is more tolerant to abiotic stress than an untransformed plant, wherein the polynucleotide is as set forth in SEQ ID NO: 4.
In still another embodiment of the present invention, there is provided a method for producing a transformed plant cell, plant, or a part thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the method comprises: transforming the plant cell, plant, or part thereof with a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, and selecting the transformed plant cell, plant or part thereof, wherein the transformed plant cell, plant, or part thereof is more tolerant to abiotic stress than an untransformed plant, wherein the abiotic stress is selected from the group consisting of dehydration stress, salinity stress, oxidative stress, osmotic stress, drought stress, and phytohormone stress.
An embodiment of the present invention provides a method for producing a transformed plant cell, plant, or a part thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the method comprises: transforming the plant cell, plant, or part thereof with a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, and selecting the transformed plant cell, plant or part thereof, wherein the transformed plant cell, plant, or part thereof is more tolerant to abiotic stress than an untransformed plant, wherein the plant is a monocotyledonous or dicotyledonous plant.
In an embodiment of the present invention, there is provided a method for producing a transformed plant cell, plant, or a part thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the method comprises: transforming the plant cell, plant, or part thereof with a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, and selecting the transformed plant cell, plant or part thereof, wherein the transformed plant cell, plant, or part thereof is more tolerant to abiotic stress than an untransformed plant, wherein the plant is selected from the group consisting of tobacco, potato, sweet potato, cassava, sugar-beet, Arabidopsis, brassica species, tomato, brinjal, capsicum, chilli, okra, cucurbit; melon, mulberry, banana, mango, papaya, alfalfa, grass, canola, sunflower, cotton, legume plant, groundnut, peanut, pea, soybean, chickpea, pigeon pea, mungbean, medicago, lotus, Petunia, rice, maize, wheat, rye, barley, oats, pearl millet, corn, sorghum, nuts, avocado, turmeric, saffron, ginger, garlic, onion, nutmeg, forage plant, fruit tree, and ornamental plant.
Another embodiment of the present invention provides a method for producing a transformed plant cell, plant, or a part thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the method comprises: transforming the plant cell, plant, or part thereof with a polynucleotide encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5, and selecting the transformed plant cell, plant or part thereof, wherein the transformed plant cell, plant, or part thereof is more tolerant to abiotic stress than an untransformed plant, wherein plant is transformed using a method selected from the group consisting of floral dip method, Agrobacterium mediated transformation method, protoplast mediated transformation, particle gun bombardment method, in planta transformation, and liposome mediated transformation method.
A transgenic plant, plant cell or progeny thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the transgenic plant, plant cell or progeny thereof is more tolerant to abiotic stress than an untransformed plant.
The transgenic plant, plant cell or progeny thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the transgenic plant comprises the polynucleotide sequence as set forth in SEQ ID NO: 4 or a recombinant DNA construct comprising the polynucleotide as claimed in claim 1; or the polynucleotide having the nucleotide sequence encoding the protein having the amino acid sequence as set forth in SEQ ID NO: 5; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 6; or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 7, wherein the polynucleotide is operably linked to a promoter.
The transgenic plant, plant cell or progeny thereof over-expressing a protein having the amino acid sequence as set forth in SEQ ID NO: 5, wherein the transgenic plant is selected from a group consisting of tobacco, potato, sweet potato, cassava, sugar-beet, Arabidopsis, Brassica species, tomato, brinjal, capsicum, chilli, okra, cucurbit, melon, mulberry, banana, mango, vitis, papaya, alfalfa, grass, canola, sunflower, cotton, legume plant, groundnut, peanut, pea, soybean, chickpea, pigeon pea, mungbean, medicago, lotus, petunia, rice, maize, wheat, rye, barley, oats, pearl millet, corn, sorghum, nuts, avocado, turmeric, saffron, ginger, garlic, onion, nutmeg, forage plant, fruit tree and ornamental plant. Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.
Cicer arietinum L., (chickpea) seedlings were grown in pots containing a 2:1 w/w mixture of soil and soilrite in an environment-controlled growth room and maintained at 25±2° C. with 50±5% relative humidity under 16 h photoperiod (270 μmol m−2 s−1 light intensity).
Genomic DNA of C. arietinum L., (chickpea) seedlings was extracted using DNeasy kit (Qiagen, USA). Aliquots of 10 μg DNA were digested with different restriction enzymes (SspI, SacI, HindIII, EcoRI and BamHI) and Southern blot was hybridized with [32P]-labelled 314-bp fragment of CaTLP1.
In a separate set of experiment, RNA was isolated using Tripure reagent (Roche Applied Science), as recommended by manufacturer. The electrophoresis of nucleic acids was performed as described by Sambrook et al., 2001 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY), blotted overnight onto Hybond-N membrane (Amersham Biosciences, UK), and fixed at 1200 j/cm for 30 s using Stratalinker UV crosslinker (Stratagene). Immobilized nucleic acids were hybridized with [32P]-labelled 314-bp CaTLP1 fragment amplified with internal primers having nucleotide sequence as set forth in SEQ ID NO: 1 and SEQ ID NO: 2. The cDNA sequence of CaTLP1 was determined by ABI Prism 3700 DNA Analyzer (Applied Biosystems, USA). The cDNA nucleotide sequence thus obtained is as set forth in SEQ ID NO: 4.
A phylogenetic tree was constructed from amino acid alignment by neighbour-joining method using the default parameters. Multiple alignments of proteins were performed with ClustalW program, and a phylogram was generated using the neighbor-joining algorithm of MEGA software, version 5.05 as shown in
The full-length CaTLP1 gene was amplified from the genomic DNA isolated from C. arietinum using forward and reverse primers of size 25 bp as set forth in SEQ ID NO:10 and SEQ ID NO:11, respectively.
The PCR product of size 1,236 bp was purified using QIAquick gel extraction kit (QIAGEN, Germany). The purified PCR product was ligated into pGEX4T-2 expression plasmid (Amersham Biosciences, UK) in-frame with GST fusion protein utilizing BamHI and NotI restriction sites engineered into 5′- and 3′-ends, respectively, using T4 DNA ligase enzyme (New England Biolabs, UK) in 1:3 molar ratio of vector to insert, to yield the recombinant vector construct pGEXCaTLP1. The successfully ligated constructs were verified by nucleotide sequencing and was used for the transformation in Escherichia coli BL21(DE3) strain.
E. coli Transformation and Expression of the Recombinant Protein
The recombinant vector pGEXCaTLP1 was introduced into the competent E. coli BL21(DE3) cells by transforming chemically competent cell (Invitrogen) and its selection on LB Agar media (Invitrogen, USA) supplemented with 50 mg ml−1 ampicillin antibiotic.
The GST-tagged protein was produced by inducing transformed cells with 0.5 mM isopropyl β-D-1-thiogalactopyranoside. The recombinant protein was purified from the bacterial lysates with chromatography using glutathione-sepharose beads (Amersham Biosciences, UK) as per the manufacturer's instructions.
The purified recombinant protein was digested using trypsin. Trypsin-digested peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (LC/MS/TOF) using a Tempo Nanoflow MDLC system coupled to Q-STAR Elite spectrometer (Applied Biosystems, USA). The spectra was analysed to identify the protein of interest using Mascot sequence matching software (Matrix Science) (
Gel-shift assays were carried out using double-stranded 20 mer d(A/T) and d(G/C) and single-stranded 20 mer d(C20) and d(T20) [32P]-labelled probes. 0.5 mg of recombinant protein was incubated in a reaction mix containing 25 mM MOPS (pH 8.0), 2 mM DTT, 120 mM KOAc, and 2 mM EDTA with 0.5 pmol of either [32P]-labelled double- or single-stranded oligonucleotides for 30 min at 30° C. The complexes were resolved on 6% polyacrylamide gel and visualized by autoradiography (
To determine the activation domain, the coding sequence of CaTLP1 (SEQ ID NO: 4) was cloned in yeast expression plasmid pGBKT7 (Clontech Laboratories, USA) at NdeI-BamHI restriction site to express CaTLP1 fused to GAL4 DNA-BD. The construct was transformed into yeast strain AH109 (Cagney, Uetz and Fields 2000) that harbour MEL1 reporter gene. The GAL4 AD domain from the plasmid pGADT7 (Clontech Laboratories, USA) was cloned in-frame with the plasmid pGBKT7 and used as positive control, while pGBKT7 plasmid transformed in AH109 cells served as negative control. Yeast colonies harbouring no vector, empty vector (pGBKT7), and recombinant pGBKT7-CaTLP1 were grown on synthetically defined medium YPDA synthetic medium deficient in tryptophan and on SD media lacking tryptophan but supplemented with X-α-gal. The transformants were then analyzed for α-galactosidase expression.
CaTLP1-Enhanced Yellow fluorescent Protein (YFP) construct in pGWB441 gateway vector were used to transform Agrobacterium. The Agrobacterium strain was grown overnight in 10 ml liquid culture at 28° C. with vigorous agitation and the resulting bacteria were suspended into a buffer solution known as infiltration medium of 10 mM MES, 10 mM MgCl2 and 150 μM acetosyringone, up to an OD600=2.0 Agrobacterium-mediated transient expression assay was carried out in epidermal cells of tobacco leaves, and the localization of chimeric CaTLP1 protein fused to YFP was analyzed. The fluorescent signals of YFP were confined to the extracellular space and nucleus of transfected cells (
Arabidopsis seedlings were transformed with the CaTLP1-YFP construct in pGWB441 gateway vector by floral-dip method (Clough S J, and Bent A F (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735-43). The bacteria were grown to stationary phase in liquid culture at 25-28° C., 250 r.p.m. in sterilized YEP (10 g yeast extract, 10 g peptone, and 5 g NaCl per liter) supplemented with spectinomycin (100 mg ml−1). Cultures were initiated with 1:100 dilution of primary overnight cultures and grown for roughly 18-20 h. Cells were harvested by centrifugation (5500 g) for 20 min at room temperature. The harvested cells were resuspended in inoculation medium [½ strength MS Medium (M-5519; Sigma Chemicals), 5.0% sucrose, 44 nM benzylamino purine (10 ml 1−1 of a 1 mg ml−1 stock in DMSO) and 0.008% Silwet L-77] pH adjusted to 5.7 to a final OD600 of approximately 2.0. Arabidopsis plants were transformed by floral-dip method (Clough and Bent 1998). The primary roots of transformants were examined under confocal microscope (Leica Microsystems). A cell wall specific dye, propidium iodide (PI), was used to stain the cell wall of the primary root cells (
Full-length coding sequence of CaTLP1 (SEQ ID NO: 4) was cloned into the polylinker site of the binary vector pBI121 (Clontech Laboratories, USA) under the control of CaMV35S promoter. The construct was mobilized into Agrobacterium tumefaciens strain EHA105. Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum L. var. Petit Havana) was carried out using standard protocol prevalent in the art (Hobbs S L, Kpodar P, DeLong C M (1990) The effect of T-DNA copy number, position and methylation on reporter gene expression in′tobacco transformants. Plant Mol Biol 15: 851-64).
Regenerated shoots were rooted on phytohormone-free rooting medium supplemented with cefotaxime (250 mg/L) and kanamycin (100 mg/L), transferred to soil, and grown in standardized greenhouse conditions. Seventeen independent transgenic events harboring CaTLP1 (SEQ ID NO: 4) were grown for further analyses. The wild-type and the transgenic tobacco plants were studied parallely in the same growth room and comparative morpho-anatomical, molecular, and physiological analyses were carried out. Morpho-anatomical studies were accomplished by scanning electron microscopy (EVO LS10, Carl Zeiss) (
Transgenic tobacco plants constitutively expressing CaTLP1 (SEQ ID NO: 5) under the control of CaMV35S promoter was evaluated for its response to stress. A total of 17 independent transgenic events were confirmed by PCR analysis with primers having sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 11, the transcripts by Northern blot analyses, and protein expression by Western blot analyses (
Immunoblot analysis was done by resolving protein extracts from wild-type and transgenic plants on a uniform 12.5% SDS-PAGE, and then electrotransferring onto nitrocellulose membrane at 150 mA for 2 h. The membranes were blocked with 5% (w/v) non-fat milk for 1 h and incubated with anti-CaTLP1 polyclonal antibody for 2 h. The antibody was raised in rabbit against an antigenic peptide having amino acid sequence as set forth in SEQ ID NO: 3. (Sigma-Genosys, USA). The blot was eventually incubated with alkaline phosphatase conjugated secondary antibody for 1 h and the signals were detected using NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) method (
None of the transgenic lines displayed any phenotypic abnormality throughout their life cycle. Two independent overexpressing lines from F2 generation, one with high (T1) and the other with moderate expression (T4) of the transgene, were selected for further characterization. Southern blot analysis revealed the integration of three and two copies of the CaTLP1 in T1 and T4 lines, respectively (
The CaTLP1-overexpressing tobacco seedlings were transplanted and grown alongside the wild-type plants under identical conditions. The transgenic plants displayed a higher growth rate, as evident by increased shoot height and biomass, when compared to wild-type plants (
To determine the stress tolerance of the transgenic plants, chlorophyll retention rate was monitored by leaf-disc assay. Leaf-disc bioassay was carried out to determine dehydration, salinity, and oxidative stress tolerance using 1 cm2 discs excised from 30-day-old, both wild-type and transgenic, tobacco plants.
Chlorosis was observed against osmotic or oxidative stress on the leaf-discs subjected to sorbitol (200 mM), NaCl (200 mM and 500 mM), H2O2 (2 mM) and menadione (5 μM and 7 μM) treatments. The discs floated on sterile distilled water served as experimental control. (b) Quantitation of chlorophyll content in leaf-discs of wild-type and transgenic plants under progressive osmotic stress of 250 mM NaCl.
For salinity experiments, the seedlings were supplemented with half-Hoagland's medium until three weeks, followed by treatment with 100, 250, and 500 mM of NaCl in the medium. The tissues were harvested 24 hours after the treatment.
Methyl jasmonate 100 μM was sprayed on three-week-old seedlings and tissues were collected at 8, 16, and 24 h for analysis.
Salicylic acid 5 mM solution was prepared in ethanol and sprayed on the leaflets of three-week-old seedlings. The harvested tissues were instantly frozen in liquid nitrogen and stored at −80° C., unless stated otherwise.
The chlorosis of the discs was physically examined after 72 h of incubation in the various stress inducer solutions. Chlorophyll retention assay was done in leaf-discs subjected to salinity stress as described earlier (Porra R J (2002). The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73: 149-156). The chlorophyll content was calculated using following formulas:
[Chl a]=(12.00 9*A663.8)−(3.11 9*A646.8); (1)
[Chl b]=(20.78 9*A646.8)−(4.88 9*A663.8); (2)
[Chl a+b]=(17.67 9*A646.8)+(7.12 9*A663.8); (3)
where A is the absorption measured at respective wavelengths.
The leaf-discs of transgenic plants could retain higher percentage (30-45% as against the wild-type plants) of chlorophyll when compared with that of wild-type plants (
To evaluate ABA-mediated stress response, 8-day-old wild-type and transgenic seedlings were transferred to medium supplemented with 1 μM ABA and allowed to grow for 3 weeks. 100 μM ABA was sprayed on three-week-old seedlings and tissues were harvested at targeted intervals. The response was measured in terms of percent relative fresh weight, i.e. fresh weight of the seedlings grown in experimental conditions relative to the fresh weight of the same line grown in control conditions. The relative fresh weight was 58% in T1, followed by 50% in T4, and 31% in the wild-type plants (
Net photosynthesis and rate of transpiration were measured using a gas exchange fluorescence system GFS3000 (Heinz Walz GmbH, Germany). The measurements were made under saturating irradiance (1000 μmol photons m−2 s−1) and constant airflow of 750 μl s−1 at 25° C. Five independent leaves of three randomly chosen plants each, from wild-type and transgenic plants, were placed directly into the measuring cell (3 cm2) for the measurements. Each experiment was replicated at least three times and values are expressed as means±SE. All mean comparisons were done using paired t-test for independent samples. The measurements for determining net photosynthetic rate and leaf conductance were analyzed by one-way analysis of variance (ANOVA).
The three-week-old seedlings were subjected to dehydration condition for a period of seven days by withholding watering of the plants and tissues were harvested at different intervals of 24 hours. The pots containing dehydrated seedlings were then rewatered and allowed to recover for a period of 24 hours, and the rehydrated tissues (R24) were harvested.
Under progressive dehydration, the photosynthetic rate in transgenic plants remained unaltered in initial 24 hours, decreased linearly up to 96 h, and sustained thereafter. In contrast, the decrease in photosynthetic rate in wild-type plants was found to be proportional to the duration of stress treatment. In wild-type plants, photosynthetic rate reduced to 12% after 120 hours of dehydration, while the rates were 60% and 50% in transgenic T1 and T4 plants, respectively (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1406/DEL/2012 | May 2012 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2013/000302 | 5/7/2013 | WO | 00 |
