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WO1998004701A1 - Procedes et compositions pour ameliorer la capacite d'une plante a absorber le phosphate du sol - Google Patents

Procedes et compositions pour ameliorer la capacite d'une plante a absorber le phosphate du sol Download PDF

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Publication number
WO1998004701A1
WO1998004701A1 PCT/US1997/013458 US9713458W WO9804701A1 WO 1998004701 A1 WO1998004701 A1 WO 1998004701A1 US 9713458 W US9713458 W US 9713458W WO 9804701 A1 WO9804701 A1 WO 9804701A1
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Prior art keywords
seq
plant
ala
promoter
gly
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PCT/US1997/013458
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English (en)
Inventor
K. G. Raghothama
Umesh S. Muchhal
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Purdue Research Foundation
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Priority to AU38218/97A priority Critical patent/AU3821897A/en
Publication of WO1998004701A1 publication Critical patent/WO1998004701A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to methods and materials in the field of molecular biology and the regulation of protein synthesis through plant genetic engineering. More particularly, the invention relates to newly-isolated nucleotide sequences, nucleotide sequences having substantial identity thereto and proteins encoded thereby.
  • the invention also involves plant nutrition, specifically, the introduction of foreign nucleotide sequences into a plant genome, wherein the introduction of the nucleotide sequence effects an increase in the plant ' s ability to take in phosphate (Pi) from the soil.
  • Plant transformants harboring an inventive DNA construct comprising a promoter operably linked to an inventive nucleotide coding sequence demonstrate increased levels of phosphate transporter protein production, rendering the plant better able to withstand a phosphate deficiency.
  • Phosphorus is one of the essential and major plant nutrients, and phosphorus availability is considered as one of the major growth limiting factors for plants in many natural ecosystems. Most commercial farming worldwide depends upon phosphatic fertilizers as a source of phosphorus. The availability of rock phosphate, a non-renewable source of phosphorus, is predicted to last only for the next 60-90 years. In view of the importance of phosphorus as a nutrient and the non-renewable nature of the raw material, there is a neet to enhance the efficiency of its uptake by plant roots. Plants have developed adaptive mechanisms to overcome Pi stress. Changes in the root growth and architecture, increased production of phosphatases and RNAases. and altered activity of several enzymes of the glycolytic pathway are among the well characterized responses to Pi deficiency in plants. In addition, an increase in phosphate uptake rate of roots and cell cultures following phosphate starvation has been observed in several plant species.
  • Phosphate is acquired by plants in an energy mediated cotransport process, driven by a proton gradient generated by plasma membrane H ⁇ -ATPases. Phosphate absorption is accompanied by H+ influx with a stoichiometry of 2 to 4 H7H : PO 4 " transported.
  • a dual-mechanism model for uptake of ions, including phosphate has been proposed. This is characterized by a high-affinity transport system operating at low ( ⁇ M) concentration and a low-affinity system functioning at high concentration (mM) of ions. Measurements of phosphate in saturation extracts of a large number of soils showed that phosphate concentration in the majority of samples ranged between 0.65 to 2.5 ⁇ M. Under these conditions, the high-affinity transport system is considered to be the major mechanism for phosphate acquisition. Additionally, the kinetic characterization of the Pi uptake system by whole plants and cultured cells indicates a high-affinity transport activity operating at low concentration ( ⁇ M range).
  • Pi transporter proteins are predicted to have a structure containing 12 membrane spanning domains separated into two groups of six by a charged hydrophilic region.
  • the present inventors have discovered, isolated and characterized four nucleotide sequences which encode phosphate transporters, two having been isolated from Arabidopsis thaliana and two having been isolated from Lycopersicon esculentum (tomato). The inventors have further discovered that these genes are induced in a tissue specific manner in response to phosphate starvation.
  • the present invention therefore, provides materials and methods for producing transgenic plants which over-express phosphate transporter proteins, preferably in their roots, these transgenic plants being more efficient in absorbing phosphate from the soil.
  • the regulated expression of efficient forms of phosphate transporters should lead to greater agricultural productivity and reduced fertilizer costs to growers. Incorporation of phosphate uptake-efficiency traits in breeding programs will be an added benefit to biotechnology and seed companies trading with tropical countries, where soils are naturally deficient in phosphorus.
  • the present invention relates to the isolation, purification and use of nucieotide sequences and proteins encoded thereby.
  • Inventive nucleotide sequences are advantageously integrated into a DNA construct which comprises a nucleotide coding sequence which encodes a phosphate transporter protein and a promoter capable of eliciting expression of the nucleotide coding sequence in a transformed plant.
  • the nucleotide sequence encoding a phosphate transporter has substantial identity to the sequence set forth in SEQ ID NO: 1 ; SEQ ID NO:2; SEQ ID NO:3; or SEQ ID NO:4.
  • the promoter is a tissue specific promoter.
  • the promoter is an inducible promoter which elicits expression of the nucleotide sequence when a plant experiences a phosphate deficiency.
  • the inventive DNA construct comprises a promoter having substantial identity to a native high-affinity phosphate transporter promoter such as, for example, AtPTl. AtPT2, LePTl or LePT2.
  • the promoter has substantial identity to the nucleotide sequence set forth in SEQ ID NO:9.
  • a phosphate transporter protein selected in accordance with the invention is an essential part of the high- affinity phosphate uptake mechanism, and over-expression thereof in the proper plant tissues results in an increased ability by the plant to survive and flourish in a low phosphate soil environment.
  • inventive DNA constructs may advantageously be used according to the invention to transform a plant, thereby providing an inventive transformed plant which over-expresses a phosphate transporter protein.
  • inventive DNA constructs refers to the production of the protein product encoded by a nucleotide coding sequence.
  • “Over-expresses” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non- transformed organisms.
  • a plant transformed in accordance with the invention produces an amount of a phosphate transporter protein that is greater than that of a non-transformed plant of the same species.
  • the present invention thus provides methods for genetically engineering plants to provide inventive transformed plants which have an increased ability to withstand phosphate deprivation by increasing the plant ' s ability to bring phosphate into the plant ' s roots. This increased ability results from an increase in the rate of expression of phosphate transporter coding sequences.
  • the invention features DNA constructs comprising a promoter sequence and a coding sequence as set forth herein, as well as DNA constructs comprising nucleotide sequences having substantial identity thereto and having similar levels of functionality. Inventive constructs may be inserted into an expression vector to produce a recombinant DNA expression vector which is also an aspect of the invention.
  • an isolated nucleic-acid construct comprising a promoter and a nucleotide sequence having substantial identity to the sequence set forth in SEQ ID NO: l (AtPTl cDNA); SEQ ID NO:2 (AtPT2 cDNA); SEQ ID NO:3 (LePTl cDNA) or SEQ ID NO:4 (LePT2 cDNA).
  • the protein encoded thereby preferably has an amino acid sequence having substantial identity to the sequence set forth in SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7 or SEQ ID NO:8, wherein the amino acid sequence may include amino acid substitutions, additions and deletions that do not alter the function of the phosphate transporter protein.
  • Figure 1 A illustrates the alignment of the deduced amino acid sequence of LePTl .
  • LePT2 AtPTl and AtPT2 with that of potato (STPT1 and STPT2) and Catharanthus roseus (PIT1) phosphate transporters.
  • Identical amino acids are indicated by asterisks (*) and conserved substitutions are indicated by dots (.).
  • the membrane spanning domains of LePTl and LePT2 as predicted by TopPred are underlined and their numbering is indicated by roman numerals (I-XII).
  • the shaded sequences are consensus sites for N-linked glycosylation; boxed sequences are consensus sites for phosphorylation by casein kinase II; and boxed and shaded sequences are consensus sites for phosphorylation by protein kinase C.
  • Figure IB is a summary of percent amino acid identity between tomato and other plant phosphate transporters.
  • Figure 2 sets forth the results of a Northern blot analysis of the expression of phosphate transporter genes in Arabidopsis.
  • the blots were exposed to x-ray film for 2 days (AtPTl), 5 days (AtPT2) or 5 hr (rDNA).
  • Figure 3 sets forth the results of a Northern blot analysis of the expression of tomato phosphate transporter genes.
  • Figure 4 sets forth the results of a Southern blot analysis of Arabidopsis genomic DNA digested with BamHI (B), EcoRl (E). or H dIII (H). The blots were hybridized with labeled cDNA inserts from AtPTl or AtPT2.
  • Figure 5 sets forth the results of a Southern blot analysis of tomato genomic DNA digested with Pstl (P), Hindlll (H). or EcoRI (E). Blots were hybridized with labeled cDNA inserts from LePTl and LePT2.
  • Figure 7 provides the results of the procedure set forth in Example 7 herein relating to the complementation of yeast high-affinity phosphate transporter mutant NS219.
  • Figure 7(a) sets forth acid phosphatase activity in NS219 transformants containing either the vector (pYES2) or the vector containing AtPTl (pYES2 + AtPTl )/AtPT2 (pYES2 + AtPT2) coding regions.
  • the cells were grown on SD medium— high-phosphate (1 ImM) plates containing 3% glycerol and .5% galactose for 4 days before staining for acid phosphatase activity. The red color indicates presence of acid phophatase activity.
  • Three independent transformants (A-C) for each construct are shown.
  • Figure 7(b) sets forth growth of NS219 transformants in SD medium — low phosphate (1 10 ⁇ m) medium containing 2% galactose and 0.5% sucrose. Values are the averages from two experiments using three independent transformants for each construct. Error bars indicate the standard deviation. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to DNA constructs that may be integrated into a plant to provide an inventive transformed plant which over-expresses a phosphate transporter protein.
  • Over-expression of a phosphate transporter protein in the proper plant tissues results in an increased uptake of phosphate from the soil in which the plant is grown. In soil environments where there is a phosphate deficiency, this results in an increased probability that the plant will survive and flourish without the addition of phosphorus-containing fertilizers.
  • the present inventors have discovered, isolated and characterized four nucleotide sequences which encode phosphate transporters, two having been isolated from Arabidopsis thaliana and two having been isolated from Lycopersicon esculentum (tomato). The inventors have further discovered that these sequences regulated by inducible promoters which are induced in a root specific manner in response to a phosphate deficiency.
  • the present invention relates to the nucleotide sequences set forth in SEQ ID NO: 1 ; SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4, which encode phosphate transporter proteins, and sequences having substantial identity thereto.
  • a nucleotide sequence selected in accordance with the invention may be operably linked to a promoter to provide a novel DNA construct which may be used to transform a plant to provide a transformed plant having the ability to withstand a phosphate deficiency.
  • a nucleotide sequence selected for use in accordance with the present invention is one that effectively expresses a functional phosphate transporter protein in tissues that are involved in taking up phosphate from the soil.
  • the nucleotide sequence encodes a protein selected from the group consisting of AtPTl , AtPT2.
  • LePTl LePT2 and proteins having substantial identity to these. It is not intended, however, that this list be limiting, but only provide examples of nucleotide sequences which may be advantageously used in accordance with the present invention to provide over-expression of a functional phosphate transporter protein in cells involved with the phosphate uptake.
  • sequences encoding phosphate transporters commonly differ to some degree between species; however, it is understood that the present invention is intended to encompass genes which encode phosphate transporter proteins in a wide variety of plant species. While nucleotide sequences encoding two phosphate transporters of the species
  • Arabidopsis thaliana are set forth in SEQ ID NO: 1 and SEQ ID NO:2 herein (AtPTl and AtPT2, respectively), and nucleotide sequences encoding two phosphate transporters of the species Lycopersicon esculentum are set forth in SEQ ID NO:3 and SEQ ID NO:4 herein (LePTl and LePT2. respectively), it is not intended that the present invention be limited to these exemplary sequences, but include sequences having substantial identity thereto and sequences from different plant species that encode phosphate transporter proteins of that species.
  • nucleotide sequence is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, and derivatives thereof.
  • encoding and coding refer to the process by which a gene, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional protein, such as. for example, an active enzyme. It is understood that the process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e..
  • a preferred DNA construct selected or prepared in accordance with the invention expresses a phosphate transporter protein, or a protein having substantial identity thereto and having a level of activity suitable to achieve the advantageous result of the invention.
  • a preferred amino acid sequence encoded by an inventive DNA construct is an amino acid sequence set forth in SEQ ID NO:5; SEQ ID NO:6 SEQ ID NO:7; SEQ ID NO:8 or a sequence having substantial identity thereto.
  • proteins and "amino acid sequence” are used interchangeably herein to designate a plurality of amino acids linked in a serial array.
  • Skilled artisans will recognize that through the process of mutation and/or evolution, proteins of different lengths and having differing constituents, e.g., with amino acid insertions, substitutions, deletions, and the like, may arise that are related to the proteins of the present invention by virtue of (a) amino acid sequence homology; and (b) good functionality with respect to phosphate transport activity.
  • a phosphate transporter protein isolated from one species and/or the nucleotide sequence encoding it. may differ to a certain degree from the sequences set forth herein, and yet have excellent functionality in accordance with the invention.
  • Such a protein and/or nucleotide sequence falls directly within the scope of the present invention. While many deletions, insertions, and, especially, substitutions, are not expected to produce radical changes in the characteristics of the protein, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect may be evaluated by routine screening assays.
  • the present invention also contemplates proteins having substantial identity thereto.
  • the term "substantial identity.” as used herein with respect to an amino acid sequence is intended to mean sufficiently similar to have suitable functionality when expressed in a plant transformed in accordance with the invention to achieve the advantageous result of the invention.
  • variants having such potential modifications as those mentioned above which have at least about 50% identity to an amino acid sequence set forth in SEQ ID NOS:5-8, are considered to have "'substantial identity" thereto. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
  • inventive proteins have at least about 75% identity to those set forth in SEQ ID NOS:5-8, more preferably at least about 85% identity and most preferably at least about 95% identity. It is believed that the identity required to maintain proper functionality is related to maintenance of the tertiary structure of the protein such that specific interactive sequences will be properly located and will have the desired activity. As such, it is believed that there are discrete domains and motifs within the amino acid sequence which must be present for the protein to retain its advantageous functionality and specificity. While it is not intended that the present invention be limited by any theory by which it achieves its advantageous result, it is contemplated that a protein including these discrete domains and motifs in proper spatial context will retain good enzymatic activity.
  • the invention also encompasses more than the specific exemplary nucleotide sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce '"silent" changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alterations in the nucleotide sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine.
  • leucine or isoleucine.
  • changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine can also be expected to produce a biologically equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein.
  • Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded products. As a related matter, it is understood that similar base changes may be present in a promoter sequence without substantially affecting its valuable functionality. Such variations to a promoter sequence are also within the purview of the invention.
  • the present invention contemplates nucleotide sequences having substantial identity to those set forth in SEQ ID NOS. 1, 2, 3 and 4.
  • the term "substantial identity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence has a sequence sufficiently similar to one of those explicitly set forth herein that it will hybridize therewith under moderately stringent conditions, this method of determining identity being well known in the art to which the invention pertains. Briefly, moderately stringent conditions are defined in Sambrook et al.. Molecular Cloning: a Laboratory Manual, 2ed. Vol. 1, pp. 101- 104.
  • nucleotide coding sequence in accordance with this embodiment is that it must encode a protein having substantially similar functionality to a phosphate transporter protein set forth in SEQ ID NOS:5-8. i.e.. one which is capable of effecting an increase in a plant ' s ability to withstand phosphate deficiency when over-expressed in the plant's tissues involved in the uptake of phosphate from the soil.
  • Suitable DNA sequences selected for use according to the invention may be obtained, for example, by cloning techniques using cDNA libraries corresponding to a wide variety of plant species, these techniques being well known in the relevant art, or may be made by chemical synthesis techniques which are also well known in the art.
  • Suitable nucleotide sequences may be isolated from DNA libraries obtained from a wide variety of species by means of nucleic acid hybridization or PCR. using as hybridization probes or primers nucleotide sequences selected in accordance with the invention, such as those set forth in SEQ ID NOS: 1, 2, 3 and 4; nucleotide sequences having substantial identity thereto; or portions thereof.
  • nucleotide sequence which encodes a phosphate transporter, or a protein having substantial identity thereto and having suitable activity with respect to phosphate uptake may be isolated and/or amplified from a wide variety of plant species.
  • Nucleotide sequences specifically set forth herein or selected in accordance with the invention may be advantageously used in a wide variety of plant species, including but not limited to the species from which it is isolated.
  • Inventive DNA sequences can be incorporated into the genome of a plant using conventional recombinant DNA technology, thereby making a transformed plant better capable of withstanding phosphorus deprivation.
  • the term "genome” as used herein is intended to refer to DNA which is present in a plant and which is heritable by progeny during propagation of the plant.
  • an inventive transgenic plant may alternatively be produced by breeding a transgenic plant made according to the invention with a second plant or selfing an inventive transgenic plant to form an FI or higher generation plant. Transformed plants and progeny thereof are all contemplated by the invention and are all intended to fall directly within the meaning of the term "transgenic plant.”
  • transformation of a plant involves inserting a DNA sequence into an expression vector in proper orientation and correct reading frame.
  • the vector contains the necessary elements for the transcription of the inserted protein-encoding sequence.
  • vector systems known in the art can be advantageously used in accordance with the invention, such as plasmids, bacteriophage viruses or other modified viruses.
  • Suitable vectors include, but are not limited to the following viral vectors: lambda vector system gtl 1, gtlO, Charon 4. and plasmid vectors such as pBI121. pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18.
  • the DNA sequences are cloned into the vector using standard cloning procedures in the art, for example, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference.
  • the plasmid pBI121 is available from Clontech Laboratories, Palo Alto, California. It is understood that related techniques may be advantageously used according to the invention to transform microorganisms such as, for example, Agrobacterium sp., yeast, E.coli and Pseudomonas sp.
  • a promoter In order to obtain satisfactory expression of a nucleotide sequence which encodes an inventive phosphate transporter in a plant, a promoter must be present in the expression vector. Promoters selected for use in accordance with one preferred aspect of the present invention effectively target phosphate transporter expression to those tissues that are involved with phosphorus uptake (i.e., in most plants, the roots). In another preferred aspect of the invention, the promoter is inducible by phosphorus deficiency in a plant. Preferably, the promoter is one isolated from a native gene which encodes a phosphate transporter protein. For example, over-expression of a phosphate transporter may preferably be obtained in target plant tissues using one of the following promoters: AtPTl , AtPT2.
  • the promoter is the AtPT2 promoter, which is set forth in SEQ ID NO:9 herein.
  • SEQ ID NO:9 promoters for certain classes of genes commonly differ between species, it is understood that the present invention includes promoters which regulate expression of phosphate transporters in a wide variety of plant species.
  • An expression vector according to the invention may be either naturally or artificially produced from parts derived from heterologous sources, which parts may be naturally occurring or chemically synthesized, and wherein the parts have been joined by ligation or other means known in the art.
  • the introduced coding sequence is under control of the promoter and thus will be generally downstream from the promoter. Stated alternatively, the promoter sequence will be generally upstream (i.e., at the 5' end) of the coding sequence.
  • enhanced production of a phosphate transporter protein may be achieved by inserting an inventive nucleotide sequence in a vector downstream from and operably linked to a promoter sequence capable of driving over-expression in a host cell.
  • Two DNA sequences (such as a promoter region sequence and a phosphate transporter-encoding nucleotide sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter region sequence.
  • RNA polymerase normally binds to the promoter and initiates transcription of a DNA sequence or a group of linked DNA sequences and regulatory elements (operon).
  • a transgene such as a nucleotide sequence selected in accordance with the present invention, is expressed in a transformed plant to produce in the cell a protein encoded thereby.
  • transcription of the DNA sequence is initiated by the binding of RNA polymerase to the DNA sequence's promoter region.
  • mRNA messenger RNA
  • the DNA sequence is transcribed into a corresponding mRNA.
  • RNA transfer RNA
  • Proteins of the present invention thus produced in a transformed host then perform an important function in the plant's ability to take in phosphate. It is well known that there may or may not be other regulatory elements (e.g., enhancer sequences) which cooperate with the promoter and a transcriptional start site to achieve transcription of the introduced (i.e., foreign) coding sequence. Also, the recombinant DNA will preferably include a transcriptional termination sequence downstream from the introduced sequence.
  • Transformation may be achieved using one of a wide variety of techniques.
  • One technique of transforming plants with a DNA construct in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising the DNA construct.
  • this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for about 48 to about 72 hours on regeneration medium without antibiotics at about 25- 28°C.
  • Bacteria from the genus Agrobacterium may be advantageously utilized to transform plant cells.
  • Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes.
  • Agrobacterium tumefaciens e.g., strains LBA4404 or EHA105
  • Another technique which may advantageously be used is vacuum-infiltration of flower buds using Agrobacterium-based vectors.
  • Another approach to transforming plant cells with a DNA sequence selected in accordance with the present invention involves propelling inert or biologically active particles at plant tissues or cells. This technique is disclosed in U.S. Patent Nos. 4.945.050. 5,036.006 and 5,100,792. all to Sanford et al.. which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector . Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA material sought to be introduced
  • Biologically active particles can also be propelled into plant cells. It is not intended, however, that the present invention be limited by the choice of vector or host cell. It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same vector expression system. However, one of skill in the art may make a selection among vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of this invention.
  • successful transformants can be screened using standard techniques such as the use of marker genes, e.g., genes encoding resistance to antibiotics. Additionally, the level of expression of the foreign DNA may be measured at the transcriptional level, as protein synthesized or by assaying to determine the level of phosphorus uptake by the plant.
  • An isolated DNA construct selected in accordance with the present invention may be utilized in an expression vector to increase phosphorus uptake capabilities in a wide variety of plants, including gymnosperms. monocots and dicots. Inventive DNA constructs are particularly useful in plant species which are commonly grown in low phosphorus soil. For example, tropical soils are known to have low phosphorus content.
  • the invention finds advantageous use. for example, in transforming the following plants: gymnosperms. rice, wheat, barley, rye. corn, potato, carrot, sweet potato, bean, pea. chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • Plant Material Arabidopsis thaliana var. Columbia seeds were germinated on a 300- ⁇ m mesh nylon screen placed on a petri plate containing solidified agar supplemented with 1/10 Murashige-Skoog salts as described by Poirier et al.
  • the nylon filter was removed along with the intact root system and transferred to a sterile floating membrane raft (Lift raft, Sigma).
  • the floating device carrying seedlings was placed in a GA-7 (Sigma) tissue culture box containing 100 ml of half- strength Hoagland II nutrient solution.
  • Tomato ⁇ Lycopersicon esculentum plants were grown in an aeroponic growth facility similar to the one described in Liu et al., 1997.
  • Tomato seeds var. OS4 were germinated in seedling trays filled with Scotts ready earth plug mix (Scotts Co.. Marysville. OH).
  • Scotts ready earth plug mix Scotts Co.. Marysville. OH.
  • roots were sprayed with a fine mist of half strength Hoagland's solution for 3 seconds every 10 minutes. Phosphorus starvation treatments were initiated one week after the plants were transferred to aeroponics.
  • RNA Isolation Total RNA was isolated from the roots and leaves of Arabidopsis plants and of tomato plants by hot phenol extraction and lithium chloride precipitation. Poly(A) " RNA was isolated by the oligo(dT) cellulose batch binding method.
  • RNA (lO ⁇ g) was electrophoretically separated on 1% denaturing formaldehyde agarose gels and blotted onto BA-S (Schleicher & Schuell) nitrocellulose membrane.
  • the nitrocellulose filters were hybridized overnight with 32 P-labeled DNA probe (10 6 cpm/ml) in a solution containing 50% formamide, 5x Denhardt's solution. 0.1% SDS, 6x SSPE, and 100 ⁇ g/ml denatured salmon sperm
  • LePTl and LePT2 are expressed in tomato plants grown either in the presence of 250 ⁇ M phosphate or no phosphate. Both probes hybridized to about 2.0 kb transcripts. Their expression was markedly increased in plants grown under Pi limiting conditions. LePTl is primarily expressed in roots, with a small amount of the message also detectable in leaves, stems and petioles of tomato plants subject to Pi starvation. LePT2 is expressed only in the roots.
  • AtPTl and AtPT2 transcripts were compared by Northern blot analysis of total RNA isolated from roots and leaves of Arabidopsis plants grown either in the presence of 250 ⁇ M phosphate or no phosphate. A 1.9-kb transcript was detected for both of these genes in roots. Their expression was markedly increased in the roots of plants subjected to phosphate starvation. The AtPTl message was more abundant compared with that of AtPT2 n phosphate-starved roots. There was no detectable message for either of the genes in leaves even under phosphate starvation. Northern blots are set forth in Figures 2 and 3. -II
  • Genomic DNA (10 ⁇ g) was digested with restriction enzymes, electrophoretically separated through 0.8% agarose gels, denatured, and transferred to a supported nitrocellulose membrane. The hybridization and washing conditions were the same as those described above for Northern blots. Results are set forth in Figures 4 and 5 herein.
  • Arabidopsis A cDNA library representing the mR A isolated from Arabidopsis roots starved for phosphate for 7 days was constructed in the EcoRl-X ol site of the Uni- ZAP XR vector according to manufacturer's instructions (Stratagene).
  • the Arabidopsis expressed sequence tag clones (stock nos. 134M11T7, 178H14T7. And ATTS2854) showing similarity to yeast PHO84 were obtained from the Arabidopsis Biological Resource Center (Columbus, OH).
  • the inserts from these clones were radio-labeled by random priming (DECAprime II. Ambion. Austin.
  • AtPTl is 1754 bp long and contains an open reading frame encoding a 524 amino acid polypeptide (molecular mass of 57.6 kDa), whereas AtPT2 is 1897 bp long and encodes a 534 amino acid polypeptide (molecular mass of 58.6 kDa).
  • the open reading frames of AtPTl and AtPT2 are flanked by 47 and 151 bp of untranslated sequence at the 5' end, and by 135 and 144 bp of untranslated sequence including the poly(A) tail at the 3' end, respectively. These two clones are 70% similar in ther nucleotide sequence within the coding region. The two polypeptides are 78% identical in their amino acid sequence.
  • LePTl is 2023 bp long and contains an open reading frame encoding a 538 aa polypeptide (molecular mass of 58.7 kDa), whereas LePT2 is 1826 bp long and encodes a 528 aa polypeptide (molecular mass 57.8 kDa).
  • the open reading frames of LePTl and LePT2 are flanked by 151 and 37 bp of untranslated sequence at the 5 ' end and by 258 and 205 bp of untranslated sequence including the poly(A) tail at the 3 " end.
  • the LePT and LePT2 polypeptides are 80% identical in their amino acid sequence. The two polypeptides share substantial similarity with the phosphate transporters from potato.
  • LePTl is more similar to potato STPT1 and Arabidopsis AtPT2, whereas LePT2 is more similar to STPT2 and AtPTl .
  • Roots of tomato plants grown in aeroponics were sprayed with nutrient solutions containing Pi (250 ⁇ M), or without Pi for 5 days. Root and leaf samples were harvested and fixed in a solution containing 3.7% (v/v) formaldehyde. 5% (v/v) acetic acid and 50% (v/v ethanol (Niu et al., 1996). Fixed tissue samples were dehydrated in an ethanoi dilution series and embedded in wax (Paraplast from Fisher Scientific Co. IL). Ten ⁇ m sections cut with a microtome were transferred to Super- Frost plus slides (Fisher Scientific Co. IL). and incubated at 42°C overnight.
  • Sense and antisense probes representing LePTl and LePT2 were transcribed by T3 or T7 RNA polymerase (Ambion, CA) from linearized pBluescript-SK-containing the cDNA.
  • the probes were labeled with digozigenin (DIG) following the procedure described by the manufacture (Boehringer Mannheim, IN).
  • DIG digozigenin
  • Tissue section pretreatment and in situ hybridization were performed as described by Niu et al., (1996). Successive sections from roots obtained from three plants were used for hybridizing with sense and anti-sense probes. After color development for 16 to 24 hr. sections were photographed using a Nikon Optiphot microscope.
  • the yeast strain NS219 contains a mutation in the PHO84 gene and thereby lacks the high-affinity phosphate transport system. As a result of this mutation, NS219 cells exhibit reduced rates of phosphate uptake and growth in low-phosphate medium. In addition, the mutant cells continue to produce an acid phosphatase when grown in high-phosphate medium, whereas this activity is repressed in wild-type cells under these conditions.
  • the coding regions of the cDNAs were ligated into a yeast expression vector pYES2 and transformed into NS219.
  • the yeast high-affinity phosphate transporter mutant NS219 was provided by Satoshi Harashima (Osaka University, Japan).
  • the coding regions of AtPTl and AtPT2 cDNAs were subcloned in yeast expression vector pYES2 (Invitrogen) downstream of the GAL 1 promoter.
  • NS219 was transformed by a Licl/PEG method.
  • the procedures and synthetic defined (SD) medium used for growth and selection of transformants were similar to those described earlier.
  • the acid phosphatase activity of the NS219 transformants was detected by a staining method based on the diazo-coupling reaction.
  • the cells transformed with only the pYES2 vector exhibited the acid phosphatase activity on high-phosphate medium as seen from their red color after staining.
  • the NS219 transformants expressing either AtPTl or AtPT2 mimicked wild-type cells, showing no acid phosphatase activity.
  • NS219 transformants expressing either AtPTl or AtPT2 were also able to grow much faster in low- phosphate ( 1 lO ⁇ M) medium than the cells containing pYES2. The average generation time of these cells was about 2.5 times higher than the control cells during the initial phases of their growth in low-phosphate medium. Results are set forth in Figure 7 herein.
  • TCGTATTTAT TACCACGTGG AAGGCGCACA AAAGCCTGGG ACTCTCCCTC CCAACGTCGC 360 AGCCGCCGTC AATGGCGTTG CCTTCTGTGG GACTCTCGCC GGTCAGCTCT TTTTCGGGTG 420
  • AAAGCAGGCA GCTTCGGACA TGTCTAAGGT TCTGCAAGTG GAGATAGAGC CAGAACAACA 960
  • CATGAGTCGC CATGGGCTTC ATTTGCTAGG CACTACATCG ACATGGTTCC TTCTCGACAT 1080
  • AAAGACTGAC GCCGGTTACC CTCCTGGCAT TGGTGTGAGG AACTCGTTGA TCGTCCTTGG 1620
  • TTGTGTTAAC TTCCTCGGTA TGCTGTTCAC ATTCTTGGTT CCAGAATCCA ATGGGAAGTC 1680
  • GAAATTTCCA TTTCTGTAAA TGCCTTAAAT TAATGGCTCT TATTTATCAA ATACGGAACA 430
  • AACCCTCTTT ACACCTTACA ACTTACGGGT ATAGGGTGTT TATTCTCCCG TACCCGTTCA 540
  • AACTACACTA TATAATAAAC CATTGACATT GTTAGACCTA TTACACATCC TGCAGTTATT 600

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Abstract

L'invention concerne des procédés et des matériaux utilisés dans le domaine de la biologie moléculaire et de la régulation de la protéogénèse par manipulation génétique des plantes. L'invention porte, plus particulièrement, sur des séquences nucléotidiques récemment isolées, sur des séquences nucléotidiques ayant une identité sensiblement identiques auxdites séquences et sur des protéines codées par celles-ci. Elle se rapporte encore à l'introduction de séquences nucléotidiques étrangères dans un génome de plante, introduction générant une augmentation de la capacité de la plante à transporter le phosphate. Les transformants de plante, abritant un produit de reconstruction de l'ADN comprenant un promoteur lié de manière active à une séquence nucléotidique selon l'invention, présentent des niveaux accrus de protéines de transport du phosphate, ce qui augmente la capacité de la plante à pousser dans un substrat à déficience en phosphate.
PCT/US1997/013458 1996-07-29 1997-07-29 Procedes et compositions pour ameliorer la capacite d'une plante a absorber le phosphate du sol WO1998004701A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998038295A1 (fr) * 1997-02-24 1998-09-03 Performance Plants, Inc. Promoteur activable par une deficience en phosphate
WO1998005760A3 (fr) * 1996-07-31 1998-10-08 Univ Kingston Proteines pouvant etre induites en cas de privation de phosphate
WO1999058657A3 (fr) * 1998-05-13 2000-07-27 Pioneer Hi Bred Int Genes transporteurs de phosphate de zea mays
US6096545A (en) * 1996-07-31 2000-08-01 Queen's University At Kingston Phosphate starvation-inducible proteins
WO2001055299A3 (fr) * 2000-01-27 2002-01-17 Yeda Res & Dev Polynucleotides isoles codant pour un nouveau transporteur de phosphates dans des plantes et procede de modulation de l'absorption de phosphates dans des plantes
KR100401007B1 (ko) * 2000-08-11 2003-10-08 윤성중 담배의 인산수송자 유전자

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
MOLECULAR AND CELLULAR BIOLOGY, June 1991, Vol. 11, No. 6, BUN-YA et al., "The PH084 Gene of Saccharomyces Cerevisiae Encodes an Inorganic Phosphate Transporter", pages 3229-3238. *
NATURE, 7 December 1995, Vol. 378, HARRISON et al., "A Phosphate Transporter From the Mycorrhizal Fungus Glomus Versiforme", pages 626-629. *
PHYSIOLOGIA PLANTARUM, May 1997, Vol. 100, LOGAN et al., "Plasma Membrane Transport Systems in Higher Plants: From Black Boxes to Molecular Biology", pages 1-15. *
PLANT PHYSIOLOGY SUPPLEMENT, 17 July 1997, Vol. 114, No. 3, OKUMURA et al., "Enhanced Phosphate Uptake in Transgenic Tobacco Cells Producing an Arabidopsis High Affinity Phosphate Transporter at High Levels", page 199, Abstract 995. *
PROC. NATL. ACAD. SCI. U.S.A., September 1996, Vol. 93, MUCHHAL et al., "Phosphate Transporters from the Higher Plant Arabidopsis Thaliana", pages 10519-10523. *
THE PLANT CELL, March 1997, Vol. 9, LEGGEWIE et al., "Two cDNAs from Potato Are Able to Complement a Phosphate Uptake-Deficient Yeast Mutant: Identification of Phosphate Transporters from Higher Plants", pages 381-392. *
THE PLANT JOURNAL, January 1997, Vol. 11, No. 1, SMITH et al., "The Cloning of Two Arabidopsis Genes Belonging to a Phosphate Transporter Family", pages 83-92. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005760A3 (fr) * 1996-07-31 1998-10-08 Univ Kingston Proteines pouvant etre induites en cas de privation de phosphate
US6096545A (en) * 1996-07-31 2000-08-01 Queen's University At Kingston Phosphate starvation-inducible proteins
WO1998038295A1 (fr) * 1997-02-24 1998-09-03 Performance Plants, Inc. Promoteur activable par une deficience en phosphate
US5922564A (en) * 1997-02-24 1999-07-13 Performance Plants, Inc. Phosphate-deficiency inducible promoter
US6175060B1 (en) 1997-02-24 2001-01-16 Performance Plants, Inc. Phosphate-deficiency inducible promoter
WO1999058657A3 (fr) * 1998-05-13 2000-07-27 Pioneer Hi Bred Int Genes transporteurs de phosphate de zea mays
WO2001055299A3 (fr) * 2000-01-27 2002-01-17 Yeda Res & Dev Polynucleotides isoles codant pour un nouveau transporteur de phosphates dans des plantes et procede de modulation de l'absorption de phosphates dans des plantes
KR100401007B1 (ko) * 2000-08-11 2003-10-08 윤성중 담배의 인산수송자 유전자

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