JP2001513326A - Controlled germination using an inducible phytic acid gene - Google Patents

Abstract

  (57) [Summary] The present invention provides a plant containing a phytic acid gene that is expressed in the plant only when desired. The invention has two properties that can be induced or switched. Its properties are (i) non-germinable seeds, and (ii) low or no phytic acid production.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This invention is based on and claims the benefit of US Provisional Application No. 60 / 055,323, filed August 11, 1997, the entire contents of which are incorporated herein by reference. Has been referred to as.

[0002] The present invention relates to a plant containing an inducible gene (phytic acid gene) that controls seed germination by regulating phytic acid content, a method for producing and using the plant, and a plant derived therefrom. Seeds, and their progeny are also provided.

BACKGROUND OF THE INVENTION [0003] Low phytin mutants in maize are known. The era of recombinant DNA is nearing financial success in the agricultural industry. Herbicide-tolerant and insect-tolerant cotton is being marketed extensively, as are soybeans and corn. Prior to the transformed plants, some crops had herbicide tolerance due to mutations in the crop. For example, IT ^TM corn Garst Seed Company production, herbicide tolerance ^{(Imazethapyr) HERBICIDE PURSUIT R (American} Cyanamid are mutant plants.

[0004] Mutant plants have also been used to create value-added properties in many crops. For example, maize uses waxy, amylose extenders and other mutants to create special starch properties, and sweet corn utilizes sweet mutants.

[0005] The low phytic acid maize mutant, lpa1-R, was created by Raboy (US Pat. No. 5,689,054, which is described in the specification as lpa-1-1).
. The market introduction of this lpa1 mutant is expected to occur during 1999. A number of companies are currently testing this new low phytic acid mutant. By using mutagens and mutagenic methods to breed maize, a number of different mutants of maize and other species can be created with this low phytin property.

[0006] Transposon labeling is a known method of gene identification and sequencing that involves mutating plants with transposable elements. In one advantage, the method provides a means to identify and sequence the gene associated with the mutation. Once a gene has been cloned, it can be transferred between species. Methods for transferring genes from one plant species to another by using gene identification, sequencing and transformation are well known in the industry. Virginia Walbo
“Strategies for Mutagenesis and Gene Cloning Using Transposon
Annu. Rev. Plant entitled “Tagging and T-DNA Insertional Mutagenesis”
Physiol. Plant Molecular Biology 1992, 43: 49-82. A specific example of the use of transposon labeling with the mu1 element for genetic isolation and cloning of the maize amylose-enhancing gene is described in Philip Stinard et al., Genetic Isolate
ation, Cloning and Analysis of a Mutator-induced, Dominant Antimorph of
Plant Cell, Vol. 5, pp. 1 entitled “The Maize Amylose Extender 1 Locus”
555-1566, (November 1993). In this article, we report a mutant plant that has been subjected to transposon labeling and identification of the mutated gene. This gene was sequenced by methods known in the art. The sequenced portion was used as a probe to identify the mutant gene, which was then cloned. Gene cloning has been performed and is well established as a technique. The techniques of DNA recombination used throughout the present invention are known to those skilled in the art and are described in Maniatis et al., MOLECULAR CLONING: a Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0007] Switchable or inducible transformation constructs are known and can be made by means known in the art. Often, after cloning a gene, it is placed in a construct used to transform a plant. This gene, or an allele, or truncated, substituted or deleted variants thereof, can be placed in a transformation vector at a sense or antisense position. Negative regulation of a native gene by antisense does not require insertion of the entire gene in the antisense orientation, but rather only that portion of the sequence that would provide negative regulation. Those skilled in the art will recognize that. Thus, fragments of a gene, such as those containing 30-100, preferably 45-65 nucleobases, may be useful in negatively controlling gene expression.

[0008] Transformation vectors for plant transformation can be purchased from Clontech and other commercial sources. The vector is preferably selected to provide materials suitable for the plant species and the transformation method used. For example, if the plant species is susceptible to Agrobacterium transformation, the vector selected may include the T-DNA portion in the vector. The promoter, intron, leader sequence, and selectable marker must be selected so that the expression of the amino acid encoded by the gene is in the desired amount in the desired plant. In addition, the vectors can be optimized for the particular host cell to be transformed by optimizing the use of specific codons.

Recently, in order to controllably activate a gene linked to a promoter,
Inducible promoters have been used. An inducible promoter activates or deactivates the gene in response to an activator.

For the purposes of the present invention, a promoter is turned on to express a gene or operably linked nucleic acid sequence (transcription only or including transcription and translation), or Can be turned off so that either the gene or the operably linked nucleic acid sequence is not expressed (including transcription only or including transcription and translation). A promoter that changes the expression level when contacted with an activator can also be used. Such promoters are well known in the art and are "switchable" or "inducible".
Called. Inducible promoters activate genes in response to heat, light, moisture, chemicals and the like. U.S. Patent No. 5,608,143 describes the use of a nucleic acid promoter that responds very well to a large number of substituted benzenesulfonamides as activators. The inducible promoter has been shown for use in recombinant DNA constructs to allow expression of the gene to be controlled by external chemical regulators that act as activators.

[0011] United States Patent No. 5,432,068 describes an externally inducible promoter for use in controlling male fertility. In WO 90/08826, a gene promoter responding to a plant herbicide safener (N, N-diallyl-2,2-dichloroacetamide) used as a gene switch to enable external regulation of gene expression Sequence (GSTII (glutathione-S-transferase isoform II)) is described. Other promoters that are inducible by external chemicals are known in the art.

[0012] Methods for transforming monocots and dicots are known. Constructs suitable for the plant species to be transformed are readily purchased or made by those skilled in the art.
These constructs are then transformed into the cells or tissues or pollen of the plant. Methods of transformation include, but are not limited to, microparticle bombardment, whiskering, electroporation, Agrobacterium, and the like. The efficiencies of these approaches are different and one of skill in the art can select the method according to the type of tissue and plant species to be transformed.

[0013] Selective breeding for the properties of interest is known, such as the creation of a commercially available Top Cross ^R strain which is a high oil corn. Once the transformants have been improved, they can be used (if necessary) in breeding methods with other plants of that type. One skilled in the art will recognize that the practice of breeding depends on the species selected. For example, soy is developed as a cultivar, while corn is produced as a hybrid. One method of producing a patented maize by Dupont does not use hybrid seeds, but rather uses male non-germinating hybrid seeds in combination with a male plant that is a pollen source. Rather than hybrid seeds, farmers plant male non-germinating hybrid seeds and male plants with high oil properties and pollen sources (maze inbred seeds). Hybrid canola has been developed. In order to gain hybrid vigor, but more importantly to reduce the germplasm risk associated with the production of plants that can reproduce from seeds, most crops that are not currently hybrid Instead, attempts have been made to migrate to hybrid seeds (separate hybrid seeds but not inbred seeds).

[0014] There is a continuing need for grains with nutritional value due to the reduction of phytic acid produced in plants. There remains a need for methods of producing lethal seeds that inhibit the growth of native plants. There is still a need for inducible lethal properties in the seeds of many plant species.

SUMMARY OF THE INVENTION It is an object of the present invention to provide seeds with very low phytic acid content, especially maize seeds.

It is another object of the present invention to provide cereals that have nutritional value by reducing phytic acid produced in plants, preferably cereals that are not maize.

Non-naturally occurring non-germinable seed, comprising at least one genetic construct, under the control of and controllably linked to an inducible promoter or other genetic element. It is yet another object of the present invention to provide non-expressible seeds, wherein the at least one genetic construct produces germinable seeds when induced. The seeds of the present invention have a non-germinable property or phenotype, and further, under the control of an inducible promoter, such that when the promoter is induced, the non-emergent property is corrected or overcome to germinate the seed. Genetic factors in

A germinable seed comprising at least one genetic construct, under control of, and operably linked to, a non-naturally occurring inducible promoter or other genetic element, It is another object of the present invention to provide a seed whose at least one genetic construct produces a seed that is not able to germinate when induced. The seeds of the present invention have a germable property or phenotype, and further under the control of an inducible promoter, such that when the promoter is induced, the germinable property is destroyed or overcome and the seed does not germinate. Genetic factors in

It is a further object of the present invention to provide nucleic acid molecules (sequences) encoding proteins involved in phytic acid synthesis, as well as the encoded phytic acid and antibodies thereto.

One object of the invention is a seed of the invention, preferably comprising a nucleic acid sequence encoding phytic acid, or an allele, or a truncated, substituted or inserted variant or fragment thereof. The present invention is to provide a vector (expression and cloning) and a method for using the same to make E.

A further object of the present invention includes providing a method for producing inducible non-germinable seeds and inducible germinable seeds.

It is a still further object of the present invention to sow and grow the germinable mature seeds of the present invention, and to apply a activating factor to the plant produced thereby to prevent daughter seeds or embryos from germinating. A method of preventing the growth of native plants from fallen seeds, including the production of

Still further objects and advantages will become apparent by consideration of the description and the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION EMS (ethyl methanesulfonate) has been used to induce a mutant phytic acid gene in maize to create low phytic acid seeds. For example, the low phytic acid maize mutant lpa1 was created by Raboy (US Pat. No. 5,689,054). In some cases, the seed may be low enough phytic acid to prevent seed germination. In other cases, low phytic acid seeds are known to germinate normally.

From these mutant plants, the genes of the present invention are reproducibly isolated and cloned. The cloned gene encodes an enzyme that affects the production of phytic acid in seeds. The genes of the present invention include allelic variants, truncated variants, deletion variants and substitution variants of the cloned gene, as well as the gene as described herein in an antisense manner. Useful fragments are also included, such as those that can be used to negatively control. The present invention provides a transformation vector comprising a novel gene operably linked to an inducible promoter so that the lethal effects of that gene can be controlled, exploited or controlled.

This vector can be used to produce seeds with very low phytic acid content, and since the seeds can only germinate under highly controlled conditions after induction, soy, wheat, barley Useful for non-hybrid cereals, such as, maize, canola and sunflower. This vector of the present invention, when present in a plant, has several advantages: That is, (i) it is useful for farmers to remove indigenous plants in the next season by making seeds unable to germinate; (ii) to prevent seeds from breeding for breeding purposes (Iii) prevent farmers from storing seeds by preventing them from breeding in the future, and (iv)
The low phytic acid content is useful in the feed and milling industry to add nutritional value to the feed or milled product. The invention is particularly useful in flour and feed cereals. These include, but are not limited to, corn, wheat, soy, sunflower, oats, rye and the like.

Thus, the present invention has been modified to include a functional gene encoding phytic acid that is operably linked to and under the control of an inducible promoter,
Provided is a method for preserving seed comprising providing a non-germinating daughter seed or embryo having low phytic acid.

Gene Targeting and Isolation The genes of the present invention can be isolated, for example, by crossing with a mutant maize maize plant having a genetic mutation that reduces the amount of phytic acid in seeds.
From this gene, wild-type or natural genes and mutant genes can be identified. Both natural or mutated phytic acid genes can be used in the present invention.

In one embodiment, the present invention provides a method for synthesizing phytic acid such that it is expected to at least drastically reduce the amount of phytic acid in seeds, even when controllably linked to a weak promoter. A method for producing and using a seed such that the phytic acid content of a seed is removed or severely reduced by inserting an antisense construct of a gene or a fragment thereof encoding a protein involved in the sense direction in the sense direction, and seed derived therefrom And plants.

In another aspect, the present invention is expected to increase seed phytic acid levels even when controllably linked to the native gene or a fragment thereof, ie, a weak promoter. Seeds of a native gene mutated to produce a low phytic acid phenotype, as known or known, such that insertion of the sense construct restores the phytic acid content of the low phytic acid mutation containing gene. Methods of making and using, and plants derived therefrom are provided.

One method of isolating the nucleic acid sequence encoding phytic acid of the present invention involves the use of transposon-labeled mutants that allow cloning of the gene by means known in the art. by. The transposable element labels the gene, in other words, the sequence adjacent to the transposon insertion site is part of the gene of interest. In the present specification, in accordance with the present invention, for example, maize as a typical example, a low phytic acid mutant inbred line is crossed so as to become a line containing the mu1 mutagenic gene, and the mu1 factor Identifies low phytic acid mutant gene by insertion of DNA. Mu1 used in an exemplary embodiment of the invention is part of the mu1-9 family, which has a common inverted repeat of the 〜20-bp terminus. Multiple copies of the transposon are included in a mutagenic strain that is an active mutagen in maize. Most of the mu insertions confer a null phenotype because transposable element insertions do not result in an altered intact protein. With targeted transposon labels, the insertion targets specific known mutations, such as the low phytate gene. Transposons occur in the range of ~ ^10-4 to ^10-6 insertions per locus per chromosome analyzed. To determine when the phytic acid gene has inserted a DNA transposon therein, identify the mutant phenotype from the insertion results and identify the restoration of partial or complete function in gametes from the excision results There is a need. The labeled mutant can then be crossed with a low phytic acid mutant plant having a recessive low phytic acid gene. If the transposable element is settled or inserted into the gene of interest, the cereal from the cross can be identified as a low phytic acid cereal or a low phytin cereal. The gene is recessive and the effect is only present if both parents have a low phytic acid gene. Allelism is confirmed in a similar breeding crossing procedure using similar known low phytic acid mutants.

When using a low-activity factor, thousands of progeny are required to find a metastasis result. The use of mu1 substantially reduces the number of progeny needed to screen the characteristics of the cereal. Depending on the transposable element used, it may be useful to backcross inbreds with the targeted mutant gene.
In this typical example of the present invention described in this specification, backcross was used because the mu1 strain was used. The mu1 line can be identified as having lost mutagenic activity through a backcrossing procedure by monitoring a reporter gene in the anthocyanin pathway that produces a purple pigment in the seed. Backcrossing to a standard maize inbred simplifies segregation analysis because the copy number is greatly reduced. Alleles are confirmed using similar known variants. The mu1 element is cloned using a transposon element as a probe for cloning a mutant allele or a probe for searching for a natural wild-type gene. By this method, natural and mutant genes that are low phytic acid in seeds can be identified and then isolated, purified and sequenced by methods known in the art.

As an effective means of cloning a functional gene, methods for finding alleles that can be mutated include a mutant labeled with an insertion factor, a clone of the factor, and a clone to be recovered. Assays and simple genetic analysis are required. Loss of reporter gene activity is used to indicate loss of mutagenic activity. Generally, this process involves a process called co-separation. This involves selecting and recovering tissues for molecular cloning; preparing DNA from the progeny of crosses of various lines; and several m / m (homozygous recessive mutant genes) from each line. ) This involves making samples from individuals and several M / M (homozygous wild-type genes) individuals. Next, Southern blots were prepared from DNA obtained from the mu-labeled material after digestion of the sample with some 5-methylcytosine-insensitive enzymes that did not cleave the mu1 element that marks the mutant; Bands present in / m individuals but not in M / M individuals are identified by localization; DNA pools of m / m individuals are prepared and three samples from each of the various strains are taken. Find and select the darkest band and the only common, diploid copy fragment. This process of co-segregation will identify the mu factor that labels the mutant gene. The results are confirmed by separating and analyzing the fragments. The fragment is identified from the Southern blot by locating the line with the fewest number of segregating bands, without any evidence of a co-migrating band. Large samples of the m / m and M / M progeny of this line are analyzed to assess the statistical significance between the band and the phenotype. Various restriction enzymes are used for confirmation. Samples that are significant in this population analysis are cut with several enzymes to screen for enzymes that do not cut the band. This is done in combination with a high resolution agarose gel, as a double cut with the first restriction enzyme and an enzyme that cuts little of the band. The m / m cleaved pool of uncleaved DNA is then replicated and the DNA is size-selected and cloned.

The gene cloned by the process described above is a gene that encodes an amino acid that produces a protein that functions to alter the amount of phytic acid in the plant. These genes are called phytate genes (phytic acid genes).

Inducible Promoters A number of compounds are useful for controlling gene expression in plants. The inducer of gene expression must be safe and must not have a detrimental effect on the desired agronomic characteristics of the plant.

[0036] Natural products, including hormones, and other chemicals that affect gene expression in plants are known. Plant growth regulators, including AAA, ethylene, abscisic acid, auxin, salicylic acid, and other plant hormones all affect gene expression. These natural chemicals appear to induce gene expression. Hormones that affect gene expression in plants, whether natural or synthetic, can be used with the present invention. However, care must be taken to avoid undesired activation of plant growth by co-activation of endogenous systems in the plant or activation of lethal genes present in the plant. Clearly, the use of hormones as inducers must be linked to hormones that will not activate metabolic systems that are detrimental to the end product.

Environmental inducers for regulating foreign genes include light, heat, cold and different amounts of gas in the atmosphere. These modulators can be used individually or in combination according to the invention. These modulators are not very practical because there are currently no known methods for controlling very many of these environmental inducers. It is not impossible to have a cool growth phase or a cloudy growth phase, in which case these inducers will not be strongly activated or will be activated in inconsistent ways. As such, these types of inducers will be useful with the present invention, but they must be carefully selected.

Other plant genes are induced by polysaccharides present during wound and pathogen infection, such as, for example, glucan induction of phenylalanine ammonia-lyase and chalcone synthase in soybean, or induction of wound-like induction in potatoes. It has been. This effort requires more effort because the plant must be damaged to induce the foreign gene.

The inducer chosen is such that it is safe and has little or no effect on the plant on which it acts, in most cases the most useful inducer. Preferred external regulation of the inducer is a substance that induces expression of the gene of interest in any tissue at any time in the life cycle of the plant. This requires a promoter that is activated or inactivated when an activator, such as a chemical, acts. This regulation is achieved by modulating this response in many plant species with little effect on plant growth. The ideal activator is a chemical that can be applied with standard field equipment in combination with others, such as herbicides, that are usually applied to the field to avoid overseeing the field repeatedly It is. Alternatively, the material could be used in the air with a crop-spreading device.

In at least one embodiment of the present invention, the production of an enzyme that protects a plant when activated by an activator commonly used with herbicides, commonly referred to as a safener. A promoter known to enable (drive) is used. It is assured that plants are more protected from herbicidal activity if the toxicity is reduced. One example of a safener is one that acts to conjugate the herbicide to glutathione. This is because the increase in glutathione-S transferase (GST) activity increases GST mRNA in plants with reduced toxicity. Thus, this safener treatment increases the gene product but does not negatively affect the plant.

Maize is treated with anhydrous nafar (na) when a sulfonylurea herbicide is used.
phal) acid, N, N-diallyl-2,2-dichloroacetamide or thyometrinil (c
It has been shown that toxicity can be mitigated by a wide variety of substances, including but not limited to yometrinil). The metabolic rates of chlosulforon and metsulfuron methyl increase within hours of application of safeners. In the present invention, it is a promoter of the GST enzyme and used as a switchable promoter linked to the phytic acid gene of the present invention. Thus, switching is achieved by using a switch-derived chemistry.

The gene inducer useful in the present description can be any number of inducers or activators. United States Patent No., clearly titled "Gene Switch"
Inducers in any of 5,608,143 (incorporated by reference) and / or WO 90/08826 (incorporated by reference herein) are useful.
Methods of using inducers for switches on genes are known in the art and can be practiced in accordance with the teachings of the present invention. These methods are also described in WO93 / 09237.

Preferred promoters of the present invention are those that are responsive to safeners used in herbicide formulations. In nature, these promoters and related genes are induced by safeners in the herbicide to protect the plant from damage that the herbicide can inflict on the plant in the absence of the safener. In the present invention, these promoters and heterologous genes are activated and switched on and off, preferably by safeners. The safener is preferably applied in the herbicide, and the marker gene can render the plant resistant to such herbicides if they were not previously resistant. Alternatively, a chemical such as a safener may be applied to the plant alone.
This may be useful if the initial application of the herbicide is significantly earlier than the emergence of the seed.

The present invention therefore provides a method for synthesizing phytic acid that is expected to at least drastically reduce the amount of phytic acid in seeds, even when controllably linked to a weak promoter. A method of producing and using a seed such that the phytic acid content of the seed is removed or severely reduced by inserting an antisense construct of a gene or a fragment thereof encoding a protein or a fragment of the protein involved in the sense direction; Seeds and plants derived therefrom; by the action of chemicals or environmental factors on the plants produced from the seeds, to produce seeds with a significantly reduced amount of phytic acid when compared to the original seeds In addition, promoters or promoters induced by chemicals or environmental factors, preferably safeners Or a construct operably linked to another genetic element.

Preferably, the seed produced from the plant that has received the factor has a control phytic acid content of less than about 5% by weight than the seed that produced the plant that has received the factor, more preferably Phytic acid having a control phytic acid content of less than about 2% by weight, more preferably less than about 1% by weight, is included.

Those skilled in the art will recognize that common means of measuring phytic acid are that these comparisons will be the most common average of an extract of a seed produced as described herein. Would admit that seed destruction is necessary. It is a further aspect of the present invention to provide seeds that are not germinable after induction of the parent plant, despite the reduced amount of phytic acid compared to the parent seed.

In another aspect, the invention is expected to increase seed phytic acid levels even when controllably linked to the native gene or a fragment thereof, ie, a weak promoter. A low phytic acid mutant-containing seed by inserting a sense construct of a native gene or a fragment thereof mutated to produce a low phytic acid mutant low phytic acid phenotype, as known or known Method of producing and using seeds that restores the phytic acid content of plants, and plants derived therefrom; by subjecting plants produced from the seeds to chemicals or environmental factors, comparing with the original seeds Induced by chemicals or environmental factors, preferably safeners, to produce daughter seeds with increased amounts of phytic acid And a construct operably linked to a promoter or other genetic element. Preferably, the seed produced from the plant that has received the factor has about 5% or more, more preferably the weight of control phytin, of the control phytic acid content greater than the seed that produced the plant that has received the factor. Phytic acid is included that is greater than about 8% of the acid content, more preferably greater than about 10% by weight of the control phytic acid content.

[0048] One of skill in the art will appreciate that the plants and seeds of the present invention with reduced or reduced phytic acid can be used alone or together with the phytic acid genes and variants described herein.
It will be appreciated that it may be produced by transformation with a nucleic acid molecule encoding phytase, preferably operably linked to an inducible promoter of the invention. Expression of any of these phytases (phytate degrading enzymes) during cereal development can also be used to degrade phytic acid synthesized in developing seeds. At the NCBI or elsewhere, the following accession numbers: P34752, gi | 464382
| Sp | P34752 | PHYA # ASPNG [464382]; P34753, gi | 464381 | sp | P34753 | PHYA
#ASPAW [464381]; 2599490 (AF029053) phyb13 precursor [Bacillus subtilis] gi
| 2599490 [2599490]; 1lHPgi | 2981783 | pdb | 1lHP | [29817383]; 3150040 (U85968) gi | 3150040 [3150040]; 2943981 (U92439) phytase [Enterobac
ter cloacae] gi | 2943981 [2943981]; P34754, gi | 464385 | sp | P34754 | PHY
B # ASPNG [464385]; P34755, gi | 464384 | sp | P34755 | PHYB # ASPAW [464384]; P07102, gi | 130735 | sp | P07102 | PPA # ECOLI [130735]; 2108356 (U59802)
Phytase [Talaromyces thermophilus] gi | 2108356 [2108356]; 2108354 (U
59804) phytase [Aspergillus fumigatus] gi | 2108354 [2108354]; 210835
2 (U59803) phytase [Emericella nidulans] gi | 2108352 [2108352]; 2300
890 (A46793) gi | 2300890 | gnl | PID | e306401 [2300890]; 2300889 (A46793) gi | 2300889 | gnl | PID | e306184 [2300889]; 230087 (A46791) gi | 230088
2300885 (A46789) gi | 2300885 | gnl | PID | e306183 [2300885]; 2300883 (A46787) gi | 2300883 | gnl | PID | e306182 [2
300883]; 2300881 (A46785) gi | 2300881 | gnl | PID | e306399 [2300881]; 23
00879 (A46783) gi | 2300879 | gnl | PID | e306181 [2300879]; 2148991 (U6041
2) Phytase [Aspergillus terreus] gi | 2148991 [2148991]; JN0715 3-phytase (EC3.1.3.8) B precursor-Aspergillus ficuum gi | 542378 | pir || JN0
715 [542378]; JN0890 acid phosphatase (EC 3.1.3.2) precursor-Aspergillus
awamori gi | 542377 | pir | | JN0890 [542377]; JN0889 3-phytase (EC3.
1.3.8) A precursor-Aspergillus awamori gi | 542376 | pir || JN0889 [542376]; JN0656 3-phytase (EC3.1.3.8) A precursor-Aspergillus niger gi | 484414 | pir || JN0656 [484414] JN0482 3-phytase (EC 3.1.3.8) A-Aspergillu
s ficuum gi | 419906 | pir || JN0482 [419906]; PQ0641 3-phytase (EC3.
1.3.8) -Aspergillus ficuum (fragment) gi | 542374 | pir || PQ0641 [542374];
S33278 phytase P2-Escherichia coli (fragment) gi | 421153 | pir || S33278 [4
21153]; B36733 acid phosphatase (EC 3.1.3.2) precursor-Escherichia coli g
i | 96267 | pir || B36733 [96267]; S18408 alkaline phosphatase (EC3.1.3
.1) rat gi | 111353 | pir || S18408 [111353]; 1943870 (U59806) phytase [Thielavia heterothallica] gi | 1943870 [1943870]; 1943868 (U59805) phytase [Aspergillus terreus] gi | 1943868 [1943868]; 1938256 (U755
31) Phytase [Zea mays] gi | 1938256 [1938256]; gi | 1831488 | pat | US | 5593963 | 20 [1831488], 408990 phytase (EC3.1.3.26) [Aspergillus fi
cuum, peptide, 441aa] [Aspergillus ficuum] gi | 408990 | bbs | 130910 [40
8990], 235916 gi | 235916 | bbs | 58159 [235916], 235915 gi | 235915 | bbs | 58160 [235915], 2393 gi | 2393 [2393]
All sequences and text of 963; gi | 912286 | pat | US | 5436156 | 32 [912286], 583196 (A19452) gi | 583196 [583196], 583194 (A19451) gi | 583194 [5831
94], 166521 (M94550) gi | 166521 [166521], 166519 (L02421) phytase [
Aspergillus niger] gi | 166519 [166519], 166482 (L02420) acid phosphatase [Aspergillus niger] gi | 166482 [166482], 304097 (L20567) phyB [Asp
ergillus niger] gi | 304097 [304097], and the sequence encoding phytase is known, as published by either of the texts. Methods and constructs useful in the present invention are also found in US Patent Nos. 5,770,413 and 5,593,963.

In a further aspect, the construct and the promoter or other genetic element are
It is operably linked to a marker gene as is known in the art.

In one aspect of the invention, the plants and seeds described in the aspects of the invention are any one of maize, soy, rice, oats, sunflower, wheat, barley, fodder and similar. Good.

Constructs The present invention includes constructs having a promoter associated with the phytic acid gene, wherein the promoter, when activated, has too little phytic acid to cause seeds to fail to germinate (ie, a plant that matures). Under external control so as not to be able to form). This construct can be transformed into any number of transformable plants. The positioning of the gene in the construct determines whether the gene is in a sense direction that is upregulated or an antisense direction that is repressed.

FIG. 1 shows a binary notation of a construct according to the invention. This construct was originally developed for use in Agrobacterium transformation. Therefore,
It is mainly developed for use in dicotyledonous plants and monocotyledonous plants capable of Agrobacterium transformation. The constructs include a right T-DNA border, a NOS promoter, an NPTII marker gene and a NOS terminator, an inducible promoter, in this case a GSTII promoter sequence, and a phytic acid gene and NOS terminator in the sense orientation and a left T- Contains DNA boundaries. This construct is ideal for soybeans and similar cultivars. The marker gene can be selected to be a herbicide resistance gene, such as a glyphosate resistance gene.

FIG. 2 shows a binary representation of a construct according to the invention developed for use in dicotyledons and Agrobacterium-transformable monocotyledons. The construct includes a right T-DNA border, a Nos promoter, an NPTII marker gene and a NOS terminator, in this case, a gene encoding the cDNA clone 5-2 described in U.S. Pat.No.5,608,143, the sequence of which is shown in FIG. Includes the indicated promoter region, the inducible promoter, and the phytic acid gene and NOS terminator in the antisense orientation and the left T-DNA border. The safener is applied to activate the promoter, thus allowing the product encoded by the gene to be expressed by the application of a herbicide containing the safener or simply by the safener.

FIG. 3 shows, in this case, the American Type Culture Collection (ATCC), Manassas
U.S. Patent No. 5,608,143 (Accession No. 67804, deposited with VA)
The cDNA clone 5- described in this patent is incorporated herein by reference).
2 shows the construct having the promoter, a promoter region from that shown in FIG. 5 therein, and the phytic acid gene and NOS terminator of the present invention. The phytic acid gene is in the sense orientation. Thus, the activator will switch on when acting on the promoter, and the gene will express its encoded amino acid.

FIG. 4 shows a construct having a promoter, in this case a GSTII promoter sequence, and a phytic acid gene and a NOS terminator of the invention. The phytic acid gene is in the antisense orientation.

These constructs can be transformed in their various forms into maize, soy, rice, oats, sunflower, wheat, barley, fodder grass and the like. The present invention includes optimized, more active enzymes or optimal antisense or co-expression for expression in each of these plants, for example, with respect to codon usage, promoters, and any other process that enhances expression. Constructs that are optimized to produce a suppressive effect are included. In addition, the constructs of the present invention can be transformed into already transformed or already mutant plants. For example, using this gene construct, existing low phytic acid variants can be externally regulated by the application of an activator. The promoter in the construct can act to induce expression of the gene, or the activator can stop expression of the gene by inducing the promoter to stop. Similarly, the orientation of the gene in the construct can be in the sense or antisense orientation, but up-regulates gene expression in the sense orientation and represses gene expression in the antisense orientation. Therefore, the same effect can be produced by switching the promoter from inducible on to inducible off, or by changing the direction of the gene.

Transformation A typical illustration of a vector according to the invention is shown in the first four figures. First two
Two figures show constructs adapted for Agrobacterium transformation. The next two figures show plasmids for use in other transformation techniques, such as microparticle bombardment. These transformation techniques are well known to those skilled in the art. in addition,
These constructs are very good for use in the whisker transformation system taught in US Pat. No. 5,302,523 and US Pat. No. 5,464,765, which are incorporated herein by reference.

[0058]

【Example】

Example 1 There are four preferred methods for producing the low phytic acid seed material of the present invention. The preferred method provides the farmer with a seed product that is low phytic acid and that is not germinable so that the seed cannot produce a mature plant. Preferred seeds of the invention terminate the seeds stored by farmers in the case of non-hybrid cultivars. Despite ongoing research to develop hybrid potential in both wheat and canola, most canola and wheat and soy are not currently sold as hybrids. If the plant is not normally sold as hybrid seeds, but instead as seeds such as wheat and soy, the material according to the invention is very easily produced.

Crossing of transformants with inbred material When the promoter is induced to turn on the gene, the gene is in the antisense orientation and seeds with very low phytic acid levels due to the action of activators. Will produce. Such seeds will not germinate. The activator is
It is preferably applied when the grain is full or shortly before. The activator may preferably take the form of a safener, such as may be in a herbicide, if the promoter is GSTII or another promoter driven by a safener.
Thus, the activator is not sprayed on the seeds while they are growing. This allows
Seeds with normal phytic acid levels can be produced. However, when seeds are sold to farmers for the production of low phytic acid grains, activators act on the plants to produce seeds that are low or no phytic acid. Due to the low phytic acid content in the seed, the activator will also prevent the seed from germinating. Therefore, the seeds of soy or other plant species will not grow as native plants in the next season,
Seeds cannot be stored for use in the next year for cultivation purposes.

In this embodiment, the invention relates to a mature seed comprising a sufficient amount of phytic acid to germinate, comprising a nucleic acid molecule encoding phytic acid in the sense orientation or a fragment thereof or an allelic variant thereof. A plant comprising an inducible promoter encoded and operably linked to a nucleic acid molecule positioned antisense to the promoter in the construct, such that the plant has grown from mature seeds. The invention provides a mature seed that produces a seed or embryo that produces a product that does not germinate when the promoter is activated by an activator. The present invention provides plants produced from the mature seeds and methods for reducing the growth of native plants and reducing the amount of germinable stored seeds, including the act of activating factors on the plants.

When a promoter is induced to turn off a gene, the gene is in the sense orientation and in the absence of the promoter and the construct containing the gene, the plant is seedless in the absence of the promoter and gene. In order to prevent germination, the seeds will have to have a mutant form of the gene that produces very low levels of phytic acid. Applying an activator to a plant produced from a mature seed containing the construct of this embodiment will produce a non-germinable, developing seed or embryo containing very low amounts of phytic acid. If the promoter is, or comprises, a fragment of the promoter shown in FIG. 5, the activator is in the form of a safener, preferably in a herbicide. The activator is preferably a substituted benzenesulfonamide. Thus, while the seeds are growing, i.e., while the progeny of the mature seed of this aspect of the invention is growing to increase the number of mature seeds of this aspect, the plants produced from the mature seed of this aspect will not Do not spray activator. However, if the mature seed of this embodiment grows and provides the seed or cereal to a farmer or end consumer for the production of a low phytic acid product, the activator may be used in this embodiment of the invention. Acting on plants produced from mature seeds to produce low phytic acid, non-germinable developing seeds (embryos or progeny seeds).
As a result, low phytic acid seeds are produced, rendering the seeds non-germinable.
Thus, the seeds of soy or other plant species will not grow as native plants in the next season and the seeds cannot be stored for use in cultivation purposes in the following year.

In this embodiment, the invention relates to a mature seed comprising a sufficient amount of phytic acid to germinate, wherein the promoter is activated by an activator in a plant grown from the mature seed. A sufficient amount of a gene construct comprising an inducible promoter operably linked to a nucleic acid molecule encoding phytic acid to produce a developing seed or embryo by a non-germinating plant. A mature seed is provided such that at least a portion of the phytic acid is produced. Mature seeds of this embodiment do not produce enough phytic acid to germinate in the absence of the construct. The present invention provides plants produced from the mature seeds and methods for reducing the growth of native plants and reducing the amount of germinable stored seeds, including the act of activating factors on the plants.

If the promoter is induced to turn on the gene and the gene is in the sense orientation, the plant must have a mutant form of the gene in the seed that produces very low amounts of phytic acid. Would. Such seeds will not be able to germinate.
Actuation of the activator of this aspect will increase the amount of phytic acid produced and thus restore germination potential. The activator is in the form of a safener, preferably in a herbicide, if the promoter is or contains a fragment of the promoter shown in FIG. The activator is preferably a substituted benzenesulfonamide. An activator may be added to the plant produced from the mature seed of this embodiment during the growth of the seed, i.e., during the growth of the mature seed progeny of this embodiment of the invention to increase the number of mature seed of this embodiment. Spray. However, if the mature seed of this embodiment grows and provides the seed or cereal to the farmer or end consumer as a low phytic acid content seed or cereal, the activator must not act. As a result, very low phytic acid seeds are produced, and the seeds also become non-germinable. Thus, the seeds of soy or other plant species will not grow as native plants in the next season and the seeds cannot be stored for use in cultivation purposes in the following year.

In this embodiment, the invention is directed to a mature seed comprising a sufficient amount of phytic acid to germinate, wherein the promoter is activated by an activator in a plant grown from the mature seed. A sufficient amount of the gene construct comprising an inducible promoter operably linked to a nucleic acid molecule encoding phytic acid so that the germinable plant produces a developing seed or embryo. In which at least a portion of the phytic acid is produced. Mature seeds of this embodiment do not produce enough phytic acid to germinate in the absence of the construct. The present invention provides plants produced from the mature seeds and methods for reducing the growth of native plants and reducing the amount of germinable stored seeds, including the act of activating factors on the plants.

If the promoter is induced to turn off the gene and the gene is in the antisense orientation, the plant without the promoter and operably linked gene will have a normal amount of seed in the seed. It would have to include normal or natural genes capable of producing phytic acid. Such seeds will not be able to germinate with some of their promoters and operably linked genes. Upon activation of the activator, production of phytic acid will be restored, so that seeds with the promoter of this embodiment and the operably linked gene will germinate normally. The activator takes the form of a safener in the herbicide if the promoter is or contains a fragment of the promoter shown in FIG. The activator is preferably a substituted benzenesulfonamide. During seed growth as described above, the activator is applied to plants produced from mature seeds of this embodiment. However, if the seed is sold to farmers or end consumers as low phytic acid as described above, the activator must not act. This results in the production of low phytic acid seeds, which also renders the seeds incapable of germination in low seed phytic acid and in the absence of activators. Thus, the seeds of soy or other plant species will not grow as native plants in the next season and the seeds cannot be stored for use in cultivation purposes in the following year.

In this embodiment, the invention relates to a mature seed comprising phytic acid in an amount sufficient to germinate, wherein the nucleic acid molecule encodes phytic acid in the sense orientation or a fragment thereof or an allelic variant thereof. A nucleic acid molecule that is located in an antisense orientation relative to the promoter in the construct and an inducible promoter operably linked thereto, wherein the promoter is grown in a plant grown from mature seeds. Provided is a mature seed comprising the genetic construct such that when activated by an activator, a developing seed or embryo that can germinate is produced. The mature seed of this embodiment, in the absence of this construct, can produce at least enough phytic acid to germinate. Due to the antisense arrangement of the nucleic acid molecule in the construct, phytic acid has been reduced to the extent that developing seeds or embryos from plants produced from this mature seed will not germinate unless an activator is applied to the plant. The amount of is reduced. The present invention provides plants produced from the mature seeds and methods for reducing the growth of native plants and reducing the amount of germinable stored seeds, including the act of activating factors on the plants.

Other combinations of gene orientation and promoter inducibility and other activators are known. Toxicants or pesticide activators are preferred to reduce the time and cost of filling the field in no more than one time. In other words, it offers the dual benefit of acting as a herbicide or pesticide and as a gene inducer.

Crossing of Transformants with Hybrid Material The orientation of the genes useful in the present invention and the number of combinations of mutants and wild-type
Increased when the plant is a hybrid plant. In the method of crossing the hybrid transformant, for example, as described above, a transforming gene or an allelic variant thereof, a substitution mutation capable of encoding an amino acid sequence necessary for producing phytic acid, Using a hybrid formed by two mutant low phytic acid inbreds, such as at least one of the mutants having a genetic construct, including a deletion mutant or a truncated mutant, preferably the construct Contains the gene encoding wild-type phytic acid in the sense orientation. During production, the lethal mutant carrying the transformed wild-type gene is exposed to a gene-inducing spray during seed production. This induces the promoter and turns on the sense gene, which produces the phytic acid necessary to maintain seed germination. This acts as a promoter for the strain and also as a maintenance of the strain. Preferably, the producer of the seed acts the activator symmetrically to the end consumer or farmer. End-users or farmers only need to plant mature seeds. If, as in the preferred embodiment, the inducible promoter is activated by a safener, one skilled in the art will recognize that many plant herbicides may have an activator that will induce the gene. Yes, it will be appreciated by end consumers or farmers that care must be taken in producing this hybrid seed to limit or prevent the use of herbicides, including safeners. Alternatively, the herbicide can be formulated free of the activator of the invention, for application independently of the activator / safener. In this case, it is preferred to apply the herbicide and the activator separately.

Another method of the invention uses a wild-type plant that has been transformed with a wild-type gene that encodes a normal amount of phytic acid when placed in the sense orientation. However, in this aspect of the invention, the wild-type gene is in the antisense orientation and is operably linked to an inducible promoter, as described herein. This gene has been placed in the antisense position to knock out the expression of the wild-type gene in the plant when the plant is exposed to the activator compound. This antisense gene is induced in the field by the action of inducing chemicals or environmental factors before the grain is produced or during the development of the developing seed or embryo.

Alternatively, a naturally occurring mutant plant with a lethal phytic acid level can be used, which, as described herein, overexpresses this gene when the promoter is induced in the plant. Modified to include a sense-oriented wild-type gene operably linked to an inducible promoter inserted to produce a daughter seed or embryo that will be expressed and germinate . Inducible promoters are deactivated by chemicals. The chemical inducer is acted upon, for example, by spraying the plant with the end consumer or farmer before the onset of plant senescence. In this way, the mature seed contains the phytic acid gene required to produce the amount of phytic acid required for germination, but the gene is turned off, no chemicals are applied, and no wild-type gene is expressed.

Thus, the present invention provides a gene switch operably linked to a nucleic acid molecule encoding an amino acid sequence required to produce phytic acid. Depending on the genetic and phenotypic background of the tissue to be transformed and the product of interest (daughter seed or embryo) to be transformed, Therefore, it can be selected by those skilled in the art.

Example 2 A method for creating low phytic acid variants useful for transposon labeling is a known method called mutagenesis. This process is described in the paper Maize Genet by Neuffer.
ic Neweletter 45: 146. Mutations were induced in inbred lines by treating pollen with ethyl methyl sulfonic acid in paraffin oil according to the procedure described by Neuffer (1974). Numerous inbreds from various plant genotypes of cereals were subjected to this treatment. In this example, we will focus on the development of maize low phytic acid mutants by this process. This mutagenesis process has been used to generate a number of cereal variants.

A general step in the process of the present invention involves treating the pollen (in this case, maize) of the inbred with ethyl methyl sulfonic acid, hereinafter “EMS”. Inbred pollen is placed in EMS in oil for 45 minutes. Using a brush, apply the pollen to the hair of the receiving corn ear. This forms mutant-1 (M1) seeds.
Such seeds are grown and self-pollinated to produce mutant-2 (M2) grains. The resulting M2 grains are analyzed for a low phytic acid phenotype.

A preferred maize mutant of the present invention, which can be used as a basis for transposon labeling and phytic acid gene isolation, is the ATCC (July 7, 1998)
No. 203034, deposited with the American Type Culture Collection, Manassas, VA)
, Maize (Zea Mays) inbred line EX1965PY. Alternatively, lpa1 described in United States Patent No. 5,689,054, deposited with the American Type Culture Collection (ATCC) on August 15, 1996 under the terms of the Budapest Treaty and assigned accession numbers ATCC 97678 and ATCC 97679, respectively. lpa1-1) and lpa2 (lpa2-
1) Zea maize seeds, which are mutant heterozygotes, can also be used. Preferred phytic acid genes of the present invention are those obtainable from these deposited materials, preferably by transposon labeling methods known in the art. Further preferred nucleic acid sequences of the invention include sequences encoding SEQ ID NO: 1, SEQ ID NO: 2 and the proteins shown in FIGS. 8 and 9, and accession numbers g3108052 and g3108.
Includes sequences published to NCBI (National Center for Biological Information) under 053. Nucleic acid sequences encoding the amino acid sequences of SEQ ID NO: 2 and those shown in FIGS. 8 and 9 were also able to encode the amino acid sequences required to produce phytic acid, allelic variants, substitution mutations thereof. And deletion mutants or truncation mutants.

Example 3 The HVPE method is a general analysis for low phytic acid variants. (Raboy, Maydic
a 35: 383 (1990)). This method relies on different migration of phosphorus compounds. After fractionation of the compounds by electrophoresis, chromatograms allow for semi-quantitative evaluation of phytic acid compared to other compounds. One alternative involves screening for higher amounts of inorganic P in the cereal. For example, cereal feed is ground (through a 2 mm net on an A Wiley mill) and either 50 mg of cereal embryos or 1 gram of endosperm is added to 15 ml of 0.4 M HCL 0.7 Na ₂ SO ₄ Can be added to Phytic acid precipitates as an iron salt. Determine phosphorus and total phosphorus in the ferric phytate precipitate. Phytic acid P (mg) is converted to phytic acid by a conversion factor of 3.5.

The principle of phytic acid measurement is known. In the method used in this study, a solution of 5-sulfosalicylic acid and FeCl ₃ (Wade reagent) forms a pink chromophore. Phytic acid binds to iron in this solution, reducing the amount of pink color. A measure of this color loss can be used as an indicator of the amount of phytic acid. Since the blanks do not contain phytic acid, all readings for samples containing phytic acid will be negative numbers. However, if there is an excess of phytic acid, the iron-phytic acid complex may precipitate as a milky substance. In this case, there is no pink color and the milky substance will absorb light and give a falsely high reading. Therefore, visual observation may be required. If the corn variety has a high amount of phytic acid, it may be necessary to use smaller aliquots (up to 25 microliters) of the corn extract.

[0077] Rapid screening procedures, such as those described below, can be used to score for expected low phytic acid seeds. In this procedure, the bulk of the tip of the grain, just hidden in the black layer, is cut off using a single-edged razor. The cut must cross the scutellum at or near the tip of the radicle. Normally, 8 grains were selected from each ear. The granules are then laid cut side up on a microplate having a surface covered with cellophane tape (adhesive side up). After cutting at least 100 lines, the staining procedure was completed.

Staining was performed using repeated pipetting to place a drop of 10 microliters of Wade reagent (as described below) on the cut surface of each grain. After a few minutes, the color disappeared as phytic acid from the scutellum bound to the iron in the Wade reagent. Observations were made on segregated families whose pink color disappeared more slowly than others (perhaps 5% of the total). These later families were re-analyzed by the quantitative procedure described herein.

Phytic acid was quantified as follows. Individual particles (7 to 10 from each line) are crushed on a steel plate of a Carver manual pump press (the wells of the plate are stuffed with glycine paper and crushed at approximately 5000 lbs. Pressure for best results). Was obtained) and placed in a 1.5 ml microcentrifuge tube. 1 ml of 0.65 N hydrochloric acid was added, and the mixture was allowed to stand overnight. The next day, the mixture was mixed by inversion and allowed to stand for 5 minutes. A 15 microliter aliquot of the supernatant was added to a centrifuge tube containing 100 microliters of Buffer A (as described herein) and mixed. Low phytic acid mutants develop a remarkable blue color due to high seed phosphorus levels. Mutants were extensively re-analyzed the next day. A 0.65 N HCl extraction solution was prepared by adding 216 ml of 12.1 N HCl to 3784 ml of water. Reagent A was prepared fresh daily and contained 20% (by volume) of deionized water, 10% (by volume) of ascorbic acid solution, 10% (by volume) of ammonium molybdate solution and 10% (by volume) H ₂ SO ₄ solution. Ammonium molybdate _{solution, 25 g (NH 4) 6MO} 7 O 24 · 4H 2 O was added, was prepared in 1L of water. The H ₂ SO ₄ solution was prepared by adding 167 ml of 36 N sulfuric acid to 833 ml of water. The ascorbic acid solution was prepared by adding 100 g L-ascorbic acid and making up to 1 L with water.
The ascorbic acid solution was stable on refrigeration for about 7 weeks, except that it was not refrigerated for about 2 hours.

A phytic acid standard was prepared by means known in the art. Maize grains (M2) free phosphoric acid were visually screened as follows. Grains were picked, the phenotype was recorded and placed in a multi-well grinding plate. Using a hydraulic press,
The grains were crushed in a double-well crush plate. The ground particles were transferred to a 1.5 ml Eppendorf centrifuge tube and 0.5 ml of reagent A was added. After reacting for 2 hours, 0.5 ml of reagent
B was added. The tube was capped and mixed by inversion. The reaction was visually scored for blueness after 1 hour using a light box if necessary, and the bluest sample was selected as the one containing the highest phosphoric acid. In most cases, the bluest sample from each line (hair) was chosen and compared for final selection. Reagent A in this assay was 5
Prepared from 0 ml DMSO and 50 ml reagent B. Reagent B was prepared fresh daily, 60
ml of deionized water; 30 ml of a 10% ascorbic acid solution (water to a total volume of 100 ml in 10 g of ascorbic acid; the ascorbic acid solution was refrigerated and stable for one week); 3.5% ammonium molybdate solution _{(2.5g (NH 4) 6 Mo} 7 O 24 · 4
H ₂ O was added water to a total volume of 100 ml); 30 ml of 6N sulfuric acid solution (170 ml
25 ml of concentrated H ₂ SO ₄ was added, and the total volume was adjusted to 200 ml with water).

Phytic acid (red test) was measured quantitatively as follows. Approximately 12 mature seeds were crushed on a steel crush plate of a Caver manual pump press. Best results were obtained when the wells of the plate were packed with weighing paper. From 5000
Crushed at a pressure of 10000 lbs. And transferred to an Eppendorf tube. 1 ml 0.65 NH
Cl was similarly added, allowed to stand overnight, and mixed the next day by inverting the tube. For the assay, 200 ml of Wade-A reagent (described below) was mixed with 10 ml of the corn / HCl juice extract obtained above in each well of a microtiter plate. Any change in color was recorded, and the sample that remained red was recorded as low phytic acid because phytic acid bound iron and the solution turned white. Quantitation can be completed by measuring at 490 nm using a spectrophotometer. The Wade-A reagent used herein was prepared by removing 25.4 g of 5-sulfosalicylic acid and 350 mg of FeCl ₃ .6H ₂ O (ground if necessary with mortar and pestel) for 1.5 L of desalination. It was prepared by adding to ionic water. Use NaOH to adjust the pH to 3.05,
The volume was adjusted to ₂ L with dH ₂ O. This reagent was stable in the refrigerator for about one month. 0.65 N HCl was adjusted by adding 216 ml HCl (12.1 N) to 3784 ml dH ₂ O.

[0082] These assays allow for the selection of maize plants of interest that contain the allele of interest. Further details of these procedures and embodiments according to the present invention are described in U.S. Provisional Application Nos. 60 / 051,854 and 60 / 051,8 filed July 7, 1997.
55, “Animal Feed with Low Phy, filed on July 7, 1998
PCT Application No. (not assigned) entitled "tic Acid, Oil Burdened and Protein Laden Grain", the entire contents of which are incorporated herein by reference. Are known and can be used to select plants.

Then, the seeds containing the desired amount of phytic acid are increased. This process has been used in the present invention and has selected a number of inbred lines with low phytic acid mutations. These included several low phytic acid inbred lines with good mating ability, such that they crossed each other to form hybrids that yielded the cereals of the present invention. Therefore, rigid stem, Lancaster and other versatile inbreeds of the present invention can be used so that the inbreds form excellent hybrid material when crossed with an appropriate hybrid tonic prototype. Produced from a prototype of heterosis. A number of developed variants of the present invention with low phytic acid are described in U.S. Patent No. 5,689,05.
It was also found that the mutants were not identical to the lpa1-R and lpa2-R mutants described as lpa1-1 and lpa2-1 respectively in FIG. In addition, seeds were screened for germination potential by standard seed germination tests. We found that some low phytic acid mutants failed to germinate while some germinated normally. Only seeds with good germination characteristics were maintained.

FIG. 6 shows an example of some data obtained from three such inbred lines screened for phytic acid content. When plotted as a frequency distribution curve, it is clear that some samples have very low phytic acid content (less than 0.5 units (weight percentage)) while most of the samples have higher phytic acid content. . The line known as the UO95 line was chosen as the starting material because of the higher protein and oil content than the average. This line produces oil-rich, protein-rich and normally germinating seeds. UO95py maintains these properties with the low phytic acid levels described above. When crossed with certain other inbred lines, the resulting hybrid produces a grain that is high in oil, high in protein, and low in phytin.

The table below shows the phytic acid content (mg / g of seed) for mutant corn according to the invention compared to wild-type seed.

[0086]

[Table 1]

The following provides examples of inbred lines in accordance with the present invention.

[0088]

[Table 2]

The protein and oil content were determined using the Dickey-John Reflectance Near Infra Red Spect
It was measured by NIR analysis using a rometer.

The cereal of the present invention can be prepared by substituting the cereal of the present invention in a usual cooking method and baking or processing to obtain corn tortilla, corn meal,
And as an alternative source for corn cereal or flour used to make corn flakes.

The cereal of the present invention may also be used as a substitute for the corn wet milling industry by substituting the cereal of the present invention for increasing milling efficiency and recoverable starch content. Animal feed produced as a by-product of the milling process also has a sufficiently reduced phytic acid content.

Example 4 The method of selecting for low phytic acid has been described above. FIG. 7 shows data obtained from low phytic acid variants isolated for low phytic acid (high phosphorus) content. Clear evidence of Mendel's law of separation is clear.

Example 5 By crossing a low phytic acid mutant with a population containing a mutagen (transposon-labeled), it is possible to identify rare examples of mutagen-tagged genes . First, homozygous low-phytic acid plants are crossed with a transposon-labeled population. The resulting seeds are then screened for low phytic acid content (in this case, phosphate content) using a biochemical assay. Using this method, it is possible to identify a transposon-labeled phytic acid gene that identifies a gene associated with a low phytic acid mutation. The results of the biochemical screening are shown in FIG. Here we display data on the frequency distribution of transposon labeled seeds assayed for phosphorus content. In this example,
One rare event was found.

Germination tests revealed that some low phytic acid mutants have acceptable germinability, while some do not germinate. Similar results can be obtained by those skilled in the art using the methods described herein.

[0095]

[Table 3]

From these preferred, low phytic acid non-germinating materials, phytic acid variants useful in the present invention can be cloned and sequenced using known methods,
It is possible to operate.

[0097] The entire contents of the references mentioned herein are incorporated by reference in their entirety.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a binary notation of a construct according to the invention developed for use in dicotyledonous plants and Agrobacterium-transformable monocotyledonous plants, comprising: , Right T-DNA border, NOS promoter, NPTII marker gene and NOS terminator, inducible promoter, in this case G
Includes the phytic acid gene and NOS terminator, which is the STII promoter sequence, and sense orientation, and the left T-DNA border.

FIG. 2 shows a binary representation of a construct according to the invention developed for use in dicotyledonous plants and Agrobacterium-transformable monocotyledonous plants. The constructs include a right T-DNA border, a Nos promoter, an NPTII marker gene and a NOS terminator, an inducible promoter, in this case described in US Pat. No. 5,608,143 and the sequence shown in FIG. Are incorporated herein by reference) and American Typ
eCulture Collection (ATCC), Manassas VA, deposited under the accession number 67804, a promoter region derived from the gene encoding cDNA clone 5-2, a phytic acid gene in the antisense direction, a NOS terminator, and a left T-DNA border. Is included.

FIG. 3 shows a promoter, in this case, US Pat.
08,143, wherein the sequence shown in FIG. 5 and the American Type Cul
1 shows a construct having a promoter region derived from the gene encoding cDNA clone 5-2 and a phytic acid gene of the present invention and a NOS terminator, deposited at Nature Collection (ATCC), Manassas VA under accession number 67804. The phytic acid gene is in the sense orientation.

FIG. 4 shows a construct having a GSTII promoter sequence, a phytic acid gene of the invention and a NOS terminator. The phytic acid gene is in the antisense orientation.

FIG. 5 shows a prior art sequence which is the nucleotide sequence of a 5-2 gene promoter from a gene designated 52.411 in US Pat. No. 5,608,143.

FIG. 6 shows an example of data obtained from three inbred lines screened for phytic acid content, plotted as a frequency distribution curve.

FIG. 7 shows data obtained from low phytic acid variants, separating for low phytic acid (high phosphorus) content.

FIG. 8 shows the nucleic acid and amino acid sequence of myo-inositol 1-phosphate synthase, accession number AF056326.

FIG. 9 shows a linear map of the disclosed sequence of maize myo-inositol 1-phosphate synthase from base 86 to base 1620.

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──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Guan, Hampin 50010, Iowa, U.S.A. 72) Inventor Wilhelm, Edward P. 50011-1061, Iowa, USA Ames, Food Sciences Building 1573, Iowa State University, Exceed Genetics LLC LLC F-term (reference) 2B030 AB03 AD08 AD09 AD14 CA17 CA19 4B024 AA08 BA80 CA04 DA01 EA04 GA11 GA25

Claims (32)

[Claims] 1. A transgenic plant comprising a genetic construct comprising a heterologous nucleic acid sequence encoding a selected gene product such that the plant regulates phytic acid production. 2. The transgenic plant of claim 1, wherein said nucleic acid sequence is operably linked to a promoter inducible by an activator. 3. The transgenic plant of claim 1, wherein said plant also comprises a mutant allele that confers a plant that forms low phytic acid levels. 4. The plant also comprises a wild-type phytic acid gene that provides a plant that forms a sufficient amount of phytic acid to produce daughter seeds in which the plant will germinate in the absence of the construct. Item 10. The transgenic plant according to Item 1. 5. The transgenic plant of claim 3, wherein said promoter is active until an activator is applied to said plant and said nucleic acid sequence is controllably linked in a sense orientation. 6. The transgenic plant of claim 3, wherein said promoter is inactive until an activator is applied to said plant and said nucleic acid sequence is controllably linked in a sense orientation. 7. The transgenic plant of claim 4, wherein said promoter is inactive until an activator is applied to said plant and said nucleic acid sequence is controllably linked in an antisense orientation. 8. The transgenic plant of claim 4, wherein said promoter is active until an activator is applied to said plant and said nucleic acid sequence is controllably linked in an antisense orientation. 9. The nucleic acid sequence is UO95py, lpa1-1 (ATCC accession number 97678), lpa2-
Mutant phytic acid gene product obtainable from a maize mutant plant selected from the group consisting of 1 (ATCC Accession No. 97679) and allelic, truncated, substituted and deleted mutants thereof The transgenic plant of claim 3, which encodes 10. The transgenic plant of claim 9, wherein said gene product is obtainable by mu transposon labeling. 11. The transgenic plant of claim 4, wherein said wild-type phytic acid gene is obtainable by a plant containing a mu transposon-labeled phytic acid. 12. The transgenic plant of claim 4, wherein said wild-type phytic acid gene is a nucleic acid sequence encoding SEQ ID NO: 2. 13. The transgenic plant of claim 4, wherein said wild-type phytic acid gene is SEQ ID NO: 1. 14. The transgenic plant of claim 1, wherein said plant is selected from the group consisting of maize, soy, rice, oats, sunflower, wheat, barley, rye, and forage pasture. 15. The activator may be 2-chloro-N- (methylaminocarbonyl) benzenesulfonamide, 1- (n-butyl) -3-methylsulfonylurea, methyl 2-[(aminocarbonyl ) Aminosulfonyl] benzoic acid, N-isopropylcarbamoylbenzenesulfonamide, N- (aminocarbonyl) -2-chlorobenzenesulfonamide and N ′-[2- (n-butylaminocarbonyl)]-6-chloro-N, N -
The transgenic plant according to claim 2, which is a compound selected from the group consisting of dimethyl-1,2-benzenedisulfonamide. 16. A recombinant gene construct encoding a gene product that regulates phytic acid production and capable of transforming a plant containing a nucleic acid sequence controllably linked to the 3 ′ nucleic acid promoter sequence. 17. The recombinant gene construct of claim 16, wherein said nucleic acid promoter sequence is as shown in FIG. 18. The recombinant gene construct of claim 16, wherein said nucleic acid promoter sequence is inducible by application of N, N-diallyl-2,2-dichloroacetamide. 19. The method according to claim 19, wherein the nucleic acid promoter sequence is N, N-diallyl-2,2-dichloroacetamide, benzyl-2-chloro-4- (trifluoromethyl) -5-thiazole-
Carboxylates, naphthalene-1,8-dicarboxylic anhydride, 2-dichloromethyl-2-methyl
17. The recombinant gene construct of claim 16, wherein the recombinant gene construct is inducible by a compound selected from the group consisting of -1,3-dioxolane. 20. The following steps: comprising an externally inducible recombinant DNA vector which can be activated by an activator, wherein said recombinant DNA vector has a lethal production of phytic acid. Planting a mature seed comprising at least one nucleic acid sequence encoding the selected gene product; activating the plant produced by the mature seed with an activator, thereby inducing a nucleic acid sequence for the selection. A method for producing seeds, comprising: producing a gene product that has been treated; and collecting daughter seeds produced from the plant after the activator is acted on. 21. The following steps: An externally inducible recombinant DN that can be inactivated by an activator
Planting a mature seed, comprising the A vector, wherein the recombinant DNA vector comprises at least one nucleic acid sequence encoding a selected gene product such that the production of phytic acid is lethally reduced; Causing an activator to act on a plant produced by the seed, thereby inducing a nucleic acid sequence to stop encoding said selected gene product; daughter seed produced from the plant after activator actuation Recovering the seeds. 22. Daughter seed produced by the transgenic plant of claim 5. 23. Daughter seed produced by the transgenic plant of claim 6. 24. Daughter seed produced by the transgenic plant of claim 7. 25. Daughter seed produced by the transgenic plant of claim 8. 26. The plant of claim 1, which is a dicotyledonous plant. 27. The plant of claim 1, which is a monocotyledonous plant. 28. The plant of claim 1, which is in the family Gramineae. 29. A hybrid plant wherein at least one parent is the plant of claim 1. 30. Daughter seed produced by the plant of claim 1. 31. A non-germinating cereal produced by the plant of claim 1. 32. Crossing a low phytic acid homozygous mutant plant with a population of plants containing a mutagen; identifying a mutagen-tagged daughter seed containing a low phytic acid content. And cloning and isolating the nucleic acid sequence encoding phytic acid from said daughter seed; and producing said nucleic acid sequence isolated and purified.