| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 09.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 14.09.99 |
| PATENT TITLE |
Soybean glucanases, compounds which encode therefor and related methods |
| PATENT ABSTRACT | The present invention provides 12 different sequences for soybean .beta.-1,3-glucanases and the proteins for which they encode. Also provided are methods for the utilization of knockout mutants of the sequences which are useful for engineering genic male-sterile plants. Other methods and materials related to these sequences are also provided |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 04.11.97 |
| PATENT REFERENCES CITED |
Napoli et al. Plant Cell 2: 279-289, Apr. 1990. Smith et al. Nature 334: 724-726, Aug. 1988. Tsuchiya et al. Plant Cell Physiol. 36(3): 487-494, 1995. Takeuchi et al. Plant Physiol. 93: 673-682, 1990. Christon et al. Proc. Natl. Acad. Sci. USA 86: 7500-7504, Oct. 1989. Vol. 83(6) Am. J. Bot. p. 42, abstract 121 (1996) Jin et al. |
| PATENT CLAIMS |
What is claimed is: 1. An isolated DNA molecule selected from the group consisting of: SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO 4; SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO 9; SEQ ID NO 10; SEQ ID NO 11 and SEQ ID NO 12. 2. A knockout construct of SEQ ID NO 7. 3. A vector comprising a knockout construct of SEQ ID NO 7. 4. A seed comprising a knockout construct of SEQ ID NO 7. 5. An embryo comprising a knockout construct of SEQ ID NO 7. 6. A plant comprising a knockout construct of SEQ ID NO 7. 7. A method to construct a male-sterile knockout mutant, comprising: transforming a plurality of plant cells with a knockout construct of SEQ ID NO 7; and inducing the plurality of plant cells to grow into a plant; and selecting those plants which are male-sterile. 8. A plant embryo comprising a vector which allows expression at a time other than the late tetrad stage of SEQ ID NO 7. 9. A plant seed comprising a vector which allows expression at a time other than the late tetrad stage of SEQ ID NO 7. 10. A plant comprising a vector which allows expression at a time other than the late tetrad stage of SEQ ID NO 7. 11. A method to express .beta.-1,3-glucanase in a plant which has a callose encasement of microspores, at a time other than the late tetrad stage, comprising growing a plant transformed with a vector which allows expression at a time other than the late tetrad stage of SEQ ID NO 7. 12. A method to degrade the callose of seeds, comprising expressing SEQ ID NO 7 so as to produce an expression product, and allowing the expression product adequate time and conditions to degrade said callose. 13. A male-sterile soybean seed comprising a knockout construct of SEQ ID NO 7. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION The present inventors recently characterized a genic male-sterile mutant of soybean: 83(6) Am. J. Bot. 42, abstract 121(1996); Jin et al., 10 Sex Plant Reprod. 13 (1997). Microscopic analysis of the mutant showed the callose encasement of the microspores was retained at an inappropriate developmental stage. The persistent callose encasement resulted in no release, and subsequent degeneration of the microspores. Other results in these publications showed that callase, the enzyme which is responsible for digestion of the callose wall, was inactive. The reason for the inactivity was not elucidated at that time. Other publications by the present inventors identified the callase as a .beta.-1,3-glucanase. Abstract P14, Genetics Society of Canada (June 1997); 114(3) Plant Physiology 172, abstract 842 (June 1997); 84(6) Am. J. Bot. 63, abstract 182 (June 1997). Soybean .beta.-1,3-glucanases have been cloned in the past. For example, U.S. Pat. No. 5,477,001 disclosed cloned .beta.-1,3- glucanases from soybean. The gene products are implicated in disease resistance. No disclosure of the involvement of .beta.-1,3- glucanases as a callase is described in the patent. Non-soybean glucanases, such as those described in U.S. Pat. No. 5,554,743 describe .beta.-1,3-glucanases involved in cell wall polysaccharide degradation. A search of the National Institutes of Health BLAST database disclosed a line of soybean "Glycine max cv. Century 84" with cloned .beta.-1,3-glucanases. None of the sequences found were identical to the presently-claimed sequences. The following is a table of the results of the search. ______________________________________ SEQ ID NO CLOSEST HOMOLOGY REFERENCE ______________________________________ 1 85% (soybean glucanase) U.S. Pat. No. 5477001, Seq 8; same as WO9413790, Seq 15 2 98% (soybean endoglucanase) none available 3 91% (Century 84) 26 Crop Science 199 (1986) 4 97% (soybean glucanase) U.S. Pat. No. 5477001, Seq 8; same as WO9413790, Seq 15 5 76% (Century 84) 26 Crop Science 199 (1986) 6 91% (Century 84) 26 Crop Science 199 (1986) 7 54% (unknown) U.S. Pat. No. 5614395, Seq 13; same as WO9413790, Seq 15 8 85% (soybean glucanase) U.S. Pat. No. 5477001, Seq Id 8; same as WO9413790, Seq 15 9 75% (Century 84) 26 Crop Science 199 (1986) 10 97% (Century 84) 26 Crop Science 199 (1986) 11 76% (Century 84) 26 Crop Science 199 (1986) 12 96% (soybean glucanase) U.S. Pat. No. 5477001, Seq Id 8; same as WO9413790, Seq 15 ______________________________________ The U.S. Pat. No. 5,477,001 does not disclose or suggest the present invention, because the sequences described herein are not disclosed. Moreover, the emphasis in the U.S. Pat. No. 5,477,001 is the use of a particular soybean glucanase to resist plant diseases. In certain aspects of the present invention, the focus is on the elimination of active soybean glucanases; the U.S. Pat. No. 5,477,001 therefore teaches away from those aspects of the present invention. Century 84 is a line of soybean which was released jointly by the Ohio Agricultural Research and Development Center, The Ohio State University and the USDA which contained over-expressed glucanase for the purpose of disease resistance. Registration Number 188,26 Crop Science 199 (1989). Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. For example, in some instances above, the publication was less than one year before the filing date of this patent application. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant at the time of filing, and does not constitute an admission as to the accuracy of the dates or contents of these documents. SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide sequences useful to engineer male-sterile soybean lines. It is a further object to provide methods to engineer male-sterile soybean lines. It is yet another object to provide male-sterile soybean seeds. It is yet another object to provide male-sterile soybean plants. In all of the above embodiments, it is an object to provide seed mixes with male-sterile and female-fertile seeds. It is also an object of the invention to provide materials such as vectors for genetic engineering male-sterile soybean lines. It is an additional object to provide a method to improve seed germination using the materials herein disclosed. Lastly, it is therefore an object of the present invention to provide sequences useful to engineer disease-resistant soybean lines. DEFINITIONS For the purposes of the present application, the following terms have the following meanings. All other terms have the meaning as generally recognized in the art. "Knockout construct" means a DNA sequence which has been altered via any known means, for example, deletion, insertion, point mutation or rearrangement, so as to eliminate the function of the naturally-occurring .beta.-1,3 glucanase of the pod and flower bud of a soybean plant gene product, but not so as to alter the ability of the DNA sequence to recombine with the naturally-occurring sequence. "Knockout mutants" are cells, embryos or plants in which a naturally-occurring .beta.-1,3-glucanase gene has been replaced through genetic engineering with a knockout construct, so as to result in a male-sterile phenotype. DETAILED DESCRIPTION OF THE INVENTION The present invention provides twelve different sequences for soybean .beta.-1,3 glucanases and the proteins for which they encode. The DNA sequences are as follows: __________________________________________________________________________ SEQ ID NO SEQUENCE __________________________________________________________________________ Vectors which comprise the above sequences are within the scope of the present invention, as are plants transformed with the above sequences. Vectors may be obtained from various commercial sources, including Clontech Laboratories, Inc. (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), New England Biolabs (Beverly, Me.) and Promega (Madison, Wis.). Preferred vectors are those which are capable of transferring the sequences disclosed herein into plant cells or plant parts. Methods to inhibit plant diseases, including use of a compound herein disclosed, according to the procedures described in U.S. Pat. No. 5,477,001, which patent is expressly incorporated by reference, are also part of the present invention. Moreover, the most commercially significant use of the present invention is in the construction of "knockout mutants" using the above sequences, or known soybean sequences, for design and construction of male-sterile mutants. In other words, the present invention is informative to those skilled in the art as to their usefulness in making the naturally-occuring gene inactive. For example, the above sequences can be mutated by any means, i.e., deletion, insertion, point mutation, rearrangement, etc, so long as the mutated version retains the ability to recombine. The mutated version of the gene is then introduced into cells of a preferred soybean line via routine methods (ie. biolistic processes, lambda phage transformation, etc.). Male-sterile mutants of the preferred line would then be selected and propagated. These "knockout" mutant embryos, seeds and plants are within the scope of the present invention, as are the knockout constructs, ie. sequences and vectors. In particular, sequences near the active sit of enzyme function, and the sit itself, would be preferred targets. For example, the codons for amino acid residues 240 through 250 would be a preferred knockout, but most preferred would be a construct wherein the codons encoding 245 through 248 were deleted. For example, the following seeds, embryos or plants transformed with knockout constructs are considered within the present invention: soybean, maize, beet, tobacco, wheat, barley, poppy, rape, sunflower, alfalfa, sorghum, rose, carnation, gerbera, carrot, tomato, lettuce, chicory, pepper, melon and cabbage. Particularly preferred are: soybean, tobacco and maize. However, any seed, embryo or plant which gives rise to a plant which has a callose encasement of the microspores is within the scope of the present invention. Of course, those in the art recognize that any seed, embryo or plant transformed with knockout constructs which are useful for producing plants for biomass are within the scope of the present invention. Transformation of cells with the compounds of the present invention can be accomplished according to known procedures. For example, infective, vector-containing bacterial strains (such as Agrobacterium rhizogenes and Agrobacterium tumefaciens) may be used for transformation. Zambryski, 43 Ann. Rev. Pl. Physiol. Pl. Mol. Biol 465 (1992). The following procedures are also well-known: Pollen-tube transformation ›Zhon-xun et al., 6 Plant Molec. Bio. 165(1988)!; direct transformation of germinating seeds ›Troepfer et al., 1 Plant Cell 133 (1989)!; polyethylene glycol or electroporation tranformation ›Christou et al., 84 Proc. Nat. Acad. Sci. 3662 (1987)!; and biolistic processes ›Yang & Cristou, Particle Bombardment Technology for Gene Transfer (1994)!. The transformed cells are also within the scope of the present invention. The transformed cells may be induced to form transformed plants via organogenesis or embryogenesis, according to the procedures of Dixon Plant Cell Culture: A Practical Approach (IRL Press, Oxford 1987). Therefore, also provided are methods for constructing sequences with the ability to knockout the above sequences, comprising one of the following techniques: inserting a foreign piece of DNA into one of the disclosed sequences; deleting a piece of DNA from one of the disclosed sequences; or creating a mutation such that the .beta.-1,3-glucanase activity is eliminated. Also provided are antisense constructs and methods to inhibit mRNA transcripts of the disclosed sequences, so as to either eliminate or reduce the amount of gene product. The procedures for antisense inhibition for mRNA are described in U.S. Pat. No. 5,554,743, which patent is expressly incorporated by reference into this application. Also provided in the present invention are methods to improve seed germination, comprising expressing or overexpressing the .beta.-1,3-glucanase(s) described herein, and using the glucanases to digest the callose wall of mature seeds ›i.e., Cucumis Speices; Yim and Bradford, 114(3) Plant Physiology 289, abstract 1506 (1997)!. The seeds can then be germinated according to traditional methods. Overexpression can be as skill of the art, in particular, according to the procedures described in U.S. Pat. No. 5,477,001. Lastly, the present invention includes methods to alter the naturally-occurring expression pattern of the .beta.-1,3-glucanase genes so as to either delay or prematurely digest the callose wall. In other words, in a none-male-sterile plant, a glucanase gene is expressed (and the callose encasement digested) at a critical point in microsporogenesis called "the late tetrad stage." If the genes which digest the callose encasement are not active at the late tetrad stage, or if they are active before the late tetrad stage, the potential for pollen formation is lost. In the instance where the .beta.-1,3-glucanase genes are expressed earlier than the late tetrad stage, the microspores would be too immature. In the case where the .beta.-1,3-glucanase genes are expressed later than the late tetrad stage, the microspores would be arrested in development. Since disease resistance is one characteristic conferred to a plant by the expression of .beta.-1,3-glucanase, an ideal method would be to activate the sequences disclosed herein (or other glucanase sequences), and have the gene constitutively expressed thereafter. However, another embodiment of this invention is to alter the expression pattern so that the .beta.-1,3-glucanase genes turn on later than the late tetrad stage. Constitutive expression thereafter may also be engineered. A preferred method in this regard is alteration of the regulatory regions of SEQ ID NO 7 so as to affect the expression of a .beta.-1,3-glucanase either earlier or later than the late tetrad stage. In particular, in order to practice the altered expression pattern aspect of the present invention, one would have to construct a vector which provided for either an early or late promoter in conjunction with the present sequences. For instance, the following promoters would be useful in early expression of the present sequences: Ogs4B (Tsuchiya et al., 36 Plant Cell Physiology 487 (1994) TA29 (Koltunow et al., 2 Plant Cell 1201 (1990) A3 & A9 (Paul et al., 19 Plant Molecular Biology 611 (1992) In order to then constitutively express the sequences described above, the construct optionally contains, for example, a 35S promoter. Therefore, the present invention provides methods to express the .beta.-1,3-glucanase genes described herein in a plant which has a callose encasement of microspores, at a time other than the late tetrad stage, comprising growing a plant transformed with a vector which allows expression at a time other than the late tetrad stage of a sequence selected from the group consisting of: SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO 4; SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO 9; SEQ ID NO 10; SEQ ID NO 11 and SEQ ID NO 12. A method as above, wherein the .beta.-1,3-glucanase genes are then consititutively expressed is preferred. Transformation of plants with these sequences would be according to known procedures as described above. Plants can be grown according to known procedures. In addition, there are provided male-sterile soybean seeds, embryos and plants comprising a knockout construct of soybean .beta.-1,3-glucanase gene, in particular, soybean seeds, embryos and plants wherein the soybean .beta.-1,3-glucanase gene knocked out is SEQ ID NO 7 are provided. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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