TH97352B - Seeds are transferred with genes containing increased lysine. - Google Patents

Abstract

DC60 (28/12/49) What is disclosed in this patent application is a gene-transferred plant containing an external novel DNA construct expressed as dihydrodipicolinic acid synthesizer and a gene-repressing molecule for lysine glutamate reductase, the action of which the construct results in increased lysine in the seeds of the plant. What is disclosed in this patent application is a gene-transferred plant containing an external novel DNA construct expressed as dihydrodipicolinic acid synthesizer and a gene-repressing molecule for lysine glutamate reductase, the action of which the construct results in increased lysine in the seeds of the plant.

Claims (37)

Revised September 15, 2017 1. DNA structure incorporating the plant expression cassette as found in plasmid pMON93093 as deposited by ATCC as PTA-6734 ------------------------------------------------- (Original) 1. A DNA construct consisting of a cassette of heterologous plant expression encoding a dihydropicolinic acid synthesite classified as resistant to feedback inhibition by free L-lysine, and an addition of DNA molecules transcribed to produce RNA molecules that suppress lysine ketoglutamate reductase/saccharopine dehydrogenase, each DNA molecule functionally linked to an active promoter in the seed. 2. The DNA construct of claim 1, in which the promoter facilitates the transcription of DNA molecules primarily located in the endosperm, or embryo, of a seed. 3. A DNA construct of claim 1 containing an expression cassette comprising a DNA molecule encoding aspartate kinase classified as resistant to feedback inhibition by L-lysine-free. 4. The DNA construct of claim 1 in which the DNA molecule that codes for the transmission signal peptide is operationally linked to the DNA molecule that codes for dihydrodipicolinic acid synthesizer. 5. The DNA construct of claim 3, in which the DNA molecule that codes for the transmission signal peptide is operationally linked to the DNA molecule that codes for aspartase kinase. 6. The DNA construct of claim 1, whereby the DNA molecule encoding dihydrodipicolinic acid synthesizer is SEQ ID NO: 1. 7. A DNA construct of claim 1 in which a transcribed DNA molecule encodes an RNA molecule whose lysine ketoglutamate reductase/saccharopine dehydrogenase is homologous to or matches SEQ ID NO: 2, or a portion of this sequence is sufficient to suppress such dehydrogenase. 8. The DNA construct of claim 3, whereby the DNA molecule encoding aspartate kinase is SEQ ID NO: 3. 9. Method for Enhancing Lysine in Seeds Containing Expression Cassettes: In exogenous plants, one or more cassettes are administered containing DNA molecules encoding dihydrodipicolinic acid syntheses resistant to feedback inhibition by free L-lysine; DNA molecules transcribed to produce RNA molecules that suppress lysine ketoglutarate reductase/saccharopine dehydrogenase; and DNA molecules encoding aspartate kinase resistant to feedback inhibition. These DNA molecules are functionally linked to one or more promoter molecules that induce transcription of one or more RNA molecules in the maize seed tissue. 10. The method of claim 9, in which the construct includes an additional component consisting of a DNA molecule encoding 5-enolpyruvilsikimate. 11. Method of claim 9, in which the DNA molecule encoding dihydrodipicolinic acid synthesizer is the structural DNA sequence is SEQ ID NO: 1. 12. Method of claim 9, in which the DNA molecule encoding aspartate kinase is SEQ ID NO: 3. 13. The method of claim 9, in which a transcribed DNA molecule produces an RNA molecule that suppresses lysine ketoglutamate reductase/saccharopine dehydrogenase, is SEQ ID NO: 9. 14. Method of claim 9, in which 5-enolpyruvilsikimate 3-phosphate synthesizer is aroA:CP4:EPSPS. 15. The method of claim 9, in which a DNA molecule has an additional segment containing a DNA sequence that codes for a signal peptide for transport into the chloroplast, which is operationally linked to a DNA sequence that codes for dihydrodipicolinic acid synthesize or aspartate kinase. 16. Method of claim 9, in which a promoter is selected from a group consisting of the promoter B32 of Zea mays, the promoter gamma-coic acid of Zea mays, and the promoter L3 of Zea mays. 17. Method of claim number 9, where the plant is the corn plant. 18. A DNA construct containing plasmic pMON 93092, which includes the plant expression cassette as archived with ATCC1 under the number PTA-6733, or plasmic pMON 93093, which includes the plant expression cassette as archived with ATCC under the number PTA-6734. 19. Method of claim 9, whereby a seed within its genome contains a cassette of at least one plant expression cassette of the plasmid pMON 93092 or pMON 93093. 20. Processed products of the seeds of claim 19, which are animal feed, flour, crushed material, or partially purified protein mixtures. 21. The processed product of the seed of claim 19, consisting of a fragment of natural DNA and a fragment of exogenous plant expression cassette. 22. In vitro maize cells containing exogenous plant expression cassettes encoding dihydropicolinic acidase-resistant DNA molecules that are resistant to feedback inhibition by L-lysine, transcribed DNA molecules that produce RNA molecules that suppress lysine ketoglutamate reductase/saccharopine dehydrogenase, and aspartate kinase-resistant DNA molecules that are resistant to feedback inhibition. These DNA molecules are functionally conjugated to one or more promoter molecules that induce transcription of one or more RNA molecules in maize seed tissue. 23. In vitro cells of claim 22, whose construct contains an additional component consisting of a DNA molecule encoding 5-enolprovilchikimate 3-phosphate synthesizer. 24. The in vitro cell of claim 22, in which the DNA molecule encoding dihydrodipicolinic acid synthesizer is the structural DNA sequence SEQ ID NO: 1. 25. The in vitro cell of claim 22, whose DNA molecule codes for aspartate kinase, is SEQ ID NO: 3. 26. The in vitro cell of claim 22, in which the DNA molecule is transcribed to produce an RNA molecule that suppresses lysine ketoglutamate reductase/saccharopine dehydrogenase, is SEQ ID NO: 9 (bfx sequence). 27. The in vitro cell line of claim 23, in which the 5-enolpyruvilchekimate 3-phosphate synthase is aroA:CP4 FPSPS. 28. In vitro cells of claim 22, in which DNA molecules have additional segments containing DNA sequences that code for signal peptides that are operationally transported into chloroplasts connected to DNA sequences that code for dihydrodipicolinic acidase or aspartate kinase. 29. In vitro cells of claim 22 where the promoter is the Zea mays B32 promoter, the Zea mays gamma-coic acid promoter, or the Zea mays L3 promoter. 30. In vitro cells of claim 22 with higher lysine content than maize seeds without exogenous plant expression cassettes. 31. In vitro cells whose genome contains a cassette of the plant expression plasmid pMON 93092 or pMON 93093. 32. Processed products consisting of intact or broken cells as defined in Claim 22, whereby such products are animal feed, crushed materials or flour. 33. A method for producing cereal seeds consisting of the following steps: a) genetically modifying plant line cells with expression cassettes of the plant plasmid pMON 93092 or pMON 93093; b) inducing these cells into reproductive plants containing the expression cassette in their genome; and c) harvesting seeds from these plants. This method results in seeds with a higher lysine content than seeds of the same plant line that have not been genetically modified. 34. The method for obtaining seeds consisting of the following steps: a) Selection of primary and secondary parent plants, each containing one or more heterologous expression cassettes chosen from a group containing (i) cassettes containing DNA molecules encoding dihydrodipicolinic acid syntheses classified as resistant to feedback inhibition by free L-lysine; (ii) RNA molecules that suppress lysine ketoglutamate reductase/saccharopine dehydrogenase; and (iii) cassettes containing DNA molecules encoding aspartate kinases resistant to feedback inhibition by lysine; all DNA molecules in each cassette are functionally ligated to the promoter molecules that induce transcription of RNA molecules in the maize seed tissue in such a way that each parent contains at least one expression cassette, no parent contains all three types of expression cassettes, and the parents are distinct. From one another in terms of cassette of expression, b) crossbreeding between selected paternal and maternal parents, and c) harvesting offspring seeds from such crossbreeding. 35. A method of providing nutrition to humans or lower animals consisting of feeding them a diet containing high-lysine maize or processed maize products containing cassettes of plant expression of pMON 93092 or pMON 93093. 36. A method of providing nutrition to humans or lower animals consisting of feeding them a diet containing maize kernels with lysine, or processed maize products containing natural DNA fragments from the kernels and fragments of plant expression cassettes that are pMON 93092 or pMON 93093. 37. The processed product of a seed consists of the following steps: a) genetic modification of plant line cells with a cassette of the plant expression plasmid pMON 93092 or PMON 93093; b) incubation of these cells into reproductive plants in which the expression cassette is present in the genome; c) harvesting the seeds from these plants; and d) processing the seeds into fodder, or as a powder, flour, or oil. In this way, the seeds have a higher lysine content than seeds of the same plant line that have not undergone such genetic modification.