TRANSGENIC CORN SEED WITH ENHANCED FREE LYSINE

Reference to Related Applications [for US]

[001] This application is a Continuation-In-Part of U. S. Patent Application Nos.

11/311,892,- filed 19 December 2005, and 11/394,567, filed 31 March 2006, incorporated herein by reference.

Incorporation of Sequence Listing

[002] A computer readable form (CRF) of the sequence listing on CD-R, containing the text file named 38-21(53489)D_SeqListing.txt, which is 37 KB (measured in MS-WINDOWS), was created on October 18, 2006 and is herein incorporated by reference.

Field of the Invention

[003] Disclosed herein are transgenic corn seeds with enhanced levels of free lysine resulting from recombinant DNA in transgenic corn cells and methods of making and using such seeds.

Background

[004] Dizigen et al. disclose in US 1005/0132437 Al high lysine corn compositions having recombinant DNA expressing dihydropicolinic acid synthase which is useful as nutritionally-enhanced animal feed.

Summary of the Invention

[005] This invention provides transgenic corn seed having at least 1300 ppm free lysine, e.g. between 1300 and 4000 ppm free lysine, resulting from expression of recombinant DNA in transgenic corn cells comprising an embryo specific promoter operably linked to DNA for suppressing production of the endogenous LKR-SDH protein and an endosperm specific promoter operably linked to DNA for suppressing production of the endogenous LKR-SDH protein. In an embodiment of the invention the transgenic corn seed further comprises DNA encoding a protein active in the biosynthesis of lysine operably linked to the endosperm specific promoter. Aspects of the invention are corn cells having recombinant DNA comprising the DNA that is between the T-DNA borders of pMON99142 or the DNA that is between the T-DNA borders of ρMON99143.

[006] Another aspect of the invention provides recombinant DNA constructs that are effective in providing transgenic corn seed with enhanced levels of free lysine.

Such a recombinant DNA construct comprises an embryo specific promoter opqrably linked to DNA for suppressing production of the endogenous LKR-SDH protein and an endosperm specific promoter operably linked to DNA for suppressing production of the endogenous LKR-SDH protein. An embodiment of the recombinant DNA further comprises DNA encoding a protein active in the biosynthesis of lysine operably linked to the endosperm specific promoter. Aspects of the recombinant DNA are illustrated by the

DNA that is between the T-DNA borders of pMON99142 or pMON99143.

[007] This invention also provides methods of producing corn kernels with enhanced levels of free lysine by propagating corn plants having transgenic cells which are progeny of a corn cell that has been transformed with a recombinant DNA construct of the invention.

Brief Description of the Drawings

[008] Figures 1 and 2 illustrate plasmid maps.

[009] Figure 3 schematically illustrates a recombinant DNA construct.

Detailed description of the Invention

[0010] As used herein LKR-SDH means the lysine catabolic protein lysine ketoglutarate reductase/saccharopine dehydrogenase. Effective gene suppression elements for use in this invention are designed for either the LKR domain or the SDH domain.

[0011] Recombinant DNA constructs can be readily prepared by those skilled in the art using commercially available materials and well-known, published methods.

Recombinant DNA constructs for gene suppression can be fabricated as illustrated and disclosed in US 2004-0029283 Al. A useful technology for building recombinant DNA constructs and vectors for transformation is the GATEWAY™ cloning technology

(available from Invitrogen Life Technologies, Carlsbad, California) uses the site specific recombinase LR cloning reaction of the Integrase att system from bacteriophage lambda vector construction, instead of restriction endonucleases and ligases. The LR cloning reaction is disclosed in U.S. Patents 5,888,732 and 6,277,608 and US Patent Application

Publications US 2001-283529 Al, US 2001-282319 Al, US 2002-0007051 Al, and US

2004-0115642 Al. The GATEWAY™ Cloning Technology Instruction Manual which is also supplied by Invitrogen also provides concise directions for routine cloning of any desired DNA into a vector comprising operable plant expression elements. [0012] An alternative vector fabrication method employs ligation-independent cloning as disclosed by Aslandis, C. et ah, Nucleic Acids Res., 18, 6069-6074, 1990 and Rashtchian, A. et al, Biochem., 206, 91-97,1992 where a DNA fragment with single- stranded 5' and 3' ends are ligated into a desired vector which can then be amplified in vivo. Methods for assembling DNA molecules in a predetermined order in a DNA construct are also disclosed in published patent application US 2006- (Serial NO.

11/298,234).

[0013] Numerous promoters that are active in plant cells have been described in the literature. Useful promoters for the transgenic seed of this invention include promoters from seed genes such as napin (U.S. Patent 5,420,034), cornL3 oleosin (U.S. Patent 6,433,252), zein Z27 (Russell et al (1997) Transgenic Res. 6(2): 157-166), globulin 1 (Belanger et al (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), peroxiredoxin antioxidant (Perl) (Stacy et al. (1996) Plant MoI Biol. 31(6):1205- 1216), and B32 (Hartings et al (1990) Plant MoI Biol, 14:1031-1040) [0014] In practice DNA is introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the herbicides to which plants of this invention may be resistant are useful agents for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable markers are illustrated in U.S.

Patents 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a ZJeta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

[0015] The invention provides transgenic seed having in its genome a recombinant DNA construct comprising (a) a plant endosperm-specific promoter operably linked to at least one first gene suppression element for suppressing the production of a lysine catabolic protein, e.g. LKR-SDH, and (b) a plant embryo-specific promoter in the opposite orientation to the plant endosperm-specific promoter and located 3' to the at least one first gene suppression element. The plant embryo-specific promoter is operably linked to a second gene suppression element for suppressing the production of the lysine catabolic protein LKR-SDH. The second gene suppression element can comprise same DNA as the first gene suppression element or different DNA, e.g. targeted to a different domain of the LKR-SDH gene. The gene suppression element can be assembled in a recombinant DNA construct in the same orientation or in opposing orientations, e.g. with adjacent promoters (head-to-head) or with adjacent terminators (tail-to-tail).

[0016] In some embodiments of the transgenic seed, the recombinant DNA construct further includes DNA encoding a lysine biosynthesis enzyme linked to the endosperm-specific promoter. In embodiments where both a gene suppression element and an expression element for expressing a protein are operably linked to one promoter, the gene suppression element can be embedded in an intron, which in many embodiments is preferably a transcription-enhancing intron, e.g., "enhancers" such as 5' introns from the rice actin 1 gene, rice actin 2 gene, the corn alcohol dehydrogenase gene, the corn heat shock protein 70 gene, and the corn shrunken 1 gene. Useful DNA for encoding a lysine biosynthesis enzyme is from an exogenous lysine-insensitive dihydrodipicolinic acid synthase (DHDPS) gene such as the Corynebacterium DHDPS gene as disclosed in U.S. Patents 5,773,691 and 6,459,019. When expressing DNA encoding a DHDPS enzyme, it is useful to have it linked to DNA encoding a transit peptide such as from a maize DHDPS transit peptide gene.

[0017] The invention provides transgenic plant cells having a recombinant DNA construct of this invention stably integrated into their genome such that the recombinant DNA is inherited by progeny plants and seeds. Certain embodiments of such transgenic plant cells are homozygous for the recombinant DNA.

[0018] This invention further provides recombinant DNA constructs for transformation of plant cells, methods for their use, and stably transgenic plant cells containing such constructs. Such recombinant DNA constructs are found on plasmids pMON99142 and pMON99143, described in more detail below. [0019] Numerous methods for transforming plant cells with recombinant DNA are known in the art and may be used to prepare transgenic maize. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods for producing transgenic corn cells are illustrated in U.S. Patents 5,550,318; 5,538,880; 6,160,208 and 6,399,861 and Agrobαcterium-modiaied transformation for producing transgenic corn cells is described in U.S. Patent 5,591,616. For Agrobαcteήum tumefαciens based plant transformation systems, additional elements present on transformation constructs include T-DNA left and/or right border sequences from Agrobαcterium tumefαciens (generally both left and right border sequences, but preferably at least one border sequence, e. g. at least a right border sequence) to facilitate incorporation of the recombinant polynucleotide into the plant genome.

[0020] Transformation of corn is preferably practiced in tissue culture on media and in a controlled environment. "Media" refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g. various media and recipient target cells, transformation of immature embryos and subsequent

regeneration of fertile transgenic plants are disclosed in U.S. Patents 6,194,636 and 6,232,526 and U.S. patent application publication US 2004-0216189 Al. [0021] The seeds of transgenic plants can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plant lines comprising the recombinant DNA construct expressing the genes suppression elements.

[0022] This invention further provides various methods for producing transgenic corn seed having an enhance level of lysine, i.e. at least 1300 ppm free lysine or more, e.g. at least 1500 ppm or higher, say, at least 2000 ppm or 300 ppm, up to 4000 ppm of free lysine. Such methods comprise transformation of a corn plant line and introgressing the recombinant DNA of this invention from a corn plant having the recombinant DNA in its cells into another corn plant line. One aspect of such methods comprises (a) selecting a first transgenic corn plant comprising in its cells a recombinant DNA construct of this invention; (b) introgressing the recombinant DNA construct into a second corn plant; (c) growing seed from the second corn plant to produce a population of progeny corn plants; (d) screening the population of progeny corn plants for progeny corn plants that produce corn seed having an increased level of lysine relative to non-transgenic corn plants; (e) selecting from the population one or more progeny corn plants that produce corn seed having an enhanced level of lysine relative to non-transgenic corn plants; (f) verifying that the recombinant DNA construct is stably integrated in the selected progeny corn plants; (g) verifying that the LKR-SDH lysine catabolism gene is silenced in the selected progeny corn plants relative to corn plants lacking the recombinant DNA construct; (h) collecting transgenic corn seed from the selected progeny corn plants.

Example 1

[0023] This example illustrates the preparation of a recombinant DNA construct useful for preparing the transgenic corn plants and seed with transgenic cells of this invention. With reference to Figure 1 and SEQ ID NO:1 (which is the nucleotide sequence of one strands of the DNA in the plasmid pMON99142) a plant transformation plasmid was prepared with two gene suppression recombinant DNA constructs inserted between left and right T-DNA borders from Agrobacterium tumefaciens. Between the T- DNA borders were

(a) an LKR gene suppression recombinant DNA construct which was prepared with DNA for a cornL3 promoter and leader (i.e. a corn embryo specific promoter and leader from cornL3 oleosin gene as disclosed in U. S. Patent 6,433,252), operably linked to DNA for an "LKR suppression element" (a stabilized anti-sense construct comprising about 947 base pairs of DNA from the LKR domain of a corn LKR-SDH gene arranged as an anti-sense oriented segment) followed by sense oriented segment and terminated with DNA for a wheat hspl7 terminator (i.e. polyadenylation site and signal from a Tήticum aestivum heat shock protein 17 gene); and

(b) an SDH gene suppression recombinant DNA construct which was prepared with DNA for a corn B32 promoter and leader (i.e. a corn endosperm specific promoter and leader from corn B32 gene disclosed as nucleotides 848 through 1259 of GenBank accession number X70153), operably linked to DNA for an "SDH suppression element" (a stabilized anti-sense construct comprising about 1254 base pairs of DNA from the SDH domain of a corn LKR-SDH gene arranged as an anti-sense oriented segment followed by sense oriented segment) and terminated with DNA for a corn Glbl terminator (i.e. polyadenylation site and signal from a Zea mays globulin 1 gene).

The plasmid also contained outside of the T-DNA borders DNA for a glyphosate resistance selectable marker, an Agrobacterium replication origin, an E. coli repressor protein, an E. coli replication origin Colεl, and a bactericidal antibiotic selectable marker (for spectromycin/streptomycin). The DNA for the glyphosate resistance marker comprised a rice actin 1 promoter, leader and intron operably linked to chimeric DNA encoding a chloroplast transit peptide and an εPSPS gene and DNA from a terminator element with a polyadenylation site and signal from A. tumefάciens nopoline synthase. Table 1 shows the position of key elements of the plasmid with respect to SεQ ID NO:1. The SDH gene suppression recombinant DNA construct and the LKR gene suppression recombinant DNA constructs were arranged tail-to-tail. Thus, in the following Table 1 the DNA in SεQ ID NO:1 identified for the elements of the LKR gene suppression recombinant DNA construct comprises nucleotides in the reverse complement of the

coding strand for those elements. The plasmid pMON99142 is deposited with the ATCC on May 12, 2006 under ATCC Accession No. PTA-7595.

Table 1

Example 2

[0024] This example illustrates the preparation of a recombinant DNA construct useful for preparing the transgenic corn plants and seed with transgenic cells of this invention. With reference to Example 1 and Figures 2 and 3 and SEQ DD NO:2 (which is the nucleotide sequence of one strands of the DNA in the plasmid pMON99143) a plant transformation plasmid was prepared with two gene suppression recombinant DNA constructs inserted between left and right T-DNA borders from Agrobacterium tumefaciens. Between the T-DNA borders were

(a) an SDH gene suppression recombinant DNA construct which was prepared with a corn B32 endosperm specific promoter and leader operably linked in series to a corn Hsp70 intron (i.e. from a Zea mays heat shock protein 70 gene) into which embedded an "SDH suppression element", where the intron was linked to DNA encoding a corn DHDPS transit peptide (i.e. from a Zea mays dihydrodipicolinic acid transit peptide gene) linked to DNA encoding Corynebacterium DHDPS (i.e. from a lysine-insensitive Corynebacterium DHDPS gene) and terminated with DNA for a wheat HSP17 terminator; and

(b) an LKR gene suppression recombinant DNA construct which was prepared with a corn L3 embryo specific promoter and leader operably linked in series to an "LKR suppression element" and terminated with DNA for a corn GIb 1 terminator.

The plasmid also contained outside of the T-DNA borders DNA for a glyphosate resistance selectable marker, an Agrobacterium replication origin, an E. coli repressor protein, an E. coli replication origin Colεl, and a bactericidal antibiotic selectable marker (for spectromycin/streptomycin). Table 2 shows the position of elements of the plasmid with respect to SεQ ID NO:2. The SDH gene suppression recombinant DNA construct and the LKR gene suppression recombinant DNA constructs were arranged tail-to-tail. Thus, in the following Table 2 the DNA in SεQ ID NO:2 identified for the elements of the LKR gene suppression recombinant DNA construct comprises nucleotides in the reverse complement of the coding strand for those elements. The plasmid pMON99143 is deposited with the ATCC on May 12, 2006 under ATCC Accession No. PTA-7596.

Table 2

Example 3

[0025] This example illustrates the use of the plasmid of Example 1 for producing transgenic corn cells and plants and seed of fertile transgenic corn with enhanced levels of free lysine. Plasmid pMON99142 is inserted into corn callus by Agrobacterium- mediated transformation to produce multiple transgenic events of corn cells that are selected as being resistant to glyphosate herbicide (based on insertion of the DNA for the glyphosate resistance selectable marker from one side of the T-DNA borders). Transgenic plants (RO) are grown from transformed cells of each of the multiple transgenic events. The RO transgenic corn plants are analyzed for the presence of the glyphosate resistance marker DNA and the gene suppression recombinant DNA constructs using fluorescent-tagged probes that hybridize to PCR-amplified segments of the plasmids. From the multiple plants representing the transgenic events plants are selected which have the glyphosate resistance selectable marker DNA at a single locus and a single copy of the T-DNA comprising the SDH and LKR gene suppression recombinant DNA constructs at a separate single locus. The single copy-single locus T- DNA plants are crossed with a non-transgenic inbred corn line to produce segregating progeny seed which is analyzed to identify seeds with gene suppression recombinant

DNA and without DNA for the glyphosate resistant selectable marker. Seed analysis is effected on DNA from a chip of endosperm. The identified seeds are grown into plants that are crossed with several inbred corn lines over several generations to introgress the T-DNA comprising the SDH and LKR gene suppression recombinant DNA constructs into the inbred corn lines to produce inbred corn lines with DNA for suppressing the lysine catabolic protein LKR/SDH in both the endosperm and embryo. [0026] Seeds from transgenic corn plants of the inbred corn lines with DNA for suppressing the lysine catabolic protein LKR/SDH in both the endosperm and embryo are analyzed for free lysine and determined to have greater than 1300 ppm lysine. Seeds from inbred corn line plants having cells with selected transgenic events are identified as having greater than 1500 ppm free lysine, greater than 2000 ppm free lysine, greater than 3000 ppm free lysine and greater than 4000 ppm free lysine.

Example 4

[0027] This example illustrates the use of the plasmid of Example 2 for producing transgenic corn cells and plants and seed of fertile transgenic corn with enhanced levels of free lysine. The transformation, analysis, selecting and introgressing of Example 3 are essentially reproduced using plasmid pMON99143 to provide inbred corn lines with DNA for suppressing the lysine catabolic protein LKR/SDH in both the endosperm and embryo as well as expressing the Corynebacterium DHDPS enzyme. Seeds from inbred corn line plants having cells with selected transgenic events are identified as having greater than 1500 ppm free lysine, greater than 2000 ppm free lysine, greater than 3000 ppm free lysine and greater than 4000 ppm free lysine.