US20210380997A1

US20210380997A1 - Glucanase production and methods of using the same

Info

Publication number: US20210380997A1
Application number: US17/355,698
Authority: US
Inventors: R. Michael Raab; Oleg Bougri; XueMei Li
Original assignee: Agrivida Inc
Current assignee: Agrivida Inc
Priority date: 2015-05-14
Filing date: 2021-06-23
Publication date: 2021-12-09
Also published as: CA2981500A1; CL2017002794A1; US11098319B2; CN107531766B; US20180295859A1; US10494640B2; AU2021201026A1; WO2016183467A1; AU2016260521A1; AU2021201026B2; CL2023001431A1; MX2017013318A; EP3294763A4; AU2016260521B2; CN114457181A; BR112017024087A2; EP3294763A1; CN107531766A; ES2951012T3; EP3294763B1

Abstract

Methods and compositions are described for producing a glucanase in transgenic plants and then incorporating parts of the transgenic plants in animal feed. The feed glucanase enzyme displays activity across a broad pH range, and tolerance to temperatures that are often encountered during the process of preparing animal feeds. Producing the enzyme in the transgenic plant enhances the thermal stability of the enzyme.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/599,543, which was filed on Oct. 11, 2019. U.S. patent application Ser. No. 16/599,543 is a continuation of U.S. patent application Ser. No. 15/569,592, which was filed on Oct. 26, 2017 as 35 U.S.C. § 371 national phase application of PCT/US2016/032418, which was filed May 13, 2016, and claimed the benefit of U.S. Provisional Patent Application No. 62/161,482 filed May 14, 2015, all of which are incorporated herein by reference as if fully set forth.
The sequence listing electronically filed with this application titled “Sequence Listing,” which was created on Jun. 23, 2021 and has a size of 146,050 bytes, is incorporated by reference herein as if fully set forth.

FIELD

This disclosure relates to transgenic plants expressing glucanases with improved thermal stability, nucleic acids encoding the same, as well as methods of processing transgenic plants and tissues, and producing and utilizing animal feed. This disclosure also relates to feed additives and grain and fiber processing additives that include glucanases.

BACKGROUND

The abundance of non-starch polysaccharides (NSPs) in the diets of monogastric and ruminant animals can adversely affect the nutritional value of feed, and also present an opportunity to improve nutritional content if they can be degraded in the diet or converted into beneficial nutritional components. NSPs are among the primary structural components of plant cell wall (cellulose, hemicellulose, xyloglucans, arabionxylans, galactans, arabinogalactans, etc.) and can also serve as carbohydrate storage reserves in some plants. Additionally, pectins and gums are considered non-cell wall NSP. Because of their various structural and biological roles, NSPs often bind or encase the starch, proteins, fats and other nutrients that are present in plant-based feed ingredients (such as cereals, legumes, silage etc.) and other ingredients, inhibiting the animal's ability to digest nutrients efficiently. Increased levels of NSPs in the diet may increase viscosity of intestinal contents, which can interfere with digestive enzymes and reduce the digestibility of nutrients, thereby increasing feed conversion (mass of feed divided by the mass of meat produced) and reducing body weight gain (Iji, P. A. 1999. The impacts of cereal non-starch polysaccharides on intestinal development and function in the broiler chickens. Worlds Poult. Sci. J. 55:375-387, which is incorporated herein by reference as if fully set forth). For example, feeding increasing levels of guar meal germ (0, 5, or 7.5%) or guar meal hulls (0, 2.5, or 5%) to broilers resulted in increasing digesta viscosity (Lee, J. T., C. A. Bailey, and A. L. Cartwright. 2003. β-Mannanase ameliorates viscosity-associated depression of growth in broiler chickens fed guar germ and hull fractions. Poult. Sci. 82:1925-1931, which is incorporated herein by reference as if fully set forth). In addition to increasing the viscosity, body weight gain and feed conversion was also worse with increasing guar meal hull, demonstrating the negative effects of high viscosity on animal performance.
NSPs have also been known to inadvertently trigger immune responses in the gut, which may further detract from efficiency of feed utilization and have implications for animal health.
In addition to the cereal components, diets now also routinely contain DDGS (dried distillers grains and solubles) that is also not easily digested. Multiple studies have shown that enzyme supplementation can increase diet metabolizable energy (ME), and, or, decrease the viscosity of diets containing high levels of wheat, barley, DDGSs, or other fibrous components. The addition of carbohydrases to corn-soybean meal-based broiler diets, when formulated to have a 3% reduction in dietary ME, has been accomplished without compromising the feed conversions of broilers reared under either hot or cool seasons. It has been determined that the hydrolyzed β-d-glucan is responsible for improved growth.

SUMMARY

In an aspect, the invention relates to a method of identifying maize event 4588.259, 4588.757 or 4588.652 in a sample. The method comprises contacting a sample with a first primer and a second primer. The method comprises amplifying a nucleic acid in the sample to obtain an amplified product. The method also comprises detecting the amplified product specific to a target sequence in maize event 4588.259, 4588.757 or 4588.652.
In an aspect, the invention relates to an animal feedstock comprising a transgenic plant or part thereof. The transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase. The glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides.
In an aspect, the invention relates to a method of producing an animal feedstock. The method includes mixing a transgenic plant or part thereof with plant material to form a mixture. The transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase. The glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides.
In an aspect, the invention relates to a method of increasing metabolizable energy of a diet. The method includes mixing a transgenic plant or part thereof with a feed ingredient. The transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase comprising an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides.
In an aspect, the invention relates to a method of enhancing production of fermentable sugars from grains. The method includes mixing grains derived from any one of the transgenic plants described herein with grains derived from a different plant to form mixed grains. The method also includes processing mixed grains into fermentable sugars. The fermentable sugars are subsequently converted into ethanol or a similar fermentation product, which may include butanol, lactic acid, citric acid, acetic acid, or other fuels or chemicals.
In an aspect, the invention relates to a transgenic plant, transgenic grain, or transgenic biomass comprising a synthetic nucleic acid encoding a glucanase. The glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6. The glucanase is capable of degrading one or more polysaccharides.
In an aspect the invention relates to a synthetic polypeptide or a fragment thereof. The synthetic polypeptide or a fragment thereof comprises an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6. The glucanase is capable of degrading one or more polysaccharides.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings particular embodiments. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

FIG. 1 illustrates the expression vector pAG4258 carrying a single feed glucanase expression unit.

FIG. 2 illustrates the expression vector pAG4588 carrying a single feed glucanase expression unit.

FIG. 3 illustrates the expression vector pAG4597 carrying a single feed glucanase expression unit.

FIG. 4 illustrates the expression vector pAG4708 carrying a single feed glucanase expression unit.

FIG. 5 illustrates the expression vector pAG4766 carrying two feed glucanase expression units.

FIG. 6 illustrated the expression vector pAG4767 carrying two feed glucanase expression units.

FIG. 7 illustrates the expression vector pAG4770 carrying three feed glucanase expression units.

FIG. 8 illustrates the expression vector pAG4771 carrying three feed glucanase expression units.

FIG. 9 is a chart illustrating the range of glucanase activity recovered from ears of the maize plants that carried pAG4588 construct.

FIG. 10 is a chart illustrating the range of glucanase activity recovered from ears of the maize plants that carried pAG4597 construct.

FIG. 11 is a diagram showing the T-DNA integration site in chromosome 7 of maize event 4588.652.

FIG. 12 is a chart illustrating glucanase activity observed in T1 plants.

FIG. 13 is a chart illustrating the glucanase activity in the seeds of hemizygous, homozygous, and hybrid plants.

FIG. 14 illustrates general design of the real-time PCR assay used to determine presence of the T-DNA locus (standard and real-time PCR) and zygosity (real-time PCR only) in transgenic events. Letters A, B, X and Y with arrows indicate primer binding sites. Rectangular boxes A+B and X+Y represent PCR products amplified from respective primer pairs.

FIG. 15 illustrates general design of the standard PCR assay used to determine presence of the T-DNA locus and zygosity in transgenic events. Letters A, B, and C with arrows indicate primer binding sites. Rectangular boxes A+B and A+C represent PCR products amplified from respective primer pairs.

FIG. 16 illustrates the standard multiplex PCR analysis of the selfed segregating 4588.652 plants.

FIG. 17 is a graph illustrating real-time PCR data to determine presence of the T-DNA locus and zygosity for maize event 4588.259 (FG259).

FIGS. 18A and 18B are charts illustrating glucanase activity in the Grower Diet (FIG. 18A) and the Starter Diet (FIG. 18B) before and after pelleting.

FIGS. 19A and 19B are charts illustrating glucanase activity in wild type (WT) flour mixed with microbial glucanase and transgenic flour producing glucanase after heat treatment at temperatures of 130° C. (FIG. 19A) and 94° C. (FIG. 19B).

FIGS. 20 and 21 are graphs illustrating the optimum pH for measuring AGR2314 activity in an assay at 37° C. (FIG. 20) and 80° C. (FIG. 21).

FIG. 22 is a graph illustrating an example of the optimum pH of the feed glucanase that is produced in transgenic flour.

FIGS. 23A and 23B are charts illustrating glucanase activity on multiple substrates at 37° C. (FIG. 23A) and 80° C. (FIG. 23B).

FIGS. 24A and 24B are charts illustrating enzymatic hydrolysis of untreated seeds fiber of transgenic maize plants expressing AGR2314. FIG. 24A shows glucose yield and FIG. 24B shows xylose yield.

FIGS. 25A and 25B are charts illustrating enzymatic hydrolysis of seed fiber of transgenic maize plants expressing AGR2314 pretreated with the dilute acid. FIG. 25A shows glucose yield and FIG. 25B shows xylose yield.

FIG. 26 is a chart illustrating the body weight gain (BWG) during the 28-day poultry feeding trial.

FIG. 27 is a chart illustrating the changes in poultry BWG per time interval during 28 day feeding trial.

FIG. 28 is a chart illustrating feed consumption during the 28-day poultry feeding trial using two different diets (corn/barley based and corn/LF-DDGS based) with (+) or without (−) a glucanase.

FIG. 29 is a chart illustrating feed conversion rate (FCR) during the 28-day poultry feeding trial with two different diets (corn/barley based and corn/LF-DDGS based diets) with (+) or without (−) a glucanase.

FIG. 30 is a chart illustrating the effect of glucanase on poultry BWF in experimental treatment.

DETAILED DESCRIPTION OF EMBODIMENTS

Certain terminology is used in the following description for convenience only and is not limiting.
“Synthetic nucleic acid sequence,” “synthetic polynucleotide,” “synthetic oligonucleotide,” “synthetic DNA,” or “synthetic RNA” as used herein refers to a nucleic acid sequence, a polynucleotide, an oligonucleotide, DNA, or RNA that differs from one found in nature by having a different sequence than one found in nature or a chemical modification not found in nature. This can include, but is not limited to, a DNA sequence created using biotechnology tools. Such tools include but are not limited to recombinant DNA technology, polymerase chain reaction (PCR), biotechnology mutagenesis techniques using PCR or recombination techniques including digestion and ligation of DNA, chemical mutagenesis techniques, chemical synthesis, or directed use of nucleases (so called “genome editing” or “gene optimizing” technologies).
“Synthetic protein,” “synthetic polypeptide,” “synthetic oligopeptide,” or “synthetic peptide” as used herein refers to a protein, polypeptide, oligopeptide or peptide that was made through a synthetic process. The synthetic process can include, but is not limited, to chemical synthesis or recombinant technology. The synthetic process may include production of a protein, polypeptide, oligopeptide or peptide by expression of a synthetic nucleic acid sequence in a living cell or by in vitro expression using a cell-free extract.
As used herein, “variant” refers to a molecule that retains a biological activity that is the same or substantially similar to that of the original sequence. The variant may be from the same or different species or be a synthetic sequence based on a natural or prior molecule.
As used herein, “alignment” refers to a plurality of nucleic acid or amino acid sequences aligned lengthwise for visual identification of commonly shared nucleotides or amino acids. The percentage of commonly shared nucleotides or amino acid is related to homology or identity between sequences. An alignment may be determined by or used to identify conserved domains and relatedness between the sequences. An alignment may be determined by computer programs such as CLUSTAL 0 (1.2.1) (Sievers et al. (2011) Molecular Systems Biology 7: 539 doi: 10. 1038/msb.2011.75).
The words “a” and “one,” as used in the claims and in the corresponding portions of the specification, are defined as including one or more of the referenced item unless specifically stated otherwise.
In an embodiment, a synthetic nucleic acid encoding a glucanase is provided. The synthetic nucleic acid may include a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 1 [AGR2314], SEQ ID NO: 2 [AGR2414], and SEQ ID NO: 3 [AGR2514]. The encoded glucanase may be capable of degrading one or more polysaccharides.
An embodiment includes a glucanase that includes a synthetic polypeptide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414], and SEQ ID NO: 6 [AGR2514]. The glucanase may be capable of degrading one or more polysaccharides. The glucanase may be modified for improved thermal stability.
A glucanase modified for thermal stability can be produced by standard molecular biological techniques and then screened. The glucanase can be subjected to mutation and then screened for thermal stability. Screening systems that can be utilized include lambda phage, yeast, or other expression systems that allow production of the protein and/or testing of its physical and/or functional characteristics. From a population of modified proteins, candidates can be isolated and analyzed further. Further analysis may include DNA sequencing, functional assays, structural assays, enzyme activity assays, and monitoring changes in thermal stability, or structure in response to elevated temperature conditions.
In an embodiment, a glucanase may be produced in a plant or plant tissue. The glucanase may be isolated from the plant or plant tissue.
An embodiment includes a composition comprising, consisting essentially of, or consisting of one or more glucanases. The composition may be, but is not limited, to a transgenic plant including the one or more glucanases, an animal feedstock or animal feed additive including the one or more glucanases or an enzyme mixture including the one or more glucanases. A glucanase in the composition may be encoded by any one of the synthetic nucleic acids described herein. As used herein, the term “glucanase” refers to an enzyme capable of catalyzing the degradation or depolymerization of complex carbohydrates.
A glucanase in the composition may be capable of degrading one or more of disaccharides, trisaccharides, and oligosaccharides into lower molecular weight saccharides. A glucanase in the composition may be capable of degrading one or more of cellooligosaccharide, lignocellulose, cellulose, hemicellulose, and pectin. A glucanase of the composition may act upon cellulose or mixed linkage beta glucans Some glucanases may have broader substrate specificities and may act on a wide range of carbohydrate polymers. A glucanase of the composition may have enzymatic activity on a range of carbohydrate polymers. Such enzymatic activit may be, but is not limited to, endoglucanase, exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase, β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, or pectate lyase activities. The glucanase of the composition may be capable of degrading one or more of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. Assays for determining activity of a glucanase for degrading various substrates are known in the art. The beta-glucosidase assay, endocellulase assay, exocellulase (cellobiohydrolase) assay, amylase assay, endoxylanase assay, pectinase assay, 1,3-beta-glucosidase assay, 1,4-beta-glucosidase assay are described herein in Example 13.
A glucanase of the composition may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514].
An embodiment includes a composition comprising, consisting essentially of, or consisting of an individual glucanase or a combination of two or more glucanases herein.
In an embodiment, a glucanase of the composition may be a variant. Variants may include conservative amino acid substitutions; i.e., substitutions with amino acids having similar properties. Conservative substitutions may be a polar for polar amino acid (Glycine (G, Gly), Serine (S, Ser), Threonine (T, Thr), Tyrosine (Y, Tyr), Cysteine (C, Cys), Asparagine (N, Asn) and Glutamine (Q, Gln)); a non-polar for non-polar amino acid (Alanine (A, Ala), Valine (V, Val), Thyptophan (W, Trp), Leucine (L, Leu), Proline (P, Pro), Methionine (M, Met), Phenilalanine (F, Phe)); acidic for acidic amino acid (Aspartic acid (D, Asp), Glutamic acid (E, Glu)); basic for basic amino acid (Arginine (R, Arg), Histidine (H, His), Lysine (K, Lys)); charged for charged amino acids (Aspartic acid (D, Asp), Glutamic acid (E, Glu), Histidine (H, His), Lysine (K, Lys) and Arginine (R, Arg)); and a hydrophobic for hydrophobic amino acid (Alanine (A, Ala), Leucine (L, Leu), Isoleucine (I, Ile), Valine (V, Val), Proline (P, Pro), Phenylalanine (F, Phe), Tryptophan (W, Trp) and Methionine (M, Met)). Conservative nucleotide substitutions may be made in a nucleic acid sequence by substituting a codon for an amino acid with a different codon for the same amino acid. Variants may include non-conservative substitutions. A variant may have 40% glucanase activity in comparison to the unchanged glucanase. A variant may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% activity, or an integer between any of the two values herein, in comparison to the unchanged glucanase.
In an embodiment, the one or more proteins having less than 100% identity to its corresponding amino acid sequence of SEQ ID NOS: 4-6 is a variant of the referenced protein or amino acid. In an embodiment, an isolated protein, polypeptide, oligopeptide, or peptide having a sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a protein having the sequence of any one of SEQ ID NOS: 4-6 may be a less than full length protein having the sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity any one of SEQ ID NO: 4-6 along 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, or 10 to all amino acids of a protein. This list of sequence lengths encompasses every full length protein in SEQ ID NOS: 4-6 and every smaller length within the list, even for proteins that do not include over 400 amino acids. For example, the lengths of 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, and 10 to all amino acids would apply to a sequence with 322 amino acids. A range of amino acid sequence lengths recited herein includes every length of amino acid sequence within the range, endpoints inclusive. The recited length of amino acids may start at any single position within a reference sequence where enough amino acids follow the single position to accommodate the recited length. The range of sequence lengths can be extended by increments of 10 to 100N amino acids, where N=an integer of ten or greater, for sequences of 1000 amino acids or larger. The fragment of the glucanase may be a subsequence of the polypeptides herein that retain at least 40% activity of the glucanase. The fragment may have 316, 317, or 322 amino acids. The fragments may include 20, 30, 40, 50, 100, 150, 200, or 300 contiguous amino acids. Embodiments also include nucleic acids encoding said amino acid sequences, and antibodies recognizing epitopes on said amino acid sequences.
A less than full length amino acid sequence may be selected from any portion of one of the sequences of SEQ ID NOS: 4-6 corresponding to the recited length of amino acids. A less than full length amino acid sequence may be selected from a portion of any one of SEQ ID NOS: 4-6 having a catalytic domain. The fragment may include a catalytic domain of a glucanase. For example, the catalytic domain of the glucanase of SEQ ID NO: 4 [AGR2314] includes the following sequence:

	(SEQ ID NO: 21)
	10 20 30 40
	-GVDPFERNKILGRGINIG ALEAPNEGDWGVVIKDEFFD

	50 60 70 80
	IIKEAGFSHVRIPIRWSTHAAFPPYKIEPSFFKRVDEVIN

	90 100 110 120
	GALKRGLAVVINI YEELMNDPEEHKERFLALWKQIADR

	130 140 150 160
	YKDYPETLFFEIL PHGNLTPEKWNELLEEALKVIRSID

	170 180 190 200
	KKHTVIIGTAEWGGISALEKLRVPKWEKNAIVTIHY NPF

	210 220 230 240
	EFT QGAE W VPGSEKWLGRKWGSPDDQKHLIEEFNFIEEW

	250 260 270 280
	SKKNKRPIYIG FGAYRKADLESRIKWTSFVVREAEKRGW

	290 300 310
	SWAY EFCSGFGVYDPLRK WNKDLLEALIGGDSIE

In the sequence of SEQ ID NO: 21, catalytic residues in active site are shown by enlarged characters in bold. Other active site residues that interact with the substrate are italicized, bold and underlined.
For example, positions 136 and 253 in SEQ ID NO: 21 are catalytic residues in the active site, and a less than full length amino acid sequence selected from SEQ ID NO: 21 may include residues 134 and 135 at any two respective, consecutive positions within the recited length. A less than full length amino acid sequence may be selected from a portion of any one of SEQ ID NO: 21 may have other active site residues that interact with the substrate. For example, positions 20, 35, 36, 135, 198, 205, 210 and 286 of SEQ ID NO: 21 are the active site residues that interact with the substrate, and a less than full length amino acid sequence selected from SEQ ID NO: 21 may include residues 20, 35, 36, 135, 198, 205, 210 and 286 at any respective, consecutive positions within the recited length.
A less than full length amino acid sequence may be selected from a portion of any one of SEQ ID NO: 21 may include amino acids 136-253. A less than full length amino acid sequence may possess the glucanase activity. A less than full length amino acid sequence may be capable of degrading polysaccharides. A less than full length amino acid sequence may contain those amino acids would contain the active site residues.
A catalytic domain may be a conserved domain. A “conserved domain” herein refers to a region in a heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. With respect to polynucleotides encoding a conserved domain is preferably at least 10 base pairs (bp) in length.
A conserved domain of any one of polypeptides described herein refers to a domain within a glucanase that exhibits a higher degree of sequence identity High degree of sequence identity may be at least 50% identity, at least 55% identity, at least 60% identity, at least 65%, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or at least 100% identity to consecutive amino acid residues of a polypeptide described herein. Conserved domains may be identified as domains of identity to a specific consensus sequence. Conserved domains may be identified by using alignment methods. Conserved domain may be identified with multiple sequence alignments of related proteins. These alignments reveal sequence regions containing the same, or similar, patterns of amino acids. Multiple sequence alignments, three-dimensional structure and three-dimensional structure superposition of conserved domains can be used to infer sequence, structure, and functional relationships. Since the presence of a particular conserved domain within a polypeptide is highly correlated with an evolutionarily conserved function, a conserved domain database may be used to identify the amino acids in a protein sequence that are putatively involved in functions such as degrading polysaccharides, as mapped from conserved domain annotations to the query sequence. For example, the presence in a protein of a sequence of SEQ ID NO: 21 that is structurally and phylogenetically similar to one or more domains in the polypeptides of the accompanying Sequence Listing is a strong indicator of a related function in plants. Sequences herein referred to as functionally-related and/or closely-related to the sequences or domains of polypeptides having sequences of SEQ ID NOS: 4-6 may have conserved domains that share at least at least ten amino acids in length and at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, or at least 99%, or about 100% amino acid identity to the sequences of AGR2314, AGR2414 and AGR2514, and have similar functions that the polypeptides of the instant description.
In an example, sequences of AGR2314, AGR2414, and AGR2514 may be aligned as shown below.
AGR2414 sequence is a sequence of Thermotoga maritima Cel5A (3AMC in Protein Data Bank and in PubMed protein sequence database). Residues that interact with the substrate are underlined and are shown in enlarged bold characters: N20, H95, H96, N135, E136 (catalytic residue), Y198, H205, W210, E253 (catalytic residue), W286. (T. Wu et al. (2011), Biochim. Biophys. Acta 1814, 1.832-1840, which is incorporated herein by reference as if fully set forth). The numbering of these residues in AGR2514 is one higher because of the presence of one additional residue at the N-terminus in this sequence.
In an example, the sequences of AGR2314 and AGR2414 have 305 residues conserved out of 317 residues and have 96% identity.

CLUSTAL O(1.2.1) multiple sequence alignment

AGR2414	MGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHA
AGR2314	MGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHA
	************************************************************

AGR2414	YAFPPYKIMDRFFKRVDEVINGALKRGLAVVINI HH YEELMNDPEEHKERFLALWKQIAD
AGR2314	QAFPPYKIEPSFFKRVDEVINGALKRGLAVVINI HH YEELMNDPEEHKERFLALWKQIAD
	***** ***********************************************

AGR2414	RYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISALE
AGR2314	RYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISALE
	******************************************:*************

AGR2414	KLSVPKWEKNSIVTIHY Y NPFEFT H QGAE W VEGSEKWLGRKWGSPDDQKHLIEEFNFIEE
AGR2314	KLRVPKWEKNAIVTIHY Y NPFEFT H QGAEWVPGSEKWLGRKWGSPDDQKHLIEEFNFIEE
	***:*************** ***************************

AGR2414	WSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREMEKRRWSWAY W EFCSGFGVYDTLRK
AGR2314	WSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRGWSWAY W EFCSGFGVYDPLRK
	********************************* * ************* *

AGR2414	TWNKDLLEALIGGDSIE (SEQ ID NO: 5)
AGR2314	QWNKDLLEALIGGDSIE (SEQ ID NO: 4)
	****************

In an example, the sequences of AGR2414 and AGR2514 below have 310 residues conserved out of 318 residues and have 97% identity.

CLUSTAL O(1.2.1) multiple sequence alignment

AGR2414	-MGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTH
AGR2514	MSGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTH
	**********************************:*******************

AGR2414	AYAFPPYKIMDRFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIA
AGR2514	AQAFPPYKIEDRFFKRVDEVINGALKRGLAVVINQ HH YEELMNDPEEHKERFLALWKQIA
	* **** ******************** ***********************

AGR2414	DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL
AGR2514	DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL
	************************************************************

AGR2414	EKLSVPKWEKNSIVTIHY Y NPFEFT H QGAE W VEGSEKWLGRKWGSPDDQKHLIEEFNFIE
AGR2514	EKLRVPKWEKNAIVTIHY Y NPFEFT H QGAE W VEGSEKWLGRKWGSPDDQKHLIEEFNFIE
	* ***:*********************************************

AGR2414	EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREMEKRRWSWAY W EFCSGFGVYDTLR
AGR2514	EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRRWSWAY W EFCSGFGVYDTLR
	********************************** *********************

AGR2414	KTWNKDLLEALIGGDSIW (SEQ ID NO: 5)
AGR2514	KTWNKDLLEALIGGDSIE (SEQ ID NO: 6)
	******************

In an example, the sequences of AGR2314 and AGR2514 have 308 residues conserved out of 318 residues and have 97% identity.

CLUSTAL O(1.2.1) multiple sequence alignment

AGR2314	-MGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTH
AGR2514	MSGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTH
	**********************************:*******************

AGR2314	AQAFPPYKEIPSFFKRVDEVINGALKRGLAVVINI HH YEELMNDPEEHKERFLALWKQIA
AGR2514	AQAFPPYKIEDRFFKRVDEVINGALKRGLAVVINQ HH YEELMNDPEEHKERFLALWKQIA
	******** ****************** **********************

AGR2314	DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISAL
AGR2514	DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL
	*******************************************:***********

AGR2314	EKLRVPKWEKNAIVTIHY Y NPFEFT H QGAE W VPGSEKWLGRKWGSPDDQKHLIEEFNFIE
AGR2514	EKLRVPKWEKNAIVTIHY Y NPFEFT H QGAE W VEGSEKWLGRKWGSPDDQKHLIEEFNFIE
	***************************** *************************

AGR2314	EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRGWSWAY W EFCSGFGVYDPLR
AGR2514	EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRRWSWAY W EFCSGFGVYDTLR
	************************************** ***********

AGR2314	KQWNKDLLEALIGGDSIE (SEQ ID NO: 4)
AGR2514	KTWNKDLLEALIGGDSIE (SEQ ID NO: 6)
	* ****************

Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and that have comparable biological and regulatory activity to the present polypeptide sequences, thus being glucanases, described herein. Sequences having lesser degrees of identity, but comparable biological activity, are considered to be equivalents.
The functionality of a glucanase, variants, or fragments thereof, may be determined using any methods. The functionality of a glucanase may be measured by any one of the assays described in Example 3.
Any one or more glucanases herein may be expressed in a plant upon introduction into the plant genome of any one more of synthetic nucleic acids described herein. The methods of introduction of synthetic nucleic acids into the plants are known in the art. The method may be transformation of the plant with a vector that includes synthetic nucleic acids.
In an embodiment, a synthetic polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of SEQ ID NO: 7 [pAG4258], SEQ ID NO: 8 [pAG4588], SEQ ID NO: 9 [pAG4597], SEQ ID NO: 10 [pAG4708], SEQ ID NO: 11 [pAG4766], SEQ ID NO: 12 [pAG4767], SEQ ID NO: 13 [pAG4770], SEQ ID NO: 14 [pAG4771], SEQ ID NO: 15 [pAG4257], SEQ ID NO: 16 [pAG4692], SEQ ID NO: 17 [pAG4693], SEQ ID NO: 18 [pAG4705] and SEQ ID NO: 19 [pAG4706] is provided. The synthetic polynucleotide may include any one of the synthetic nucleic acids described herein that encode glucanase and that are capable of degrading a polysaccharide.
In an embodiment, a vector is provided. The vector may include any one of the synthetic polynucleotides or nucleic acids described herein.
In an embodiment, synthetic nucleic acids are provided having a sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. In an embodiment, isolated nucleic acids having a sequence that hybridizes to a nucleic acid having the sequence of any nucleic acid listed herein or the complement thereof are provided. In an embodiment, the hybridization conditions are low stringency conditions. In an embodiment, the hybridization conditions are moderate stringency conditions. In an embodiment, the hybridization conditions are high stringency conditions. The hybridization may be along the length of the synthetic nucleic acid. Examples of hybridization protocols and methods for optimization of hybridization protocols are described in the following publications: Molecular Cloning, T. Maniatis, E. F. Fritsch, and J. Sambrook, Cold Spring Harbor Laboratory, 1982; and, Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl, Volume 1, John Wiley & Sons, 2000 (standard protocol) and Amersham Gene Images AlkPhos Direct Labeling and Detection System (GE Healthcare UK, Ltd), which are incorporated by reference in their entirety as if fully set forth.
In an AlkPhos Direct Labeling and Detection System, moderate conditions may be as follows: membranes loaded with DNA samples are prehybridized for at least 15 minutes at 55° C. in the hybridization buffer (12% (w/v) urea, 0.5M NaCl, 4% (w/v) blocking reagent). The labeled probe is added to the same solution and hybridization is carried overnight at 55° C. The membranes are washed for 10 minutes at 55° C. in the primary wash solution (2M urea, 0.1% (W/v) SDS, 50 mM of 0.5M Na phosphate pH 7.0, 150 mM NaCl, 1 mM of 1.0 M Mg Cl₂and 0.2% (w/v) of blocking reagent). The washing procedure is repeated. The membranes are placed in a clean container and washed for 5 minutes in a secondary buffer (1M Tris base, and 2M NaCl). The washing in the secondary solution is performed two more time. Chemoluminescence was detected using CDP-STAR® substrate for alkaline phosphatase. Low stringency refers to hybridization conditions at low temperatures, for example, between 37° C. and 60° C. High stringency refers to hybridization conditions at high temperatures, for example, over 68° C.
In the standard protocol, moderate conditions may be as follows: filters loaded with DNA samples are pretreated for 2-4 hours at 68° C. in a solution containing 6×citrate buffered saline (SSC; Amresco, Inc., Solon, Ohio), 0.5% sodium dodecyl sulfate (SDS; Amresco, Inc., Solon, Ohio), 5×Denhardt's solution (Amresco, Inc., Solon, Ohio), and denatured salmon sperm (Invitrogen Life Technologies, Inc. Carlsbad, Calif.). Hybridization is carried in the same solution with the following modifications: 0.01 M EDTA (Amresco, Inc., Solon, Ohio), 100 μg/ml salmon sperm DNA, and 5-20×10⁶cpm ³²P-labeled or fluorescently labeled probes. Filters are incubated in hybridization mixture for 16-20 hours and then washed for 15 minutes in a solution containing 2×SSC and 0.1% SDS. The wash solution is replaced for a second wash with a solution containing 0.1×SSC and 0.5% SDS and incubated an additional 2 hours at 20° C. to 29° C. below Tm (melting temperature in ° C.). Tm=81.5+16.61 Log₁₀[Na⁺]/(1.0+0.7[Na⁺]))+0.41(%[G+C])−(500/n)−P-F. [Na+]=Molar concentration of sodium ions. %[G+C]=percent of G+C bases in DNA sequence. N=length of DNA sequence in bases. P=a temperature correction for % mismatched base pairs (1° C. per 1% mismatch). F=correction for formamide concentration (=0.63° C. per 1% formamide). Filters are exposed for development in an imager or by autoradiography. Low stringency conditions refers to hybridization conditions at low temperatures, for example, between 37° C. and 60° C., and the second wash with higher [Na⁺] (up to 0.825M) and at a temperature 40° C. to 48° C. below Tm. High stringency refers to hybridization conditions at high temperatures, for example, over 68° C., and the second wash with [Na+]=0.0165 to 0.0330M at a temperature 5° C. to 10° C. below Tm. In an embodiment, synthetic nucleic acids having a sequence that has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity along its length to a contiguous portion of a nucleic acid having any one of the sequences set forth herein or the complements thereof are provided. The contiguous portion may be the entire length of a sequence set forth herein or the complement thereof.
In an embodiment a synthetic nucleic acid may encode the fragment of a glucanase that have 316, 317, or 322 amino acids. The synthetic nucleic acids may encode the fragments that include 20, 30, 40, 50, 100, 150, 200, or 300 contiguous amino acids and retain at least 40% activity of the glucanase. The functionality of a glucanase, variants, or fragments thereof, may be determined using any methods. The functionality of a glucanase may be measured by any one of the assays described in Example 3.
Determining percent identity of two amino acid sequences or two nucleic acid sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity can be measured by the Smith Waterman algorithm (Smith T F, Waterman M S 1981 “Identification of Common Molecular Subsequences,” Journal of Molecular Biology 147: 195-197, which is incorporated by reference in its entirety as if fully set forth).
In an embodiment, synthetic nucleic acids, polynucleotides, or oligonucleotides are provided having a portion of the sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. These isolated nucleic acids, polynucleotides, or oligonucleotides are not limited to but may have a length in the range from 10 to full length, 10 to 800, 10 to 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, or 20 to 30 nucleotides or 10, 15, 20 or 25 nucleotides. A synthetic nucleic acid, polynucleotide, or oligonucleotide having a length within one of the above ranges may have any specific length within the range recited, endpoints inclusive. In an embodiment, a hybridization probe or primer is 85 to 100%, 90 to 100%, 91 to 100%, 92 to 100%, 93 to 100%, 94 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% complementary to a nucleic acid with the same length as the probe or primer and having a sequence chosen from a length of nucleotides corresponding to the probe or primer length within a portion of a sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, a hybridization probe or primer hybridizes along its length to a corresponding length of a nucleic acid having the sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, the hybridization conditions are low stringency. In an embodiment, the hybridization conditions are moderate stringency. In an embodiment, the hybridization conditions are high stringency.
In an embodiment, a transgenic plant comprising a synthetic nucleic acid encoding any one or more of the glucanases described herein is provided. The one or more glucanases expressed in the transgenic plant herein may have activity at a pH ranging from 2.0 to 10.00. The pH may be 2.0, 3.0, 4.0, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 9.0, 9.5, or 10, or a pH within a range between any two of the foregoing pH values (endpoints inclusive). The one or more glucanases expressed in a transgenic plant herein may have activity when exposed to a temperature in the range of 25° C. to 130° C., endpoints inclusive. The temperature may be 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120° C., 125° C., 130° C., 25° C., to 30° C., 25° C. to 35° C., 25° C. to 40° C., 25° C. to 45° C., 25° C. to 50° C., 25° C. to 55° C., 25° C. to 60° C., 25° C. to 65° C., 25° C. to 70° C., 25° C. to 75° C., 25° C. to 80° C., 25° C. to 85° C., 25° C. to 90° C., 25° C. to 95° C., 25° C. to 100° C., 25° C. to 105° C., 25° C. to 110° C., 25° C. to 115° C., 25° C. to 120° C., 25° C. to 125° C., or less than 130° C. The glucanase expressed in the transgenic plant may have the improved activity compared to the glucanase having an identical amino acid sequence but expressed in a bacterial cell. The glucanase may have improved thermal stability compared to the activity of the glucanase expressed in the bacterial cell.
The one or more glucanase may be produced in any transgenic plant. The transgenic plant may be but is not limited to wheat, maize, soybean, barley, and sorghum.
In an embodiment, a method of making a transgenic plant that includes a glucanase is provided. The method may include contacting a plant cell with any one of the synthetic nucleic acids herein. The synthetic nucleic acids may be part of any one of the vectors described herein. The vector may include a synthetic nucleic acid encoding a glucanase. The glucanase may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514]. The method may also include regenerating a transgenic plant from the transgenic plant cell. The method may include selecting the transgenic plant expressing a glucanase.
The transgenic plant herein is also referred to as an “event.” An event is characterized by presence of the transgene comprising a synthetic nucleic acid encoding a glucanase. The term “event” also refers to the genomic region of the transformed parent comprising the inserted synthetic nucleic acid sequence and the parent genomic sequences flanking the insertion. The term “event” also refers to progeny produced by crossing of the transgenic plant and a non-transgenic plant of the same genetic background. The term “line” also refers to progeny produced by crossing of the transgenic plant and a non-transgenic plant with any genetic background. After repeated crosses, the transgene and the flanking sequences of the originally transformed parent may be present in a progeny plant in the same location in the genome or on the same chromosome as in the transformed parent.
The transgenic plant may be homozygous for the transgene comprising a synthetic nucleic acid encoding a glucanase.
The transgenic plant may be hemizygous for the transgene comprising a synthetic nucleic acid encoding a glucanase. To produce homozygous plants expressing a glucanase, a hemizygous transgenic plant may be self-crossed. Progeny may be obtained from such crosses. The progeny may include homozygous, hemizygous and wild type plants. A hemizygous plant may be phenotypically indistinguishable from the wild type plants. The method may include analyzing the progeny for the presence of the transgene and selecting a progeny plant that includes the transgene. A method of identifying the homozygous event by PCR is described herein in Example 8.
In an embodiment, the method may further include crossing a hemizygous transgenic plant to another transgenic plant hemizygous for the same transgene. The method may include selecting a first progeny plant that is homozygous for the transgene. The method may further include crossing the transgenic plant to a wild type plant of the same, or different, genetic background. Progeny may be obtained from such crosses. The progeny may include hemizygous and wild type plants. The method may include selecting a first progeny plant that is hemizygous for the transgene. The method may further include selfing the first hemizygous progeny plant and selecting a second progeny plant that is homozygous for the transgene comprising a synthetic nucleic acid sequence encoding a glucanase.
The glucanase may have activity and improved thermal stability when exposed to high temperatures as here described.
It has been unexpectedly discovered that expression and accumulation of an enzyme in a plant provides the enzyme with additional thermal stability relative to the same enzyme that is produced microbially.
In an embodiment, the method of making a transgenic plant includes transformation. For transformation, the nucleic acid may be introduced into a vector. Suitable vectors may be cloning vectors, transformation vectors, expression vectors, or virus-based vectors. The expression cassette portion of a vector may further include a regulatory element operably linked to a nucleic acid encoding a glucanase. In this context, operably linked means that the regulatory element imparts its function on the nucleic acid. For example, a regulatory element may be a promoter, and the operably linked promoter would control expression of the nucleic acid.
The expression of a nucleic acid encoding a glucanase from the expression cassette may be under the control of a promoter which provides for transcription of the nucleic acid in a plant. The promoter may be a constitutive promoter or, tissue specific, or an inducible promoter. A constitutive promoter may provide transcription of the nucleic acid throughout most cells and tissues of the plant and during many stages of development but not necessarily all stages. An inducible promoter may initiate transcription of the nucleic acid sequence only when exposed to a particular chemical or environmental stimulus. A tissue specific promoter may be capable of initiating transcription in a particular plant tissue. Plant tissue may be, but is not limited to, a stem, leaves, trichomes, anthers, cob, seed, endosperm, or embryo. The constitutive promoter may be, but is not limited to the maize ubiquitin promoter (ZmUbil), Cauliflower Mosaic Virus (CAMV) 35S promoter, the Cestrum Yellow Leaf Curling Virus promoter (CMP), the actin promoter, or the Rubisco small subunit promoter. The tissue specific promoter may be the maize globulin promoter (ZmGlb1), the rice glutelin promoter (prGTL), the maize zein promoter (ZmZ27), or the maize oleosin promoter (ZmOle). The promoter may provide transcription of a synthetic polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of SEQ ID NO: 7 [pAG4258], SEQ ID NO: 8 [pAG4588], SEQ ID NO: 9 [pAG4597], SEQ ID NO: 10 [pAG4708], SEQ ID NO: 11 [pAG4766], SEQ ID NO: 12 [pAG4767], SEQ ID NO: 13 [pAG4770], SEQ ID NO: 14 [pAG4771], SEQ ID NO: 15 [pAG4257], SEQ ID NO: 16 [pAG4692], SEQ ID NO: 17 [pAG4693], SEQ ID NO: 18 [pAG4705] and SEQ ID NO: 19 [pAG4706] and expression of glucanase that is capable of degrading a polysaccharide.
In an embodiment, the transformation in the method of making a transgenic plant may be stable transformation, wherein the nucleic acid encoding the glucanase integrates into the genome of the transformed plant. The transformation may be Agrobacterium-mediated transformation using a vector suitable for stable transformation described herein. The method of making a transgenic plant may include any other methods for transforming plants, for example, particle bombardment, or protoplast transformation via direct DNA uptake. The transgenic plant may include any synthetic nucleic acid, amino acid sequence, or vector herein.
In an embodiment, the method of making a transgenic plant may include transient transformation to transiently express the recombinant protein. The term “transient expression” refers to the expression of an exogenous nucleic acid molecule delivered into a cell: e.g., a plant cell, and not integrated in the plant's cell chromosome. Expression from extra-chromosomal exogenous nucleic acid molecules can be detected after a period of time following a DNA-delivery. Virus-based vectors may be used to carry and express exogenous nucleic acid molecules. Virus-based vectors may replicate and spread systemically within the plant. Use of virus based vectors may lead to very high levels of glucanase accumulation in transgenic plants.
Methods of making a transgenic plant, methods of increasing utilization of non-starch polysaccharides in an animal, methods for enhancing production of fermentable sugars from grains, methods for increasing metabolizable energy of a diet, methods preparing and animal feedstock and methods for producing genetically engineered plants homozygous for a synthetic nucleic acid that encodes a glucanase may comprise a method of detection herein as part of making transgenic plants and/or identifying plants, plant biomass or animal feed that comprise a synthetic nucleic acid herein.
An embodiment comprises a kit for identifying maize event 4588.259, 4588.757 or 4588.652 in a sample. The kit may comprise a first primer and a second primer.
The first primer and the second primer may be capable of amplifying a target sequence specific to an event. The target sequence may include a nucleic acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from SEQ ID NOS: 51-55. The target sequence may be a sequence included in a junction between a genomic sequence of a transformed plant and a sequence of the T-DNA insertion. The target sequence may be included in a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from SEQ ID NOS: 22-31.
The kit may comprise the first primer comprising a nucleic acid sequence selected from SEQ ID NOS: a nucleic acid sequence selected from SEQ ID NOS: 38, 41, and 47. The kit may comprise the second primer comprising a nucleic acid sequence selected from SEQ ID NOS: 39, 42, 43, 45, and 46. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 38 and the second primer comprising the nucleic sequence of SEQ ID NO: 39. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 41 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 42. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 41 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 43. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 47 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 45. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 47 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 46. The first primer and the second primer may be capable of amplifying the target sequence to produce an amplified product. The amplified product or the target sequence may be capable of hybridizing to the sequence of the nucleic acid comprising a sequence of SEQ ID NO: 40, or SEQ ID NO: 44 under conditions of high stringency. The target sequence may be used as a probe for diagnosing any one of the events described herein.
A sample may include any sample in which nucleic acids from plant matter are present. A sample may be a protein sample. A protein sample may include any sample in which proteins from plant matter are present. The sample or protein sample may include any plant matter. The plant matter may derive from a plant or part thereof. The plant material may derive from an animal feed or food.
In an embodiment, a method of identifying maize event 4588.259, 4588.757 or 4588.652 in a sample is provided. The method may include contacting a sample with a first primer and a second primer. The method may include amplifying a synthetic polynucleotide comprising a target sequence specific to the maize event. The target sequence may be any target sequence described herein. The first primer and the second primer may be capable of amplifying the target sequence to produce an amplified product. The amplified product may be used to determine whether a plant resulted from a sexual crossing or selfing contains one or more of the target sequences and diagnose specific events. The length of the amplified product from the sample of the maize event may differ from the length of the amplified product from the sample of wild type plant of the same genetic background. The amplified product from the event sample may be further used as probe that hybridizes to a synthetic polynucleotide comprising a specific region encoding a glucanase under conditions of high stringency. The method may include further detecting hybridization of the at least one probe to the specific region of the target sequence.
In an embodiment, an animal feedstock comprising any one or more of the transgenic plants described herein or parts of the transgenic plants is provided. The term “animal feedstock” refers to any food, feed, feed composition, diet, preparation, additive, supplement, or mixture suitable and intended for intake by animals for their nourishment, maintenance, or growth. The glucanases included in the transgenic plants and in the animal feedstock may be active in the gastrointestinal or rumen environment of animals. The animal may be a monogastric animal. The animal may be a ruminant animal. The monogastric animal may be but is not limited to a chicken, a turkey, a duck, a swine, a fish, a cat, or a dog. The ruminant animal may be but is not limited to cattle, a cow, a steer, a sheep, or a goat. The glucanases may be active after preparation of the animal feed. The temperatures which feeds are exposed to during ensiling may be within range of 20° C. to 70° C. The temperatures which feeds are exposed to during pelleting may be within range of 70° C. to 130° C. The glucanases may have improved thermal stability and may retain activity after being exposed to high temperatures during feed pelleting. The glucanase with improved thermal stability may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514].
In an embodiment, a glucanase may be isolated from the transgenic plant prior to being included into the animal feedstock. The glucanase may be any one of the glucanases described herein.
In an embodiment, the animal feedstock may further include a feed supplement. The feed supplement may be any plant material. The plant material may be a non-transgenic plant or a part thereof. The plant material may include an engineered plant or a mutant plant. The feed supplement may be a mineral. The mineral may be a trace mineral. The mineral may be a macro mineral. The feed supplement may be at least one vitamin. The at least one vitamin may be a fat-soluble vitamin. The feed supplement may include one or more exogenous enzymes. The one or more exogenous enzymes may include a hydrolytic enzyme. The hydrolytic enzyme. The hydrolytic enzyme may be an enzyme classified under EC3.4 as hydrolase. The hydrolytic enzymes may be but are not limited to xylanases, mannanases, carbohydrases, proteases, peptidases, phytases, cellulases, lipases, phospholipases, pectinases, galactosidases, laccases, amylases, hemicellulases, or cellobiohydrolases. The hydrolytic enzymes may be expressed in the engineered plants or parts thereof included in the feed supplement. The feed supplement may include purified hydrolytic enzymes. The feed supplements may be but are not limited to growth improving additives, coloring agents, flavorings, stabilizers, limestone, stearine, starch, saccharides, fatty acids, or a gum. The coloring agents may be carotenoids. The carotenoids may be but are not limited to cantaxanthin, beta-carotene, astaxanthin, or lutein. The fatty acids may be polyunsaturated fatty acids. The polyunsaturated fatty acids may include but are not limited to arachidonic acid, docosohexaenoic acid (DHA), eicosapentaenoic acid (EPA) or gamma-linoleic acid. The feed supplement may be a non-transgenic plant or a part thereof. The non-transgenic plant or part thereof may include at least one component selected from the group consisting of: barley, wheat, rye, oat, corn, rice, triticale beet, sugar beet, spinach, cabbage, quinoa, corn meal, corn pellets, corn oil, distillers grains, forage, wheat meal, wheat pellets, wheat grain, barley grain, barley pellets, soybean meal, soybean oilcake, lupin meal, rapeseed meal, sorghum grain, sorghum pellets, rapeseed, sunflower seed, and cotton seed.
The feed supplement may include at least one component selected from the group consisting of: soluble solids, fat, vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, monensin sodium COBAN® PREMIX, vitamin premix, inorganic feed phosphates, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate, monodicalcium phosphate, rock phosphate, selenium premix, choline chloride, sodium chloride, and mineral premix.
The feed supplement may include fish meal, fish oil, bone meal, feather meal and animal fat. The feed supplement may include yeast or yeast extract.
In an embodiment, a method of producing an animal feedstock is provided. The method may comprise including a transgenic plant that includes any one or more glucanase described herein in the animal feedstock. The animal feedstock may comprise, consist essentially of or consist of the transgenic plant. The method may further include combining the transgenic plant with a feed supplement. The feed supplement may be a non-transgenic plant or a part thereof. The transgenic plant may be produced by any one of the methods described herein. The feed supplement may be a mineral. The supplement may include one or more exogenous enzymes. The exogenous enzymes may be but are not limited to xylanases, mannanases, carbohydrases, proteases, peptidases, phytases, cellulases, lipases, phospholipases, pectinases, galactosidases, laccases, amylases, hemicellulases, or cellobiohydrolases.
In an embodiment, a method of meat production is provided. The method may include feeding an animal feedstock or one produced by any of the methods described herein to the animal. The method may include preparing an animal feedstock that includes a transgenic plant expressing a glucanase.
In an embodiment, a method of feeding an animal is provided. The method may include feeding an animal feedstock or one produced by any of the methods described herein to the animal. The method may include preparing an animal feedstock that includes a transgenic plant expressing a glucanase.
In an embodiment, a method of increasing utilization of non-starch polysaccharides in an animal is provided. The method may include feeding an animal with an animal feedstock that includes any one or more of the transgenic plants described herein. The method may include preparing the animal feedstock.
In an embodiment, a method of decreasing gastrointestinal viscosity in an animal is provided. The method may include feeding an animal with an animal feedstock that includes any one or more of the transgenic plants described herein. The method may include preparing the animal feedstock.
Addition of exogenous enzymes collectively known as carbohydrases may ameliorate the effects of non-starch polysaccharides (NSPs) in the diet of an animal. An animal feedstock that includes any one or more of glucanases described herein may increase utilization of NSPs by the animal that ingested the feedstock, or may decrease the anti-nutritional effects of the NSP on the animal that ingested the feedstock, and improve growth of the animal. Preparing the animal feedstock may include combining one or more transgenic plant herein with any other feed supplement. The glucanase may be isolated, purified and added to the animal feedstock as a pure glucanase. The glucanase may be added to the animal feedstock in admixture with other feed supplements. The transgenic plant including the glucanase or the purified glucanase may be combined with other feed supplements to form premixes.
An animal feedstock may be produced as mash feed. The animal feedstock may be produced as pellets. The milled feed stuffs may be mixed with the premix that includes any one of the transgenic plants that include a glucanase. The milled feed stuffs may include the plant material and the feed supplements described herein. The feed supplements may include one or more exogenous enzymes described herein. Enzymes may be added as liquid or solid formulations. For mash feed, a solid or liquid enzyme formulation may be added before or during the mixing step. For pelleted feed, the enzyme preparation may be added before or after the pelleting step. The glucanase may be included in a premix. The premix may also include vitamins and trace minerals. Macro minerals may be added separately to animal feedstock.
In an embodiment, a method of increasing metabolizable energy of a diet is provided. Metabolizable energy (ME) refers to the net energy of a diet or feed that is available to an animal after the utilization of some energy in the processes of digestion and absorption and the loss of some of the material as being undigested or indigestible. Metabolizable energy may be apparent metabolizable energy (AME) measured as the difference between the calories of the feed consumed by an animal and excrements collected after feed consumption. Metabolizable energy may be true metabolizable energy (TME), which is similar to AME except that it also takes into account endogenous energy. Energy contents in a diet or feed ingredients may be determined using one of several methodologies (NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, D.C., which is incorporated herein by reference as if fully set forth). Gross energy (GE) is direct measurement using an adiabatic bomb calorimeter, which measures the heat of combustion of a sample within a high oxygen atmosphere. Apparent digestible energy (DE) is GE of a feed or feedstuff minus GE of feces only. Apparent metabolizable energy (AME) is GE of a feed or feedstuff minus GE of feces, urine, and gaseous products from digestion. For poultry, the gaseous release is very low, and typically neglected due to its very small value, and the urine and feces are excreted together and are not collected separately in most cases. True metabolizable energy (TME) accounts for only the GE from excreta that is from the feed or feedstuff origin, by subtracting the endogenous energy loss from non-feed origin (i.e. sloughing of intestinal tract cells). Another energy measurement used for feedstuffs in animals is net energy (NE) which adjusts for heat increment. Since heat increment is dependent on level of productivity, which fluctuates in poultry because of short lifespan, this variable is not frequently used in poultry.
The TME rooster assay may be used to account for endogenous (non-feed) losses of GE by including a fasted rooster and collecting excreta to correct the GE from the fed (feed/feedstuff) rooster. See Sibbald, 1976, Poultry Science 55: 303-308, which is incorporated herein by reference as if fully set forth. This assay has commonly been used for determining TME of individual feedstuffs rather than complete feed, and requires cecetomized roosters (ceca surgically removed) to always be on hand. The assay involves force-feeding (into the crop) a known quantity of an ingredient (in birds that were previously fasted 24-48 hr) and then collect feces for a 24-48 hour period. The equation used to calculate TME is given as TME={(GE_f×FI)−[(GE_e×EO)⁺−(GE_e×EO)⁻]}/FI, where Gross Energy (GE) is determined by bomb calorimetry in kcal/kg; FI is feed intake (kg); EO is excreta output fed birds (kg); GE_eis the Gross Energy of the excreta content; GE_fis the Gross Energy of the feed; “k” signifies the quantity is from the fed birds energy output; and “−” signifies that the quantity is from the fasted birds energy output. The roosters (or turkeys) used in TME assays are adult birds with a fully developed digestive tract. Research has shown that there are differences in ME determinations using roosters (layer breeds), turkeys and broilers when analyzing same feed ingredients (Cozannet et al, 2010 J. Anim, Sci., 88(7):2382-2392, which is incorporated herein by reference as if fully set forth). So determining TME or AME using rooster model may not be equivalent to what is observed in a young broiler, but is a commonly used proxy in research and industry.
For broilers, the AME assay may be used for determining complete feed and some energy supplying feedstuffs, as well as the effect from adding feed ingredients that aid in digestion. There are two common methods for determining ME: 1) doing a total excreta collection and weighing and record feed consumption during the time period (Equation 1 below) or 2) using an indigestible marker in feed (chromic oxide, titanium oxide or acid insoluble ash) and taking a subsample of feces with no weighing required (Equation 2 below). The marker method of AME determination may be used, in which no weighing of feed consumption or total fecal collection and no need to separate feed spilled from feces pan are required. With the marker method, birds are fed the marker for at least two days (but preferably five or more days). Feces are collected over several days (e.g., three days) with daily collection composited into one sample.
AME using the total collection method (Equation 1) is calculated as follows:
AME=[(GE _f ×FI)−(GE _e ×EO)]/FI,
where Gross Energy (GE) is measured in bomb calorimetry (kcal/kg); FI is feed intake (kg); EO is excreta output (kg); e refers to excreta content; and f refers to the feed content. AME using the marker method is calculated as AME=[(GE_e/M_e)−(GE_f/M_f)]/(GE_e/M_e), where Gross Energy is GE (kcal/kg); M is the marker (ppm or %); “e”=excreta content; “f”=feed content.
Another method that may be used to determine AME of feed when investigating feed additives that aid in digestion is Ileal digestible energy (IDE). This method uses the AME marker method (described above), but the birds are euthanized and a section of ileum excised and contents removed, dried and analyzed for GE and the marker. The IDE method may be used effectively for testing and comparing feed additives used to improve digestion/absorption of feed energy. The benefit of IDE, is no cages with collection pans are required and can collect during a floor-pen study. With the marker method, birds are fed the marker for at least two days (and preferably five or more days).
AME using the IDE marker method (Equation 2) is calculated as follows:
AME=GE _f−(GE _d ×M _f /M _d),
where GE (kcal/kg); M represents the marker; “_d” represents the digesta content; and “_f” signifies the feed content.
AME and TME may be corrected for nitrogen retention (AMEn and TMEn). To adjust, the grams of N are multiplied by 8.22 kcal/g (GE of uric acid; primary excretory product of protein tissue oxidized for energy), which also is subtracted off of the GE consumed. See Hill, F. W., and D. L. Anderson, 1958, “Comparison of metabolizable energy and productive energy determinations with growing chicks.” J. Nutr. 64:587-603, which is incorporated herein by reference as if fully set forth. Calculations for total collection of marker method for AMEn are shown in Equation 3 and Equation 4 below, respectively. Equation 3: AMEn, total collection:
AMEn={(GE _f ×FI)−(GE _e ×EO)−[8.22×(N _f −N _e)]}/FI,
where GE=kcal/kg; FI=feed intake (kg); EO=excreta output (kg); N=nitrogen (g); e=excreta content; f=feed content.
Equation 4: IDEn, marker method:
AMEn=GE _f−(GE _d ×M _f /M _d)−{8.22×[N _f−(Nd×M _f /M _d)]},
where GE=kcal/kg; M=marker; N=nitrogen (g/kg) “d”=digesta content; “f”=feed content.
While the TME method may be used for determining ME of individual ingredients, the AME (IDE) method may be used with broilers to measure ME in individual ingredients or total diet and testing effects improving ME by use of enzymes or other feed additives.
A diet or feed may include any feed ingredient or mixture of ingredients including water. The diet may be any food, feed, feed composition, diet, preparation, additive, supplement, or mixture included in an animal feedstock described herein. The diets are known in the art and described at least in the following publications: Nutrient Requirements of Poultry, 1994, National Research Council, National Academy Press, Washington, D.C.; Broiler Performance and Nutrition Supplement, Cobb-500™, L-2114-07EN, July, 2015; Broiler Performance and Nutrition Supplement, Cobb-700™, L-21124-13EN, Dec. 21, 2012; Broiler Performance and Nutrition Supplement, CobbAvian™ L-2144-04EN, April, 2012; Broiler Performance and Nutrition Supplement, CobbSasso™, L-2154-01, May 7, 2008; Ross 308 Broiler: Nutrition Specifications, 2014 Aviagen, 0814-AVNR-035; Ross Nutrition Supplement 2009, Aviagen; Ross 708 Broiler: Nutrition Specification, 2014 Aviagen, 0814-AVNR-036; Ross PM3 Brioler Nutrition Specification, 2014 Aviagen, 0814-AVNR-037; Arbor Acres Plus Broiler Nutrition Specifications, 2014 Aviagen, 1014-AVNAA-043; Arbor Acres Broiler Nutrition Supplement, 2009 Aviagen; and Association of American Feed Control Officials (AAFCO) 2015 Official Publication, Nutrient Requirements for Poultry, all of which are incorporated herein by reference as if fully set forth.
In an embodiment, the diet may be a diet for broilers. The diet for broilers may be composed of one or more of the following ingredients: 51.49% (w/w)-61.86% (w/w) corn, 25.45% (w/w)-35.03% (w/w) soybean meal, 5.00% (w/w) corn distillers dry grains plus soluble solids, 2.00% (w/w) vermiculite, 0.30% (w/w)-1.99% (w/w) dicalcium phosphate, 1.00% (w/w) poultry fat, 0.81% (w/w)-4.01% (w/w) limestone, 0.24% (w/w)-0.50% (w/w) salt (NaCl), 0.13% (w/w)-0.45% (w/w) DL-methionine, 0.20% (w/w) choline chloride 60, 0.20% (w/w) mineral premix, 0.05% (w/w) vitamin premix, 0.13% (w/w)-0.23% (w/w) L-lysine, 0.08% (w/w)-0.14% (w/w) L-threonine, 0.05% (w/w) coban, 0.05% (w/w) selenium premix, 0.15% (w/w) sodium bicarbonate and 0.10% (w/w) sand. Digestible lysine in the diet may be 1.00% (w/w) to 1.20% (w/w). Digestible methionine in the diet may be 0.47% (w/w) to 0.54% (w/w). Digestible methionine and cysteine in the diet may be 0.98% (w/w) to 1.10% (w/w). Digestible threonine in the diet may be 0.68% (w/w) to 0.84% (w/w). Digestible tryptophan in the diet may be 0.17% (w/w) to 0.22% (w/w). Calcium in the diet may be 0.71% (w/w) to 0.96% (w/w). Available phosphorus in the diet may be 0.17% (w/w) to 0.46% (w/w). Sodium in the diet may be 0.17% (w/w) to 0.19% (w/w). The concentration of each ingredient within any one of the ranges herein may be any value between any two of the concentration points included in the range. In an embodiment, the diet may be the diet for broilers composed of one or more of the following ingredients: 30.00% (w/w)-75.00% (w/w) corn, 5.00% (w/w)-75.00% (w/w) wheat; 5.00% (w/w)-65.00% (w/w) barley; 5.00% (w/w)-30.00% (w/w) sorghum, 5.00% (w/w)-50.00% (w/w) millet, 10.00% (w/w)-45.00% (w/w) soybean meal, 5.00% (w/w)-20.00% (w/w) Canola (Rapeseed) meal, 2.00% (w/w)-15.00% (w/w) corn gluten meal, 5.00% (w/w)-15.00% (w/w) sunflower meal, 5.00% (w/w)-30.00% (w/w) corn distillers dry grains plus soluble solids, 1.00% (w/w)-8.00% (w/w) poultry/porcine/bovine meat and bone meal, 1.00% (w/w)-8.00% (w/w) fish meal, 0.10% (w/w)-2.1% (w/w) dicalcium or monocalcium or defluorinated phosphate, 0.50% (w/w)-6.00% (w/w) soy oil or vegetable oil or animal fat or grease or combination, 0.81% (w/w)-2.00% (w/w) limestone, 0.50% (w/w)-7.00% soy hulls, 0.24% (w/w)-0.50% (w/w) salt (NaCl), 0.13% (w/w)-0.50% (w/w) DL-methionine, 0.01% (w/w)-0.20% (w/w) choline chloride 60, 0.10% (w/w)-0.20% (w/w) mineral premix, 0.05% (w/w)-0.25% (w/w) vitamin premix, 0.05% (w/w)-0.30% (w/w) L-lysine, 0.10% (w/w)-0.30% (w/w) DL-Methionine or methionine analog (MHA), 0.05% (w/w)-0.20% (w/w) L-threonine, 0.05% (w/w) coban, 0.05% (w/w) selenium premix, 0.05% (w/w)-0.15% (w/w) sodium bicarbonate and 250 FTU/kg-2000 FTU/kg Phytase. Metabolizable energy of the diet may be 1225 (kcal/lb)-1491 (kcal/lb). Crude protein (CP) in the diet may be 15% (w/w) to 25% (w/w). Digestible lysine in the diet may be 0.85% (w/w) to 1.30% (w/w). Digestible methionine in the diet may be 0.45% (w/w) to 0.70% (w/w). Digestible methionine and cystine in the diet may be 0.65% (w/w) to 1.10% (w/w). Digestible threonine in the diet may be 0.60% (w/w) to 0.84% (w/w). Digestible tryptophan in the diet may be 0.10% (w/w) to 0.25% (w/w). Calcium in the diet may be 0.68% (w/w) to 1.10% (w/w). Available phosphorus in the diet may be 0.17% (w/w) to 0.50% (w/w). Sodium in the diet may be 0.17% (w/w) to 0.19% (w/w). Phytase in the diet may be 500 FTU/kg (w/w) to 8,000 FTU/kg (w/w). The concentration of each ingredient within any one of the ranges herein may be any value between any two of the concentration points included in the range. Variations in the concentrations of these ingredients may also be used in a diet.
The method may include mixing a transgenic plant or part thereof with a feed ingredient to obtain a mixture. The feed ingredient may be one or more ingredients included in the diet described herein. The transgenic plant or part thereof may be any transgenic plant or part thereof described herein. The method may include feeding an animal with the mixture. The body weight gain (BWG) in an animal fed with the mixture comprising a glucanase may be higher than the BWG in a control animal fed with identical feed ingredients not mixed with a transgenic plant including a glucanase. In an embodiment, the BWG in an animal fed with the mixture comprising a glucanase may be similar to the BWG in a control animal fed with a high energy diet or a diet that includes more or higher concentrations of the ingredients compared to the mixture including a glucanase. In an embodiment, the feed conversion ratio (FCR) in an animal fed with the mixture comprising a glucanase may be lower than the FCR in a control animal fed with identical feed ingredients not mixed with a transgenic plant including a glucanase. The FCR is defined as the mass of the feed eaten by the animal divided by the animal's mass. In an embodiment, the FCR in an animal fed with the mixture comprising a glucanase may be similar to the FCR in a control animal fed with a high energy diet or a diet that includes more or higher concentrations of the ingredients compared to the mixture including a glucanase.
In an embodiment, a method of enhancing thermal stability of a glucanase is provided. The method may include producing a transgenic plant that includes a synthetic nucleic acid encoding the glucanase. The synthetic nucleic acid may include any one the sequences described herein. The glucanase may be thermally stable upon exposure to temperatures in the range of 70° C. to 130° C., endpoints inclusive.
The glucanase may be thermally stable upon exposure to temperatures in the range of 25° C. to 130° C., endpoints inclusive. The glucanase may be thermally stable upon exposure to temperatures in the range from 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120° C., 125° C., 130° C., 25° C., to 30° C., 25° C. to 35° C., 25° C. to 40° C., 25° C. to 45° C., 25° C. to 50° C., 25° C. to 55° C., 25° C. to 60° C., 25° C. to 65° C., 25° C. to 70° C., 25° C. to 75° C., 25° C. to 80° C., 25° C. to 85° C., 25° C. to 90° C., 25° C. to 95° C., 25° C. to 100° C., 25° C. to 105° C., 25° C. to 110° C., 25° C. to 115° C., 25° C. to 120° C., 25° C. to 125° C., or less than 130° C. The glucanase may be thermally stable upon exposure to temperatures in the range of 70° C. to 130° C., endpoints inclusive. The glucanase may be thermally stable upon exposure to temperatures in the range from 70° C. to 75° C., 70° C. to 80° C., 70° C. to 85° C., 70° C. to 90° C., 70° C. to 95° C., 70° C. to 100° C., 70° C. to 105° C., 70° C. to 110° C., 70° C. to 115° C., 70° C. to 120° C., or 70° C. to 130° C., endpoints inclusive.
The above mentioned synthetic nucleic acids may be provided in embodiments herein alone, as part of another nucleic acid, as part of a vector or as stated above as part of a transgenic plant.
In an embodiment, the transgenic plant may be derived from one of corn, rye, switchgrass, miscanthus, sugarcane or sorghum. The transgenic plant may be made by Agrobacterium mediated transformation using a vector having a nucleic sequence as set forth above.
In an embodiment, a method for enhancing production of fermentable sugars from grains is provided. The method may include mixing grains derived from any one of the transgenic plants described herein with grains from a different plant to form mixed grains. The different plant may be a non-transgenic plant. The different plant may be an engineered plant that includes a synthetic nucleic acid encoding at least one hydrolytic enzyme. The hydrolytic enzyme may be but is not limited to xylanase, an amylase, an endoglucanase, an exoglucanase, a feruloyl esterase, a glucoamylase, an intein-modified amylase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, a protease, an intein-modified protease, a phytase, or an intein-modified phytase. The method may include processing the mixed grains. The processing may include one or more operations selected from the group consisting of harvesting, baling, grinding, milling, chopping, size reduction, crushing, pellitizing, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, ensiling, mixing with silage or other biomass and ensiling, fermentation, chemical conversion, and chemical catalysis. The biomass may be but is not limited to hay, straw, stover, silage, compressed and pelleted feeds, soybeans, sprouted grains, legumes, feed grains, maize, rice, barley or wheat grains. The biomass may be any biomass derived from agricultural waste. The method may include converting fermentable sugars into a biochemical product. The biochemical product may be but is not limited to ethanol, butanol, lactic acid, citric acid, and acetic acid.
In an embodiment, a method for reducing the viscosity of a grain mixture is provided. The method may include mixing grains derived from any one of the transgenic plants described herein with grains from a different plant to form mixed grains. Water may be added to the mixed grains to form the grain mixture. The viscosity of the grain mixture may be lower when it includes any one of the glucanases described herein. The viscosity may be intestinal viscosity, which is typically measured from an intestinal sample removed from a bird or pig after euthanization. In this method, the digesta sample is centrifuged and the viscosity of supernatant is analyzed using a viscometer. For example, as describe by Lee et al., ileal digesta was centrifuged for 10 min at 3,500× gravity and 0.5 ml of supernatant was put in a Brookfield Cone and Plate Viscometer¹with a CPE-40 spindle. See Lee, J. T., C. A. Bailey, and A. L. Cartwright. 2003. β-Mannanase ameliorates viscosity-associated depression of growth in broiler chickens fed guar germ and hull fractions. Poult. Sci. 82:1925-1931, which is incorporated herein by reference as if fully set forth. Samples are analyzed for 30 sec at 40° C. and 5 rpm, to determine centipoise (cP units) readings. The higher the cP, the higher the viscosity of the sample.
The different plant may be a non-transgenic plant. The different plant may be an engineered plant that includes a synthetic nucleic acid encoding at least one hydrolytic enzyme. The hydrolytic enzyme may be but is not limited to xylanase, an amylase, an endoglucanase, an exoglucanase, a feruloyl esterase, a glucoamylase, an intein-modified amylase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, a protease, an intein-modified protease, a phytase, or an intein-modified phytase. The method may include processing the grain mixture. The processing may include one or more operations selected from the group consisting of harvesting, grinding, milling, size reduction, crushing, heating, gelotinzing, liquefaction, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, saccharifying, fermentation, chemical conversion, and chemical catalysis.
In an embodiment, a method for enhancing ethanol production from grains is provided. The method includes performing any one of the methods for enhancing production of fermentable sugars described herein.
The following list includes particular embodiments of the present invention. But the list is not limiting and does not exclude alternate embodiments, as would be appreciated by one of ordinary skill in the art.

EMBODIMENTS

1. A transgenic plant comprising a synthetic nucleic acid encoding a glucanase, wherein the glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides.
2. The transgenic plant of embodiment 1, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
3. The transgenic plant of any one or both of the preceding embodiments, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 4.0 to 10.0.
4. The transgenic plant of any one or more of the preceding embodiments, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25° C. to 130° C.
5. The transgenic plant of any one or more of the preceding embodiments, wherein the glucanase activity has improved stability upon expression in the plant compared to the activity of a glucanase having an identical amino acid sequence and expressed in a bacterial cell.
6. The transgenic plant of any one or more of the preceding embodiments, wherein a plant is selected from the group consisting of: wheat, maize, barley, rice, and sorghum.
7. A transgenic plant comprising a synthetic nucleic acid including a sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3, wherein the glucanase is capable of degrading one or more polysaccharides.
8. The transgenic plant of embodiment 7, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
9. The transgenic plant of any one or more of embodiments 7-8, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 4.0 to 10.0.
10. The transgenic plant of any one or more of embodiments 7-9, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25° C. to 130° C.
11. The transgenic plant of any one or more of embodiments 7-10, wherein the glucanase activity has improved stability upon expression in the plant compared to the activity of a glucanase having an identical amino acid sequence and expressed in a bacterial cell.
12. The transgenic plant of any one or more of embodiments 7-11, wherein the transgenic plant is a plant is selected from the group consisting of: wheat, maize, barley, rice, and sorghum.
13. The transgenic plant of any one or more of embodiments 7-12, which comprises the nucleic acid sequence of SEQ ID NO: 1 and produces an amplicon for diagnosing event 4588.259, 4588.757, or 4588.652.
14. A synthetic nucleic acid comprising a sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3, wherein the glucanase is capable of degrading one or more polysaccharides.
15. The synthetic nucleic acid of embodiment 14, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
16. A synthetic polynucleotide comprising a sequence with at least 70% identity to a reference sequence selected from the group consisting of SEQ ID NO: 7-19, wherein the synthetic polynucleotide comprises a synthetic nucleic acid encoding a glucanase that is capable of degrading one or more polysaccharides.
17. The synthetic polynucleotide of embodiment 16, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
18. A vector comprising a synthetic polynucleotide, or a fragment of a synthetic polynucleotide, of embodiment 17.
19. A method of making a transgenic plant that includes a glucanase comprising:
contacting a plant cell with a synthetic nucleic acid encoding an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3, wherein the glucanase is capable of degrading one or more polysaccharides;

- regenerating a transgenic plant from the transgenic plant cell; and

selecting the transgenic plant expressing a glucanase, wherein the glucanase is active and thermally stable upon exposure to a temperature in the range from 25° C. to 130° C.
20. The method of embodiment 19, wherein the one or more polysaccharide is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
21. The method of any one or both of embodiments 19-20, wherein the synthetic nucleic acid is part of a vector of embodiment 13.
22. An animal feedstock comprising a transgenic plant or part thereof of any one or more of embodiments 1-13, the product of any one or more of embodiments, 19-21, or a synthetic polypeptide of any one or more of embodiments 51-54.
23. The animal feedstock of embodiment 22 further comprising a feed supplement or feed additive.
24. The animal feedstock of any one or both of embodiments 22-23, wherein the feed supplement is plant material.
25. The animal feedstock of any one or more of embodiments 22-24, wherein the plant material is a non-transgenic plant.
26. The animal feedstock of any one or more of embodiments 22-24 wherein the plant material is an engineered plant.
27. The animal feedstock of any one or more of embodiments 22-26, wherein the feed supplement includes one or more exogenous enzymes.
28. The animal feedstock of embodiment 27, wherein the one or more exogenous enzyme includes a hydrolytic enzyme selected from the group consisting of: xylanase, endoglucanase, cellulase, exoglucanase, feruloyl esterase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified cellulase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, mannanase, amylase, an intein-modified amylase, phytase, an intein-modified phytase, protease, and an intein-modified protease.
29. The animal feedstock of any one or more embodiments 22-28, wherein the plant material includes at least one component selected from the group consisting of: forage, biomass, corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, barley grain, barley pellets, soybean meal, soybean oilcake, silage, sorghum grain and sorghum pellets.
30. The animal feedstock of any one or more of embodiments 23-29, wherein the feed supplement includes at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, and mineral premix.
31. A method of producing an animal feedstock comprising mixing 1) a transgenic plant or part thereof of any one or more of embodiments 1-13, 2) the product of any one or more of embodiments 19-41, or 3) a synthetic polypeptide of any one or more of embodiments 51-54 with plant material.
32. The method of embodiment 31 further comprising pelletizing the mixture.
33. The method of embodiment 32 further comprising adding a feed supplement to the mixture.
34. The method of embodiment 33, wherein the feed supplement includes at least one exogenous enzyme.
35. The method of embodiment 34, wherein the at least one exogenous enzyme includes a hydrolytic enzyme selected from the group consisting of xylanase, endoglucanase, cellulase, exoglucanase, feruloyl esterase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified cellulase, an intein-modified feruloyl esterase, amylase, an intein-modified amylase, mannanase, phytase, and protease.
36. A method of increasing utilization of non-starch polysaccharides in an animal comprising feeding an animal with an animal feedstock 1) including a transgenic plant of any one or more of embodiments 1-13, 2) of any or more of embodiments 22-30, 3) produced by the method of any one or more of embodiments 31-35, or 4) including a synthetic polypeptide of any one or more of embodiments 51-54.
37. The method of embodiment 36 further comprising preparing the animal feedstock.
38. The method of any or both of embodiments 36-37, wherein the animal is a monogastric animal or a ruminant animal.
39. A method of enhancing thermal stability of a glucanase comprising producing a transgenic plant that includes a synthetic nucleic acid comprising, consisting essentially of, or consisting of an amino acid a sequence having 70% identity to a reference sequence of selected fromthe group consisting of: SEQ ID NOS: 4-6, wherein the sequence encodes a glucanase capable of degrading one or more polysaccharides.
40. The method of embodiment 39, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
41. The method of any or both of embodiments 39-40, wherein expression of the nucleic acid produces the glucanase and the glucanse is thermally stable upon exposure to a temperature in the range of 25° C. to 130° C.
42. A method for enhancing production of fermentable sugars from grains comprising:
mixing grains derived from a transgenic plant of any one of any one or more of embodiments 1-13 with grains derived from a different plant to form mixed grains; and
processing the mixed grains.
43. The method of embodiment 42, wherein the different plant is an engineered plant that includes a synthetic nucleic acid encoding at least one hydrolytic enzyme.
44. The method of any or both of embodiments 42-43, wherein the at least one hydrolytic enzyme is selected from the group consisting of: xylanase, an endoglucanase, an exoglucanase, cellulase, a feruloyl esterase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified cellulase, an intein-modified feruloyl esterase, amylase, phytase and protease.
45. The method of any one or more of embodiments 42-43, wherein the processing includes at least one operations selected from the group consisting of harvesting, baling, grinding, milling, chopping, size reduction, crushing, pellitizing, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, ensiling, fermentation, chemical conversion, and chemical catalysis.
46. The method of embodiment 45 further comprising producing a biochemical product.
47. The method of embodiment 46, wherein the biochemichal product is selected from the group consisting of ethanol, butanol, lactic acid, citric acid, and acetic acid.
48. A method for enhancing ethanol production from grains comprising performing a method of any one or more of embodiments 42-47.
49. A method for enhancing ethanol production from a transgenic plant comprising:
mixing a transgenic plant or part thereof of any one or more of embodiments 1-13 with a different plant or part thereof to form mixed plant material;
converting the mixed plant material into fermentable sugars; and
processing the fermentable sugars into ethanol.
50. The method of embodiment 49, wherein the plant material includes fiber, grain, or a combination thereof.
51. A synthetic polypeptide that includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and capable of degrading one or more polysaccharides.
52. The synthetic polypeptide of embodiment 51, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
53. A synthetic polypeptide that includes an amino acid sequence comprising a contiguous amino acid sequence having at least 90% identity to 50 to 100, 50 to 150, 50 to 200, 50 to 250, 50 to 300, 50 to 322, or 50 to all contiguous amino acid residues of a glucanase having the sequence of any of SEQ ID NOS: 4-6, wherein the glucanase is capable of degrading one or more polysaccharides.
54. The synthetic polypeptide of embodiment 51, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
55. A method of increasing metabolizable energy of a diet comprising mixing a transgenic plant or part thereof with a feed ingredient, wherein the transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase comprising an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 4-6, and and capable of degrading one or more polysaccharides.
56. The synthetic polypeptide of embodiment 55, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
57. The method of any one or both of embodiments 55-56, wherein the synthetic nucleic acid comprises a sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3.
58. The method of any one or more of embodiments 55-57, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 5.0 to 10.0.
59. The method of any one or more of embodiments 55-58, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25° C. to 130° C.
60. The method of any one or more of embodiments 55-59, wherein the feed ingredient includes at least one component selected from the group consisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, wheat middlings, barley grain, barley pellets, soybean meal, soy hulls, dried distillers grain, soybean oilcake, sorghum grain and sorghum pellets.
61. The method of any one or more of embodiments 55-60, wherein the feed ingredient includes at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, mineral premix, and one or more exogenous enzymes.
62. A method for producing an animal feedstock comprising mixing a transgenic plant or part thereof of any one or more of embodiments 1-13 with plant material. The method may also comprise the method for producing a plant that includes a glucanase of any one or more of embodiments 63-68.
63. A method for producing a plant that includes a glucanase comprising crossing a plant with a transgenic plant comprising event 4588.259, 4588.757 or 4588.652, and selecting a first progeny plant comprising event 4588.259, 4588.757 or 4588.652 and capable of degrading one or more polysaccharides.
64. The method of embodiment 63, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.
65. The method of any one or more of embodiments 63-64 further comprising selfing the first progeny plant and selecting a second progeny plant comprising event 4588.259, 4588.757 or 4588.652 and capable of degrading one or more polysaccharides.
66. The method of embodiment 65, wherein the second progeny plant is homozygous for event 4588.259, 4588.757 or 4588.652.
67. The method of embodiment 65, wherein the second progeny plant is heterozygous for event 4588.259, 4588.757 or 4588.652.
68. The method of embodiment 67 further comprising selfing the second progeny plant and selecting a third progeny plant homozygous event 4588.259, 4588.757 or 4588.652 and capable of degrading one or more polysaccharides.
69. A kit for identifying maize event 4588.259, 4588.757 or 4588.652 in a sample comprising a first primer and a second primer, wherein the first primer and the second primer are capable of amplifying a target sequence specific to maize event 4588.259, 4588.757 or 4588.652.
70. The kit of any one or more of embodiment 69 wherein, the first primer comprises a nucleic acid sequence selected from SEQ ID NOS: 38, 41, and 47.
71. The kit of any one or more of embodiments 69-70, wherein the second primer comprises a nucleic acid sequence selected from SEQ ID NOS: 39, 42, 43, 45, and 46.
72. The kit of any one or more of embodiments 69-71, wherein the target sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 51-55.
73. The kit of any one or more of embodiments 69-72, wherein the target sequence is capable of hybridizing to the sequence of the nucleic acid comprising a sequence of SEQ ID NOS: 40 or 44 under conditions of high stringency.
74. The kit of any one or more of embodiments 69-73, wherein the sample comprises plant matter derived from a transgenic plant of any one or more of embodiments 1-13.
75. A method of identifying maize event 4588.259, 4588.757 or 4588.652 in a sample comprising:
contacting a sample with a first primer and a second primer of the kit of any one or more of embodiments 69-74;
amplifying a nucleic acid in the sample to obtain an amplified product; and
detecting an amplified product specific to a target sequence in maize event 4588.259, 4588.757 or 4588.65.
76. The method of embodiment 75, wherein the target sequence comprises a sequence selected from SEQ ID NOS: 51-55. The method of identifying may be added to any one or more of embodiments 63-68.
77. The method of embodiment 75, wherein the target sequence is at least one sequence selected from the group consisting of SEQ ID NOS: 22-31.
78. The method of embodiment 75, wherein the step of detecting comprises hybridizing the amplified product to the nucleic acid comprising a sequence of SEQ ID NOS: 40 under conditions of high stringency, and selecting the amplified product specific to maize event 4588.259.
79. The method of embodiment 75, wherein the step of detecting comprises hybridizing the amplified product to the nucleic acid comprising a sequence of SEQ ID NOS: 44 under conditions of high stringency, and selecting the amplified product specific to maize event 4588.652.
80. A method for reducing the viscosity of a grain mixture comprising combining grains from a transgenic plant of any one or more of embodiments 1-13, a different plant, and liquid to form a grain mixture.
81. The method of embodiment 80, wherein the different plant is a non-transgenic plant.
82. The method of embodiment 80, wherein the different plant is a genetically engineered plant.
83. The method of embodiment 80 and 82, wherein the genetically engineered plant comprises a synthetic nucleic acid encoding at least one hydrolytic enzyme.
84. The method of embodiment 83, wherein the at least one hydrolytic enzyme is selected from the group consisting of: xylanase, an amylase, an endoglucanase, an exoglucanase, a feruloyl esterase, a glucoamylase, an intein-modified amylase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, a protease, an intein-modified protease, a phytase, or an intein-modified phytase.
85. The method of any one or more of embodiments 80-84 further comprising processing the grain mixture.
86. The method of embodiment 85, wherein the step of processing includes one or more operations selected from the group consisting of harvesting, grinding, milling, size reduction, crushing, heating, gelatinizing, liquefaction, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, saccharifying, fermentation, chemical conversion, and chemical catalysis.
Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein.

EXAMPLES

The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and/or one or more element from an embodiment may be substituted with one or more detail from one or more example below.

Example 1. Feed Glucanase Expression Vectors

A codon optimized nucleotide sequence for expression of the AGR2314 feed glucanase in maize was synthesized. For generating initial plant transformation constructs, single AGR2314 expression cassettes were assembled in vectors pAG4000 (pAG4258) or pAG4500 (pAG4588, pAG4597, and pAG4708). The vector pAG4000 has been created by replacing the rice ubiquitin 3 promoter with the first intron by the maize ubiquitin 1 promoter containing its own first intron for driving expression of the selectable marker gene encoding E. coli phosphomannose isomerase (PMI). The vector pAG4500 represents further improvement of pAG4000 and contains three modifications such as 1) insertion after the first maize ubiquitin intron of a 9 bp sequence (ATCCAGATC) representing the first three codons of the ubiquitin monomer with ATG converted into ATC; 2) insertion of the maize Kozak element (TAAACC) after the 9 bp sequence ubiquitin monomer; 3) replacement of the old multiple cloning site (MCS) by a new MCS that was synthesized by PCR and that was designed to contain multiple sites for several rare cutting enzymes (NotI, PacI, FseI, SwaI, AscI, AsiSI) to facilitate cloning of up to 4-5 expression cassettes on one T-DNA.
Sequence of the new MCS in pAG4500 (PmeI-KpnI fragment):

(SEQ ID NO: 20)

GTTTAAACTGAAGGCGGGAAACGACAACCTGATCATGAGCGGAGAATTAA

GGGAGTCACGTTATGACCCCCGCCGATGACGCGGGACAAGCCGTTTTACG

TTTGGAACTGACAGAACCGCAACGTTGAAGGAGCCACTCAGCCTAAGCGG

CCGCATTGGACTTAATTAAGTGAGGCCGGCCAAGCGTCGATTTAAATGTA

CCACATGGCGCGCCAACTATCATGCGATCGCTTCATGTCTAACTCGAGTT

ACTGGTACGTACCAAATCCATGGAATCAAGGTACC.

FIGS. 1-4 illustrate the expression vectors pAG4258, pAG4588, pAG4597, and pAG4708, respectively, carrying a single feed glucanase expression unit. The vector pAG4258 (FIG. 1; SEQ ID NO 7) has been cloned by assembling an expression cassette that was composed of the maize Glb1 promoter fused to the maize codon optimized AGR2314 sequence in KpnI-AvrII sites of pAG4000. The vectors pAG4588 (FIG. 2; SEQ ID NO 8) and pAG4597 (FIG. 3; SEQ ID NO 9) were developed by assembling their corresponding AGR2314 expression cassettes in KpnI-EcoRI sites of pAG4500, while the vector pAG4708 (FIG. 4; SEQ ID NO 10) was produced by cloning AGR2314 expression cassette into XmaI-AvrII sites of pAG4500. FIGS. 5-6 illustrate the expression vectors pAG4766 and pAG4767, respectively, carrying two feed glucanase expression units. FIGS. 7-8 illustrate the expression vectors pAG4770 and pAG4771, respectively, carrying three feed glucanase expression units. The unique rare cutting restriction sites that are available within the MCS of the pAG4500 were subsequently used in order to develop additional expression constructs containing either double AGR2314 expression units, such as pAG4766 (FIG. 5; SEQ ID NO 11) and pAG4767 (FIG. 6; SEQ ID NO 12), or triple AGR2314 expression units, such as pAG4770 (FIG. 7; SEQ ID NO 13) and pAG4771 (FIG. 8; SEQ ID NO 14), on the same T-DNA. The constructed vectors for expression of AGR2314 glucanase in plants are listed in Table 1. E. coli strains carrying the expression vectors were used for conjugation with Agrobacterium and subsequent transformation of maize.

TABLE 1

Description of Sequences

SEQ
ID			Sequence
NO	Construct	Description	Type

1		AGR2314 maize—optimized protein	DNA
		coding sequence (including C-terminal
		ER-retention signal “SEKDEL”
2		AGR2414 coding sequence	DNA
3		AGR2514 coding sequence	DNA
4		AGR2314 Mature protein sequence	Amino acid
		(including C-terminal ER-retention
		signal “SEKDEL”)
5		AGR2414 protein	Amino acid
6		AGR2514 protein	Amino acid
7	pAG4258	Glb1:mZ27:AGR2314:SEKDEL:NOS	DNA
8	pAG4588	Glu1:mZ27:AGR2314:SEKDEL:T35S	DNA
9	pAG4597	mZein:mZ27:AGR2314:SEKDEL:T35S	DNA
10	pAG4708	Ole:mZ27:AGR2314:SEKDEL:NOS	DNA
11	pAG4766	Glu1:mZ27:AGR2314:SEKDEL:NOS,	DNA
		Glb1:mZ27:AGR2314:SEKDEL:NOS
12	pAG4767	mZein:mZ27:AGR2314:SEKDEL,	DNA
		Glb1:mZ27:AGR2314:SEKDEL:NOS
13	pAG4770	mZein:mZ27:AGR2314:SEKDEL:NOS,	DNA
		Glu1:mZ27:AGR2314:SEKDEL:NOS,
		Glb1:mZ27:AGR2314:SEKDEL:NOS
14	pAG4771	Glu1:mZ27:AGR2314:SEKDEL:NOS,	DNA
		mZein:mZ27:AGR2314:SEKDEL:NOS,
		Glb1:mZ27:AGR2314:SEKDEL:NOS
15	pAG4257	mZein:mZ27:AGR2514:SEKDEL:NOS	DNA
16	pAG4692	Glu1:mZ27:AGR2414:SEKDEL:T35S	DNA
17	pAG4693	mZein:mZ27:AGR2414:SEKDEL:T35S	DNA
18	pAG4705	Glu1:mZ27:AGR2514:SEKDEL:T35S	DNA
19	pAG4706	Ole:mZ27:AGR2514:SEKDEL:NOS	DNA

Expression cassettes for related beta glucanases, AGR 2414 and AGR 2514, were prepared using similar strategies, and sequences are provided for these expression cassettes as they are found in the expression vectors pAQ4257 pAQ4692, pAQ4693, pAQ4705, pAQ4706, pAQ4766, pAQ4257, pAQ4692, pAQ4693, pAQ4705, and pAQ4706.

Example 2. Feed Glucanase Protein Extraction Procedure

Flour was prepared from about 20 transgenic seeds by milling in an Udy cyclone mill or knife mill with 0.5 mm or 1 mm screen. About 0.5 ml of protein extraction buffer (100 mM sodium phosphate, pH 6.5, 0.01% Tween 20) was added to 20 mg flour in a 2 ml tube. In some cases, 2 g, 10 g, or 20 g ground samples was mixed with 10 ml, 50 ml or 100 ml of the extraction buffer in 15 ml tubes or 250 ml bottles. Larger masses and volumes can be used by scaling these amounts appropriately. After vortexing, the tubes were placed on a rotating platform in a 60° C. incubator and rotated for 1 hour for protein extraction. After centrifugation at 16,000×g for 10 min in a tabletop centrifuge, the supernatant was diluted 20-fold for enzyme assay by adding 20 μl supernatant to 380 μl protein extraction buffer. In some cases, other dilution factors were used, as necessary.

Example 3. Feed Glucanase Activity Measurement

Colorimetric Assay. Fifty microliters of the diluted (20-fold to 360-fold) protein extract was mixed with 450 μl of 100 mM sodium phosphate buffer, pH 6.5, 0.01 % Tween 20 and 1 tablet of β-glucazyme from Megazyme (Wicklow Ireland), and then incubated at 80° C. for 1 hour before adding 1 ml of 2% Tris base. After centrifugation at 3000×g for 10 min, 100 μl of supernatant was transferred to a microplate for absorbance measurement at 590 nm (A590). The activity was recorded as A590/mg flour after multiplying the dilution factors: A590xAx(500/50)/20 mg, where A is protein extraction dilution factor; 500 is the volume (ml) of buffer used for protein extraction; 50 is the volume of protein extraction (ml) used in the activity test.
Unit Activity Measurement. The assay involves the quantitation of reducing sugars that are released during a time course digestion of a model substrate (barley-β-glucan) obtained from Megazyme (Wicklow, Ireland).
Hydrolysis of model substrate. Test 2 ml tubes were labeled with “+” sign, and 5 mg barley-β-glucan substrate (reaction) was added to each test tube; no substrate was added to control tubes (control). Four hundred fifty microliters of 100 mM sodium phosphate buffer, pH 6.5, was added to each tube (reactions and controls), and tubes were placed into a Thermo-shaker with temperature set at 80° C. and shaking speed set at 1000 rpm. Tubes were shaken at 1000 rpm at 80° C. for 20 min until the substrate was completed dissolved. A tube with 2 ml of diluted grain protein extract, extracted as described above was placed in the Thermo-shaker to be pre-warmed.
Fifty microliters of the pre-warmed sample were added to the control and reaction tubes. Shaking was resumed and a timer was started. After 15 minute of shaking at 80° C., 50 μl of each of the reaction and control samples were removed and mixed with 10 μl of 0.5N HCl in separate microplates. Shaking of the samples was resumed until all samples were removed and mixed with acid.
BCA quantification of glucose reducing equivalents. Glucose standards were prepared in protein extraction buffer at the following concentrations: 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM. BCA reagent (from Thermo Scientific) was prepared by mixing reagent A with reagent B in a ratio of 50:1. To make a glucose standard curve, 75 μl of buffer were dispensed into the first well of row A (A1) in a microplate and 75 μl of each glucose standard were dispensed into wells A2 through A7. To detect the reducing sugars from the feed glucanase reaction and control samples, 25 μl from each reaction were dispensed into rows of the microplate in the order of their incubation time with barley-β-glucan (e.g., row B1-B2: 15 min-30 min), then added 25 μl of corresponding control to another row of the microplate (e.g., row C1-C2: 15 min-30 min). Subsequently, 50 μl of sodium phosphate buffer were dispensed in each well in these two rows (reaction and control), and 175 μl BCA reagent were added to each well using a multichannel pipette. Mixing was achieved by pipetting up and down. The microplate was sealed and incubated at 80° C. in a heat block. After 10 min incubation, the microplate was chilled on ice for 10 minutes and centrifuged to bring down condensate. Subsequently, the absorbance at 560 nm of each well was measured on a microplate reader.
Calculating units of feed glucanase activity from A560. The absorbance from the reagent blank was subtracted from the absorbance values for each of the glucose standards, and the resulting values were plotted according to their glucose concentrations. Linear regression was then used to calculate the “best fit” line through the data set. To determine glucose reducing equivalents in glucanase/barley-β-glucan reactions, for each time point, the absorbance value from the control sample was subtracted from the reaction sample, and the resulting value was used to calculate the concentration of reducing sugars by comparison to the glucose standard curve. One unit (U) of glucanase activity is the amount of enzyme required to release 1 μmol glucose reducing equivalents from 1% Barley-β-glucan per minute at 80° C., pH 5.3, using the BCA method of quantitation.
Unit Activity Measurement (semi-high throughput method). As described herein, the method detects the reducing sugars such as glucose released from the model substrate (barley-β-glucan) by glucanase treatment at 80° C. for 40 minutes or 90 minutes. When protein extract from grain or feed is appropriately diluted, the initial velocity is detected within 40 minutes (grain product) or 90 minutes (feed sample) of the reaction. The reactions were carried out in 96-well block (Costar, Cat #3960) or strip tubes (VWR, Cat #29442-610).
Substrate preparation: Barley-β-glucan (low viscosity) was weighed based on the number of reactions, e.g., 10 samples, 4 dilutions for each sample needed a total of 40 reactions. Each reaction needed 5 mg substrate, therefore, at least 40×5=200 mg of barley-β-glucan was required. The substrate was completely dissolved with the extraction buffer at 80° C. water bath for 20 minutes, and vortexed at every 5 to 10 minutes.
The cluster tubes were used for 90 minutes endpoint activity unit assay of feed samples. Protein extract was diluted to 2-, 6-, 10- and 20-fold dilutions.
Purified protein diluted 100-fold was used as a positive control for assay validation. Purified glucanase protein (200,000 ppb) was stored in 50 mM MES, 150 mM sodium chloride, pH6.3 buffer plus 40% glycerol at −20° C. Ten microliters of protein were mixed with 990 μl of the extraction buffer, and 50 μl were used for activity assay.
Barley-β-glucan digestion by feed glucanase was carried out at a water bath set at 80° C. In the block of cluster tubes, 450 μl of the substrate were dispensed into tubes of A2 to D12 referring Table 2. These rows served as the reaction.
Four hundred fifty microliters of the extraction buffer (no substrate) were added to each control tubes from rows E2 to H12, which served as blank to correct protein content detected by BCA method for each reaction as described in Table 2 (A2 to D12).
Fifty microliters of the diluted sample extract including the negative control and positive control were added first to each blank tube, E2 to H12, and then to each reaction tube, A2 to D12, as described in Table 2.

TABLE 2

Example of enzyme hydrolysis of feed samples in cluster tubes

	1	2	Columns 1 to 11	12

A		Neg. Ctr,	Sample_X, 2x dilution	Pos. Ctr	Reaction
		2x
B		Neg. Ctr,	Sample_X, 6x dilution	Pos. Ctr
		6x
C		Neg. Ctr,	Sample_X, 10x dilution	Pos. Ctr
		10x
D		Neg. Ctr,	Sample_X, 20x dilution	Pos. Ctr
		20x
E		Neg. Ctr,	Sample_X, 2x dilution	Pos. Ctr	Blank
		2x
F		Neg. Ctr,	Sample_X, 6x dilution	Pos. Ctr
		6x
G		Neg. Ctr,	Sample_X, 10x dilution	Pos. Ctr
		10x
H		Neg. Ctr,	Sample_X, 20x dilution	Pos. Ctr
		20x

The tubes were covered with Corning™ Storage Mat III, the Corning Storage Mat Applicator was used to seal the tubes. The plate was shaken at a low speed. The block was placed in the water bath at 80° C. for the 90 minutes incubation period. The reaction was terminated by adding 100 μl of 0.5 N HCl to each well and cooling the block on ice.
BCA quantification of glucose reducing equivalents. Glucose standards were prepared in 100 mM sodium phosphate buffer, pH6, at the following concentrations: 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM. BCA reagent (from Thermo Scientific) was prepared by mixing reagent A with reagent B in a ratio of 50:1. To make a glucose standard curve in column 1 on a microplate, 75 μl of buffer was dispensed into the first well of row A (A1) and 75 μl of each glucose standard were dispensed into wells A2 through A7. To detect the reducing sugars from the feed glucanase reaction and control samples, 25 μl from each reaction were dispensed into rows of the microplate according to the order displayed on Table 2. Subsequently, 50 μl of the extraction buffer was dispensed in each sample well (reaction and blank) referring to Table 2 from A2 to H12 to make a total volume 75 μl. One hundred seventy five microliters of the BCA reagent were added to each well and mixed. The microplate was sealed and incubated at 80° C. on a heat block. After 10 min incubation, the microplate was chilled on ice for 10 min and centrifuged to bring down condensate. Subsequently, the absorbance at 560 nm of each well was measured on a microplate reader.
Calculating units of feed glucanase activity from A560. The absorbance value for the reagent blank was subtracted from the absorbance values for each of the glucose standards, and the resulting values were plotted according to their glucose concentrations. Linear regression was then used to calculate the “best fit” line for the data set. To determine glucose reducing equivalents in the glucanase/barley-β-glucan reactions, the absorbance value from the control sample was subtracted from absorbance value of the corresponding reaction sample, and the resulting value was used to calculate the concentration of reducing sugars by comparison to the glucose standard curve. One unit (U) of glucanase activity equals 1 μmol glucose reducing equivalents released from 1% barley-β-glucan per minute at 80° C., using the BCA method of quantitation.
Calculating units of positive controls from A560 to validate the assay. The value of absorbance for blank samples (E12, F12, G12, H12) was subtracted from the value of absorbance for each reaction sample (A12, B12, C12, D12). The regression equation for the glucose standard was used to calculate the glucose content (μmol). To determine the amount of reducing units produced per minute (A), the value for the amount of glucose (μmol) released from barley-β-glucan in the reaction was divided by the reaction time, for example 90, if the reaction time was 90 minutes. The unit value of positive controls equals the dilution x (A)/mg of protein in the assay. The dilution factor in the assay described herein equals 24. The dilution factor of 24 was determined by comparing the ratio of the total reaction volume to the portion of the reaction that was used in the BCA assay. In the assay, the total reaction volume was 600 μl including 500 μl reaction and 100 μl of HCl used to stop the reaction. The portion of the reaction that was used in the BCA assay was 25 μl. Therefore, the dilution factor of 24 was calculated by dividing 600 μl by 25 μl.
The amount of protein in the assay was calculated as follows. The concentration of the positive control was 2000 ng/ml, 50 μl was the aliquot of the positive control used in the test (or 50/1000 if calculated in mL). The amount of protein calculated in nanograms was 2000×(50/1000), or 2000×(50/1000)/1000000 if calculated in milligrams.

Example 4. Glucanase Activity in Seed from Transgenic Maize

Silks on untransformed (wild type) maize plants were pollinated with pollen from individual transgenic maize plants that carried the pAG4588 construct. Mature, dried seeds were harvested from the resulting ears and assayed for activity via the colorimetric assay. FIG. 9 illustrates the range of activities recovered from 42 independent ears. In this figure, the numbers along the abscissa correspond to individual event identifiers. The highest activity was observed in the event 259. In the T0 transgenic maize plants 757 that also carried the pAG4588 construct, the activity was about 25 A590/mg. The average activity of the homozygous seeds derived from the first generation of the selfed plants was approximately 116 f 15 A590/mg. The activity of heterozygous seeds from this population was about 59 f 18 A590/mg.
Silks on untransformed (wild type) maize plants were pollinated with pollen from individual transgenic maize plants that carried the pAG4597 construct. Mature, dried seed were harvested from the resulting ears and assayed for activity via the colorimetric assay. FIG. 10 illustrates the range of activities recovered from 15 independent ears. In this figure, the numbers along the abscissa correspond to individual event identifiers. The highest activity was observed in the event 460.

Example 5. Maize Genomic Sequences Flanking T-DNA Integration Sites in Transgenic Events 4588.259, 4588.757 and 4588.652

Event 4588.259: The event 4588.259 carries two independent T-DNA integration sites that are located on the maize chromosomes 4 and 8. The chromosomal locations of the T-DNA integration sites were determined through BLASTN searches, in which the maize genomic DNA sequences that are contained in OB-2880, OB-2832 and OB-3252 sequences isolated from T-DNA insertion sites at the right and left T-DNA borders, were used as the queries for screening publicly available maize B73 genome sequence databases, such as the Maize Genetics and Genome Database, htt)://www.maizegdb.org/(Accessed May 8, 2016) See also Andorf, C M, Cannon, E K, Portwood, J L, Gardiner, J M, Harper, L C, Schaeffer, M L, Braun, B L, Campbell, D A, Vinnakota, A G, Sribalusu, V V, Huerta, M, Cho, K T, Wimalanathan, K, Richter, J D, Mauch, E D, Rao, B S, Birkett, S M, Richter, J D, Sen, T Z, Lawrence, C. J. (2015) MaizeGDB 2015: New tools, data, and interface for the maize model organism database. Nucleic Acids Research doi: 10.1093/nar/gkv1007; Lawrence, C J, Seigffried, T E, and Brendel, V. (2005) The Maize Genetics and Genomics Database. The community resource for access to diverse maize data. Plant Physiology 138:55-58; Lawrence, C J, Dong, Q, Polacco, M L, Seigfried, T E, and Brendel, V. (2004) Maize GDB, the community database for maize genetics and genomics. Nucleic Acids Research 32:D393-D397, all of which incorporated herein by reference as if fully set forth. Because both loci segregate independently, plants carrying both loci and each individual locus were evaluated.
In the flanks OB-2880, OB-2832 and OB-3252, which are provided below, the maize genomic DNA is shown in the uppercase letters, while the pAG4588 vector sequences are indicated in the lowercase letters and are underlined.
Integration Site on the Maize Chromosome 4:
The T-DNA integration site on the maize chromosome 4 is characterized by the 795 bp right T-DNA border flanking sequence OB-2880, which contains 677 bp of maize genomic. The isolated 677 bp maize genomic DNA flank has 99.3% sequence identity to the sequence derived from the antisense DNA strand of the maize chromosome 4 (nucleotide coordinates 56612593-56612026).

>OB-2880

(SEQ ID NO: 22)

CTTAGATTAGAGAATGAAAATTTGATTGCTAAGGCCCAAGATTTTGATGT

TTGCAAAGATACAATTACCGATCTTAGAGATAAGAATGATATACTTCGTG

CTAAGATTGTTGAACTTACACCACAACCTTCTATGCCTTCTGTGACATTA

ACATTACGTCACAAACAATAGTATTTTTGTCATACCTTACATGTTGGTGA

CGTGATTGTGACGAAAATCACATCGTCACAGAAGGTGCGTGTTAAATGGT

GTACTATGACGAATAACAAAAAAACGTCATAATAGTTTATGACGCAAACT

ACAAACGTCACTAATCTATGACACTCGAATTCGTCACTAATTATGTCTAA

ATACGTCACAATTCATGTAGTCGTGCCTTGCCACGTGGCTGATTACGTGG

CGAGATGACATGGCAGTTGACGTGGCAGGTGATGTGGCGAAAATGTTGTG

ACGAGTTCATTCGTCACAGATGTTATGACGTGGCATGCCACATGGCAGAT

GATGTGGCAAAATTATGTGACAAAAATATTTGTCATAAATATCAATGAGG

TGGCAATATATGTGTGACGAAATTTTTCATCACAAAGTACGATGACGTTG

CAATATATTTATGACGAATTGTTCATCATAAGGCGTGATGAATTCATAGC

GTCATGGAATATTATGAAATCACATGCtcaaacactgatagtttaaactg

aaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacg

ttatgacccccgccgatgacgcgggacaagccgttttacgtttgg

Integration site on the maize chromosome 8:
The T-DNA insertion site on the chromosome 8 is characterized by the sequences OB-2832 and OB-3252 that represent, accordingly, left and right flanks for the T-DNA integrated into this locus.
The 1211 bp OB-2832 sequence contains 864 bp of maize genomic DNA. The isolated 864 bp maize genomic DNA flank has 99.65% sequence identity to the sequence derived from the maize chromosome 8 with nucleotide coordinates 100613054-100613915.

>OB-2832

(SEQ ID NO: 23)

tcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccg

gtcttgcgatgattatcatataatttctgttgaattacgttaagcatgta

ataattaacatgtaatgcatgacgttatttatgagatgggtttttatgat

tagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc

gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagat

cgggaattggcgagctcgaattaattcagtacattaaaaacgtccgcaat

gtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatACT

AAAAAAACTCAAGGATCTGTCTCCAGAAAGGCCTTGCAGGGTTTGGCCAC

GCCCACGGACATTCCATCTCAGAGCCATGATTAGAACGAAAAACACATGA

GAGCCGTCGTTGCTAGGAGTCGGTTTCATATGTTCGCTAAAACAAGAGAT

TTGTTTTTTTTCTCTCTCGTACATACACGAGTCAGCCCTTTTAATCTCAG

GTTGACGTGCAATGTCGCTCGTCTAAGCAGAACATTTTGAGAACAAATGT

GTTGTACATGAGAGTTTTGTGTACATGGTACGTACATTAAAACATCATCA

TTTATCTTAGATCTAACATCTCTACTTGCTTGTTATATATTTTTTTTGTA

AAATAACATCTTTCACCACTTTATATGGTGTTGTTTGCAAAATATACAGA

GCAATTAGAGACGTTAGATTTGAGATGGACGGTGATAATTTAATACATGC

ATAATGTACAAGAAAATCCTAACTGCACTAGATATGTTGTCAAACATTTT

ACCTTTGTTACAAAAAGAAATGAATAGATGTTGAACGGTTGTCTTTCAAG

CCTGTTCGCTGCGGCTTTAATTCACCAACTGCAATGAACAACCTGAAAGG

TGATCGTTGCCGAACACATGCTGTTTGGCAAAGCTAGTAGTACCTTTTTT

GTCTGTCACCTGGAATGATGAGAAAGGAGACAAGAGGAGAGGGCTGGCCA

TTGTTTATATATATACGTATTTCCATTGCTTTGTGGCATGCAACAGTTCA

AGGGTCCAAACTGGCAGGTTTTCAGCCCCGACAAATATAATAAAAAAACT

ACAAAAAAAAAAGGTCCGTTTACATTCCTTTTTTGACAACGCTAGTCCGT

GCGGAGCGAGC

The 696 bp OB-3252 sequence contains 95 bp of maize genomic DNA. The OB-3252 does not contain left T-DNA border sequence. The isolated 95 bp flank has 100% sequence identity to the sequence derived from the antisense DNA strand of the maize chromosome 8 with nucleotide coordinates 100613034-100612940.

>OB-3252

(SEQ ID NO: 24)

Ggtgaaacaaggtgcagaactggacttcccgattccagtggatgattttg

ccttctcgctgcatgaccttagtgataaagaaaccaccattagccagcag

agtgccgccattttgttctgcgtcgaaggcgatgcaacgttgtggaaagg

ttctcagcagttacagcttaaaccgggtgaatcagcgtttattgccgcca

acgaatcaccggtgactgtcaaaggccacggccgtttagcgcgtgtttac

aacaagctgtaagagcttactgaaaaaattaacatctcttgctaagctgg

gagctctagatccccgaatttccccgatcgttcaaacatttggcaataaa

gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataa

tttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgac

gttatttatgagatgggtttttatgattagagtcccgcaattatacattt

aatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcg

cgcgcggtgtcatctatgttactagatcgggaattggcgagctcgaatta

aTTCAAGTGTCTTCGTACAAACTGGGGGATGGGGCAGACCGCCAGGTTCA

AACCGTTTGACTAGATGCGGCTGGCAGGCTACTTTGCAGTGCATGC

The maize genomic DNA flanks in sequences OB-2832 and OB-3252 are separated by 20 nucleotides on the maize chromosome 8, which indicates that during T-DNA integration 20 bp of the original maize genomic DNA sequence were replaced by the inserted T-DNA sequences.
There is also an OB-2861 sequence and an OB-2868 sequence within the 259 event. The 970 bp OB-2861 sequence consists of the re-arranged pAG4588 sequences including a partial 223 bp Nos terminator sequence (uppercase letters, nucleotides 3290-3512 in pAG4588); the 73 bp sequence near the left T-DNA border with the first 3 bp of the processed left T-DNA border sequence (italicized lowercase letters, nucleotides 3513-3585); the 299 bp sequence near the right T-DNA border with 5 bp of the processed right T-DNA border sequence and polylinker sequence with multiple cloning sites (lowercase letters, nucleotides 9647-9945); the 359 bp 5′ sequence of the rice glutelin promoter prGTL-03 (uppercase letters, nucleotides 9946-10304). The underlined are 18 bp of a duplicated sequence that has been created during T-DNA integration process. The OB-2861 sequence is as follows:

(SEQ ID NO: 25)

GAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATT

ACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGA

TGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGA

AAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCAT

CTATGTTACTAGATCGGGAATTGgcgagctcgaattaattcagtacatta

aaaacgtccgcaatgtgttatt aagttgtctaagcgtcaa tttgttatca

agttgtctaagcgtcaaacactgatagtttaaactgaaggcgggaaacga

caacctgatcatgagcggagaattaagggagtcacgttatgacccccgcc

gatgacgcgggacaagccgttttacgtttggaactgacagaaccgcaacg

ttgaaggagccactcagcctaagcggccgcattggacttaattaagtgag

gccggccaagcgtcgatttaaatgtaccacatggcgcgccaactatcatg

cgatcgcttcatgtctaactcgagttactggtacgtaccaaatccatgga

atcaaggtaccTCCATGCTGTCCTACTACTTGCTTCATCCCCTTCTACAT

TTTGTTCTGGTTTTTGGCCTGCATTTCGGATCATGATGTATGTGATTTCC

AATCTGCTGCAATATaAATGGAGACTCTGTGCTAACCATCAACAACATGA

AATGCTTATGAGGCCTTTGCTGAGCAGCCAATCTTGCCTGTGTTTATGTC

TTCACAGGCCGAATTCCTCTGTTTTGTTTTTCACCCTCAATATTTGGAAA

CATTTATCTAGGTTGTTTGTGTCCAGGCCTATAAATCATACATGATGTTG

TCGTATTGGATGTGAATGTGGTGGCGTGTTCAGTGCCTTGGaTTTGAGTT

TGATGAGAGTTGCTTCTGGG

The 1127 bp OB-2868 sequence consists of re-arranged pAG4588 sequences including the 595 bp 3′ sequence of the PMI marker gene (uppercase letters, nucleotides 2594-3188 in pAG4588); the 48 bp sequence between PMI and Nos terminator (lowercase letters, nucleotides 3189-3236); the 276 bp Nos terminator sequence (uppercase letters, nucleotides 3237-3512); the 73 bp sequence near the left T-DNA border with the first 3 bp of the processed left T-DNA border sequence (italicized lowercase letters, nucleotides 3513-3585); the 119 bp sequence near the right T-DNA border with 5 bp of the processed right T-DNA border and a partial polylinker sequence (lowercase letters, nucleotides 9647-9765). The underlined are 18 bp of a duplicated sequence that has been created during T-DNA integration process. The OB-2868 sequence is as follows:

(SEQ ID NO: 26)

AAAATCCCGCGCGCTGGCGATTTTAAAATCGGCCCTCGATAGCCAGCAGG

GTGAACCGTGGCAAACGATTCGTTTAATTTCTGAATTTTACCCGGAAGAC

AGCGGTCTGTTCTCCCCGCTATTGCTGAATGTGGTGAAATTGAACCCTGG

CGAAGCGATGTTCCTGTTCGCTGAAACACCGCACGCTTACCTGCAAGGCG

TGGCGCTGGAAGTGATGGCAAACTCCGATAACGTGCTGCGTGCGGGTCTG

ACGCCTAAATACATTGATATTCCGGAACTGGTTGCCAATGTGAAATTCGA

AGCCAAACCGGCTAACCAGTTGTTGACCCAGCCGGTGAAACAAGGTGCAG

AACTGGACTTCCCGATTCCAGTGGATGATTTTGCCTTCTCGCTGCATGAC

CTTAGTGATAAAGAAACCACCATTAGCCAGCAGAGTGCCGCCATTTTGTT

CTGCGTCGAAGGCGATGCAACGTTGTGGAAAGGTTCTCAGCAGTTACAGC

TCAAACCGGGTGAATCAGCGTTTATTGCCGCCAACGAATCACCGGTGACT

GTCAAAGGCCACGGCCGTTTAGCGCGTGTTTACAACAAGCTGTAAgagct

tactgaaaaaattaacatctcttgctaagctgggagctctagaTCCCCGA

ATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAAT

CCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGT

TAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGG

TTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAAC

AAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTAT

GTTACTAGATCGGGAATTGgcgagctcgaattaattcagtacattaaaaa

cgtccgcaatgtgttatt aagttgtctaagcgtcaa tttgttatcaagtt

gtctaagcgtcaaacactgatagtttaaactgaaggcgggaaacgacaac

ctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatg

acgcgggacaagccgttttacgtttgg

Event 4588.757: The event 4588.757 carries one T-DNA integration site that is located on the maize chromosome 8.
The chromosomal location of the T-DNA integration site was determined through BLASTN searches, in which the maize genomic DNA sequences that are contained in OB-3170 and OB-3237 sequences isolated accordingly from T-DNA insertion sites at the right or left T-DNA borders, were used as the queries for screening publicly available maize B73 genome sequence databases, such as http://www.maizegdb.org/. See also Andorf, C M et al. (2015) Nucleic Acids Research doi: 0.10.1093/nar/gkv1007; Lawrence, C J et al, (2005) Plant Physiolgy 138:55-58; Lawrence, C J et al., (2004) Nucleic Acids Research 32:D393-D397, all of which incorporated herein by reference as if fully set forth.
In the flanks OB-3170 and OB-3237, which are provided below, the maize genomic DNA is shown in the uppercase letters, while the pAG4588 vector is indicated in the lowercase underlined letters.
The 1303 bp OB-3170 right T-DNA border flanking sequence consists of the 975 bp maize genomic DNA attached to the 328 bp of the pAG4588 vector borderless sequence proximal to the right T-DNA border site. The isolated 975 bp maize genomic DNA flank has 99.38% sequence identity to the sequence derived from the maize chromosome 8 with nucleotide coordinates 62661042-62662016.
The OB-3170 sequence is as follows:

(SEQ ID NO: 27)

TTGGGGTTCCTTATCCTGTTGTCGGAGTTGTGCCATTATCCTTTCCATGG

TTGACCTGAGCTTTAGCCTGTACACTGTAGACTCTACTAGAGGTTTACCT

GAGGCTGAATTCCCGCTGCTAAGATGTGATGTTCCCGGCCATAAGCAAAG

ATGCAGGTTGTCTTTGCTTTGTAAAGATGAAGGTTGTCTTTGTTTTGTAA

TCGAAAAAAAAACCCTCCGACTTCGATAGCAATCCATTTCTTGAAACGAT

ATAGCTATAAGCTGCAGCCACACCTTGCGTTGATGATGCCAAAGCTTTCT

TTCGAGTGCGATGCATGCACTGGCCTGTTGAGATCTTATCAATATGGCAA

ACAGTAACCTAACGTATATGACTACATGGTCTTCATGCTTTTGAGAGGTG

CCTCATAGGAAACAGTCAGGCCAATGATTTTAGGGAATACAATATATTTT

TGCTGTTTTTTTTTTGCAAATTGTCCATATTATTACAAAAAAAACTAAAC

ATGCCCAAAGGCAATAGCTTTCTAAATAAAAATGAATAACGGTCCACTTA

TATATGTTGGCCAGTAATCAATTCTGAGGCCTGACAAACCATGCATATAT

TAACAGTAGGTTAATGGCCGTGCGTGAAAAAATTTCAATACAACAAGAGA

TTGAAAAAAAAGAGTGTCTTACCAATATGTTATTTTATAAGTACCAAATG

TGTAGGAAACTTGCATTCATTTTTTCCCTGAGAATGGAAAAAAACAAGAC

ATACTCATTTTCAAGTTGAATTGTCATAGCAACACACATGTTGTATCTGC

CGGTTCATGCAATTGTGCCAACCAAAATATCTAAATGAGATATTCAAGAC

TCAACAGAATTAAAGTATGGAATAGGGTGTATATACACTCAACCATTATT

AAATGGTATAATCATCTATCTATATCACTATAAAATCTACCAGTTTAAAC

TTCACAAAACTCATCTAGCTAATGGaggcgggaaacgacaacctgatcat

gagcggagaattaagggagtcacgttatgacccccgccgatgacgcggga

caagccgttttacgtttggaactgacagaaccgcaacgttgaaggagcca

ctcagcctaagcggccgcattggacttaattaagtgaggccggccaagcg

tcgatttaaatgtaccacatggcgcgccaactatcatgcgatcgcttcat

gtctaactcgagttactggtacgtaccaaatccatggaatcaaggtacct

ccatgctgtcctactacttgcttcatccccttctacattttgttctggtt

ttg

The 960 bp OB-3237 left T-DNA border flanking sequence consists of the 620 bp of the maize genomic DNA attached to the 340 bp pAG4588 vector sequence (nucleotides 3260-3599 in pAG4588), which includes 253 bp Nos terminator sequence and 70 bp sequence upstream of the 17 bp processed left T-DNA border sequence. The isolated 620 bp maize genomic DNA flank has 100% sequence identity to the sequence derived from the maize chromosome 8 with nucleotide coordinates 62662037-62662642.
The OB-3237 sequence is as follows:

(SEQ ID NO: 28)

Aaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttg

cgatgattatcatataatttctgttgaattacgttaagcatgtaataatt

aacatgtaatgcatgacgttatttatgagatgggtttttatgattagagt

cccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaa

actaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaa

ttggcgagctcgaattaattcagtacattaaaaacgtccgcaatgtgtta

ttaagttgtctaagcgtcaatttgtttacaccacaatataAAATCTACCT

GTTCGCTGATAAGCCGTTAGGTTGACTATGTGACTGTTGGGCGGCAAAAT

GACCACGCGGACGGTCTAGCCCCAAAGCCGGACGGTCCGCGGTCCAGACA

GTCTGCACTGGTGGTGTCGGCGTTTCGACCCCGGGGGGTCCCTGGACCGA

CGAGTAAATTGTCGCTGCGTGTCCCAGCCCAGATGGGTCCGCGCGAGACG

GAACGCGAAGATGGGAAAACAGCAAAGGGGAACCCGCGGCCTTCGTGTTG

TCCTGCGCCCAGGTCGGGTGCGCTTGCAGTAGGGGGTTACAACCGTTCGC

GTGGGAGAGACAGAGAGAGAGCGAGAGCCTTATGCGTCGGCCCGTTCTCC

CGCGCGGCCAACCCTCTCGTACGAGAGCCCTGGACCTTCCTTTTATAGAC

GTAAGGAGAGGGCCCAGGTGTACAATGGGGGGTGTAGCAGAGTGCTAACG

TGTCTAGCAGAGAGGAGCCGGAGCCCTAAGTACATGTCGTCGTGGCTGTC

GGAGAGGTTTTGGCGCCCTGTTCATGTGATGTCGTGGCCGTCGGAGGAGC

GCTTGAGCCCCGTGGAAGTACAGCTGTCGGGGCTGTCGGATCCTTGCTGA

CGTCTCCTTG

The maize genomic DNA flanks in sequences OB-3170 and OB-3237 are separated by 21 nucleotides on the maize chromosome 8, which indicates that during T-DNA integration 21 bp of the original maize genomic DNA sequence were replaced by the inserted T-DNA sequences.
Event 4588.652: FIG. 11 illustrates a diagram showing positions of the characterized flanking sequences in 4588.652.
Sequences isolated at the T-DNA insertion site in 4588.652. T-DNA in the event 4588.652 has integrated into chromosome 7 of the maize genome in BxA genotype, which was used for maize transformation with the pAG4588 construct. The T-DNA insertion occurred between nucleotides 141683320-141683357 of the publicly available reference B73 maize genome. The T-DNA integration displaced 38 bp of the native maize genomic sequence at this site. This 38 bp DNA is underlined and the sequences of pAG4588 are underlined and shown in bold characters in the sequences of the T-DNA insertions shown below. The diagram illustrated in FIG. 11 depicts locations of the sequences in the locus in 4588.652. The right and the left border T-DNA flanking sequences, OB-4448 and OB-4451 respectively, were isolated from multiple 4588.652 progeny using a PCR-based genome walking approach. The entire genomic regions between the right and the left border flanks were isolated and sequence characterized from WT genotypes BxA, 19545 (E), 15009 (G) as well as the nulls BC2ES2_512x and BC1GS2_518x. The following wild type maize genomic DNA sequences were used for reference: the WT_BxA (OB-4541; SEQ ID NO: 32), the WT_E sequence (OB-4545, OB-4546; SEQ ID NO: 33), the WT_G sequence (OB-4547, OB-4548; SEQ ID NO: 34), the Null_BC2ES2_512x sequence (OB-4578 to OB-4580; SEQ ID NO: 35), the Null_BC1GS2_518x sequence (OB-4582 to OB-4584; SEQ ID NO: 36), and the WT_B73Chr7_141681606-141685147 reference sequence (SEQ ID NO: 37). Furthermore, the right and the left border flanks were additionally isolated and entirely sequenced from the more advanced 4588_652 transgenic progeny BC2ES2_472x. The entire 4044 bp BxA genomic sequence containing the right and the left border flanking sequences have high BLASTN identity hits to two nucleotide positions 141681606-141682538 and 141682550-141685147 on the chromosome 7 in the maize B73 genome.
Analysis of Nucleotide Sequences in the Left T-DNA Border Flank
The left T-DNA border flank OB-4451 has 98.66% BLASTN sequence identity to nucleotides 141683358-141685147 on the maize chromosome 7 in B73 genome. Multiple sequence alignment of the left border specific sequences from the wild type genotypes B73, BxA, 19545 (E), 15009 (G), nulls BC2ES2_512x and BC1GS2_518x as well as 4588_652 transgenic progenies 116_F1G and BC2ES2_472x and the reference public sequence of B73 genome revealed that these 1.8 kb sequences are nearly 100% identical between all genotypes.
Analysis of Nucleotide Sequences in the Right T-DNA Border Flank
The 2218 bp right border flank OB-4448 has high BLASTN sequence identity to two nucleotide positions 141681606-141682538 and 141682550-141683319 on the maize chromosome 7 in B73 genome. Multiple sequence alignment of the right border specific sequences from 4588_652 transgenic progenies 116_F1G and BC2ES2_472x with the WT sequence of BxA revealed that these three 2.2 kb sequences are nearly 100% identical.
A 521 bp “unique” sequence that is specific to the right T-DNA border flank has originated from genotype BxA, which was used for transformation with the pAG4588 construct. No BLASTN sequence identity hits to this sequence were identified at the T-DNA integration site within the reference B73 genome. On the other hand, the 521 bp sequence has multiple BLASTN identity hits on different chromosomes in maize B73 genome indicating that this sequence is highly repetitive. The 521 bp sequence is shown in italicized lowercase letters.
Sequences characterized at 4588_652 T-DNA integration site. The OB-4448 sequence (extended right border flank in 4588_652 isolated from F1G of 4588_652 is as follows:

(SEQ ID NO: 29)

CACCCTCGCTGTTGGTAAACGTGCGCCTTGGGTATGTCCTCACCTGCATG

ATACGACATGTTGAAAAAGGTACATGGCTGGGCGGATTTAAACAGTAGAA

TGAAAAGGTGCCACAAGAAAACTCGTCAAAGAATTGACTACGCGTCAATG

TTCCATAGTTAAAAAGACTTGAACTCTGGATCAGGGACTTTCAAACAAGG

ATAGCTGCCTGGTCACCAGTCATTAACTGTAATGTAATGGCCATAGATGA

TGCATGAGTACAATAATAAAAAAACACCATCCAGCCAAATATATACTCCC

TGTCACAAATGAAAATTCGTTTTAGATAATTAGTGGATTCATACAATATT

TGTTGTATGTGTTTTATGTGTCTAGATTCATCATCCTCTATTTGAATATA

GACAGAAAAATCATAACTAAAACGAATACTATTTGGGAACGGAGGGAGTA

CTACTTTGGCAGAATGCCCCCAGGAAAGTACCAGTTTCAGGGGTAGTTTG

GAAGGCTAAACCTAGGGAGGGAAAACCCCCCACATGTAACTAAATATCTT

ATTCAAATGTTACCCCTAGGGATTACTCACCCTGGGAAATGAGAAGGGTC

CCAAGGGGATTTCGGTTTCTATTATTTTTTCTGCAAACCATTTCAGAGCA

ATGATATGAAACCAAGCTAACTACTTATAACATTTCTTAAGAATATCAGA

CATAGGAAAGTGATGGCCTGGAACCAAAGTAAGACTGGTAGATAAATAGA

TCACTAGAATAAACCCTGACAGTTCATAGCCTTCATAGAAGCAAAAGGAA

ACACTACGGGAGCAATTGGTTGCTTGCACTAGCAATTCACTGCATTGGGT

CTAATGCAGGATAGACTAAGCCAGCATAAGTGTGCGCAATGTGTTTGTGT

TTGGTTGCCATGTTATAAGTAAGTTGCATTTGCTAATATctttctcctga

ctctaatgagtccacttttgctgactggtgggcgaaagtaagtaagcaag

tgcacaaatccaaaagaagaggctttaacagtatcatcatcttgggggct

tggtgtttatggcttcatcgtaataaggtggtttttgatggtgtcagtcc

ttcaattattggcataaaggcaatttttttggatgaagttgaattctgga

ggcttgccggtgctaggcatcttgaggctttggttcctggtgctggaatt

tttaggtcaagggttcttttgggtgattagtgaagagcaggtgtgtgtgg

tctgctcgcactttttgttgttcgttctcctattgcgtgctgttgtttcc

aggcgcatttatggaggctgcagttttgtgcgcagcagaagttggtggtt

ttgtgttttgtgttttgcctattttggcattgtactttggtccattttgg

actgttttcttctcttaatttaatgatgtgcagctctcctgcgcgtttaa

gaaaaaaaaaAGTTGGCTGTTTTGTATTTCTTGTGATCACCCATGCTTGT

TGTGGTCAGATTAAACTCTCACGTTTAATGCTACAGAAGCATCCATGAGA

CAATGAAACACCGCTCAAAAGCCACGTAGTAGCATACCCTGACTTATGAA

TAAAGCAACTCGATCTGATTTATTTGAGAAAACAGGAAACTGACAAGTTA

TTTTTAACACAAAATTTCATTAAAAACGAATGGTAGACAATTACCAATCT

GTAGGTCCCTGGCTTGCAAGTCCTCCCAATGTCTAAGAAATCAAATAGGA

ACTGCAGGCAAGCCAGCAAGAAAGTATTAATCACTGGATATAAAATATAA

AGAAAAAAGAAGGAAAGACGGCTACTCGGCTAGCATATGTTTTTGTTAGG

GGTGAAAATGGATACTTATTCAGAAATCATTTTTGATCTTTTTTCTTTAA

TTAGGAATAAATAGGATATAGAATATGCTAAGCAAATTCATATTCTTGTT

CTTAGCATTGGGCTTGTAAAGATTCATAAAAGGTAAATCTCAAATTTATC

ATATATCTTAAATGGTAGATATAAAATTCAGATACAAATATTTTTCAACT

TTTTTGTTGTAGGGAACAAATTATATTAAAAAAAATTATGCACAATTCTA

TTCTTATTTGTAATAATGTGCTTGATAACATAATAAAAGATTACCATCAA

ATTTCACACACACCCACCCACCCACCCACCCCTGCACGCACGCGCGCGCA

CACACACTATATGTGTGTtcaaacactgatagtttaaactgaaggcggga

aacgacaacctgatcatgagcggagaattaagggagtcacgttatgaccc

ccgccgatgacgcgggacaagccgttttacgtttggaactgacagaaccg

caacgttgaaggagccactcagcctaagcggccgcattggacttaattaa

gtgaggccggccaagcgtcgatttaaatgtaccacatggcgcgccaacta

tcatgcgatcgcttcatgtctaactcgagttactggtacgtaccaaatcc

atggaatcaaggtacctccatgctgtcctactacttgcttcatccccttc

tacattttgttctggtttttggcctgcatttcggatcatgatgtatgtga

tttccaatctgctgcaatatgaatggagactctgtgctaaccatcaacaa

catgaaatgcttatgaggcctttgctgagcagccaatcttgcctgtgttt

atgtcttcacaggccgaattcctctgttttgtttttcaccctcaatattt

ggaaacatttatctaggttgttttgtgtccaggcctataaatcataaatg

atgttgtcgtattggatgtgaatgtggtggcgtgttcagtgccttggatt

tgagt

The RB_BC2ES2_472x sequence (extended RB flank isolated from the advanced progeny of 4588_652) is as follows:

(SEQ ID NO: 30)

CACCCTCGCTGTTGGTAAACGTGCGCCTTGGGTATGTCCTCACCTGCATG

ATACGACATGTTGAAAAAGGTACAAGGCTGGGCGGATTTAAACAGTAGAA

TGAAAAGGTGCCACAAGAAAACTCGTCAAAGAATTGACTACGCGTCAATG

TTCCATAGTTAAAAAGACTTGAACTCTGGATCAGGGACTTTCAAACAAGG

ATAGCTGCCTGGTCACCAGTCATTAACTGTAATGTAATGGCCATAGATGA

TGCATGAGTACAATAATAAAAAAACACCATCCAGCCAAATATATACTCCC

TGTCACAAATGAAAATTCGTTTTAGATAATTAGTGGATTCATACAATATT

TGTTGTATGTGTTTTATGTGTCTAGATTCATCATCCTCTATTTGAATATA

GACAGAAAAATCATAACTAAAACGAATACTATTTGGGAACGGAGGGAGTA

CTACTTTGGCAGAATGCCCCCAGGAAAGTACCAGTTTCAGGGGTAGTTTG

GAAGGCTAAACCTAGGGAGGGAAAACCCCCCACATGTAACTAAATATCTT

ATTCAAATGTTACCCCTAGGGATTACTCACCCTGGGAAATGAGAAGGGTC

CCAAGGGGATTTCGGTTTCTATTATTTTTTCTGCAAACCATTTCAGAGCA

ATGATATGAAACCAAGCTAACTACTTATAACATTTCTTAAGAATATCAGA

CATAGGAAAGTGATGGCCTGGAACCAAAGTAAGACTGGTAGATAAATAGA

TCACTAGAATAAACCCTGACAGTTCATAGCCTTCATAGAAGCAAAAGGAA

ACACTACGGGAGCAATTGGTTGCTTGCACTAGCAATTCACTGCATTGGGT

CTAATGCAGGATAGACTAAGCCAGCATAAGTGTGCGCAATGTGTTTGTGT

TTGGTTGCCATGTTATAAGTAAGTTGCATTTGCTAATATctttctcctga

ctctaatgagtccacttttgctgactggtgggcgaaagtaagtaagcaag

tgcacaaatccaaaagaagaggctttaacagtatcatcatcttgggggct

tggtgtttatggcttcatcgtaataaggtggtttttgatggtgtcagtcc

ttcaattattggcataaaggcaatttttttggatgaagttgaattctgga

ggcttgccggtgctaggcatcttgaggctttggttcctggtgctggaatt

tttaggtcaagggttcttttgggtgattagtgaagagcaggtgtgtgtgg

tctgctcgcactttttgttgttcgttctcctattgcgtgctgttgtttcc

aggcgcatttatggaggctgcagttttgtgcgcagcagaagttggtggtt

ttgtgttttgtgttttgcctattttggcattgtactttggtccattttgg

actgttttcttctcttaatttaatgatgtgcagctctcctgcgcgtttaa

gaaaaaaaaaAGTTGGCTGTTTTGTATTTCTTGTGATCACCCATGCTTGT

TGTGGTCAGATTAAACTCTCACGTTTAATGCTACAGAAGCATCCATGAGA

CAATGAAACACCGCTCAAAAGCCACGTAGTAGCATACCCTGACTTATGAA

TAAAGCAACTCGATCTGATTTATTTGAGAAAACAGGAAACTGACAAGTTA

TTTTTAACACAAAATTTCATTAAAAACGAATGGTAGACAATTACCAATCT

GTAGGTCCCTGGCTTGCAAGTCCTCCCAATGTCTAAGAAATCAAATAGGA

ACTGCAGGCAAGCCAGCAAGAAAGTATTAATCACTGGATATAAAATATAA

AGAAAAAAGAAGGAAAGACGGCTACTCGGCTAGCATATGTTTTTGTTAGG

GGTGAAAATGGATACTTATTCAGAAATCATTTTTGATCTTTTTTCTTTAA

TTAGGAATAAATAGGATATAGAATATGCTAAGCAAATTCATATTCTTGTT

CTTAGCATTGGGCTTGTAAAGATTCATAAAAGGTAAATCTCGAATTTATC

ATATATCTTAAATGGTAGATATAAAATTCAGATACAAATATTTTTCAACT

TTTTTGTTGTAGGGAACAAATTATATTAAAAAAAATTATGCACAATTCTA

TTCTTATTTGTAATAATGTGCTTGATAACATAATAAAAGATTACCATCAA

ATTTCACACACACCCACCCACCCACCCACCCCTGCACGCACGCGCGCGCA

CACACACTATATGTGTGTtcaaacactgatagtttaaactgaaggcggga

aacgacaacctgatcatgagcggagaattaagggagtcacgttatgaccc

ccgccgatgacgcgggacaagccgttttacgtttgg

The OB-4451 sequence (extended left border flank in 4588_652 is as follows:

(SEQ ID NO: 31)

gggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgt

gttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcag

acacgttctgattgctaacttgccagtgtttctctttggggaatcctggg

atggctctagccgttccgcagacgggatcgatttcatgattttttttgtt

tcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccg

tgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgt

gatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgt

TACCCACTTTCATCCCTAGTTTTTGTTCTGGATTCAAGCATCTCAAAATT

GTTTACCTGAAGTTTATCAGTTTTGAGAAAGCGGCGCCCCTGTCGACTAC

CATCAGGCATTCGGACTACAACTGTCACAGCACCCTCTGCGTCTGGAGAC

GGTTCCGGTGGTAATGATGCTTGCTTCGAAGTGAGACTGGACTCTAGCTC

CTATTTAATCAAAACATCAGGGACAACATGACAAATAGTAGTCAAATATC

CAGGCAAGAAAAAAAAACCATAAACAATGAAAATACTGATCAAAAGTCCT

GTTTGGATCTCCTAAGAAAAATGAGAATGAGATCCAAACAATTGGATTCT

AGAATCCAGCTATCTATCCCAAACCCATTATTTGGCGAGATTTTCACTAT

GCAGAGGCAATGATCACTATAAGAATAAGATTCAAACACCCACTTATTAT

TTTTTTAATCCAGAAACCAGATTCTACATTCACTATAGAATCCAGAACTT

CAATATGGGAATGAGATCCAAATAGACCCTAAGCCAAAATGAAATTGGTG

AGATGAAGTGGCTAGTTGTCATAACCTCCTGTAAAGAAGACAGCGGTTTA

CAGTCCCAACACCCAAATAAACATGACATTAATATAATGACTACAACTCA

CAACCTAAACCTAAACCAATATACATCCAAACATAAGACAAAAGGAGAAC

TGAGTTTTATATGATCACACTGATGAACTGATGCTGTAGTCTAGCATTCA

AGTGTTTAAGATAGTTGACTATAAACCCTTCACCTTGCAGATTACATGTG

ACAGAAAGATACCTCTTCCTCAAGTTGTTTTTTACGCCTTTCCTCCTCCT

CTTGCTTCTGTTTCTCAAGAACAGCTTCTCTCGCAGCTGTTTCTTCAAGG

CGACGGAGCTCAGCCTCCTGTAGGGCCTTTAACTCCTTTTCTTGATCAGC

TTGTAGCGATGCAAGGTACTCATCGTCCTACAAATTTAAAATTTATAAAA

GTGCTCACCCATAGTGGCAATTATGAACAATGGATAAATCTTAGACCTCA

AACCTGCTGCTCTCGTAATAACCGCTGTTCAGTTAATGCTGGTGATGGAG

AATGAGATATTGGGGGATAATAAGTAGAGGTTCTGTGAGAAGGCATAGAG

AAAGGATATGTTGGTCCACCAAACATTGCAGCCTCAAGCATAACAGCTTC

ATCATGTTCCTCAGAAGAAATGCCACCCCACTTTATTGTAAAAAAAGACG

TCAGAAATTAAACAAATCCATCTAATGTCTTAGCGCACATTGGAACCACA

GATTATAATACCTCAGATGGGAAATCATCTCCATTATACTGGTGATTGTT

CAGAACAGGGCTAGCACCTGGGTGCACTATTTTTGGTAATTCATTGTCCT

CAGAAGGGGCACGCCGAGAACGACGCCTAACTAACGGCTGTTCTTCTACA

TCTTCAGCCTCCTCCTGGAAGCTCTCGTCATCTATTGGTTGCCTAGATGT

TCCAGCCTTTCCTGAAGCTAGTCCTTGCCTCCAAAAGGAAATTGCATGTA

TAAGGAATCAAATGATACTGTAGTAGGGTAGCCTGAGTGAAGAGGTGGGT

AGTAAAGTTAACATTCACCTCTCAACTATTCCATTTGCGGTTTCCATGCC

TGCTTTATCAGAAGAATGGTCCCTCAAATTCACTTCCTCTTGATGCTGGC

CTCCTTGCTCGACTATCTGATGATTATTGAAAAATTAGAGATGACATCAA

GATAGGTTCAAGTAAGCATGTTGGGGA

Example 6. Feed Glucanase Expression in Subsequent Generations

Several “T1” progeny from original “T0” transgenic maize plants were grown, and individual ears were either self-pollinated or pollinated with pollen from wild-type maize plants. Mature seed from the resulting ears was then assayed for feed glucanase activity via the colorimetric assay. FIG. 12 illustrates that glucanase activity was observed in T1 events. In this figure, the numbers along the abscissa correspond to the event identifiers of the original T0 plants from which the progeny were derived. The highest activity was observed for seeds of T1 plant derived from the 4597_69 event.

Example 7. Feed Glucanase Expression in Multi-Generations of Hemizygous, Homozygous and Hybrid Seeds

Progeny from original “T0” transgenic maize plants were grown and backcrossed (pollinated with pollen from the wild type maize parents or pollinated onto the wild type parents) for 4 generations (in maize inbred line E (BC4E), or in maize inbred line G (BC4G)). At each generation, some individual ears were self-pollinated. PCR method was applied to select homozygous plants as described in Example 8. Hybrid ears were made by cross-pollinating transgenic line G plants with transgenic line E plants, or vice versa.
FIG. 13 illustrates that glucanase activity in the hemizygous, homozygous and hybrid ears of event 4588.259. The homozygous and hybrid ears contained average activity of 190 units/g, which was approximately double of ears from hemizygous plants.

Example 8. PCR Assays for Identifying and Determining Zygosity of the Glucanase Events 4588.259, 4588.652, and 4588.757

Maize glucanase events 4588.259, 4588.652, and 4588.757 carry transgenes that result in seed-specific expression of glucanase enzyme. Event 4588.259 originally carried two T-DNA insertions at independently segregating loci, but subsequently a single genetic locus was selected for propagation and development. Events 4588.652 and 4588.757 carry two or more T-DNAs at a single genetic locus. Molecular identification and tracking of these transgenes can be done using standard PCR analysis (visually scoring an endpoint in a gel-based electrophoresis and staining of the PCR products) or real-time PCR. In addition to determining whether a plant is carrying a transgene, some of these PCR assays can also determine whether a plant is hemizygous (carrying one copy of the insertion) or homozygous (carrying two copies of the insertion).
FIG. 24 illustrates general real-time PCR assay design used to determine T-DNA locus presence (standard and real-time PCR) and zygosity (real-time PCR only).
In FIG. 14, the standard and real-time PCR assays include, for each T-DNA locus, one primer (Primer A) that binds to a maize genomic region that is adjacent to where the T-DNA insert is located and one primer (Primer B) that binds to a region in the T-DNA that is close to Primer A. To determine zygosity in the real-time PCR assay, a second reference gene (GWD, glucan water dikinase) is amplified (X and Y primers) along with the locus primers. Real-time PCR amplification of product from Primers A+B would indicate that the T-DNA locus is present and its fluorescence relative to the GWD reference (ref) fluorescence from amplification of product from Primers X+Y would determine whether it is hemizygous (one-copy) or homozygous (two-copy).
The standard multiplex PCR assay includes Primer A and B, as described above, but also another primer (Primer C) that binds to a maize genomic region on the other side of the T-DNA, opposite Primer A, and would be close to Primer A if the T-DNA insertion was not present, as in a wild type (WT) locus. FIG. 15 illustrates general standard PCR assay design used to determine T-DNA locus presence and zygosity. When the T-DNA insertion is present, the distance between Primer A and Primer C would be too large to amplify a product under our PCR amplification conditions and therefore absence of this amplification product is used to determine zygosity. PCR amplification of products from Primer A+B and Primer A+C indicates that the T-DNA locus is present and is hemizygous (one-copy). PCR amplification of product from Primer A+B, but not Primer A+C, indicates that the T-DNA locus is present and is homozygous (two-copy). PCR amplification of product from Primer A+C only, indicates that no T-DNA is present and the plant is WT at this locus. Primers and probes for all of these assays are listed in Tables 3 and 4.

TABLE 3

Standard and real-time (RT) PCR primers and probes used
to determine T-DNA locus presence and
ocus presence and zygosity of 4588.259, 4588.652, and 4588.757 events

			Primer/
PCR		Primer	Probe
Assay	Event	or Probe	ID (type)	Primer Sequence	Fluor*	Quencher

Standard/RT	4588.259	Primer	509 (A)	GAATTGTTCATCATAAGGCGTGA
				(SEQ ID NO: 38)

Standard/RT	4588.259	Primer	516 (B)	AACGTGACTCCCTTAATTCTCC
				(SEQ ID NO: 39)

RT	4588.259	Probe	PB5	AAACTGAAGGCGGGAAACGACAAC	HEX	BHQ1
				(SEQ ID NO: 40)

Standard/RT	4588.652	Primer	750 (A)	GAGATGCTTGAATCCAGAACAAA
				(SEQ ID NO: 41)

Standard/RT	4588.652	Primer	751 (B)	TTGTCTTGGTTGTGATGATGTG
				(SEQ ID NO: 42)

Standard	4588.652	Primer	749 (C)	GATTACCATCAAATTTCACACACAC
				(SEQ ID NO: 43)

RT	4588.652	Probe	PB17	TAGAACGACCGCCCAACCAGAC	HEX	BHQ1
				(SEQ ID NO: 44)

Standard	4588.757	Primer	513 (B)	AAACGTCCGCAATGTGTTATT
				(SEQ ID NO: 45)

Standard	4588.757	Primer	608 (C)	TCATGCAATTGTGCCACC
				(SEQ ID NO: 46)

Standard	4588.757	Primer	609 (A)	ACATAGTCAACCTAACGGCTTAT
				(SEQ ID NO: 47)

RT	GWDref	Primer	371 (X)	GGTTATAAGCCCGGTTGAAGTA
				(SEQ ID NO: 48)

RT	GWDref	Primer	525 (Y)	CTATTCCTTGCTCGGACTGAC
				(SEQ ID NO: 49)

RT	GWDref	Probe	PB2	CACCTGATATGCCAGATGTTCTGTCTCA	FAM	BHQ1
				(SEQ ID NO: 50)

*Fluor = fluorophore.

TABLE 4

4588.259, 4588.652, and 4588.757 event-specific PCR primer
combinations and PCR product sizes

	Primer	Primer	Primer	PCR Product
Event	A	B	C	(bp)	Assay Identifies

4588.259	509	516		137	T-DNA locus:
					OB-2880
					(SEQ ID NO: 51)
4588.652	750	751		107	T-DNA locus:
					OB-4451
					(SEQ ID NO: 52)
4588.652	750		749	174	WT locus
					(SEQ ID NO: 53)
4588.757	609	513		100	T-DNA locus:
					OB-3237
					(SEQ ID NO: 54)
4588.757	609		608	218	WT locus: B73ref
					(SEQ ID NO: 55)

PCR assay using primers X (371) and Y(525) identifies the ZmGWDref locus (SEQ ID NO: 56).
DNA Extraction
These PCR assays will work with any DNA extraction method that yields DNA that can be amplified with PCR. A standard DNA extraction method (10X TE+Sarkosyl) that was used in this example is as follows: leaf tissue (standard 1 cm hole punch) is sampled into a 96 deep-well block, metal beads are added, and the block is frozen at −80° C. for at least 30 min. The block is then ground for 45 sec in a Kleco Pulverizer, centrifuged at 4,000 RPM for 3 min, the lid is removed, 300 μl of 10XTE+Sarkosyl is added, the block is resealed, and the block is mixed at room temperature for 10-20 min. After incubation, the block is centrifuged at 4,000 RPM for 5 min, 165 μl of upper aqueous phase is removed and added to a 96-well PCR block, the PCR block is sealed, and the block is incubated at 90° C. for 30 min. After incubation, 20 μl of extract is added to 180 μl of sterile water in a 96-well plate (1:10 dilution) to create the final DNA sample for PCR.
PCR
Events 4588.259, 4588.652, and 4588.757 standard and real-time PCR primers are listed in Table 3 and standard PCR primer combinations with expected PCR product sizes are listed in Table 4.
Standard PCR is performed with 2 μl of DNA extract and GoTaq (Promega) or Kapa 3G (Kapa Biosystems) PCR Mix in 30 μl reaction volumes with the following components and conditions for each event:
Event 4588.259 Standard PCR:
Components (final concentration) were as follows: PCR Mix with buffer, MgCl₂, nucleotides, and enzyme (1×); primer 509 (400 nM) and primer 516 (400 nM). Conditions were as follows: 95° C., 3 min; 33 cycles (95° C., 30 sec; 55° C., 30 sec; 72° C., 30 sec); 72° C., 8 min.
Event 4588.652 Standard PCR:
Components (final concentration) were as follows: PCR Mix with buffer, MgCl₂, nucleotides, and enzyme (1×), primer 749 (400 nM), primer 750 (400 nM), and primer 751 (400 nM). Conditions were as follows: 95° C., 3 min; 33 cycles (95° C., 30 sec; 55° C., 30 sec; 72° C., 30 sec); 72° C., 8 min.
Event 4588.757 Standard PCR:
Components (final concentration) were as follows: PCR Mix with buffer, MgCl₂, nucleotides, and enzyme (1×), primer 513 (400 nM), primer 608 (400 nM), and primer 609 (400 nM). Conditions were as follows: 95° C., 3 min; 33 cycles (95° C., 30 sec; 55° C., 30 sec; 72° C., 30 sec); 72° C., 8 min.
Standard PCR was analyzed by running approximately 15 μl of PCR product on a 3% agarose gel at 95V for 30 min. An example of results from a standard PCR analysis of the 4588.652 selfed segregating plants is shown in FIG. 16. Referring to this figure, ten PCR reactions from 10 independent plants were separated on a 3% agarose gel stained with ethidium bromide. Expected locus and zygosity band sizes are indicated on the right side of the image.
Locus presence and zygosity is scored by visualizing specific bands in each lane.
Real-Time PCR was performed with 2 μl of DNA extract in 20 μl reaction volumes with the following components and conditions for each event:
Event 4588.259 Real-Time PCR:
Components (final concentration) were as follow: PCR Mix with buffer, MgCl₂, nucleotides, and enzyme (1×), primer 509 (400 nM, primer 516 (400 nM), primer 371 (400 nM), primer 525 (400 nM), probe PB5 (200 nM) and probe PB2 (200 nM). Conditions were as follows: 95° C., 4 min; 40 cycles (95° C., 5 sec; 60° C., 45 sec)
Event 4588. 652 Real-Time PCR:
Components (final concentration) were as follows: PCR Mix with buffer, MgCl₂, nucleotides, and enzyme (1×), primer 750 (400 nM), primer 751 (400 nM), primer 371 (400 nM), primer 525 (400 nM), probe PB17 (200 nM) and probe PB2 (200 nM). Conditions were as follows: 95° C., 4 min; 40 cycles (95° C., 5 sec; 60° C., 45 sec)
Event 4588. 259 Real-Time PCR. Real-Time PCR can be analyzed by any real-time PCR machine and software capable of four-channel fluorescence detection. A Bio-Rad CFX96 real-time PCR machine and CFX Manager Software were used to run an example of the 4588.259 real-time PCR assay on a selfed segregating population of 4588.259 plants. FIG. 17 illustrates an example of real-time PCR data for 4588.259 to determine locus presence and zygosity. In this figure, “RFU” refers to relative fluorescence units; “ntc” refers no target control. Presence of the 4588.259 locus and zygosity was scored by the clustering of data points on the graph.

Example 9. Germination Rates Among Seed from Independent Transgenic Plants that Express Feed Glucanase

Silks on wild-type plants were pollinated with pollen from individual transgenic plants (WT×Transgenic), or silks on transgenic plants were pollinated with pollen from wild-type plants (Transgenic×WT). Mature, dried seed were collected from the resulting ears and planted into soil. Following 1-2 weeks of incubation, germination rates were calculated. In some cases, this test was repeated following a second generation of growth and pollination (T2). Examples of results from such germination tests are shown in Table 5.

TABLE 5

Germination rates among seed expressing beta glucanase

						Germi-
		WT ×	Transgenic ×			nation
Vector	Event	Transgenic	WT	Generation	Sow	%

4588	54	x		T1	50	92
4588	17	x		T1	50	82
4588	11	x		T1	30	80
4588	161		x	T1	15	67
4588	162		x	T1	11	27
4588	215	x		T1	13	62
4588	219		x	T1	10	80
4597	18		x	T1	30	37
4597	54		x	T1	10	100
4597	56		x	T1	10	100
4597	69		x	T1	14	14
4597	69	x		T1	30	73
4588	161		x	T1	20	100
4597	101		x	T1	33	90
4597	104		x	T1	30	70
4588	259	x		T2	17	100
4588	251	x		T2	17	100
4588	54	x		T2	17	47
4597	101		x	T2	20	100
4597	104		x	T2	20	100

The germination rate in T1 and/or T2 for events 4597_54, 4597_56, 4588_161, 4588_252, 4588_259, 4597_101, and/or 4597_104 was observed to be 100%.

Example 10. Survival of Feed Glucanase Activity During Preparation of Poultry Feed Pelleting

Milled grain from transgenic plants expressing the feed glucanase was mixed with starter and grower corn-soy diets that were formulated for broiler chickens. The basal corn-soy diet for starter broilers was composed as follows: 54.89% corn 08-2012, 32.81% soybean oilcake, 5.00% distillers dry grains plus soluble solids, 2.00% vermiculite, 1.99% dicalcium phosphate, 1.00% poultry fat, 0.81% limestone fine, 0.50% plain salt (NaCl), 0.20% DL-methionine, 0.20% choline chloride 60, 0.20% mineral premix, 0.13% L-lysine, 0.12% L-threonine, 0.05% vitamin premix, 0.05% coban, and 0.05% selenium premix. The basal corn-soy diet for grower broilers was composed as follows: 58.53% corn, 26.63% soybean oilcake, 8.00% distillers dry grains plus soluble solids, 2.00% vermiculite, 1.69% dicalcium phosphate, 1.00% poultry fat, 0.76% limestone fine, 0.50% plain salt (NaCl), 0.20% mineral premix, 0.20% choline chloride 60, 0.13% DL-methionine, 0.13% L-lysine, 0.08% L-threonine, 0.05% vitamin premix, 0.05% coban, and 0.05% selenium premix.
Fine corn was ground using the hammermill screens: no. 4/4 for the starter diet and no. 6/6 for the grower and finisher diets. Coarse corn (5% of total corn) was ground with the roller mill with 0/100 gap openings.
Four diets were formulated for the pelleting trial as follows: Diet A was a basal diet, Diet D was the basal diet mixed with a control enzyme, Diet E was the basal diet mixed with the milled transgenic corn grain containing a high level of glucanase, and Diet F was the basal diet mixed with the milled transgenic corn grain containing a low level of glucanase.
Milled grain from transgenic plants expressing the feed glucanase was mixed with the basal diets at a ratio of approximately 1 lb transgenic grain per 2000 lbs basal diet mixture. For the low dose diet, transgenic grain was first mixed with non-transgenic grain at a weight ratio of 1:4 (1 gram of transgenic grain per 4 grams non-transgenic grain) to dilute the enzyme concentration prior to adding this ingredient to the basal diets.
All feed diets were pelleted at 175-180° F. into 4.4 mm pellets, and the starter diets were crumbled.
FIGS. 18A and 18B illustrate glucanase activity before and after pelleting in the Grower Diet (FIG. 18A) and the Starter Diet (FIG. 18B). Referring to these figures, samples from the resulting mixture were then removed before and after pelleting of the feed. These samples were then tested via the colorimetric glucanase assay to determine whether the enzyme survived the pelleting process. The identity of the various diets shown in FIGS. 18A and 18B were as follows: A, basal control diet (no external enzyme); D, positive control diet (commercially available enzyme added); F, low-dose diet (including milled grain from plants that express the feed glucanase); E, high-dose diet (including milled grain from plants that express the feed glucanase). It was observed that glucanase activity was high in the high-dose diet for both the Starter Diet and the Grower Diet and survived pelleting.

Example 11. Thermal Stability of Grain-Expressed Feed Glucanase

Feed glucanase was prepared via microbial expression and purification, and suspended in SEC buffer (100 mM MES, 300 mM NaCl, pH6.3). Five microliters of this preparation was mixed with 20 mg milled grain from wild-type maize. In parallel, 5 μl of SEC buffer was mixed with 20 mg milled grain from transgenic plants that express feed glucanase. Replicates from each of these two sets of samples were incubated at 94° C. or −130° C. for various periods of time, then allowed to cool to room temperature. Subsequently, residual glucanase activity was measured via the colorimetric assay. FIGS. 19 A and 19B illustrate glucanase activity after heat treatment. FIG. 19A illustrates glucanase activity after treatment at 130° C. FIG. 19B illustrates glucanase activity after treatment at 94° C. In these experiments, more activity survived when the feed glucanase was produced in the grain itself than when it was added to the grain exogenously. This finding demonstrates that expression and accumulation of the enzyme in grain effectively provides the enzyme with additional thermal stability relative to the same enzyme that is produced microbially. In this particular instance both the grain-expressed and the microbially-expressed enzymes have the same primary amino acid sequence. Therefore, the enhanced thermal stability that was observed in the flour from transgenic grain is a function of the expression host.

Example 12. Activity of Microbially-Produced AGR2314 at Various pH Values

AGR2314 activity was measured at several pH values between 3 and 8.5. Each assay (500 μL) contained Britton-Robinson polybuffer (40 mM sodium phosphate, 40 mM sodium borate, and 40 mM sodium acetate), 0.01% (v/v) Tween 20, one Beta-glucazyme substrate tablet (Megazyme, Wicklow, Ireland), and 20 nM of AGR2314 in a 2 mL Eppendorf tube. Samples were incubated for 1 hour at 37° C. or 80° C. Reactions were terminated by the addition of 1 mL of 2% (w/v) tris base. Samples were centrifuged at 15,000×g for 10 minutes, and 100 μL of each supernatant (37° C. assays) or 5 μL supernatant plus 100 μL of water (80° C. assays) was transferred to a flat-bottomed 96-well microplate. Absorbances were read at 590 nm. Assays at each pH value were performed in triplicate. Single blank assays (containing no enzyme) were performed at each pH, and these absorbance values were subtracted from the assays containing enzyme.
FIGS. 20 and 21 illustrate the optimum pH for measuring AGR2314 activity at 37° C. (FIG. 20) and 80° C. (FIG. 21). Referring to FIG. 20, the optimum pH was determined to be 7-7.5 at 37° C. FIG. 21 illustrates that the optimum was determined to be 6 at 80° C.
FIG. 22 shows an example of pH optimum of the feed glucanase that is produced in transgenic flour.
To determine the relationship between the pH of the assay conditions and the activity of the enzyme that was derived from transgenic grain, 5 ml of water containing 0.2% Tween-20 was mixed with 200 mg of flour from transgenic seed on a rotating platform for 1 hour at 60° C. Following centrifugation at 1500×g for 20 minutes in a clinical centrifuge, the supernatant was transferred to a 15 mL Eppendorf tube. This sample was centrifuged at 1500×g for 10 minutes in a tabletop centrifuge. Aliquots of this protein extract were diluted 20-fold in assays to test each pH condition by mixing 50 μl of extract with 950 μl of Britton-Robinson polybuffer (40 mM sodium phosphate, 40 mM sodium borate, and 40 mM sodium acetate) that had been prepared at pH 2-10. The pH of each reaction mixture was checked using a pH strip. Five hundred microliters from each mixture was transferred to a 96 deep-well plate for the assay. One beta-glucazyme tablet was added to each well and mixed by gentle vortexing, the plate was sealed and incubated at 80° C. for 1 hour. The reactions were stopped by adding 1 mL of 2% (w/v) Tris-base to each well. The 96-well plate was centrifuged at 3000×g for 10 minutes in a clinical centrifuge, then 100 μL of the supernatant from each of the samples was transferred to wells in a flat-bottom microplate, and the absorbance at 590 nm was determined on a microplate spectrophotometer. As shown in FIG. 22, the seed-produced enzyme has a pH optimum between pH6 and pH7, but still retains a large fraction of its activity at a pH as high as 10.

Example 13. Activity of Microbially-Produced AGR2314 and AGR2414 on Various Substrates

All reactions used 5 nM of AGR2314, AGR2414, or 5 μL of control enzyme at the indicated concentrations, in 200 mM sodium phosphate, 0.01% (v/v) Tween 20, pH 6.5. Reactions were carried out for one hour at either 37° C. or 80° C. and terminated as described.
Beta-glucosidase assays: the substrate was 1 mM pNP-D-glucopyranoside (Sigma Chemical Co. catalog # N:7006) and the positive control enzyme was Rhizobium etli beta-glucosidase (Prozomix, catalog No. PRO-E0110; 315.9 Unit/mL). Reaction volumes were 500 μL; reactions were terminated by the addition of 500 L of 2% (w/v) tris base. After centrifugation at 3000×g for 10 minutes, 100 μL of supernatant was transferred to a microplate and the absorbance at 405 nm was recorded.
Endocellulase assays: each assay contained one tablet of Cellazyme C substrate (Megazyme, catalog No, T-CCZ) in 500 μL buffer. Reactions were terminated by the addition of 1 mL of 2% tris base. Samples were centrifuged for 10 minutes at 15,000×g, 100 μL of supernatant was transferred to a microplate, and the absorbance at 590 nm was recorded.
Exocellulase (cellobiohydrolase) assays: the substrate was 1 mM pNP-D-cellobioside (Sigma catalog No. N5759) and the positive control enzyme was CBHI from Trichoderma longibrachiatum (Megazyme catalog No. E-CBHI; 0.5 Units/μL). Reaction conditions were as described above for the beta-glucosidase assays.
Anylase assays: the substrate was Red Starch (Megazyme catalog No. S-RTAR; prepared as directed by the manufacturer) and the positive control enzyme was α-amylase from Bacillus licheniformis (Megazyme catalog No. E-BLAAM: 3000 Units/mL). 245 μL buffer, 5 μL of enzyme, and 125 μL of Red Starch reagent were mixed and incubated as described above. Reactions were terminated by the addition of 625 μL ethanol. After incubating at room temperature for 10 minutes, samples were centrifuged for 10 minutes at 3000×g, and 100 μL of supernatant was transferred to a microplate; absorbance at 510 nm was recorded.
Endoxylanase assays: each assay contained one tablet of Xylazyme AX substrate (legazyme catalog No, No. XAX-1000) and the positive control enzyme was 100 mg/mL of Thermomyces lanuginosis xylanase (Sigma catalog #X2753) in assay buffer. Reactions were carried out as described above for the endocellulase assays.
Pectinase assays: the substrate was 25 mg/mL pectin (Sigma catalog No. P7536) in assay buffer and the positive control enzyme was pectinase from Aspergillus niger (Sigma catalog No. 17389) at 100 mg/mL in assay buffer. Five p L of enzyme was added to 35 PL of pectin solution and incubated as described above. Reactions were terminated by the addition of 60 μL of DNS stop/reagent solution (Wicher et al. [2001], Appl. Microbiol. Biotechnol. 55, p. 578) followed by heating at 950° C. for 15 minutes. Samples were centrifuged at 3000×g for 10 minutes, 20 μL supernatant was mixed with 100 μL water in a microplate, and the absorbance at 550 nm was recorded.
1,3-beta-glucosidase assays: each assay contained one tablet of 1,3-beta-glucazyme HS substrate (Megazyme catalog No. ET-CUR200) and the positive control enzyme was Trichoderma sp. 1,3-β-D-glucanase (Megazyme catalog No. E-LAMSE; 50 Units/mL). Reactions were carried out as described above for the endocellulase assays.
1,4-beta-glucosidase assays: each assay contained one tablet of Beta-glucazyme substrate (Megazyme catalog No. TBGZ-1000T). Reactions were carried out as described above for the endocellulase assays, except that 5 μL of supernatant was mixed with 100 PL of water in a microplate for recording of absorbance.
FIGS. 23A and 23B illustrate the glucanase activity for hydrolyzing starch, cellobiose (pNP-D-cellobioside), xylan (Xylazyme AX), HE-cellulose (Cellazyme C), barley-B-glucan (Beta-glucazyme), pectin and PNP-D-gluopyranoside at 37° C. (FIG. 23A) and 80° C. (FIG. 23B). Referring to these figures, it was observed that both AGR2314 and AGR2414 enzymes were highly active in hydrolyzing cellobiose and HE-cellulose at 37° C. and 80° C.

Example 14. Glucanase Activity on Seed Fiber

Glucose release from untreated seed fiber 20 mg seed fiber was digested at pH 5.0 with 5 μM AGR2314 protein for 72 hours at 55 C. A commercial enzyme cocktail was used at full loading (FCT) as a positive control. After enzymatic hydrolysis, the soluble sugars in reaction supernatant were hydrolyzed into monomers via acid hydrolysis at 121° C. FIGS. 24A and 24B illustrate release of monomeric sugars after enzymatic hydrolysis of the seed fiber. FIG. 24A shows glucose yield and FIG. 24B shows xylose yield. Pre-acid hydrolysis (light gray bars) and total (dark gray bars) sugars were separated and quantified via HPLC using a Bio-Rad Aminex HPX-87-P ion-exclusion column. AGR2314 does not release monomeric glucose or xylose from untreated seed fiber. However, this enzyme was able to solubilize untreated seed fiber into oligosaccharides which account for approximately 80% of the total sugars released by a commercially-available cellulase-enzyme cocktail.
Glucose release from dilute acid-pretreated seed fiber 20 mg seed fiber was pretreated at 80 C in 0.5% H₂SO₄for 16 hours, then neutralized to pH 5.0. The pretreated seed fiber was digested at pH 5.0 with 2 μM AGR2314 and a suite of glucanase, cellobiohydrolase, endoglucanase, and beta-glucosidase (all at 2 μM loading) for 72 hours at 55° C. A commercial enzyme cocktail (Accellerase XY, Genencor) was used at full loading (FCT) as a positive control. After enzymatic hydrolysis, the soluble sugars in reaction supernatant were hydrolyzed into monomers via acid hydrolysis at 121° C. FIGS. 25A and 25B illustrate release of monomeric sugars after enzymatic hydrolysis of the seed fiber. FIG. 25A shows glucose yield and FIG. 25B shows xylose yield. Pre-acid hydrolysis (light gray bars) and total (dark gray bars) sugars were separated and quantified via HPLC using a Bio-Rad Aminex HPX-87-P ion-exclusion column.
AGR2314 did not release sugars from pretreated seed fiber at greater levels than the pretreatment itself. When combined with other cell-wall degrading enzymes, approximately 90% of total sugars were released as compared to a commercially-available cellulase-enzyme cocktail.

Example 15. The Use of Glucanase Enzymes on Broiler Live Performance

The chemical energy contained within an animal's diet, and its availability to the animal eating the diet, are critical characteristics influencing the nutritional value of any diet. Diets rich in energy, provide adequate nutrition and promote rapid growth to higher levels than diets that are deficient in energy. Therefore, determining the energy within a diet, and altering energy availability by using glucanase enzymes, provides an important set of tools to improve animal nutrition and therefore animal performance.
To demonstrate the use of glucanase enzymes in broiler production, metabolizable energy and nutrient digestibility, male broilers were fed with alternative feed ingredients (wheat, barley and low-fat DDGS) with or without supplemental glucanase. Broiler body weight gain, feed consumption and feed conversion rate were determined and feed glucanase enzyme was evaluated.
Dietary Treatments and Procedures
Day-old male broiler were obtained from a commercial hatchery and randomly allocated to 64 battery cages in groups of 10. Experimental diets were fed from 0 to 28 d of ages. Initial group weights were obtained and equalized amongst the treatments. Feed disappearance and body weight were measured weekly (7, 14, 21, and 28 d of age) to calculate live performance parameters (feed consumption, body weight gain, and feed conversion ratio). In addition, excreta were collected twice to determine apparent metabolizable energy (AME) of the diets at 14 and 29 d of age.
Dietary treatments were fed in a 4×2 factorial design and further delineated below. Four different diets (corn/soybean meal based, corn/wheat based, corn/barley based, and corn/LF-DDGS based diets) and two levels of glucanase (with or without) were fed. Diets were formulated to be isocaloric and all nutrients, with the exception of energy, were formulated to meet or exceed the nutrient requirements. The enzyme treatments had the enzyme added on top of the diet. In addition, titanium dioxide was added to the diets as an indigestible marker to determine AME and nutrient digestibility. Table 6 describes dietary treatments.

TABLE 6

Treatment delineation

Trt	Diet	Enzyme

1	Corn-soybean meal	−
2	Corn-soybean meal	+
3	Corn-soybean meal-wheat	−
4	Corn-soybean meal-wheat	+
5	Corn-soybean meal-LF-	−
	DDGS

6	Corn-soybean meal-LF-	+
	DDGS
7	Corn-soybean meal-barley	−
8	Corn-soybean meal-barley	+
	Total	640 male broilers

Total Males needed: 8 treatments × 8 reps × 10 birds/cage = 640 male broilers + 60 for equating = 700 male broilers

Temperature
Battery cages: 92° F. from placement to 4 d, 90° F. from 5 to 9 d, 84° F. from 10 to 15 d, 80° F. from 16 to 24 d, 78° F. from 25 to 29 d,
Room Setup
Prior to bird placement, lighting and temperature in the battery cage rooms were set 48 hours in advance. Wire bottoms were added to the battery cages until day 0 to 4. Unless noted in the schedule, for collection periods, excreta pans were scrapped on a regular basis to avoid any pest and odor issues.
Lighting Program
A 23 light:1 dark with a lighting intensity of 3.0 ft²was implemented from placement until 7 d of age. A 23 light: 1 dark lighting schedule was implemented from 8-21d with a lighting intensity of 1.0 ft candles. A 23 light: 1 dark lighting schedule was implemented from 22-28 d of age with a lighting intensity of 0.3 ft candles.
Special Instructions
Wire bottoms were placed into all battery cages prior to the start ofthe experiment or day-old chicks will fall through the floors. Wire bottoms were removed at d 7. In addition, cage doors were modified to accommodate smaller birds from 0 to 7 d of age.
Any mortality was not replaced.
All diets were fed in mash form. Mix sheets were forthcoming.
To avoid cross contamination of the enzyme, separate feed scoops were needed. Table 7 describes experimental timeline.

TABLE 7

Timeline of dietary treatments

	Age of
Day	birds	Experimental Timeline	Lighting	Temperature

Tue	−2		23L: 1D	92° F.
			3.0 ft²
Thu	0	Place 720 male broilers in
		battery cages
		Equate weights
		Start experimental diets
Tue
	5			90°
Thu	7	Weigh all birds and feed
		Remove wire bottoms
Fri
	8		23L: 1D
			1.0 ft²
Sat	9
Sun	10			84°
Mon	11	Clean excreta pans
		Start 14 d collection period
Thu
	14	Weigh all birds and feed
		Collect excreta for 14 d
		collection period
Sat
	16			80°
Sun	17
Wed	20			80°^B-east
Thu	21	Weigh all birds and feed
Fri	22		23L: 1D
			0.3 ft²
Tues	25	Clean excreta pans		78°
		Start d 29 collection period
Fri
	28	Weigh all birds and feed		75°
		Collect excreta for 29 d
		collection period
		END OF EXPERIMENT

FIG. 26 illustrates the body weight gain (BWG) during the 28-day poultry feeding trial. Referring to FIG. 26, four different diets, corn/soybean meal based, corn/barley based, corn/wheat based, and corn/LF-DDGS based, with (+) or without (−) a glucanase were tested. Still referring to FIG. 26, average of BWG with or without glucanase across the four diets was shown. It was observed, that body wait gain was on average higher in chickens fed with the diets that included a glucanase.
FIG. 27 illustrates the changes in poultry BWG per time interval during 28 day feeding trial. Referring to FIG. 27, initial group weights were obtained and equalized amongst the treatments. BWG was measured weekly (7, 14, 21, and 28 d of age) for broilers feed using the corn/LF-DDGS diet with (+) or without (−) a glucanase. Still referring to FIG. 27, it was observed across all treatments that body wait gain in chickens fed with the glucanase including diets was higher than in chickens fed with diets without glucanase.
FIG. 28 illustrates feed consumption during the 28-day poultry feeding trial using two different diets (corn/barley based and corn/LF-DDGS based) with (+) or without (−) a glucanase. Referring to FIG. 28, it was found that feed consumption was higher for the diets that included a glucanase.
FIG. 29 illustrates the feed conversion rate (FCR) during the 28-day poultry feeding trial with two different diets (corn/barley based and corn/LF-DDGS based diets) with (+) or without (−) a glucanase. The feed conversion rate refers to the feed consumption required for gaining the body weight for a tested animal. The FCR is calculated by dividing the value of feed consumption by the value of body weight gain. Referring to FIG. 29, it was observed that the FCR was lower for the diets that included a glucanase. These data indicates that diets that included a glucanase facilitated digestion and feed consumption in the tested animals.

Example 16. The Use of Glucanase Enzymes for Broiler Live Performance

To demonstrate the effect of glucanase enzymes in broiler production, 936 male broilers were feed with varies glucanase concentrations in a 17 day battery trial. Birds were weighed at day 17. Feed composition includes corn, soybean meal and fat (soybean oil). Table 8 describes experimental details of the 17 day battery trial.

TABLE 8

Experimental Treatments: (diets were fed
through day 17)—total of 9 TRTs

Trt
Code	Description	Dose

1	Positive Control (PC)	—
2	Negative Control	—
	(NC, less 50-60 kcal/lb of PC)
3	NC + Industry Std Enzyme_1*	0.25 lb/ton
4	NC + Industry Std Enzyme_2**	0.2 lb/ton
5	NC + Glucanase	5
6	NC + Glucanase	50
7	NC + Glucanase	100
8	NC + Glucanase	250
9	NC + Glucanase	500

*Industry Std Enzyme_l refers to ENSPIRAT ™ (JBS United)

**Industry Std Enzyme_2 refers to HOSTAZYM X ® (Huvepharma)

No. of treatments	9	Broilers per replicate	8
Replicates per treatment	13	Broilers per treatment	104
Total No. of replicates	117	Total No. of broilers	936

Referring to FIG. 30, the inclusion of beta-glucanase in treatments 7 (Trt_7) and 8 (Trt_8) significantly (p-value <0.05) increased body weight gain compared to the positive control (PC), negative control (NC), and treatments 3 (Trt_3) and 4 (Trt_4), which contained a commercial NSPase inclusion. The glucanase inclusion in treatments 5 (Trt_5), 6 (Trt_6), and 9 (Trt_9) produced intermediate results.

REFERENCES

Leeson, S. and L. Caston. 2000. Commercial enzyme and their influence on broilers fed wheat or barley. J. Appl. Poult. Res. 9:242-251.
Yu, B. and T. K. Chung. 2004. Effects of multiple-enzyme mixtures on growth performance of broilers fed corn-soybean meal diets. J. Appl. Poult. Res.
13:178-182.

The references cited throughout this application, are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.
It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.

Claims

What is claimed is:

1. A transgenic plant, part or seed thereof comprising at least one synthetic nucleic acid encoding a glucanase and at least one synthetic polynucleotide producing a diagnostic amplicon for identifying event 4588.652, wherein the at least one nucleic acid comprises a sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8, and the at least one synthetic polynucleotide comprises a sequence with at least 90% identity to the sequence selected from the group consisting of SEQ ID NOS: 29-31.

2. The transgenic plant or part thereof of claim 1, wherein the glucanase is capable of degrading one or more polysaccharides selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.

3. The transgenic plant or part thereof of claim 1, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 5.0 to 10.0.

4. The transgenic plant or part thereof of claim 1, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25° C. to 130° C.

5. The transgenic plant or part thereof of claim 1, wherein the glucanase activity has improved stability upon expression in the plant compared to the activity of a glucanase having an identical amino acid sequence and expressed in a bacterial cell.

6. The transgenic plant or part thereof of claim 1, wherein a plant is selected from the group consisting of: wheat, maize, barley, and sorghum.

7. An animal feedstock comprising the transgenic plant, part or seed thereof of claim 1.

8. The animal feedstock of claim 7 further comprising a feed supplement.

9. The animal feedstock of claim 8, wherein the feed supplement is plant material selected from the group consisting of: a non-transgenic plant, another transgenic, mutant and engineered plant.

10. The animal feedstock of claim 8, wherein the feed supplement comprises one or more exogenous enzymes selected from the group consisting of: a hydrolytic enzyme selected from the group consisting of: xylanase, endoglucanase, cellulase, protease, phytase, amylase and mannanase.

11. The animal feedstock of claim 8, wherein the feed supplement comprises at least one component selected from the group consisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, barley grain, barley pellets, soybean meal, soybean oilcake, sorghum grain and sorghum pellets.

12. The animal feedstock of claim 8, wherein the feed supplement comprises at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, and mineral premix.

13. A method of producing an animal feedstock comprising mixing the transgenic plant, part or seed thereof of claim 1 with other plant material to form a mixture.

14. The method of claim 13, wherein the method further comprises pelletizing the mixture.

15. A method of increasing metabolizable energy of a diet, wherein the method comprises mixing the transgenic plant, part, or seed thereof of claim 1 with a feed ingredient.

16. The method of 15, wherein the feed ingredient comprises at least one component selected from the group consisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, wheat middlings, barley grain, barley pellets, soybean meal, soy hulls, dried distillers grain, soybean oilcake, sorghum grain and sorghum pellets.

17. The method of claim 15, wherein the feed ingredient comprises at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, mineral premix, and one or more exogenous enzymes.