OA20529A - Novel intergenic sequence regions and uses thereof - Google Patents
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Abstract
The invention provides recombinant DNA molecules comprising novel synthetic Intergenic Sequence Regions for use in plants to reduce the interaction of a first transgene expression cassette on a second transgene cassette when inserted between the first transgene cassette and second transgene cassette. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising the novel synthetic Intergenic Sequence Regions. The invention also provides methods to reduce the interaction between transgene expression cassettes using the novel synthetic Intergenic Sequence Regions
Description
Titre : Novel intergenic sequence régions and uses thereof.
O Abrégé :
Mandataire : S.C.P AKKUM, AKKUM & Associates, Quartier Mballa II, Dragages, B.P. 4966, YAOUNDE (CM).
Control without Enhancer
The invention provides recombinant DNA molécules comprising novel synthetic Intergenic Sequence Régions for use in plants to reduce the interaction of a first transgene expression cassette on a second transgene cassette when inserted between the first transgene cassette and second transgene cassette. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising the novel synthetic Intergenic Sequence Régions. The invention also provides methods to reduce the interaction between transgene expression cassettes using the novel synthetic Intergenic Sequence Régions.
Planche II
25_________27 28
| P-Gm.Sphas | GUS-2 | T-AC145767 |
T 26^-L-Gm.Sphas FIG. 2A
Control with Enhancer
22^-L-Ph.Dnak L-Gm.Sphas-. ^g
FIG. 2B
Insertion of ISR between first and second transgene expression cassette
FIG. 2C
O.A.P.I. - B.P. 887, YAOUNDE (Cameroun)-Tel. (237) 222 20 57 00-Site web: http:/www.oapi.int- Email: oapi@oapi.int
NOVEL INTERGENIC SEQUENCE REGIONS AND USES THEREOF
REFERENCE TO RELATED APPLICATION
[01] This application daims the benefit of United States Provisional Application No. 62/875,752, filed July 18, 2019, which is herein incorporated by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[02] The file named “MONS472WO_ST25.txt” containing a computer-readable form of the Sequence Listing was created on June 9, 2020. This file is 38,698 bytes (measured in MSWindows®), is contemporaneously filed by electronic submission (usîng the United States Patent Office EFS-Web filing system), and is incorporated into this application by reference in its entirety.
FIELD OF THE INVENTION
[03] The invention relates to the field of plant molecuiar biology and plant genetic engineering. More specifically, the invention relates to DNA molécules useful for reducîng the influence of one transgene cassette on the expression of another transgene cassette în plants.
BACKGROUND OF THE INVENTION
[04] Intergenic Sequence Régions (“ISRs”) are DNA sequences that, when placed between two or more transgene cassettes, reduce the interaction of one transgene cassette on another transgene cassette, preventing the alteration of the expression pattern of transgene cassettes due to expression element interaction between cassettes.
[05] Expression éléments in an expression cassette such as promoters, introns, and 3' untranslated régions (3' UTRs) contain cis-acting éléments that hâve the potential to influence expression of an adjacent or neighborîng expression cassette. For example, a plant viral promoter such as that of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S) is comprised of enhancer domains that can influence the transcription of nearby genes, activating genes up to 4.3 Kb upstream or downstream from the site of insertion (Gudynaite-Savitch et al. (2009) Strategies to mitigate transgene-promoter interactions. Plant Biotechnology Journal, 7: 47220529
485: Benfey et al. (1990) Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. The EMBO Journal, 9:1677-1684). For example, in one instance a transgene cassette subcloned into a plant transformation vector comprising a sélection cassette using the CaMV 35S promoter to drive a selectable marker coding sequence was 5 affected by the presence of the CaMV 35S promoter, which altered the tissue-specific expression of the transgene cassette to a more constitutive pattern (Yoo et al. (2005) The 358 promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta, 221: 523-530).
[06] Increasingly, in the field of plant biotechnology, vectors comprising multiple transgene 10 cassettes are being used to transform plants to introduce several agronomically important characteristics in a single vector stack. The advantage to this process is that several agronomie traits can be comprised in a single genetic locus, allowing for a more efficient and Iess costly breeding process when breeding lhe vector stacked plant with another transgenic plant comprising additional agronomie characteristics. However, as more expression cassettes are 15 cloncd into a vector, there is the potential for expression éléments from one expression cassette to alter or influence the expression profile of another expression cassette in the vector stack. An expression cassette designed to provide a spécifie pattern of tissue expression, such as expression in the seed, may change expression as a resuit of the interaction between the expression éléments of a neighboring expression cassette in the vector stack, altering Lhe seed-spccific expression 20 pattern to one more closely resembling the neighboring expression cassette. This can negatively affect lhe intended phenotype of lhe seed-spécifie expression cassette. The refore, there is a need in plant biotechnology for DNA sequences that can reduce or prevent the interaction of adjacent and neighboring expression cassettes in a vector stack.
[07] Thus, the inventer discloses herein novei synthetic ISRs that minimize the interaction of 25 expression cassettes in a vector stack in transgenic plants. These ISRs can be placed between adjacent expression cassettes in a single vector stack to prevent interaction between the expression éléments of individual cassettes, thus maintaining the intended expression pattern and level of expression of each expression cassette within the vector stack.
φ
SUMMARY OF THE INVENTION
[08] The invention provides novel synthetic Intergenic Sequence Régions or ISRs for use in plants. The invention also provides recombinant DNA constructs comprising the ISRs. The présent invention also provides transgenic plant cells, plants, and seeds comprising the ISRs. In 5 one embodiment, the ISRs are inserted between expression cassettes in a vector stack. The présent invention also provides methods for using the ISRs and making and using the recombinant DNA constructs comprising the ISRs, and the transgenic plant cells, plants, and seeds comprising the ISRs.
[09] Thus, in one aspect, the invention provides a recombinant DNA molécule comprising a 10 DNA sequence selected from the group consisting of; (a) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs:l-6; and (b) a sequence comprising any of SEQ ID NOs: 1-6. In spécifie embodiments, the recombinant DNA molécule comprises a DNA sequence having at least about 85 percent, at least about 86 percent, at least about 87 percent, at least about 88 percent, at least about 89 percent, at least about 90 percent, at least 91 percent, at least 92 15 percent, at least 93 percent, at least 94 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent sequence identity to the DNA sequence of any ofSEQ ID NOs:l-6.
[010] In another aspect, provided herein are transgenic plant cells comprising recombinant DNA molécule comprising a DNA sequence selected from the group consisting of: (a) a 20 sequence with at least 85 percent sequence identity to any of SEQ ID NOs; 1-6; and (b) a sequence comprising any of SEQ ID NOs: 1-6. In certain embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.
[011] In still yet another aspect, further provided herein is a transgenic plant, or part thereof, 25 comprising a recombinant DNA molécule comprising a DNA sequence selected from the group consisting of: (a) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs:Ιό; and (b) a sequence comprising any of SEQ ID NOs: 1-6. In spécifie embodiments, the transgenic plant is a progeny plant of any génération that comprises the recombinant DNA molécule. A transgenic seed comprising the recombinant DNA molécule that produces such a 30 transgenic plant when grown is also provided herein.
[012] In another aspect, the invention provides a method of producing a commodity product comprising obtaining a transgenic plant or part thereof containing a recombinant DNA molécule of the invention and producing the commodity product therefrom. In one embodiment, the commodity product is seeds, processed seeds, protein concentrate, protein isolate, starch, grains, plant parts, seed oil, biomass, flour and meal.
[013] In still yet another aspect, the invention provides a method for reducing the interaction of a first transgene expression cassette with a second transgene expression cassette within a transgenic plant transformed with a vector stack, said method comprising transformîng a plant cell with a vector stack comprising a recombinant DNA molécule comprising: (a) a first transgene cassette; (b) a second transgene cassette: (c) a DNA molécule comprising a sequence selected from the group consisting of: (î) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs:l-6; and (ii) a sequence comprising any of SEQ ID NOs:l-6; wherein the DNA molécule is inserted between the first transgene expression cassette and the second transgene expression cassette; and (d) regenerating a transgenic plant from the transformed plant cell. In certain embodiments, the vector stack is comprised of more than two expression cassettes. In further embodiments, the DNA moiecule of any of SEQ ID NOs:l-6 are inserted between each of the expression cassettes within the vector stack.
BRIEF DESCRIPTION OF THE SEQUENCES
[014] SEQ ID NO:1 is a DNA sequence of Intergenic Sequence Région ISR4_Stop which comprises the ISR4 (SEQ ID NO:4) and three stop codons on both the 5' and 3' ends.
[015] SEQ ID NO:2 is a DNA sequence of Intergenic Sequence Région ISR89.
[016] SEQ ID NO:3 is a DNA sequence of Intergenic Sequence Région ISR2.
[017] SEQ ID NO:4 is a DNA sequence of Intergenic Sequence Région ISR4.
[018] SEQ ID NO:5 is a DNA sequence of Intergenic Sequence Région ISR97.
[019] SEQ ID NO:6 is a DNA sequence of Intergenic Sequence Région ISR69
[020] SEQ ID NO:7 is a DNA sequence of Intergenic Sequence Région ISR88.
[021] SEQ ID NO:8 is a DNA sequence of Intergenic Sequence Région 1SR86.
[022] SEQ ID NO;9 is a DNA sequence of Intergenic Sequence Région ISR_X.
[023] SEQ ID NO:10 is a DNA sequence of an enhancer, E-CaMV.35S.2xAl-B3-l:l:l, presented in Figures la-c as “E-CaMV.35S.”
[024] SEQ ID NO:11 is a DNA sequence of a promoter, P-Os.Actl:67, presenled in Figures la- c as “P-Os.Actl.”
[025] SEQ ID NO: 12 is a DNA sequence of a leader or 5' UTR, L-Ta.Lhcbl:l, presented in Figures la-c as “L-Ta.Lhcbl.”
[026] SEQ ID NO: 13 is a DNA sequence of an intron, I-Os.Actl-l:l:19, presented in Figures la-c as “I-Os.Actl
[027] SEQ ID NO: 14 is a DNA sequence encoding neomycîn phosphotransferase, CR-Ec.nptlITn5-l:l:3, presented in Figures la-c as “nptll-l.”
[028] SEQ ID NO:15 is a DNA sequence of a 3' UTR, T-Ta.Hspl7-l:l;l, presented in Figures la-c as “T-Ta.Hspl7.”
[029] SEQ ID NO: 16 is a DNA sequence of a promoter, P-Zm.39486-l:l:l, presented in 15 Figures la-c as “P-Zm.39486.”
[030] SEQ ID NO: 17 is a DNA sequence of leader or 5 UTR, L- Zm .39486-1:1:1, presented in Figures la-c as “L- Zm.39486.”
[031] SEQ ID NO: 18 is a DNA sequence of an intron, I-Zm.DnaK:l, presented in Figures la-c as “I-Zm.DnaK.”
[032] SEQ ID NO: 19 is a DNA sequence of synthetic codîng sequence optimîzed for plant expression for β-glucuronidase (GUS-1: GOI-Ec.uidA+St.LSl.nno:l) with a processable intron derived from the potato light-inducibie tissue-specific ST-LS1 gene (Genbank Accession: X04753), presenled in Figures la-c as “GUS-1.”
[033] SEQ ID NO:20 is a DNA sequence of a 3' UTR, T-Os.Mth-l:l:l, presented in Figures 25 la-c as “T-Os.Mth.”
[034] SEQ ID NO:21 is a DNA sequence of a promoter, P-FMV.35S-enh-l:l:2, presented in Figures 2a-c as “P-FMV.35S.”
[035] SEQ ID NO:22 is a DNA sequence of a leader or 5 UTR, L-Ph.DnaK-1:1:3, presented in
Figures 2a-c as “L-Ph.DnaK.”
[036] SEQ ID NO:23 is a DNA sequence encoding neomycin phosphotransferase, CR-Ec.nptlI-
Tn5-l:l:2, presented in Figures 2a-c as “nptII-2.”
S [037] SEQ ID NO:24 is a DNA sequence of a 3' UTR, T-Mt.AC139600vl6:l, presented in
Figures 2a-c as “T-AC139600.”
[038] SEQ ID NO:25 is a DNA sequence of a promoter, P-Gm.Sphasl:14, presented in Figures
2a-c as “P-Gm.Sphas.”
[039] SEQ ID NO:26 is a DNA sequence of a leader or 5' UTR, L-Gm.Sphasl-l:l:l, presented in Figures 2a-c as “L-Gm.Sphas.”
[040] SEQ ID NO:27 is a DNA sequence of synthetic coding sequence for β-glucuronidase (GUS-2: GOI-GUS:1:2) with a processable intron derived from the potalo light-inducible tissuespecific ST-LS1 gene (Genbank Accession: X04753), presented in Figures 2a-c as “GUS-2.”
[041] SEQ ID NO:28 is a DNA sequence of a 3' UTR, T-Mt.AC145767v28:3, presented in 15 Figures 2a-c as “T- AC145767.”
BRIEF DESCRIPTION OF THE DRAWINGS
[042] Figures la-c are diagrammatic représentations of vector stacks used to assay the effectiveness of synthetic Intergenic Sequence Régions (“ISRs”) in reducing the interaction of 20 two transgene expression cassettes in a single vector stack on each other’s expression in slably transformed corn plants. The reference numbers in the figures indîcate the corresponding sequence identifier for each genetic element as presented in the Brief Description of the Sequences. Figure la shows the transgene expression cassette configuration for a control vector stack, Control without Enhancer, The Control without Enhancer is comprîsed of two transgene 25 expression cassettes cloned in divergent orientation. A first transgene cassette is comprîsed of a promoter, P-Os.Actl:67 (SEQ ID NO: 11), operably linked 5' to a leader, L-Ta.Lhcbl:l (SEQ ID
NO: 12), operably linked 5' to an intron, I-Os.Actl-l:l:19 (SEQ ID NO:13), operably linked 5' to a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-l:l:3 (SEQ ID
NO:14), operably linked 5' to a 3' UTR, T-Ta.Hspl7-l:l:l (SEQ ID NO:15). A second transgene cassette, cloned in a divergent direction relative to the first transgene cassette, is comprised of a seed-specific promoter, P-Zm.39486-l:l:l (SEQ ID NO: 16), operably linked 5' Lo a leader, L- Zm.39486-l:l:l (SEQ ID N0:17), operably linked 5' to an intron, I-Zm.DnaK:l 5 (SEQ ID NO: 18), operably linked 5' to a coding sequence encodîng GUS-1, GOIEc.uidA+St.LSl.nno:l (SEQ ID NO:19), operably linked 5' to a 3' UTR, T-Os.Mth-l:l:l (SEQ ID NO:20). Figure lb shows the transgene expression cassette configuration for a control vector stack, Control with Enhancer. The Control with Enhancer is comprised of a strong enhancer, ECaMV.35S.2xAl-B3-l:l:l (SEQ ID NO: 10) comprising tandem repeats of spécifie enhancer 10 régions derived from the Cauliflower mosaîc virus 35S promoter, operably linked 5' to a promoter, P-Os.Actl:67 (SEQ ID NO: 11), operably linked 5 ' to a leader, L-Ta.Lhcbl:l (SEQ ID NO: 12), operably linked 5' to an intron, l-Os.Actl-l:l:19 (SEQ ID NO: 13), operably linked 5' to a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-l:l:3 (SEQ ID NO: 14), operably linked 5' to a 3' UTR, T-Ta.Hspl7-l:l:l (SEQ ID NO:15). A second 15 transgene cassette, cloned in a divergent direction relative to the first transgene cassette is comprised of a seed-specific promoter, P-Zm.39486-l:l:l (SEQ ID NO: 16), operably linked 5' to a leader, L- Zm.39486-l:l;l (SEQ ID NO: 17), operably linked 5' to an intron, I-Zm.DnaK:l (SEQ ID NO: 18), operably linked 5' to a coding sequence encodîng GUS-1, GOIEc.uidA+St.LSl.nno:l (SEQ ID NO: 19), operably linked 5' to a 3' UTR, T-Os.Mth-l:l:l (SEQ 20 ID NQ:20). The Control with Enhancer in Figure la lacks an ISR between the first and second transgene expression cassettes. As a resuit, the enhancer from the first transgene expression cassette interacts with and alters the expression of the seed-specific promoter in the second transgene expression cassette, changing the expression of the second expression transgene cassette from seed-specific to constitutive. In Figure le, an ISR is cloned between the first and 25 second transgene expression cassettes of the Control with Enhancer. If the ISR is effective, then it will reduce the interaction of the enhancer in the first transgene expression cassette on the expression of the promoter in the second expression transgene cassette, reducing expression in non-seed tissues relative to the Control with Enhancer.
[043] Figures 2a-c are a diagrammatic représentation of vector stacks used to assay the 30 effectiveness of ISRs in reducing the interaction of two transgene expression cassettes in a single vector stack on each other’s expression în stably transformed soy plants. The reference numbers in the figures indicate the corresponding sequence identifier for each genetic element as presented in the Brief Description of the Sequences. Figure 2a shows the transgene expression cassette configuration for a control vector stack, Control without Enhancer. The Control without Enhancer (Figure 2a) is comprised of a seed-specific promoter, P-Gm.Sphasl:14 (SEQ ID 5 NO:25), operably linked 5' to a leader, L-Gm.Sphasl-l:l:l (SEQ ID NO:26), operably linked 5' to a coding sequence encoding GUS-2, GOI-GUS:1:2 (SEQ ID NO:27), operably linked 5' to a 3' UTR, T-Mt.AC145767v28:3 (SEQ ID NO:28). The seed-specific promoter is able to drive GUS expression primarily in the seed of the soybean plant in the Control without Enhancer. Figure 2b shows the transgene expression cassette configuration for a control vector stack, 10 Control with Enhancer. The Control with Enhancer is comprised of two transgene expression cassettes in divergent orientation. A first transgene cassette is comprised of a strong promoter derived from the Figwort mosaic virus 35S promoter with a rearranged and duplicated enhancer, P-FMV.35S-enh-l:l:2 (SEQ ID NO:21), operably linked 5' to a leader, L-Ph.DnaK-l:l:3 (SEQ ID NO;22), operably linked 5' to a coding sequence for neomycin phosphotransferase, CR15 Ec.nptII-Tn5-l:l:2 (SEQ ID NO:23), operably linked 5' to a 3' UTR, T-Mt.AC139600vl6:l (SEQ ID N0:24). A second transgene cassette, cloned in a divergent direction relative to the first transgene cassette is comprised of a seed-specific promoter, P-Gm.Sphasl:14 (SEQ ID NO:25), operably linked 5' to a leader, L-Gm.Sphasl-l:l:l (SEQ ID NO:26), operably linked 5' to a coding sequence encoding GUS-2, G0I-GUS:l:2 (SEQ ID NO:27), operably linked 5' to a 20 3' UTR, T-Mt.AC145767v28:3 (SEQ ID NO:28). The Control with Enhancer iacks an ISR between the first and second transgene expression cassette. As a resuit, the seed-specific promoter expression in the second transgene expression cassette is affected by the enhancer région of the Figwort mosaic virus 35S promoter in the first transgene expression cassette, changing the expression of the second expression transgene cassette from seed-specific to 25 constitutive. In Figure 2c, an ISR is cloned between the first and second transgene expression cassette of the Control with Enhancer. If the ISR is effective, then it will reduce the interaction of the enhancer région of the Figwort mosaic virus 35S promoter in the first transgene expression cassette with the promoter in the second transgene expression cassette, reducing expression in non-seed tissues relative to the Control with Enhancer.
DETAILED DESCRIPTION OF THE INVENTION
[044] The invention provides novel synthetic Intergenic Sequence Régions (“ISRs”) for use in transgenic plants. The nucléotide sequences of these novel synthetic ISRs are provided as SEQ ID NOs:l-6. These synthetic ISRs reduce the interaction of expression éléments in a first 5 transgene expression cassette on the expression of a second transgene cassette in a transgenic plant when inserted between the first transgene cassette and second transgene. The invention also provides transgenic plant cells, plants, and seeds comprising the ISRs. The invention also provides methods for using the ISRs and making and using the recombinant DNA molécules comprising the ISRs.
[045] The following définitions and methods are provided to belter define the présent invention and to guide those of ordinary skill in the art in the practice of the présent invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
ISRs and the Interaction of a First Transgene Expression Cassette with a Second
Transgene Expression Cassette
[046] As used herein, the term “interaction” refers to the effect of one or more éléments in a first transgene expression cassette on the expression pattern of a second transgene expression cassette when provided in close proximity to each other in a transgenic plant, in certain embodiments having been transfonned using a vector slack.
[047] The regulatory éléments within each transgene expression cassette are comprised of various cis-elements that are bound by trans-acting factors which effect transcription of a transgene. For example, a plant promoter is comprised of cis-elements that are essential for the initiation of transcription and efficiency of transcription. In addition, a plant promoter is often comprised of other cis-element motifs that can modulate transcription in response to a particular stimulus such as stress (ABRE and AB 14), pathogen (W Box), or light (GT1-motif). Other ciselements can provide tissue-spécifie or tissue-preferred expression (Porto et al. (2014) Plant Promoters: An Approach of Structure and Function, Mol. Biotechnol 56: 38-49). For example, the Cauliflower mosaic virus 35S promoter comprises an enhancer région made of two domains. The downstream domain, domain A, confers expression princîpally in the roots. A cis-element within a twenty-two base pair région within Domain A, as-1 is primarily responsibie for this expression. The upstream domain, domain B, confers expression in most cell types of leaf and stem as well as in vascular tissue of the roots (Benfey et al. (1990) Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant, development. The EMBO Journal, 9:1677-1684).
[048] When two transgene expression cassettes are adjacent to each other in the plant genome, there is the potential for the expression éléments of one transgene expression cassette to alter the expression of the other transgene expression cassette. This “interaction” of one transgene expression cassette with an adjacent transgene expression cassette in transgenic plants is demonstrated in Examples 2 and 3 by the Control with Enhancer.
[049] “Leakiness” is the term used to describe the level of average expression change in tissues caused by the interaction of expression éléments in a first expression cassette on the expression profile of a second expression cassette. Leakiness is determined by comparing the expression profile of a Control with Enhancer to the expression profile of the test vector stack with an ISR (which is comprised of the Control with Enhancer with an ISR inserted between the two transgene cassettes). The leakiness of the Control without Enhancer compared to the Control with Enhancer is 100%. Leakiness of the constructs comprising an ISR is determined by dividing the average GUS expression in the non-target tissues in the test construct by the average GUS expression in the non-target tissues of the Control with Enhancer construct and multiplying by one-hundred. The percent réduction in leakiness is determined by subtracting the percent 20 leakiness from one-hundred percent.
[050] “Intergenic Sequence Région” or “ISR” is a synthetic nucléotide sequence that is designed to minimize the interaction of expression éléments in neighboring transgenic cassettes on each other’s expression. The Intergenic Sequence Régions disclosed herein were computationally-designed and assayed for the ability to reduce the interaction of a first transgene 25 expression cassette on a second transgene expression cassette in a vector stack used to transform plant cells, thus preserving the expression profile of each transgene expression cassette as that when observed individually in a transgenic plant.
[051] A synthetic nucléotide sequence or “artificial nucléotide sequence” is a nucléotide sequence that is not known to occur in nature or that is not naturally occurring. The Intergenic 30 Sequence Région éléments of the present invention comprise synthetic nucléotide sequences.
Preferably, synthetic nucléotide sequences share iittle or no extended homology to natural sequences. Extended homology in this context generally refers to 100% sequence identîty extending beyond about 25 nucléotides of contiguous sequence.
[052] In Example 2, control corn plants were transformée! using two vector stacks comprised of 5 two transgene expression cassettes in a divergent orientation. One control vector stack comprised a first transgene expression cassette comprising a rice actin one promoter (Control without Enhancer, see Figure la) driving expression of an antibiotic résistance gene and a second transgene expression cassette that comprised a seed-preferred promoter driving GUS expression. Corn plants transformed with this vector stack demonstrated seed-preferred expression of GUS. 10 The other control vector stack (Control with Enhancer) comprised a first transgene expression cassette comprising a strong enhancer derived from CaMV 35S operably linked to the rice actin one promoter driving expression of an antibiotic résistance gene and a second transgene expression cassette that comprised a seed-preferred promoter driving GUS expression. Corn plants transformed with the Control with Enhancer demonstrated high levels of GUS expression 15 in roots, leaves, anther, silk, and seed. Thus, in the Control with Enhancer, the first transgene expression cassette enhancer modifîed the expression pattern of the second expression transgene cassette’s expression profile, changing the expression of the second expression transgene cassette from seed-preferred to constitutive.
[053] Certain computationally-designed ISRs were inserted between the first and second 20 transgene cassettes of the Control with Enhancer, as demonstrated in Figure le. The percent leakiness in the interaction of the first transgene expression cassette’s expression pattern on the second transgene expression cassette’s expression pattern was 16%, 8%, and 6%, rcspectively, when the ISRs ISR4_Stop (SEQ ID NO:1), ISR89 (SEQ ID NO:2), and 1SR97 (SEQ ID NO:5) were inserted between the first and second transgene expression cassettes. Thus, these ISRs 25 reduced the interaction of the first transgene expression cassette with the second transgene expression cassette by 84%, 92%, and 94%, respectively.
[054] In Example 3, a similar experimental design was used to test the effectiveness of certain ISRs in soybeans. Insertion of ISR2 (SEQ ID NO:3), ISR4 (SEQ ID NO:4), ISR69 (SEQ ID NO:6) between the first transgene expression cassette and second transgene expression cassette 30 of the Control with Enhancer resulted in a réduction of the effcct of the first transgene expression cassette’s expression pattern on the second transgene expression cassette’s with only 3%, 4%, and 5% leakiness, respectively. This resuited in a réduction in interaction of the expression éléments in the first transgene expression cassette on the second transgene expression cassette’s expression pattern by 97%, 96%, and 95%, respectively.
[055] As demonstrated in the Examples, not al] computationally-designed Intergenic Sequence
Régions were as efficacïous in reducing interaction. Further, even ISRs which resuited in réduction of interaction did so to varying degrees. For example, ISR88 (SEQ ID NO:7) and ISR86 (SEQ ID NO:8) only reduced interaction by 39% and 68%, respectively in transgenic corn plants with a leakiness of 61% and 32%, respectively. This réduction in the interaction was much less when compared to 84% for ISR4_Stop, 92% for 1SR89, and 94% for ISR97. Likewise, in transgenic soybeans ISR_X (SEQ ID NO:9) only reduced the interaction by 76% (percent leakiness, 24%) in coniparison to 97% for ISR2, 96% for ISR4, and 95% for ISR69. Thus, each computationally designed ISR is unique, and different ISRs can be used in conjunction with different expression cassettes to reach the desired expression profiles for one or more genes of interest.
DNA Molécules
[056] As used herein, the term “DNA” or “DNA molécule” refers to a double-stranded DNA molécule of genomic or synthetic origin, Le., a polymer of deoxyrîbonucleotide bases or a DNA molécule, read from the 5' (upstream) end to the 3 ' (downstream) end. As used herein, the term 20 “DNA sequence” refers to the nucléotide sequence of a DNA molécule. The nomenclature used herein corresponds to that of Title 37 of the United States Code of Fédéral Régulations § 1.822, and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
[057] As used herein, a “heterologous molécule” is a molécule comprising a combination of DNA molécules that would not naturally occur together without human intervention. For 25 instance, a heterologous molécule may be a DNA molécule thaï is comprised of at least two
DNA molécules heterologous with respect to each other, a DNA molécule that comprises a DNA sequence that devîntes from DNA sequences that exist in nature, a DNA molécule that comprises a synthetic DNA sequence or a DNA molécule that has been incorporated into a host cell’s DNA by genetic transformation or gene editing.
[058] Reference in this application to an “isolated DNA molécule” or an équivalent term or phrase is intended to mean that the DNA molécule is one that is présent alone or in combination with other compositions, but not within ils natural environment. For example, nucleic acid éléments such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found. However, each of these éléments, and subparts of these éléments, would be “isolated” within the scope of this disclosure so long as the element is not within the genome of the organism and at the location within the genome in which it is naturally found. Similarly, a nucleotîde sequence encodîng an insecticidal protein or any naturally occurring insecticidal variant of that protein would be an isolated nucleotîde sequence so long as the nucléotide sequence was not within the DNA of the bacterium from which the sequence encoding the protein is naturally found. A synthetic nucleotîde sequence encoding the amîno acid sequence of the naturally occurring insecticidal protein would be considered to be isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotîde sequence, i.e., the nucleotîde sequence of the DNA inserted into the genome of the cells of a plant or bacterium, or présent in an extrachromosomal vector, would be considered to be an isolated nucleotîde sequence whether it is présent within the plasmid or similar structure used to transform the cells, within the genome of the plant or bacterium, or présent in détectable amounts in tîssues, progeny, bîological samples or commodity products derived from the plant or bacterium.
[059] As used herein, the tenn “sequence identity” refers to the extent to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is created by manually alîgning two sequences, e.g., a reference sequence and another sequence, to maximize the number of nucleotîde matches in the sequence alignment with appropriate internai nucleotîde insertions, délétions, or gaps. As used herein, the tenu “reference sequence” refers to a DNA sequence provided as SEQ ID NOs: 1-6.
[060] As used herein, the term “percent sequence identity” or “percent identity” or “% identity” is the identity fraction multiplied by 100. The “identity fraction” for a sequence optimally aligned with a reference sequence is the number of nucleotîde matches in the optimal alignment, divided by the total number of nucléotides in the reference sequence, e.g., the total number of nucléotides in the full length of the entire reference sequence. Thus, one embodiment of the invention provides a DNA molécule comprising a sequence that, when optîmally aligned to a reference sequence, provided herein as SEQ ID NOs:l-6, has at least about 85 percent identity, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent 5 identity, at least about 89 percent identity, at least about 90 percent identity, at least about 91 percent identity, at least about 92 percent identity, at least about 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, at least about 99 percent identity, or at least about 100 percent identity to the reference sequence. In certain embodiments, 10 a sequence having a given percent identity to any of SEQ ID NOs: 1-6 maintains the general functionaiity of any of SEQ ID NOs: 1-6, i.e., exhibits the same or similar capacîty to reduce the influence of a first transgene expression cassette on the expression of a second transgene cassette in a transgenic plant. In certain embodiments, a sequence having a given percent identity to any of SEQ ID NOs: 1-6 has the activity of any of SEQ ID NOs: 1-6 with respect to reducing the 15 influence of a first transgene expression cassette on the expression of a second transgene cassette in a transgenic plant.
Regulatory Eléments
[061] Regulatory éléments such as promoters, leaders (also known as 5’ UTRs), enhancers, introns, and transcription te imination régions (or 3' UTRs) play an intégral part in the overall 20 expression of genes in living cells. The term “regulatory element,” as used herein, refers to a
DNA molécule having gene-regulatory activity. The term “gene-regulatory activity,” as used herein, refers to the ability to affect the expression of an operably iinked transcribable DNA molécule, for instance by affecting the transcription and/or translation of the operably Iinked transcribable DNA molécule. Regulatory éléments, such as promoters, leaders, enhancers, 25 introns and 3' UTRs that function in plants are useful for modifying plant phenotypes through genetic engineering.
[062] Regulatory éléments may be characterized by their gene expression pattern, e.g., positive and/or négative effects such as constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, palhological, cell cycle, and/or chemically responsive 30 expression, and any combination thereof, as well as by quantitative or qualitative indications. As used herein, a “gene expression pattern” is any pattern of transcription of an operably iinked DNA molécule into a transcribed RNA molécule, The transcribed RNA molécule may be translated to produce a protein molécule or may provide an antisensc or other regulatory RNA molécule, such as a double-stranded RNA (dsRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), a small interfering RNA (siRNA), and the like,
[063] As used herein, the term “protein expression” is any pattern of translation of a transcribed RNA molécule into a protein molécule. Protein expression may be characterized by its temporal, spatial, developmental, or morphological qualities, as well as by quantitative or qualitative indications.
[064] A promoter is useful as a regulatory element for modulatîng the expression of an operably Iinked transcribable DNA molécule. As used herein, the term “promoter” refers general ly to a DNA molécule that is involved in récognition and binding of RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. A promoter may be initially isolated from the 5' untranslated région (5' DTR) of a genomic copy of a gene. Alternately, promoters may be synthetically produced or manîpulated DNA molécules. Promoters may also be chimeric. Chimeric promoters are produced through the fusion of two or more heterologous DNA molécules. Promoters useful in demonstrating the présent invention include promoter cléments provided as SEQ ID NOs:ll, 16, 21, and 25.
[065] As used herein, the term “leader” refers to a DNA molécule isolated from the untranslated 5' région (5' UTR) a gene and defined generally as a nucléotide segment between the transcription start site (TSS) and the protein coding sequence start site. Alternately, leaders may be synthetically produced or manîpulated DNA éléments. A leader can be used as a 5' regulatory element for modulatîng expression of an operably Iinked transcribable DNA molécule. Leader molécules may be used with a heterologous promoter or with their native promoter. Leaders useful in demonstrating the présent invention include SEQ ID NOs: 12, 17, 22, and 26.
[066] As used herein, the term “intron” refers to a DNA molécule that may be isolated or identified from a gene and may be defined generally as a région spliced out during messenger RNA (mRNA) processing prior to translation. Alternately, an intron may be a synthetically produced or manîpulated DNA element. An intron may contain enhancer éléments thaï effect the transcription of operably linked genes. An intron may be used as a regulatory element for modulating expression of an operably linked transcribable DNA molécule. A construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcribable DNA molécule. Introns useful in demonstratîng the présent invention are presented 5 as SEQ ID NOs:13 and 18.
[067] As used herein, the terms “3' transcription termination molécule,” “3' untranslated région” or “3' UTR” refer to a DNA molécule that is used during transcription to the untranslated région of the 3' portion of an mRNA molécule. The 3' untranslated région of an mRNA molécule may be generated by spécifie cleavage and 3' poly adénylation, also known as a 10 polyA tail. A 3' UTR may be operably linked to and located downstream of a transcribable
DNA molécule and may include a polyadenylation signal and other regulatory signais capable of affecting transcription, mRNA processing, or gene expression. PolyA tails are thought to function in mRNA stability and in initiation of translation.
[068] As used herein, the term “enhancer” or “enhancer element” refers to a cw-acting 15 regulatory element, a.k.a. cfr-element, which confers an aspect of the overall expression pattern, but is usually insufficient alone to drive transcription, of an operably linked transcribable DNA molécule. Unlike promoters, enhancer éléments do not usually include a transcription start site (TSS) or TATA box or équivalent DNA sequence. A promoter or promoter fragment may naturally comprise one or more enhancer éléments that affect the transcription of an operably 20 linked DNA sequence. An enhancer element may also be fused to a promoter to produce a chimeric promoter c A-element, which confers an aspect of the overall modulation of gene expression.
[069] As used herein, the term “variant” refers to a second DNA molécule that is in composition similar, but not identical to, a first DNA molécule. For example, a variant of one of 25 the ISRs disclosed herein would hâve a slightly different sequence composition but would maintain the capacity to rcduce the influence of a first transgene expression cassette on the expression of a second transgene cassette in a transgenic plant in the same manner as the ISR from which ît was derived. A variant may be a shorter or truncated version of the first DNA molécule or an altered version of the sequence of the first DNA molécule, such as one with 30 different restriction enzyme sites and/or internai délétions, substitutions, or insertions. A “variant” can also encompass an ISR having a nucléotide sequence comprising a substitution, délétion, or insertion of one or more nucléotides of a reference sequence, wherein the dérivative Intergenic Sequence Région element has more or less or équivalent capacity to reduce the influence of a first transgene expression cassette on the expression of a second transgene cassette in a transgenic plant. In the present invention, a polynucléotide sequence provided as SEQ ID NOs:l-6 may be used to create variants that are similar in composition, but not identical to, the DNA sequence of the original ISR, while still maintaining the general functionality, i.e., the same or similar capacity to reduce the influence of a first transgene expression cassette on the expression of a second transgene cassette in a transgenic plant. In certain embodiments, a variant of any of SEQ ID NOs: 1-6 has the activity of any of SEQ ID NOs: 1-6 with respect to reducîng the influence of a first transgene expression cassette on the expression of a second transgene cassette in a transgenic plant. Production of such variants of the invention is weil within the ordinary skill of the art in light of the disclosure and is encompassed within the scope of the invention.
[070] In certain examples, a variant of an ISR may be a fragment of any of SEQ ID NOs: 1-6. Fragments of SEQ ID NOs: 1-6 may comprise at least about 50 contiguous nucléotides, at least about 100 contiguous nucléotides, at least about 150 contiguous nucléotides, at least about 200 contiguous nucléotides, at least about 250 contiguous nucléotides, at least about 300 contiguous nucléotides, at least about 350 contiguous nucléotides, at least about 400 contiguous nucléotides, at least about 450 contiguous nucléotides, at least about 500 contiguous nucléotides, at Ieast about 550 contiguous nucléotides, at Ieast about 600 contiguous nucléotides, at least about 650 contiguous nucléotides, at Ieast about 700 contiguous nucléotides, at least about 750 contiguous nucléotides, at least about 800 contiguous nucléotides, at least about 850 contiguous nucléotides, at least about 900 contiguous nucléotides, at least about 950 contiguous nucléotides, at least about 1000 contiguous nucléotides, at least about 1100 contiguous nucléotides, at least about 1200 contiguous nucléotides, at least about 1300 contiguous nucléotides, at least about 1400 contiguous nucléotides, contiguous nucléotides, contiguous nucléotides, contiguous nucléotides, contiguous nucléotides, at Ieast about 1500 at least about 1700 at least about 1900 at least about 2100 at least about 2300 contiguous nucléotides, contiguous nucleotîdes, contiguous nucleotîdes, contiguous nucleotîdes, contiguous nucleotîdes, at least about 1600 at least about 1800 at least about 2000 at least about 2200 at least about 2400
contiguous contiguous contiguous contiguous nucléotides, at least nucléotides, at least nucléotides, at least nucléotides, or more about 2500 about 2700 contiguous nucléotides, at least about 2600 contiguous nucléotides, at least about 2800 about 2900 contiguous nucléotides, at least about 3000 of any of SEQ ID NOs: 1-6. In certain embodiments, a fragment of any of SEQ ID NOs; 1-6 has the activity of any of SEQ ID NOs: 1-6 with respect to reducing the influence of a first transgene expression cassette on the expression of a second transgene cassette in a transgenic plant.
Constructs
[071] As used herein, the term “construct” means any recombinant DNA molécule such as a 10 plasmid, cosmid, virus, phage, or linear or circular DNA or RNA molécule, derived from any source, capable of genomic intégration or autonomous réplication, comprising a DNA molécule where at least one DNA molécule has been linked to another DNA molécule in a functionally operative manner, Le. operably linked. As used herein, the term “vector” means any construct that may be used for the purpose of transformation, i.e., the introduction of heterologous DNA or 15 RNA into a host cell. A “vector stack” is a vector comprised of two or more cassettes stacked together for transformation. Two or more transgene expression cassettes in a vector stack are separated by fragments of DNA sequence which can be as few as approximateiy 10 nucléotides to approximateiy several hundred nucléotides, or several thousand nucléotides, or more, depending upon the method of cloning or synthesis that was used to construct the vector stack.
As used herein, an “expression cassette” refers to a DNA molécule comprising at least a transcribable DNA molécule operably linked to one or more regulatory éléments, typically at least a promoter and a 3' UTR.
[072] As used herein, the term “operably linked” refers to a first DNA molécule joined to a second DNA molécule, wherein the first and second DNA molécules are so arranged that the 25 first DNA molécule affects the function of the second DNA molécule. The two DNA molécules may or may not be part of a single contiguous DNA moiecule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable DNA moiecule if the promoter modulâtes transcription of the transcribable DNA moiecule of interest in a cell. A leader, for example, is operably linked to DNA sequence when it is capable of affecting the transcription or 30 translation of the DNA sequence.
•
[073] Methods are known in the art for assembling and introducing construits into a cell in such a manner that the transcribable DNA molécule is transcribed into a functional rnRNA molécule that is translated and expressed as a protein. For the practice of the invention, conventional compositions and methods for preparing and using construits and host iells are 5 well known to one skilled in the art. Typieal vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN transfer control vector.
[074] Various regulatory éléments may be included in a construit, including any of those provided herein. Any such regulatory éléments may be provided in combination with other 10 regulatory éléments. Such combinations can be designed or modified to produce désirable regulatory features. In one embodiment, construits of the invention comprise at least one regulatory element operably linked to a transcribable DNA molécule operably linked to a 3' UTR.
[075] Constructs of the invention may include any promoter or leader provided herein or 15 known in the art. For example, a promoter of the invention may be operably linked to a heterologous non-translated 5' leader such as one derived from a heat shock protein gene. Alternatively, a leader of the invention may be operably linked to a heterologous promoter such as the Cauliflower Mosaic Virus 35S transcript promoter.
Transcribable DNA molécules
[076] As used herein, the tenu ‘‘transcribable DNA molécule” refers to any DNA molécule capable of being transcribed into a RNA molécule, including, but not limited to, those having protein coding sequences and those producing RNA molécules having sequences useful for gene suppression. The type of DNA molécule can include, but is not limited to, a DNA molécule from the same plant, a DNA molécule from another plant, a DNA molécule from a different organism, or a synthetic DNA molécule, such as a DNA molécule containing an antisense message of a gene, or a DNA molécule encoding an artificial, synthetic, or otherwise modified version of a transgene. Exemplary transcribable DNA molécules for incorporation into constructs of the invention include, e.g., DNA molécules or genes from a species other than the specîes into which the DNA molécule is incorporated or genes that originale from, or are présent in, the same species, but are incorporaled into récipient cells by genetîc engineering methods railler than classical brecding techniques.
[077] A “transgene” refers to a transcribable DNA molécule heterologous to a host cell at least with respect to its location in the host cell genome and/or a transcribable DNA molécule artifîciaily incorporated into a host celFs genome in the current or any prior génération of the cell. In certain embodiments, a transgene comprises a gene of agronomie interest, such as a gene capable of providing herbicide résistance in plants, or a gene capable of providing plant pest résistance in plants.
[078] A regulatory element, such as a promoter, may be operably linked to a transcribable DNA 10 molécule that is heterologous with respect to the regulatory element. As used herein, the term “heterologous” refers to the combination of two or more DNA molécules when such a combination is not normally found in nature. For example, the two DNA molécules may be derived from different species and/or the two DNA molécules may be derîved from different genes, e.g., different genes from the same species or the same genes from different species. A 15 regulatory element is thus heterologous with respect to an operably linked transcribable DNA molécule if such a combination is not normally found in nature, Le., the transcribable DNA molécule does not naturally occur operably linked to the regulatory element.
[079] As used herein, a “recombinant DNA molécule” is a DNA molécule comprising a combination of DNA molécules that would not naturally occur together without human intervention. For instance, a recombinant DNA molécule may be a DNA molécule that is comprîsed of at least two DNA molécules heterologous with respect to each other, a DNA molécule that comprises a DNA sequence that deviates from DNA sequences that exist in nature, a DNA molécule that comprises a synthetic DNA sequence or a DNA molécule that has been incorporated into a host cell’s DNA by genetîc transformation or gene editing.
[080] The transcribable DNA molécule may generally be any DNA molécule for which expression of a transcript is desired. Such expression of a transcript may resuit in translation of the resulting mRNA molécule, and thus protein expression. Altematively, for example, a transcribable DNA molécule may be designed to ultimately cause decreased expression of a spécifie gene or protein. In one embodiment, this may be accomplished by using a transcribable
DNA molécule that is oriented in the antisense direction. One of ordinary skill in the art is familiar with using such antisense technology. Any gene may be negatively regulated in this manner, and, in one embodiment, a transcribable DNA molécule may be designed for suppression of a spécifie gene through expression of a dsRNA, siRNA or miRNA molécule.
Selectable Markers
[081] Selectable marker transgenes may also be used with the regulatory éléments of the invention. As used herein the terrn “selectable marker transgene” refers to any transcribable DNA molécule whose expression in a transgenic plant, tissue or cell, or lack thereof, can be sercened for or scored in some way. Selectable marker genes, and their associated sélection and screening techniques, for use in the practice of the invention are known in the art and include, 10 but are not limited to, transcribable DNA molécules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic résistance, and proteins that confer herbicide tolérance. Examples of selectable marker transgenes is provided as SEQ ID NOs:18 and 26.
Cell Transformation
[082] The invention is also direcled to a method of producing transformed cells and plants that comprise one or more regulatory éléments operably linked to a transcribable DNA molécule.
[083] The terrn “transformation” refers to the introduction of a DNA molécule into a récipient host. As used herein, the tenu “host” refers to bacteria, fungi, or plants, including any cells, tissues, organs, or progeny of the bacteria, fungi, or plants. Plant tissues and cells of particular 20 interest include protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.
[084] As used herein, the terrn “transformed” refers to a cell, tissue, organ, or organism into which a foreign DNA molécule, such as a construct or a vector stack, has been introduced. The inlroduced DNA molécule may be integrated into the genomic DNA of the récipient cell, tissue, 25 organ, or organism such that the introduced DNA molécule is inherited by subséquent progeny.
A “transgenic” or “transformed” cell or organism may also include progeny of the cell or organism and progeny produced from a breeding program employing such a transgenic organism as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a foreign DNA molécule. The introduced DNA molécule may also be transiently introduced into the 30 récipient cell such that the introduced DNA molécule is not inherited by subséquent progeny.
The term “transgenic” refers to a bacterium, fungus, or plant containing one or more heterologous DNA molécules.
[085] There are many methods well known to those of skill in the art for introducing DNA molécules into plant cells. The process generally comprises the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining the transformed host cell. Methods and materials for transforming plant cells by introducing a plant construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct deiivery of DNA (e.g., by PEG-medîated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with Silicon Carbide fibers, and accélération of DNA coaled parti cl es), and gene editing (e.g., CRISPR-Cas Systems), among others.
[086] Host cells may be any cell or organism, such as a plant cell, algal cell, algae, fungal cell, fungî, bacterial cell, or insect cell. In spécifie embodiments, the host cells and transformed cells 15 may include cells from crop plants.
[087] A transgenic plant subsequently may be regeneraled from a transgenic plant cell of the invention. Using conventional breeding techniques or self-pollination, seed may be produced from this transgenic plant. Such seed, and the resulting progeny plant grown from such seed, will contain the recombinant DNA molécule of the invention, and therefore will be transgenic.
[088] Transgenic plants of the invention can be self-pollinated to provide seed for homozygous transgenic plants of the invention (homozygous for the recombinant DNA molécule) or crossed with non-transgenic plants or different transgenic plants to provide seed for heterozygous transgenic plants of the invention (heterozygous for the recombinant DNA molécule). Both such homozygous and heterozygous transgenic plants are referred to herein as “progeny plants.”
Progeny plants are transgenic plants descended from the original transgenic plant and containing the recombinant DNA molécule of the invention. Seeds produced using a transgenic plant of the invention can be harvested and used to grow générations of transgenic plants, Le., progeny plants of the invention, comprising the construct of this invention and expressing a gene of agronomie interest. Descriptions of breeding methods that are commonly used for different crops can be found in one of several reference books, see, e.g., Allard, Principles of Plant Breeding, John •
Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987);
Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), lowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).
[089] The transformed plants may be analyzed for the presence of the gene or genes of interest and the expression level and/or profile conferred by the régulâtory éléments of the invention. Those of skill in the art are aware of the numerous methods available for the analysis of 10 transformed plants. For example, methods for plant analysis include, but are not limited to, Southern biots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field évaluations, and immunodiagnostic assays. The expression of a transcribable DNA molécule can be measured using TaqMan® (Applied Biosystems, Foster City, CA) reagents and methods as described by the manufacturer and PCR cycle limes 15 determined using the TaqMan® Testing Matrix. Alternativeiy, the Invader® (Third Wave
Technologies, Madison, WI) reagents and methods as described by the manufacturer can be used to evaluate transgene expression.
[090] The invention also provides for parts of a plant of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. Plant parts of 20 the invention may be viable, nonviable, regenerable, and/or non-regenerable. The invention also includes and provides transformed plant cells comprising a DNA molécule of lhe invention. The transformed or transgenic plant cells of the invention include regenerable and/or non-regenerable plant cells.
[091] The invention also provides a commodity product that is produced from a transgenic 25 plant or part thereof containing the recombinanl DNA molécule of the invention. Commodity products of the invention contain a délectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID N0s:l-6. As used herein, a “commodity product” refèrs to any composition or product which is comprised of material derived from a transgenic plant, seed, plant cell, or plant part containing the recombînant DNA molécule of the invention. 30 Commodity products include but are not limited to processed seeds, grains, plant parts, and meal.
A commodity product of the invention will contain a détectable amount of DNA corresponding to the recombinant DNA molécule of the invention. Détection of one or more of this DNA in a sample may be used for determining the content or the source of the commodity product. Any standard method of détection for DNA molécules may be used, including methods of détection disclosed herein.
[092] The invention may be more readîly understood through reference to the following Examples, which are provided by way of illustration, and are not intended to be limitîng of the invention, unless specified. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill în the art should, in light of the présent disclosure, appreciate that many changes can be made in the spécifie embodiments that are disclosed and still obtain a like or sîmilar resuit without departîng from the spirît and scope of the invention, therefore ail matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limitîng sense.
EXAMPLES
Exampie 1 Design, Synthesîs, and Cloning of the Intergenic Sequence Région Eléments
[093] Synthetic Intergenic Sequence Région éléments (“ISRs”) were computationally-designed through algorithme methods. Each ISR was designed to not contain any potentiai Open Reading Frames (ORF) that could iiiadvertently lead to the production of unwanted proteins after insertion into the plant genome. In addition, many of the ISRs were designed to contain stop codons at the 5' and 3' ends of the ISR, positioned in a manner to provide stop codons in ail six reading frames.
[094] Once designed, the ISRs were chemically synthesized and cloned between transgene expression cassettes in a heterologous vector stack. Well over 100 synthetic Intergenic Sequence Région éléments were designed and assayed in stably transformed corn and soybean plants to identify those synthetic ISRs that reduced the interaction of a first transgene cassette with a second transgene cassette.
[095] Certain designed and tested ISRs are presented in Table 1. ISR4_Stop is a variant of
ISR4, wherein stop codons were appended to the 3' and 5' ends of 1SR4.
Table 1. Synthetic Intergenic Sequence Région Eléments.
| Description | SEQ ID NO: | Size (bp) | ORFs Présent | Stop codons in ail 6 frames |
| ISR4_Stop | 1 | 1219 | No | Yes |
| ISR89 | 2 | 1024 | No | Yes |
| ISR2 | 3 | 1195 | No Ί | No |
| ISR4 | 4 | 1195 | No | No |
| ISR97 | 5 | 3024 | No | Yes |
| ISR69 | 6 | 1035 | No | Yes |
| ISR88 | 7 | 1024 | No | Yes |
| ISR86 | 8 | 1024 | No | Yes |
| ISR_X | 9 | 1219 | No | Yes |
[096] The synthetic Intergenic Sequence Région éléments presented as SEQ ID NOs:l-6 demonstrated the ability to reduce the interaction of a first transgene cassette on a second transgene cassette in a veclor stack in stably transformed corn and soybean plants as presented in 10 Examples 2 and 3.
Example 2
Réduction of Transgene Expression Cassette Interaction by ISR4_Stop, ISR89, and ISR97 in Stably Transformed Corn Plants •
[097] This Example demonstrates the ability of the ISRs ISR4_Stop, ISR89, and ISR97 to reduce transgene expression cassette interaction when insertcd between a first transgene expression cassette and a second transgene expression cassette of a vector stack used to stably transform corn plants.
[098] Corn plants were transformed with binary plant transformation vector stacks comprising two transgene expression cassettes in divergent orientation with an ISR between the two transgene expression cassettes to assess the ability of the ISR to reduce transgene expression cassette interaction. Two control vector stacks were also transformed into corn plants and tested.
[099] One control vector stack (Figure la, Control without Enhancer) comprised a first 10 transgene expression cassette which comprised a promoter, P-Os.Actl:67 (SEQ ID NO:11), operably linked 5' to a leader, L-Ta.Lhcbl:l (SEQ ID NO:12), operably linked 5' to an intron, IOs.Act 1-1:1:19 (SEQ ID NO: 13), operably linked 5' to a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-l:l:3 (SEQ ID NO: 14), operably linked 5' to a 3' UTR, TTa.Hspl7-l:l:l (SEQ ID NO; 15). The second transgene expression cassette cloned in a 15 divergent orientation relative to the first transgene expression cassette comprised a seed-specific promoter, P-Zm.39486-1:1:1 (SEQ ID NO; 16), operably linked 5 to a leader, L- Zm.394861:1:1 (SEQ ID NO:17), operably linked 5' to an intron, I-Zm.DnaK:l (SEQ ID NO:18), operably linked 5' to a coding sequence encoding GUS-1, GOI-Ec.uidA+St.LSl.nno:l (SEQ ID NO:19), operably linked 5' to a 3' UTR, T-Os.Mth-l:l:l (SEQ ID NO;20). The Control without 20 Enhancer vector stack also comprised an additional transgene expression cassette which was used for sélection of the transformed cells using glyphosate sélection.
[0100] The other control vector stack (Figure 1b, Control with Enhancer) comprised a first transgene expression cassette which comprised a strong enhancer, E-CaMV.35S.2xAl-B3-l:l:l (SEQ ID NO: 10) comprising tandem repeats of spécifie enhancer régions derived from the 25 Cauliflower mosaic virus 35S promoter, operably linked 5' to a promoter, P-Os.Actl:67 (SEQ
ID NO: 11), operably linked 5' to a leader, L-Ta.Lhcbl:l (SEQ ID NO: 12), operably linked 5' to an intron, I-Os.Act 1-1:1:19 (SEQ ID NO: 13), operably linked 5' to a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-l:l:3 (SEQ ID NO: 14), operably linked 5' to a 3 UTR, T-Ta.Hspl7-l:l:l (SEQ ID NO: 15). The second transgene expression cassette cloned 30 in a divergent orientation relative to the first transgene expression cassette comprised a seed spécifie promoter and was the same transgene expression cassette as described above. The Control with Enhancer vector stack also comprised an additional transgene expression cassette which was used for sélection of the transformed cells using glyphosate sélection.
[0101] To assay the effectiveness of an ISR in reducing the interaction between a first and 5 second transgene expression cassette, the ISRs ISR4_Stop (SEQ ID NO;1), ISR89 (SEQ ID
NO:2), ISR97 (SEQ ID NO:5), ISR88 (SEQ ID NO:7), and ISR86 (SEQ ID NO:8) were cloned between the first and second transgene expression cassettes of the Control with Enhancer vector stack, as depicted in Figure le. Variety LH244 corn plant cells were transformed using an Agrobactenum-mediated transformation method similar to Lhose known in the art with the two 10 control vector stacks and the five vector stacks comprising the ISRs. The transformed plant cells were induced to form whole plants.
[0102] Qualitative and quantitative GUS analysis was used to evaluate expression element activity in selected plant organs and tîssues in the transformed plants. For qualitative analysis of GUS expression by histochemical staining, whole-mount or sectioned tissues were incuba ted 15 with GUS staining solution containing 1 mg/mL of X-Gluc (5-bromo-4-chloro-3-indolyl-bglucuronide) for 5 h at 37° C and de-stained with 35 % EtOI-I and 50 % acetic acid. Expression of GUS was qualitatively determined by visual inspection of selected plant organs or tîssues for blue coloration under a dîssecting or compound microscope. For quantitative analysis of GUS expression by enzymatîc assays, total protein was extracted from selected tissues of transformed 20 corn plants. One to two micrograms of total protein was incubated with the fluorogenic substrate, 4-methyleumbelliferyl-p-D-glucuronîde (MUG) at 1 mM concentration in a total reaction volume of 50 microliters. After 1 h incubation at 37° C, the reaction was stopped by adding 350 microliters of 200 mM sodium bicarbonate solution. The reaction product, 4methlyumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is 25 ionized. Addition of the basic sodium carbonate solution simultaneously stops the assay and adjusts the pH for quantifying the fluorescent product 4-MU. The amount of 4-MU formed was estimated by measuring its fluorescence using a FLUOstar Oméga Microplate Reader (BMG LABTECH) (excitation at 355 nm, émission at 460 nm). GUS activity values are provided in nmoles of 4-MU /hour/mg total protein.
[0103] The following tissues were sampled for GUS expression in the Ro génération: V3 stage Leaf and Root; V7 stage Leaf and Root; VT stage Leaf, Root, and Anther and Silk; and R3 stage Seed Embryo and Seed Endosperm 21 days after pollination (DAP). Table 2 shows the mean GUS expression in the végétative, reproductive, and seed tissues, wherein “bdl” îndicates GUS 5 expression was below the levels of détection. Table 3 shows the average GUS expression in the végétative and reproductive tissues. The Control with Enhancer is considered to represent the full interaction of the first transgene expression cassette enhancer with the seed-specific promoter of the second transgene expression cassette. Therefore, the average végétative and reproductive tissue expression from the GUS cassette which was drîven by the P-Zm.3948610 1:1:1, seed-specific promoter affected by the strong constitutive enhancer of the first transgene expression cassette represents a leakiness of 100 percent. The percent leakiness of the vector stacks comprising an ISR were determined by dividing the average GUS expression în the végétative and reproductive tissues of plants transformed with constructs comprising an ISR by the average GUS expression in the végétative and reproductive tissues of the Control with 15 Enhancer, and multiplying the resuit by one-hundred.
Table 2. Mean GUS expression in végétative, reproductive, and seed tissues of LH244 stable transformed corn plants.
| Control/ISR | SEQ ID NO: | V3root | V3- leaf | V7root | V7- leaf | VTroot | VTleaf | VT- Anther | VTsilk | 21- DAP- Em | 21- DAP- Endo |
| Control without Enhancer | 23 | bdl | 46 | 11 | bdl | bdl | 19 | bdl | 13 | 215 | |
| Control with Enhancer | 1469 | 1310 | 1847 | 1698 | 367 | 946 | 323 | 603 | 68 | 2651 | |
| ISR4_Stop | 1 | 409 | 71 | 255 | 84 | 169 | 60 | 296 | 31 | 197 | 1169 |
| ISR89 | 2 | 140 | 75 | 146 | 39 | 110 | 27 | 113 | 41 | 24 | 560 |
| ISR97 | 5 | 35 | 39 | 22 | 78 | 38 | 41 | 146 | 41 | 27 | 269 |
| ISR88 | 7 | 789 | 169 | 142 | 217 | 1671 | 456 | 1349 | 426 | 62 | 3033 |
| ISR86 | 8 | 370 | 355 | 144 | 90 | 712 | 380 | 628 | 85 | 40 | 1645 |
Table 3. Average Végétative and Reproduction GUS Expression and Mean Percent Leakiness of ISRs compared to Controls.
| Contre 1/ISR | SEQ ID NO: | Average Végétative and Reproductive Expression | % Leakiness |
| Control without Enhancer | 12 | 1% | |
| Control with Enhancer | 1070 | 100% | |
| ISR4_Stop | 1 | 172 | 16% |
| ISR89 | 2 | 87 | 8% |
| ISR97 | 5 | 55 | 6% |
| ISR88 | 7 | 652 | 61% |
| ISR86 | 8 | 346 | 32% |
[0104] As can be seen in Table 2, the Control with Enhancer demonstrated high GUS expression in ail tissues of stably transformed corn plants when compared to the Control without Enhancer. This demonstrates that the strong enhancer in the first transgene expression cassette modified the seed-specific expression pattern of the second transgene expression cassette to a more constitutive expression pattern.
[0105] As shown in Table 2, the interaction of the strong enhancer in the first transgene expression cassette on the second transgene expression cassette was reduced when the ISRs ISR4_Stop, ÏSR89, and ISR97 were inserted between the cassettes. The average GUS expression of the végétative and reproductive tissues in the vector stacks with ISR4_Stop, ISR89 and ISR97 were mu ch less than that of the Control with Enhancer vector. The percent leakiness of ISR4_Stop, ISR89, and ISR97 was 16%, 8%, and 6%, respectively, thus providing a réduction in the interaction between the two transgene expression cassettes by 84%, 92%, and 94%, respectively. In comparison, ISR88 and ISR86 were much leakier (61% and 32%, respectively), and only reduced the interaction between the two transgene expression cassettes by 39% and 68%, respectively.
[0106] ISR4_Stop (SEQ ID NO:1), ISR89 (SEQ ID NO:2), and 1SR97 (SEQ ID NO:5) demonstrated the ability to reduce the interaction of a first transgene expression cassette with a second transgene expression cassette in a vector stack în stably transformed corn plants.
Exampîe 3
Réduction of Transgene Expression Cassette Interaction by ISR2 and ISR4 in Stably Transformed Soybean Plants
[0107] This Example demonstrates the ability of the Intergenic Sequence Région éléments, ISR2 and ISR4 to reduce transgene expression cassette interaction when inserted between a first transgene expression cassette and a second transgene expression cassette of a vector stack used to stably transform soybean plants.
[0108] Soybean plants were transformed with binary plant transformation vector stacks comprising two transgene expression cassettes in divergent orientation with an ISR between the two transgene expression cassettes to assess the ability of the ISR to reduce transgene expression cassette interaction. Two control vector stacks were also transformed înto soy plants and tested.
[0109] One control vector stack (Figure 2a, Control without Enhancer) comprised a single transgene expression cassette comprised of a seed-specific promoter, P-Gm.Sphasl;14 (SEQ ID NO:25), operably linked 5' to a leader, L-Gm.Sphasl-l:l:l (SEQ ID NO:26), operably linked 5' to a coding sequence encodîng GUS-2, GOI-GUS:1:2 (SEQ ID NO:27), operably linked 5' to a N UTR, T-Mt.AC145767v28:3 (SEQ ID NO:28). The Control without Enhancer vector stack also comprised an additional transgene expression cassette which was used for sélection of the transformed cells using antibiotic sélection.
[0110] The other control vector stack, (Figure 2b, Control with Enhancer) comprised two transgene expression cassettes in divergent orientation. The first transgene cassette comprised a strong promoter derived from the Figwort mosaic virus 35S promoter with a rearranged and duplicated enhancer, P-FMV.35S-enh-l:l:2 (SEQ ID NO:21), operably linked 5' to a leader, LPh.DnaK-l:l:3 (SEQ ID NO:22), operably linked 5' lo a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-l:l:2 (SEQ ID NO:23), operably linked 5' to a 3' UTR, TMt.AC139600vl6:l (SEQ ID NO:24). The second transgene expression cassette was the same as the seed-specific transgene expression cassette described above. The Control with Enhancer vector stack also comprised an additional transgene expression cassette which was used for sélection of the transformed cells using antibiotic sélection.
[0111] To assay the effectiveness of an ISR in reducing the interaction between a first and second transgene expression cassette, the ISRs ISR2 (SEQ ID NO:3), ISR4 (SEQ ID NO:2), ISR69 (SEQ ID NO:6), and ISR_X (SEQ ID NO:8) were cloned between the first and second transgene expression cassettes of the Control with Enhancer vector stack as depicted in Figure 2c. Variety A3555 soybean plant cells were transformed using an Agrobacterium-mediated transformation method similar to those known in the art with the Control without Enhancer, the Control with Enhancer, and the three vector stacks comprising the ISRs. The transformed plant cells were induced to form whole plants.
[0112] Qualitative and quantitative GUS analysis was performed as previously described in Example 2. The following tissues were sampled for GUS expression in the Ro génération: Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, RI Flower, R3 Immature seed, R3 Pod, R5 Cotylédon, Yellow Pod (YP) Embryo, and Yellow Pod (YP) Cotylédon.
[0113] The Control with Enhancer is considered to represent the full interaction of the first transgene expression cassette enhancer with the seed-specific promoter of the second transgene expression cassette. Therefore, the average végétative and reproductive tissue expression from the GUS cassette which was driven by the P-Gm.Sphasl:14, seed-specific promoter, affected by the strong constitutive enhancer of the first transgene expression cassette, represents a leakiness of 100 percent. The percent leakiness of the constructs comprising an ISR were detennined by dividing the average GUS expression in the Vn5, RI, and R3 tissues of plants transformed with constructs comprising an ISR by the average GUS expression of the Vn5, RI, and R3 tissues of the Control with Enhancer, and multiplying the resuit by one-hundred.
[0114] The Mean GUS expression of the Vn5, RI, and R3 tissues is presented in Table 4, wherein “nd” indicates not determined. The Mean GUS expression of R5 and Yellow Pod tissues, the average Vn5, RI, and R3 tissue expression, and the percent leakiness is presented in Table 5.
Table 4. Mean GUS expression of Vn5, RI, and R3 tissues in stably transformed A3555 soybean piants.
| Control/ISR | SEQ ID NO: | Vn5 Root | Vn5 Sink Leaf | Vn5 Source Leaf | RI Source Leaf | RI Petiote | RI Flower | R3 Immature Seed | R3 Pod |
| Control No Enhancer | 10 | 3 | 4 | 0 | 0 | 0 | 11 | 8 | |
| Control With Enhancer | 3072 | 2524 | 1939 | 2722 | 6369 | 2434 | 520 | 6236 | |
| ISR2 | 3 | 113 | 69 | 107 | 39 | 155 | 56 | 45 | 112 |
| ISR4 | 4 | 207 | 33 | 86 | 28 | 349 | 84 | 31 | 97 |
| ISR69 | 6 | 108 | 74 | 62 | 76 | 488 | 311 | 113 | 64 |
| 1SR_X | 9 | 391 | 107 | 121 | 103 | 974 | 179 | nd | 3654 |
Table 5. Mean GUS expression of R5 and Yellow Pod tîssues, the average Vn5, RI, and R3 tissue expression, and the percent leakiness in stably transformed A3555 soybean plants.
| Controi/ISR | SEQ ID NO: | R5 Cotylédon | Yellow Pod Embryo | Yellow Pod Cotylédon | Average Vn5, RI, and R3 | % leakiness |
| Control No Enhancer | 47 | 1445 | 4264 | 5 | 0% | |
| Control With Enhancer | 2673 | 6746 | 6294 | 3227 | 100% | |
| ISR2 | 3 | 3330 | 6308 | 6703 | 87 | 3% |
| ISR4 | 4 | 10066 | 3881 | 6267 | 114 | 4% |
| ISR69 | 6 | 3223 | 4114 | 5432 | 162 | 5% |
| ISR_X | 9 | 5049 | 11495 | 11767 | 790 | 24% |
[0115] As can be seen in Table 4, very little GUS expression is observed in the Vn5, RI, and R3 tissues in plants transformed with the Control without Enhancer. Plants transformed with the Control with Enhancer demonstrate a constitutive expression pattern, with high GUS expression observed in the Vn5, RI, and R3 tissues. Likewise, as seen in Table 5, plants transformed with the Control without Enhancer only demonstrate high GUS expression in the Yellow Pod Embryo and Cotylédon, consistent with the known seed-specîfic expression pattern of P-Gin.Sphasl:14. Very little expression is observed in the R5 Cotylédon wherein expression is seen to increase slîghtly relative to R3 Immature Seed. Plants transformed with the Control with Enhancer show high levels of expression in the R5 cotylédon and an increase in the Yellow Pod Embryo and Cotylédon relative to Plants transformed with the Control without Enhancer. Thus, the strong enhancer comprised in the P-FMV.35S-enh-l:l:2 promoter of the first transgene expression cassette of the Control with Enhancer interacted with, and changed the seed-specific expression of P-Gm.Sphasl:14 of the second transgene expression cassette, to a constitutive expression pattern.
[0116] As dcmonstrated in Table 5, the Intergenic Sequence Régions ISR2, ISR4, and ISR69 were able to reduce the interaction of the first transgene expression cassette on the second transgene expression cassette of the Control with Enhancer configuration by 97%, 96%, and 95%, respectively (were only 3%, 4%, and 5% leaky). The ISR_X was not as effective in reducing the interaction of the first transgene expression cassette on the second transgene expression cassette of the Control with Enhancer configuration and dcmonstrated a leakîness of 24%. ISR_X only reduced the interaction by 76% in comparison to 97%, 96%, 95% for ISR2, ISR4, and ISR69.
[0117] ISR2 (SEQ ID NO:3), ISR4 (SEQ ID NO:4), and ISR69 (SEQ ID NO:6) demonstrated the ability to reduce the interaction of a first transgene expression cassette with a second transgene expression cassette in stably transformed soybean plants.
[0118] Having illustrated and described the principles of the présent invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim ail modifications that are within the spirit and scope of the claims. Ail publications and published patent documents cited herein are hereby incorporated by reference to the same extent as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.
Claims (15)
1. A recombinant DNA molécule comprising a DNA sequence selected from the group consisting of:
a. a sequence with at least 85 percent sequence identity to any of SEQ ID NOs:l6; and
5 b. a sequence comprising any of SEQ ID NOs: 1-6.
2. The recombinant DNA molécule of claim 1, wherein the DNA sequence is inserted between a first expression cassette and a second expression cassette in a vector stack.
3. The recombinant DNA molécule of claim 1, wherein the DNA sequence has at least 90 percent sequence identity to the DNA sequence of any of SEQ ID NOs: 1-6.
10
4. The recombinant DNA molécule of claim 1, wherein tbe DNA sequence has at least 95 percent sequence identity to the DNA sequence of any of SEQ ID NOs: 1-6.
5. The recombinant DNA molécule of claim 1, wherein tbe DNA sequence comprises any of SEQ ID NOs:l-6.
6. A transgenic plant cell comprising the recombinant DNA molécule of claim 1.
15
7. The transgenic plant cell of claim 6, wherein said transgenic plant cell is a monocotyledonous plant cell.
8. The transgenic plant cell of claim 6, wherein said transgenic plant cell is a dicotyledonous plant cell.
9. A transgenic plant, or part thereof, comprising the recombinant DNA molécule of claim 1.
20
10. a progeny plant of the transgenic plant of claim 9, or a paît thereof, wherein the progeny plant or paît thereof comprises the recombinant DNA molécule.
11. A transgenic seed, wherein the seed comprises the recombinant molécule of claim 1.
12. A method of producing a commodity product comprising obtaining a transgenic plant or paît thereof according to claim 9 and producing the commodity product therefrom.
25
13. The method of claim 12, wherein the commodity product is seeds, processed seeds, protein concentrate, protein isolate, starch, grains, plant parts, seed oil, biomass, tlour, and mcal.
14. A method for reducing the interaction of a first transgene expression cassette with a second transgene expression cassette within a transgenic plant transformed with a vector stack, said method comprising transforming a plant cell with a vector stack comprising a heterologous transfer DNA (T-DNA) comprising:
5 a. a first transgene expression cassette;
b. a second transgene cassette;
c. the recombinant DNA molécule of claim 1, wherein the DNA molécule is inserted between the first transgene expression cassette and the second transgene expression cassette; and
10 d. regenerating a transgenic plant from the transformed plant cell.
15. The method of claim 14, wherein the DNA molécule of any of SEQ ID NOs: 1-6 are inserted between the first transgene expression cassette and the second transgene expression cassette within the vector stack.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US62/875,752 | 2019-07-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA20529A true OA20529A (en) | 2022-09-30 |
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