Novel intergenic sequence regions and uses thereof

Abstract

To provide DNA molecules useful for reducing the influence of one transgene cassette on the expression of another transgene cassette in plants.SOLUTION: 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 for reducing the interaction between transgene expression cassettes using the novel synthetic intergenic sequence regions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application is the benefit of U.S. Provisional Application No. 62 / 875,752, filed July 18, 2019. The benefit of this application is claimed, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference "MONS472WO_ST25, which contains a sequence listing in computer readable format. The file, named "txt", was created on June 9, 2020. , 38,698 bytes (measured on MS-Windows®), (U.S. Patent Concurrently filed by electronic filing (using the Office's EFS-Web Filing System) and referenced and the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to the fields of plant molecular biology and plant genetic engineering. More specifically, the present invention The aim is to determine the effect of one transgene cassette on the expression of another transgene cassette in a plant. The present invention relates to DNA molecules useful for reducing the effects of [Background technology]

[0004] Intergenic sequence regions ("ISR") are placed between two or more transgene cassettes. In this case, the interaction of one transgene cassette with another transgene cassette is reduced. The expression pattern of the transgene cassettes is determined by the interaction of the expression elements between the cassettes. It is a DNA sequence that prevents mutations in the gene.

[0005] In the expression cassette, such as the promoter, intron, and 3' untranslated region (3'UTR), Expression elements in a gene may affect the expression of adjacent or nearby expression cassettes. For example, the cauliflower mosaic virus 35S promoter contains a cis-acting element having the sequence Plant virus promoters such as the CaMV 35S promoter control the transcription of nearby genes. can affect and activate genes up to 4.3 Kb upstream or downstream from the insertion site, It consists of an enhancer domain (Gudynaite-Savitch et al (2009) Strategies to mitigate transgene- promoter interactions.Plant Biotechnolog y Journal,7:472-485, Benfey et al.(1990)T issue-specific expression from CaMV 35S enhancer subdomains in early stages of p lant development.The EMBO Journal,9:1677 For example, one example is a Ca2+ gene that drives the coding sequence of a selectable marker. Subcloned into a plant transformation vector containing a selection cassette using the MV 35S promoter. The cloned transgene cassette is driven by the presence of the CaMV 35S promoter. This results in tissue-specific expression of this transgene cassette in a more constitutive pattern. (Yoo et al. (2005) The 35S promoter used in a selectable marker gene of ap lant transformation vector affects the e xpression of the transgene.Planta,221:52 3-530).

[0006] In the field of plant biotechnology, several agricultural Using vectors containing multiple transgene cassettes to introduce traits of environmental importance Plant transformation is becoming more common. The advantage of this process is the ability to express several agronomic traits. The advantage of this is that it allows the vector stack to be used in plants with a single locus. transgenic plants containing additional agronomic traits. However, this allows for a more efficient and less expensive breeding process. When a set is cloned into a vector, an expression cassette is Expression elements from one expression cassette alter or modify the expression profile of another expression cassette. It may affect the expression of certain genes in a particular tissue, such as expression in seeds. The expression cassettes designed to The seed-specific expression pattern is determined by the interaction between neighboring elements. This can be achieved by using an expression cassette to change the expression pattern to closely resemble that of a seed-specific expression cassette. Therefore, in plant biotechnology, In this case, the interaction between adjacent expression cassettes in a vector stack is reduced or prevented. There is a need for DNA sequences that can

[0007] Therefore, the present inventors have herein described the use of vectors in transgenic plants. We disclose novel synthetic ISRs that minimize interactions of expression cassettes within the stack. ISRs can be placed between adjacent expression cassettes in a single vector stack to separate individual cassettes. This prevents interactions between the expression elements of each expression group within the vector stack. The intended expression pattern and expression levels of the set can be maintained. Summary of the Invention

[0008] The present invention provides novel synthetic intergenic sequence regions or ISRs for use in plants. The present invention also provides a recombinant DNA construct comprising an ISR. Also provided are transgenic plant cells, plants, and seeds comprising the ISR. The ISR is inserted between the expression cassettes in the vector stack. Methods for using ISRs, as well as recombinant DNA constructs containing ISRs, and ISRs Methods for making and using transgenic plant cells, plants, and seeds containing R are also provided. provide.

[0009] Therefore, in one aspect, the present invention provides a method for detecting a nucleotide sequence of at least 8 to any one of SEQ ID NOs: 1 to 6. (b) a sequence having 5% sequence identity with any of SEQ ID NOs: 1 to 6; The present invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: In an embodiment, the recombinant DNA molecule has at least one sequence similar to any of the DNA sequences of SEQ ID NOs: 1 to 6. 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 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 It includes DNA sequences that have sequence identity to the target gene.

[0010] In another embodiment, (a) a sequence identical to any of SEQ ID NOs: 1-6 by at least 85 percent (b) a sequence having sequence identity with the target sequence; and (c) a sequence including any one of SEQ ID NOs: 1 to 6. and a transgenic plant cell containing a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: In certain embodiments, the transgenic plant cells are single-celled. In other embodiments, the transgenic plant cell is a monocotyledonous plant cell. or dicotyledonous plant cells.

[0011] In yet another aspect, (a) a nucleic acid sequence comprising at least 85% of any of SEQ ID NOs: 1-6 (b) a sequence having a sequence identity of 1 to 6; and (c) a sequence having a sequence identity of 1 to 6. A transgenic recombinant DNA molecule comprising a DNA sequence selected from the group consisting of Further provided herein are plants, or parts thereof. In certain embodiments, A transgenic plant is a descendant plant of any generation that contains a recombinant DNA molecule. a transgenic plant containing the recombinant DNA molecule, Also provided herein are seeds.

[0012] In another aspect, the present invention provides a transgenic plant or a plant comprising a recombinant DNA molecule of the present invention. the production of commodity products, including the taking of the goods or parts thereof and the production of commodity products from them. In one embodiment, the commodity product is a seed, processed seed, protein Concentrates, protein isolates, starches, grains, plant parts, seed oils, biomass, flours, and coarse flour.

[0013] In yet another aspect, the present invention provides a transgenic plant transformed with a vector stack. The interaction of the first transgene expression cassette with the second transgene expression cassette in a transgenic plant is A method for reducing interactions is provided, the method comprising: (a) a first transgene cassette; b) a second transgene cassette; and (c) (i) at least one of SEQ ID NOs: 1 to 6. and (ii) a sequence having 85% sequence identity with any of SEQ ID NOs: 1 to 6. a DNA molecule comprising a sequence selected from the group consisting of a sequence comprising inserted between one transgene expression cassette and a second transgene expression cassette, Transforming plant cells with a vector stack containing a recombinant DNA molecule containing the DNA molecule. (d) regenerating a transgenic plant from the transformed plant cell. In certain embodiments, the vector stack comprises more than two expression cassettes. In a further embodiment, the DNA molecule of any of SEQ ID NOs: 1 to 6 is It is inserted between each of the expression cassettes in the master stack.

[0014] A brief description of arrays SEQ ID NO: 1 contains ISR4 (SEQ ID NO: 4) and the nucleotide sequences at both the 5' and 3' ends. This is the DNA sequence of the intergenic region ISR4_Stop, which contains three stop codons.

[0015] SEQ ID NO: 2 is the DNA sequence of the intergenic sequence region ISR89.

[0016] SEQ ID NO: 3 is the DNA sequence of the intergenic sequence region ISR2.

[0017] SEQ ID NO: 4 is the DNA sequence of the intergenic sequence region ISR4.

[0018] SEQ ID NO: 5 is the DNA sequence of the intergenic sequence region ISR97.

[0019] SEQ ID NO: 6 is the DNA sequence of the intergenic sequence region ISR69.

[0020] SEQ ID NO: 7 is the DNA sequence of the intergenic sequence region ISR88.

[0021] SEQ ID NO: 8 is the DNA sequence of the intergenic sequence region ISR86.

[0022] SEQ ID NO: 9 is the DNA sequence of the intergenic sequence region ISR_X.

[0023] SEQ ID NO: 10 is the E-Ca This is the DNA sequence of the enhancer called MV.35S.2xA1-B3-1:1:1.

[0024] SEQ ID NO: 11 is presented as "P-Os.Act1" in Figures 1A-C, P-Os. This is the DNA sequence of the promoter Act1:67.

[0025] SEQ ID NO: 12 is the L-Ta, presented as "L-Ta.Lhcb1" in Figures 1A-C. .Lhcb1:1 is the DNA sequence of the leader or 5'UTR.

[0026] SEQ ID NO: 13 is presented as "I-Os.Act1" in Figures 1A-C, I-Os. This is the intron DNA sequence Act1-1:1:19.

[0027] SEQ ID NO: 14 is the CR-Ec.n, presented as "nptII-1" in Figures 1A-C. ptII-Tn5-1:1:3, which encodes neomycin phosphotransferase. It is a DNA sequence.

[0028] SEQ ID NO: 15 is presented as "T-Ta.Hsp17" in Figures 1A-C. This is the DNA sequence of the 3'UTR of Hsp17-1:1:1.

[0029] SEQ ID NO: 16 is presented in Figures 1A-C as "P-Zm.39486". The promoter DNA sequence is .39486-1:1:1.

[0030] SEQ ID NO: 17 is presented in Figures 1A-C as "L-Zm.39486". The DNA sequence of the leader or 5'UTR is .39486-1:1:1.

[0031] SEQ ID NO: 18 is presented as "I-Zm.DnaK" in Figures 1A-C, I-Zm. This is the DNA sequence of an intron called DnaK:1.

[0032] SEQ ID NO: 19 is a gene encoding the potato light-inducible GUS-1 gene, presented as "GUS-1" in FIGS. 1A-C. The process originates from the tissue-specific ST-LS1 gene (Genbank accession: X04753). Optimized synthesis of β-glucuronidase with a synchronizable intron for plant expression DNA of coding sequence (GUS-1:GOI-Ec.uidA+St.LS1.nno:1) It is an A array.

[0033] SEQ ID NO: 20 is presented as "T-Os.Mth" in Figures 1A-C. This is the DNA sequence of the 3'UTR called th-1:1:1.

[0034] SEQ ID NO: 21 is P-FMV, presented as "P-FMV.35S" in Figures 2A-C. The promoter DNA sequence is .35S-enh-1:1:2.

[0035] SEQ ID NO: 22 is presented as "L-Ph.DnaK" in Figures 2A-C, L-Ph. The DNA sequence of the leader or 5'UTR is DnaK-1:1:3.

[0036] SEQ ID NO: 23 is the CR-Ec.n, presented as "nptII-2" in Figures 2A-C. ptII-Tn5-1:1:2, which encodes neomycin phosphotransferase. It is a DNA sequence.

[0037] SEQ ID NO: 24 is presented as "T-AC139600" in Figures 2A-C, The DNA sequence of the 3'UTR is AC139600v16:1.

[0038] SEQ ID NO: 25 is presented as "P-Gm.Sphas" in Figures 2A-C. This is the DNA sequence of the promoter called Sphas1:14.

[0039] SEQ ID NO: 26 is presented as "L-Gm.Sphas" in Figures 2A-C. .Sphas1-1:1:1 is the DNA sequence of the leader or 5'UTR.

[0040] SEQ ID NO: 27 is a potato light-inducible gene encoding GUS-2, presented as "GUS-2" in Figures 2A-C. The process originates from the tissue-specific ST-LS1 gene (Genbank accession: X04753). A synthetic coding sequence for β-glucuronidase (GUS-2) with a syntenic intron :GOI-GUS:1:2) DNA sequence.

[0041] SEQ ID NO: 28 is the T-Mt The DNA sequence of the 3'UTR is AC145767v28:3. [Brief explanation of the drawings]

[0042] [Figure 1]Figures A-C are diagrammatic representations of vector stacks used to assay the effectiveness of synthetic intergenic sequence regions ("ISRs") in reducing the interaction of two transgene expression cassettes within a single vector stack on each other's expression in stably transformed corn plants. Reference numbers in these figures indicate the corresponding sequence identifier for each genetic element as presented in the sequence brief description. Figure A shows the transgene expression cassette configuration of a control vector stack, an enhancer-less control. The enhancer-less control consists of two transgene expression cassettes cloned in opposite orientations. The first transgene cassette is composed of a 3'UTR called T-Ta.Hsp17-1:1:1 (SEQ ID NO: 15), operably linked to the 5' end of the coding sequence for neomycin phosphotransferase called CR-Ec.nptII-Tn5-1:1:3 (SEQ ID NO: 14), operably linked to the 5' end of the intron called I-Os.Act1-1:1:19 (SEQ ID NO: 13), operably linked to the 5' end of the leader called L-Ta.Lhcb1:1 (SEQ ID NO: 12), and operably linked to the 5' end of the promoter called P-Os.Act1:67 (SEQ ID NO: 11). The second transgene cassette, cloned in the opposite orientation to the first, consists of a 3'UTR called T-Os.Mth-1:1:1 (SEQ ID NO:20), operably linked at the 5' end to a coding sequence encoding GUS-1 called GOI-Ec.uidA+St.LS1.nno:1 (SEQ ID NO:19), operably linked at the 5' end to an intron called I-Zm.DnaK:1 (SEQ ID NO:18), operably linked at the 5' end to a leader called L-Zm.39486-1:1:1 (SEQ ID NO:17), and operably linked at the 5' end to a seed-specific promoter called P-Zm.39486-1:1:1 (SEQ ID NO:16). Panel B shows the transgene expression cassette configuration of the control vector stack, which is a control with an enhancer.The enhancer control consisted of a 3'UTR called T-Ta.Hsp17-1:1:1 (SEQ ID NO: 15), operably linked at the 5' end to the neomycin phosphotransferase coding sequence called CR-Ec.nptII-Tn5-1:1:3 (SEQ ID NO: 14), operably linked at the 5' end to an intron called I-Os.Act1-1:1:19 (SEQ ID NO: 13), operably linked at the 5' end to a leader called L-Ta.Lhcb1:1 (SEQ ID NO: 12), operably linked at the 5' end to a promoter called P-Os.Act1:67 (SEQ ID NO: 11), and operably linked at the 5' end to a strong enhancer called E-CaMV.35S.2xA1-B3-1:1:1 (SEQ ID NO: 10), which contains tandem repeats of a specific enhancer region derived from the cauliflower mosaic virus 35S promoter. The second transgene cassette, cloned in the opposite orientation to the first, consists of a 3'UTR called T-Os.Mth-1:1:1 (SEQ ID NO:20), operably linked at the 5' end to a coding sequence encoding GUS-1 called GOI-Ec.uidA+St.LS1.nno:1 (SEQ ID NO:19), operably linked at the 5' end to an intron called I-Zm.DnaK:1 (SEQ ID NO:18), operably linked at the 5' end to a leader called L-Zm.39486-1:1:1 (SEQ ID NO:17), and operably linked at the 5' end to a seed-specific promoter called P-Zm.39486-1:1:1 (SEQ ID NO:16). The enhancer-containing control in A lacks an ISR between the first and second transgene expression cassettes. As a result, 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 transgene expression cassette from seed-specific to constitutive. In C, an ISR has been cloned between the first and second transgene expression cassettes in the enhancer-containing control.If the ISR is effective, it will reduce the interaction of the enhancer in the first transgene expression cassette with the expression of the promoter in the second expression transgene cassette, resulting in reduced expression in non-seed tissues compared to the enhancer-containing control. [Figure 2]Figures A-C are diagrammatic representations of vector stacks used to assay the effectiveness of ISR in reducing the interaction of two transgene expression cassettes within a single vector stack with each other's expression in stably transformed soybean plants. Reference numbers in these figures indicate the corresponding sequence identifiers for each genetic element as provided in the sequence brief description. Figure A shows the transgene expression cassette configuration of a control vector stack, an enhancer-less control. The enhancer-less control (A) is composed of a 3'UTR, T-Mt.AC145767v28:3 (SEQ ID NO:28), operably linked at its 5' end to a coding sequence encoding GUS-2, GOI-GUS:1:2 (SEQ ID NO:27), operably linked at its 5' end to a leader, L-Gm.Sphas1-1:1:1 (SEQ ID NO:26), and operably linked at its 5' end to a seed-specific promoter, P-Gm.Sphas1:14 (SEQ ID NO:25). The seed-specific promoter can drive GUS expression primarily in soybean plant seeds in the enhancer-less control. Figure B shows the transgene expression cassette configuration of the control vector stack, which is an enhancer-containing control. The enhancer-containing control consists of two transgene expression cassettes in opposite orientations. The first transgene cassette consists of the 3'UTR T-Mt.AC139600v16:1 (SEQ ID NO:24), operably linked at the 5' end to the neomycin phosphotransferase coding sequence CR-Ec.nptII-Tn5-1:1:2 (SEQ ID NO:23), operably linked at the 5' end to the leader L-Ph.DnaK-1:1:3 (SEQ ID NO:22), and operably linked at the 5' end to the strong promoter derived from the Striga mosaic virus 35S promoter P-FMV.35S-enh-1:1:2 with a reconstituted, duplicated enhancer (SEQ ID NO:21).The second transgene cassette, cloned in the opposite orientation to the first transgene cassette, is composed of a 3'UTR, T-Mt.AC145767v28:3 (SEQ ID NO:28), operably linked at the 5' end to a coding sequence encoding GUS-2, GOI-GUS:1:2 (SEQ ID NO:27), operably linked at the 5' end to a leader, L-Gm.Sphas1-1:1:1 (SEQ ID NO:26), and operably linked at the 5' end to a seed-specific promoter, P-Gm.Sphas1:14 (SEQ ID NO:25). The enhancer-containing control lacks an ISR between the first and second transgene expression cassettes. As a result, expression of the seed-specific promoter in the second transgene expression cassette is influenced by the enhancer region of the Striga mosaic virus 35S promoter in the first transgene expression cassette, changing expression of the second transgene cassette from seed-specific to constitutive. In C, an ISR is cloned between the first and second transgene expression cassettes of the enhancer-containing control. If the ISR is effective, it will reduce the interaction between the enhancer region of the Scrophulariac mosaic virus 35S promoter in the first transgene expression cassette and the promoter in the second transgene expression cassette, resulting in reduced expression in non-seed tissues compared to the enhancer-containing control. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention provides novel synthetic intergenic sequence regions for use in transgenic plants. The nucleotide sequences of these novel synthetic ISRs are shown in SEQ ID NO: These synthetic ISRs are provided as Nos. 1-6. When inserted between a transgene and a first transgene in a transgenic plant The relative position of the expression elements in the transgene cassette relative to the expression of the second transgene cassette. The present invention also provides transgenic plant cells, plants, and The present invention also provides methods for using the ISR, as well as methods for producing plants containing the ISR. Methods for making and using the recombinant DNA molecules are also provided.

[0044] The following definitions and methods will better define the present invention and guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are used in accordance with the conventional understanding of the terminology by those skilled in the relevant art. It shall be understood according to usage.

[0045] ISR and interaction between the first transgene expression cassette and the second transgene expression cassette for As used herein, the term "interaction" refers in certain embodiments to a vector adjacent to each other in transgenic plants transformed using a star stack. one or more elements in the first transgene expression cassette when provided This refers to the effect of a transgene on the expression pattern of a second transgene expression cassette.

[0046] The regulatory elements within each transgene expression cassette regulate the transcription of the transgene. It is composed of various cis-elements to which cis-acting factors bind. For example, plant promoters It is composed of cis elements that are essential for the initiation and efficiency of transcription. Promoters are often regulated by stress (ABRE and AB14), pathogens (W box), or other systems that can regulate transcription in response to specific stimuli such as light (GT1-motif). Other cis elements are tissue-specific or tissue-preferential. (Porto et al. (2014) Plant Promote rs:An Approach of Structure and Function Mol.Biotechnol 56:38-49). For example, cauliflower mosaic The viral 35S promoter contains an enhancer region made up of two domains. The downstream domain, domain A, confers expression primarily in roots. The as-1 cis-element located within the base-paired region is primarily responsible for this expression. Domain B is expressed in most cell types in leaves and stems, as well as in the vascular tissue of roots. (Benfey et al. (1990) Tissue-specific cific expression from CaMV 35S enhancer subdomains in early stages of plant development lopment.The EMBO Journal,9:1677-1684).

[0047] When two transgene expression cassettes are adjacent to each other in the plant genome, one The expression elements of the transgene expression cassettes modulate the expression of the other transgene expression cassettes. A transgene expression cassette in a transgenic plant and This "interaction" with the adjacent transgene expression cassette is not observed with the enhancer-containing control. This is shown in Examples 2 and 3.

[0048] "Leakiness" refers to the lack of expression elements in the first expression cassette. the interaction with the expression profile of the second expression cassette, Leakage is a term used to describe the level of average expression change in tissue. The expression profile of the control with ISR was compared with the test vector stack (two (composed of an enhancer-containing control with an ISR inserted between the transgene cassettes) The expression profile of the enhancer-containing control is compared to that of the control with the enhancer. The leakage of the enhancer-free control is 100%. The leakage of the ISR-containing construct is The average GUS expression levels in non-target tissues for the test constructs were compared with those of the enhancer-containing control construct. Determined by dividing by the average GUS expression level in non-target tissues and multiplying by 100. The percent leakage reduction is calculated by subtracting the percent leakage from 100 percent. It is decided.

[0049] "Intergenic sequence region" or "ISR" refers to the region of interest in adjacent transgenic cassettes. Synthesis of expression elements designed to minimize interactions with each other on expression The intergenic sequence regions disclosed herein are computationally designed nucleotide sequences. 1. First transgene expression in the vector stack used to transform plant cells Reduced interaction of the cassette with the second transgene expression cassette, thus increasing the Expression profiles of gene expression cassettes are observed individually in transgenic plants The expression profiles of the nucleotides were assayed for their ability to be preserved as they were when the nucleotides were transfected.

[0050] "Synthetic nucleotide sequence" or "artificial nucleotide sequence" means a nucleotide sequence that is not naturally occurring. The intergenic sequences of the present invention are nucleotide sequences that are not known or do not occur in nature. The sequence region element comprises a synthetic nucleotide sequence. Preferably, the synthetic nucleotide sequence The sequence shares little or no extended homology with the native sequence. Extended homology generally means that 100% sequence identity is found over a contiguous sequence of about 25 nucleotides. It refers to extending beyond the column.

[0051] In Example 2, two transgene expression cassettes were constructed in opposite orientations. The vector stack was used to transform control corn plants. The stack contains the rice actin 1 promoter, which drives the expression of antibiotic resistance genes. The first transgene expression cassette contains the enhancer-less control (Figure 1A), and the second transgene contains the GUS expression cassette. The second transgene expression cassette contained a seed-preferred promoter driving Maize plants transformed with this vector stack exhibit seed-preferential expression of GUS. The other control vector stack (enhancer-containing control) showed antibiotic resistance. operably linked to the rice actin 1 promoter, which drives expression of the gene, CaMV The first transgene expression cassette contains a strong enhancer derived from 35S, and the second transgene expression cassette contains a strong enhancer derived from GU. A second transgene expression cassette containing a seed-preferred promoter driving S expression was included. The maize plants transformed with the enhancer control showed no changes in roots, leaves, anthers, silk, or High levels of GUS expression were observed in the silk and seeds. wherein the enhancer of the first transgene expression cassette is The expression profile of the target gene is altered to allow expression of a second transgene cassette. changed from seed-dominant to constitutive.

[0052] As shown in Figure 1C, one particular computationally designed ISR was engineered to contain an enhancer. The control was inserted between the first and second transgene cassettes. R4_Stop (SEQ ID NO: 1), ISR89 (SEQ ID NO: 2), and ISR97 (SEQ ID NO: 5) ISR is used to separate the first transgene expression cassette and the second transgene expression cassette. When inserted between the first transgene expression cassette, the expression pattern of the second transgene The percent leakage in the interaction of the expression cassettes with the expression pattern was 16 %, 8%, and 6%. Therefore, these ISRs were and the second transgene expression cassette by 84%, 92%, and 94%, respectively. It decreased.

[0053] In Example 3, a similar experimental design was used in soybean to examine the effectiveness of certain ISRs. The first transgene expression cassette with an enhancer and the second transgene were tested. Between the expression cassettes ISR2 (SEQ ID NO: 3), ISR4 (SEQ ID NO: 4), and ISR69 (SEQ ID NO: 6) insertions were found to be the first derivatives with only 3%, 4%, and 5% leakage, respectively. The expression pattern of the first transgene expression cassette and the expression pattern of the second transgene expression cassette This resulted in a reduction in the effect of the first transgene expression cassette on the expression of the first transgene. of the interaction of the elements on the expression pattern of the second transgene expression cassette, These resulted in reductions of 97%, 96%, and 95%, respectively.

[0054] As shown in these examples, all computationally designed intergenic sequence regions Furthermore, it was not effective in reducing the interaction. Even for ISRs that were reduced, the degree of reduction varied. ) and ISR86 (SEQ ID NO: 8) had 61% and 32% leakage, respectively, resulting in trans The interaction was reduced by 39% and 68%, respectively, in transgenic maize plants. This reduction in interaction was 84% ​​for ISR4_Stop and 92% for ISR89. , and was much lower compared to 94% for ISR97. In soybean, ISR_X (SEQ ID NO: 9) is 97% of ISR2 and 1% of ISR4. 96% and 95% for ISR69, respectively ( Leakage percentage, 24%). Therefore, each computationally designed ISR is unique and has a different I SR can be used in conjunction with different expression cassettes to generate desired expression vectors for one or more genes of interest. The current profile can be reached.

[0055] dna molecule As used herein, the term "DNA" or "DNA molecule" refers to a 5' (upper) Double-stranded DNA of genomic or synthetic origin that is read from the 3' (downstream) end to the 4' (upstream) end This refers to a molecule (i.e., a polymer of deoxyribonucleotide bases or a DNA molecule). As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein conforms to the nomenclature of 37 CFR 1.822. The corresponding rules are set out in Annex 2, Tables 1 and 3 of WIPO Standard ST.25(1998).

[0056] As used herein, "heterologous molecules" refer to molecules that do not occur together in nature without human intervention. For example, heterologous molecules are molecules that contain combinations of DNA molecules that would not otherwise exist. A DNA molecule that is composed of at least two DNA molecules that are heterologous in nature. DNA molecules containing DNA sequences that deviate from the intended DNA sequence, DNA molecules containing synthetic DNA sequences molecules or incorporated into the host cell's DNA by genetic transformation or gene editing The DNA molecule may be a DNA molecule.

[0057] Any reference in this application to an "isolated DNA molecule" or equivalent term or phrase means that The DNA molecule may exist alone or in combination with other compositions, but not in its natural environment. is intended to mean a non-existent DNA molecule. For example, a coding sequence, Biosynthetic sequences such as intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc. A nucleic acid element that is naturally found within the DNA of the genome of an organism is one that is found in the genome of the organism. "Isolated" refers to a sequence of an element that is present in a genome and is in the location in the genome where the element is found in nature. However, each of these elements, and the The subpart of an element is a subpart of an element that is not present in the genome of an organism and that is naturally occurring. A gene will not be considered "isolated" within the scope of this disclosure unless it is in a location within the genome where it is found. Similarly, the insecticidal protein or any naturally occurring insecticidal barrier of that protein A nucleotide sequence encoding a polypeptide is a sequence that encodes a polypeptide that encodes a protein. An isolated nucleotide sequence is not a nucleotide sequence unless the sequence is within the bacterial DNA in which it is naturally found. The synthetic nucleotides encoding the amino acid sequences of naturally occurring insecticidal proteins A sequence of the polypeptide chain will be considered isolated for the purposes of this disclosure. Any transgenic nucleotide sequence, i.e., the genomic DNA of a plant or bacterial cell, The nucleotide sequence of the DNA inserted into the genome or present in an extrachromosomal vector whether it is present in a plasmid or similar construct used to transform the cell , present in the genome of a plant or bacterium or in tissue derived from a plant or bacterium; whether present in detectable amounts in progeny, biological samples, or commercial products. These will be considered to be isolated nucleotide sequences.

[0058] As used herein, the term "sequence identity" refers to the difference between two optimally aligned sequences. The polynucleotide sequences or two optimally aligned polypeptide sequences are Optimal sequence alignment refers to the degree to which two sequences, e.g., a reference sequence and a Manually align with another sequence to remove any appropriate internal nucleotide insertions, deletions, or deletions. by maximizing the number of nucleotide matches in a sequence alignment with gaps. As used herein, the term "reference sequence" refers to SEQ ID NO: 1. 6.

[0059] As used herein, "percent sequence identity" or "percent identity" or or "% identity" refers to the percentage of identity multiplied by 100. The "percent identity" for optimally aligned sequences is calculated based on the optimal alignment. The number of nucleotide matches in the reference sequence is compared to the total number of nucleotides in the reference sequence, e.g., the total length of the reference sequence. divided by the total number of nucleotides in the entire string. When optimally aligned to the reference sequences provided as SEQ ID NOs: 1 to 6 in At least about 85 percent identity to the reference sequence, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity, at least at least about 91 percent identity, at least about 92 percent identity, at least about 93 percent identity percent identity, at least about 94 percent identity, at least about 95 percent at least about 96 percent identity, at least about 97 percent identity sex, at least about 98 percent identity, at least about 99 percent identity, or provides DNA molecules containing sequences with at least about 100 percent identity. In certain embodiments, a given percent identity to any of SEQ ID NOs: 1-6 The sequence retains the general function of any of SEQ ID NOs: 1 to 6, i.e., Expression of a first transgene expression cassette in a transgenic plant. In certain embodiments, the sequences Sequences with a given percent identity to any of numbers 1-6 are transgenic. The first transgene expression cassette is expressed in the cloned plant. It has the activity of any of SEQ ID NOs: 1 to 6 in reducing the effect on the immune system.

[0060] Regulatory elements Promoter, leader (a.k.a. 5'UTR), enhancer, intron, and transcription Regulatory elements such as the termination region (or 3'UTR) regulate the overall expression of genes in living cells. As used herein, the term "regulatory element" refers to a gene that plays an essential role in the regulation of a gene. The term "gene regulatory activity" refers to a DNA molecule that has gene regulatory activity. The term "transcriptional activity" refers to, for example, the transcription of an operably linked transcribable DNA molecule. and / or by affecting translation of operably linked transcribable DNA fragments. This refers to the ability of promoters, leaders, and enzymes to function in plants. Regulatory elements such as enhancers, introns, and 3'UTRs can be engineered into genetically engineered plants. It is useful for modifying the phenotype of

[0061] Regulatory elements affect gene expression patterns, e.g., their positive and / or negative effects, For example, constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, or pathological , cell cycle, and / or chemical responsiveness, and any combination thereof, as well as It may be characterized by quantitative or qualitative indicators. "Pattern of gene expression" refers to the sequence of operably linked DNA molecules into transcribed RNA molecules. Any pattern of transcription. Transcribed RNA molecules are translated to produce protein molecules. In some cases, the target gene may be an antisense RNA or other regulatory RNA molecule, e.g., a double-stranded RNA. NA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA ), microRNA (miRNA), small interfering RNA (siRNA), etc. There are also.

[0062] As used herein, the term "protein expression" refers to the expression of a transcribed RNA molecule. Protein expression is any pattern of translation of a gene into a protein molecule. By spatial, developmental, or morphological properties, as well as by quantitative or qualitative indicators. It can be characterized as:

[0063] A promoter is a molecule that regulates the expression of an operably linked transcribable DNA molecule. As used herein, the term "promoter" is used to refer to a gene that is useful as a regulatory element. The term generally refers to the use of RNA polymerase II and other proteins (e.g., A promoter is a DNA molecule involved in the recognition and binding of a transcription factor (e.g., a trans-acting transcription factor). The gene may be first isolated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. Alternatively, the promoter may be a synthetically produced or engineered DNA molecule. The promoter may also be chimeric. A chimeric promoter is a promoter that contains two or more different Promoters useful in demonstrating the present invention include: The promoter elements provided as SEQ ID NOs: 11, 16, 21, and 25 are included. do.

[0064] As used herein, the term "leader" refers to the untranslated 5' region (5') of a gene. The sequence is isolated from the UTR and is separated from the transcription start site (TSS) and the protein coding sequence start site. It refers to a DNA molecule, generally defined as the segment of nucleotides between the A leader may be a synthetically produced or engineered DNA element. 5' regulatory elements for regulating expression of an operably linked transcribable DNA molecule. Leader molecules can be used as promoters for heterologous or their native promoters. Leaders useful in demonstrating the present invention may include those having SEQ ID NO: Includes Nos. 12, 17, 22, and 26.

[0065] As used herein, the term "intron" refers to a molecule isolated or identified from a gene. can be spliced ​​out during pre-translational messenger RNA (mRNA) processing Alternatively, an intron may refer to a region of a DNA molecule that can be generally defined as a region that is omitted. They may be synthetically produced or engineered DNA elements. It may also contain enhancer elements that effect transcription of genes to which it is functionally linked. The gene is a regulatory element for regulating the expression of an operably linked transcribable DNA molecule. The construct may also include an intron, which intron may be used as a It may or may not be heterologous to the transcribable DNA molecule. Introns useful in this regard are provided as SEQ ID NOs: 13 and 18.

[0066] As used herein, the term "3' transcription terminator," "3' untranslated region," or "3' The term 'UTR' is used during transcription to the untranslated region of the 3' part of the mRNA molecule The 3' untranslated region of an mRNA molecule is a DNA molecule that undergoes specific cleavage and 3' polyadenylation. The 3'UTR can be generated by poly(A) tailing. It may be operably linked to and located downstream of the Polyadenylation signals and other regulatory signals that can affect gene expression The poly(A) tail is believed to function in mRNA stability and translation initiation. It is thought that...

[0067] As used herein, the term "enhancer" or "enhancer element" The term cis-acting regulatory element (also known as a cis-element) refers to an operative An aspect of the overall expression pattern of a transcribable DNA molecule linked to it (but usually not alone) Unlike promoters, enhancer elements confer a signal that is insufficient to drive transcription. The nucleotide sequence usually contains a transcription start site (TSS) or TATA box, or equivalent D The promoter or promoter fragment does not include the NA sequence. naturally contain one or more enhancer elements that affect transcription of the DNA sequence. Alternatively, an enhancer element may be fused to the promoter to regulate the entire gene expression. Chimeric promoter cis elements may be created that confer aspects of systemic regulation.

[0068] As used herein, the term "variant" refers to a variant of a DNA molecule that has a different composition from the first DNA molecule. refers to a second DNA molecule that is similar, but not identical, to the DNA molecules disclosed herein. One variant of the ISR has a slightly different sequence composition, but is identical to the I from which it is derived. The first transgene expression cassette in the transgenic plant was expressed in the same manner as in the SR. The variant maintains the ability to reduce the effect of the 2 transgene cassette on expression. a shorter or truncated version of the first DNA molecule, or a sequence of the first DNA molecule Modified versions, e.g., with different restriction enzyme sites and / or internal deletions, substitutions, or A "variant" may also be a variant of one or more of the reference sequences. ISRs with nucleotide sequences containing multiple nucleotide substitutions, deletions, or insertions are also wherein the derivative intergenic sequence region element is incorporated into the transgenic plant. Influence of a first transgene expression cassette on the expression of a second transgene cassette In the present invention, The polynucleotide sequences provided as SEQ ID NOs: 1-6 were used to identify the original ISR. While similar but not identical to DNA sequences, they share a common function: transduction. Expression of a first transgene expression cassette in a transgenic plant. Create variants that still maintain the same or similar ability to reduce the impact on the environment. In certain embodiments, a variant of any of SEQ ID NOs: 1 to 6 may be Transgenic plants expressing a first transgene expression cassette in a second transgene cassette. The activity of any of SEQ ID NOs: 1 to 6 in reducing the effect on the expression of ribonucleotides was The production of such variants of the present invention is well within the skill of those in the art in light of this disclosure. and are included within the scope of the present invention.

[0069] In certain instances, the variant of the ISR is a fragment of any of SEQ ID NOs: 1-6. The fragment of SEQ ID NO: 1 to 6 may be a sequence of at least about 50 consecutive sequences of any of SEQ ID NO: 1 to 6. consecutive nucleotides, at least about 100 consecutive nucleotides, at least about 150 consecutive nucleotides consecutive nucleotides, at least about 200 consecutive nucleotides, at least about 250 consecutive nucleotides consecutive nucleotides, at least about 300 consecutive nucleotides, at least about 350 consecutive nucleotides consecutive nucleotides, at least about 400 consecutive nucleotides, at least about 450 consecutive nucleotides contiguous nucleotides, at least about 500 contiguous nucleotides, at least about 550 contiguous nucleotides consecutive nucleotides, at least about 600 consecutive nucleotides, at least about 650 consecutive nucleotides consecutive nucleotides, at least about 700 consecutive nucleotides, at least about 750 consecutive nucleotides at least about 800 consecutive nucleotides, at least about 850 consecutive nucleotides consecutive nucleotides, at least about 900 consecutive nucleotides, at least about 950 consecutive nucleotides consecutive nucleotides, at least about 1000 consecutive nucleotides, at least about 1100 consecutive nucleotides at least about 1200 contiguous nucleotides, at least about 130 0 contiguous nucleotides, at least about 1400 contiguous nucleotides, at least about 1 500 contiguous nucleotides, at least about 1600 contiguous nucleotides, at least about 1700 contiguous nucleotides, at least about 1800 contiguous nucleotides, at least At least about 1900 contiguous nucleotides, at least about 2000 contiguous nucleotides, At least about 2100 contiguous nucleotides, at least about 2200 contiguous nucleotides , at least about 2300 contiguous nucleotides, at least about 2400 contiguous nucleotides nucleotides, at least about 2500 contiguous nucleotides, at least about 2600 contiguous nucleotides nucleotides, at least about 2700 contiguous nucleotides, at least about 2800 contiguous nucleotides, at least about 2900 contiguous nucleotides, at least about 3000 In certain embodiments, the sequence may comprise 10 or more consecutive nucleotides. Any of the fragments in column numbers 1 to 6 is used for the expression of the first introduced gene in a transgenic plant. Reducing the effect of the current cassette on the expression of the second transgene cassette: It has the activity of any one of SEQ ID NOs: 1 to 6.

[0070] construct As used herein, the term "construct" refers to a construct derived from any source and derived from a genome. capable of integration or autonomous replication, with at least one DNA molecule merging with another It comprises functionally operatively linked, i.e., operably linked, DNA molecules, Any recombinant DNA molecule, such as a plasmid, cosmid, virus, phage, or As used herein, refers to a linear or circular DNA or RNA molecule. The term "vector" refers to a vector used to transform, i.e., introduce heterologous DNA or RNA into a host cell. "Vector stack" means any construct that can be used to introduce A vector consisting of two or more cassettes stacked together for transformation Two or more transgene expression cassettes in a vector stack construct a vector stack Depending on the cloning or synthesis method used to create the From nucleotides to about hundreds, or thousands, or even more As used herein, the term "sequence" refers to a sequence of DNA fragments separated by fragments of DNA sequence, which fragments may be nucleotides. When used in a gene expression cassette, an "expression cassette" includes one or more regulatory elements, typically at least one promoter. At least one transcribable DNA operably linked to a promoter and a 3'UTR. It refers to a DNA molecule containing the molecule.

[0071] As used herein, the term "operably linked" refers to a first DNA sequence. The first DNA molecule binds to a second DNA molecule, and this first DNA molecule affects the function of the second DNA molecule. This means that the first and second DNA molecules are arranged so as to exert an effect on the A DNA molecule may or may not be part of a single continuous DNA molecule. For example, a promoter may or may not be able to express the desired transcription factor in a cell. If the promoter regulates the transcription of a transcribable DNA molecule, it will act on the transcribable DNA molecule. For example, a leader may be a gene that influences the transcription or translation of a DNA sequence. A DNA sequence is operably linked if it can be expressed in a manner that is capable of expressing the DNA sequence.

[0072] Transcribable DNA molecules are transformed into functional mRNA molecules that are translated and expressed as proteins. Methods for assembling and introducing constructs into cells in a manner that allows them to be transcribed are known in the art. For the practice of the present invention, methods for preparing and using constructs and host cells are known in the art. Conventional compositions and methods are well known to those skilled in the art. Typical vectors are well known in the art and include Agrobacterium Vectors derived from the Ti plasmid of C. tumefaciens and the pCaMVCN transfer system Contains a control vector.

[0073] A variety of regulatory elements, including any of the regulatory elements provided herein Any such regulatory element may be combined with other regulatory elements. Such combinations may be designed or adapted to produce the desired accommodation function. In one embodiment, the construct of the present invention comprises a nucleotide sequence operably linked to the 3'UTR. at least one regulatory element operably linked to the transcribable DNA molecule include.

[0074] The constructs of the present invention can be prepared using any promoter provided herein or known in the art. For example, the promoters of the present invention may comprise a heterologous, untranslated 5′ promoter or leader. operably linked to a leader (e.g., from a heat shock protein gene) Alternatively, the leader of the present invention may be driven by a heterologous promoter, e.g., a cauliflower promoter. It may be operably linked to a mosaic virus 35S transcript promoter.

[0075] transcribable DNA molecule As used herein, the term "transcribeable DNA molecule" refers to a molecule that can be transcribed into an RNA molecule. refers to any DNA molecule that can be transcribed, including those containing protein-coding sequences. and those that produce RNA molecules with sequences useful for gene suppression. Types of DNA molecules include, but are not limited to, DNA molecules from the same plant, DNA molecules from different plants, DNA molecules from different organisms, or synthetic DNA molecules, e.g. Artificial, synthetic or transgene DNA molecules containing antisense messages can include, but are not limited to, DNA molecules encoding otherwise modified versions of the Exemplary transcribable DNA molecules for incorporation into constructs of the invention include, for example, DNA DNA molecules or genes originating from a species other than the species into which the molecule is incorporated, or from the same species origin or exists in the same species, but developed by genetic engineering methods rather than classical breeding techniques. Thus, genes that are integrated into recipient cells are included.

[0076] A "transgene" is a gene that is different to the host cell at least with respect to its location within the host cell genome. The species, the transcribable DNA molecule, and / or the current or any past generation of cells refers to a transcribable DNA molecule that is artificially integrated into the genome of a host cell. In embodiments, the transgene is a gene of agronomic interest, for example, a gene that confers herbicide resistance in plants. or genes that can provide plant pest resistance in plants. Contains genes that

[0077] A regulatory element such as a promoter may be a transcriptionally active D As used herein, the term "heterologous" refers to a heterologous nucleic acid molecule that may be operably linked to a NA molecule. The term refers to a combination of two or more DNA molecules in a manner that is not normally found in nature. For example, the two DNA molecules may be from different species and / or Alternatively, the two DNA molecules may be derived from different genes (e.g., from the same species). (different genes that share the same gene, or the same gene from different species). Thus, regulatory elements For operably linked transcribable DNA molecules, such combinations are not normally found in nature. If not expressed, i.e., the transcribable DNA molecule is operably linked to a regulatory element If it is not naturally occurring, it is heterologous.

[0078] As used herein, a "recombinant DNA molecule" refers to a molecule that does not occur naturally without human intervention. A DNA molecule that contains a combination of DNA molecules that would not occur together. For example, a recombinant DNA molecule A molecule is composed of at least two DNA molecules that are heterologous to each other. NA molecules, DNA molecules containing DNA sequences that deviate from naturally occurring DNA sequences, synthetic DNA molecules containing DNA sequences or those derived from genetic transformation or gene editing of host cells It may also be a DNA molecule integrated into the DNA of the cell.

[0079] A transcribable DNA molecule is generally any DNA molecule from which expression of a transcript is desired. Such expression of the transcript may result in translation of the resulting mRNA molecule, and thus transcription. Alternatively, for example, the transcribable DNA molecule may be a transcription factor that encodes a specific It can be designed to ultimately cause a decrease in gene or protein expression. In some embodiments, this can be achieved by using a transcribable DNA molecule oriented in the antisense direction. Those skilled in the art are familiar with the use of such antisense technology. In one embodiment, the transcribable DNA molecule is to suppress specific genes through the expression of sRNA, siRNA, or miRNA molecules. It may be designed as follows.

[0080] Selectable Markers Selectable marker transgenes may also be used with the regulatory elements of the present invention. As used herein, the term "selectable marker transgene" refers to a transgenic The expression, or lack of expression, in a plant, tissue, or cell is scripted in some way. The term "scanned DNA" refers to any transcribable DNA molecule that can be scanned or scored. Selectable marker genes and their associated selection and screening methods for use in Leaning techniques are known in the art and include the use of β-glucuronidase (GU). S), green fluorescent protein (GFP), proteins that confer antibiotic resistance, and herbicides These include transcribable DNA molecules that encode proteins that confer drug resistance. Examples of selectable marker transgenes are provided as SEQ ID NOs: 18 and 26. will be done.

[0081] Cell transformation The present invention also provides a method for producing a transcribable DNA molecule comprising the steps of: Also covered are methods for producing transformed cells and plants containing the element.

[0082] The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" refers to any bacterial, fungal, or bacterial species, including any cell, tissue, or organ. or plants, or the progeny of bacteria, fungi, or plants. and cells, including protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen. Includes:

[0083] As used herein, the term "transformed" refers to the transfer of a foreign DNA molecule (e.g., refers to a cell, tissue, organ, or organism into which a specific gene (e.g., a construct or vector stack) has been introduced. Integrating the introduced DNA molecule into the genomic DNA of the recipient cell, tissue, organ, or organism The introduced DNA molecule may be integrated and passed on to subsequent generations. "Transgenic" or "transformed" cells or organisms also refer to cells or organisms that are "transgenic" or "transformed" progeny, and those resulting from breeding programs using such transgenic organisms as parents in crosses. and progeny that exhibit an altered phenotype resulting from the presence of the foreign DNA molecule. Alternatively, the introduced DNA molecule can be transiently introduced into recipient cells to induce the expression of the introduced DNA. It is also possible to prevent the A molecule from being passed on to subsequent generations. The term refers to a bacterium, fungus, or plant that contains one or more heterologous DNA molecules.

[0084] There are many methods known to those skilled in the art for introducing DNA molecules into plant cells. The process generally involves the steps of selecting a suitable host cell and transforming the host cell with a vector. and obtaining the transformed host cell. for transforming plant cells by introducing a plant construct into the plant genome - Patents.com The methods and materials may include any of the well-known and proven methods. These include, inter alia, bacterial infection (e.g., Agrobacterium), binary BAC Vectors, direct delivery of DNA (e.g., PEG-mediated transformation, desiccation / inhibition-mediated DNA Uptake, electroporation, stirring with silicon carbide fibers, and DNA coating particle acceleration), and gene editing (e.g., CRISPR-Cas systems). , but not limited to these.

[0085] A host cell can be any cell or organism, for example, a plant cell, an algae cell, an alga, a fungal cell, The host cell may be a fungal, bacterial, or insect cell. In certain embodiments, the host cell and the Transformed cells can include cells from crop plants.

[0086] Transgenic plants are then regenerated from the transgenic plant cells of the invention. Seeds can be produced from this transgenic plant using conventional breeding techniques or self-pollination. Such seeds and the resulting progeny plants grown from such seeds may produce offspring. The organism contains a recombinant DNA molecule of the present invention and is therefore transgenic.

[0087] Transgenic plants of the present invention may be self-pollinated to produce plants homozygous for the recombinant DNA molecule. The present invention also provides seeds of homozygous transgenic plants of the present invention (i.e., plants that are homozygous for the transgenic plants of the present invention). By crossing the transgenic plant with another transgenic plant, (recombinant D Seeds of heterozygous transgenic plants of the present invention (heterozygous for the NA molecule) are cultured in a manner similar to that described above. Such homozygous and heterozygous transgenic plants can also be provided. Both are referred to herein as "progeny plants." Progeny plants are plants that are derived from the original transgenic A transgenic plant is a plant derived from the plant and contains a recombinant DNA molecule of the present invention. Seeds produced using the transgenic plants of the invention are harvested and used to Transgenic plants containing the constructs of the invention and expressing genes of agricultural interest, i.e. Thus, generations of progeny plants of the present invention can be grown. Descriptions of breeding methods can be found in one of several references, e.g., Al lard,Principles of Plant Breeding,John W. iley & Sons,NY,U.of CA,Davis,CA,50-98(19 60), Simmonds, Principles of Crop Improvement ent, Longman, Inc., NY, 369-399 (1979), Sneep and Hendriksen,Plant breeding Perspectiv es,Wageningen(ed),Center for Agricultura l Publishing and Documentation (1979), Feh r,Soybeans:Improvement,Production and Us es, 2nd Edition, Monograph, 16:249 (1987), F ehr,Principles of Variety Development,Th eory and Technique,(Vol.1)and Crop Speci es Soybean(Vol.2),Iowa State Univ.,Macmi See, Illan Pub. Co., NY, 360-376 (1987).

[0088] Transformed plants are characterized for the presence of the gene(s) of interest, as well as the characteristics of the preparations of the present invention. The expression levels and / or profiles conferred by the nodal elements are analyzed. Those skilled in the art will recognize the numerous methods available for analyzing transformed plants. For example, methods for plant analysis include Southern or Northern blots, PCR-based approaches, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic approaches. The expression level of a transcribable DNA molecule can be determined by a method such as, but not limited to, TaqMan. an® (Applied Biosystems, Foster City, CA)) can be measured using the reagents and methods as described by the manufacturer. The PCR cycle time was determined using the TaqMan® Testing Matrix. Alternatively, the determination can be made using Invader® (Third Reagents from Wave Technologies (Madison, WI) and its manufacturers The expression level of the transgene can be assessed using methods such as those described by.

[0089] The present invention also provides plant parts of the present invention. Plant parts include leaves, stems, roots, tubers, Plant parts of the present invention include, but are not limited to, seeds, endosperm, ovules, and pollen. The cells may be viable, non-viable, regrowable, and / or non-regrowable. Also included are transformed plant cells containing the DNA molecules of the present invention. Transformed or transgenic plant cells include regenerable and / or non-regenerable plant cells. Contains biological cells.

[0090] The present invention also relates to transgenic plants or parts thereof comprising a recombinant DNA molecule of the present invention. Commercial products produced from the sequences are also provided. The commercial products of the present invention comprise sequences of SEQ ID NOS: 1 to 6. As used herein, the term "detectable amount of DNA" refers to a DNA sequence selected from the group consisting of: In this context, a "commercial product" refers to a transgenic plant containing a recombinant DNA molecule of the present invention. , any composition composed of material derived from seeds, plant cells, or plant parts; Commodity products include processed seeds, grains, plant parts, and meal. Commercial products of the present invention include, but are not limited to, those corresponding to the recombinant DNA molecules of the present invention. Determine the content or source of commercial products. The detection of one or more of these DNAs in a sample may be used to Any standard method for detecting DNA molecules may be used, including the detection methods disclosed in the literature. stomach.

[0091] The present invention may be more readily understood by reference to the following examples, the implementation of which is further illustrated by the following examples. Examples are provided by way of illustration only and are not intended to limit the invention unless expressly stated. Those skilled in the art will appreciate that the techniques disclosed in the following examples will be readily apparent to those skilled in the art from the following detailed description of the invention. It is understood that this represents a technique discovered by the inventors to work well. However, those skilled in the art will appreciate that, in light of this disclosure, Many changes may be made in the specific embodiments disclosed without departing from the spirit and scope of the invention. It should be understood that the same or similar results may be obtained. All matter set forth or shown in the accompanying drawings shall be interpreted by way of illustration and not in a limiting sense. shall be interpreted as follows. [Example]

[0092] Example 1 Design, synthesis, and cloning of intergenic sequence region elements Synthetic intergenic sequence region elements ("ISRs") are computationally synthesized using algorithmic methods. Each ISR was designed to inadvertently suppress the production of unwanted proteins after insertion into the plant genome. does not contain any potential open reading frames (ORFs) that may lead to In addition, most of the ISRs were designed to have 6 nucleotides at the 5' and 3' ends of the ISR. and positioned in such a way as to provide stop codons in all reading frames. The sequence was designed to contain a stop codon.

[0093] Once designed, these ISRs are chemically synthesized and integrated into heterologous vector stacks. Well over 100 synthetic intergenic sequences were cloned between the transgene expression cassettes. The region elements are designed to define the relationship between the first transgene cassette and the second transgene cassette. To identify synthetic ISRs that reduce interactions, we used stably transformed maize and Assays were carried out in soybean plants.

[0094] A specific designed and tested ISR is presented in Table 1. ISR4_Stop A variant of ISR4 in which stop codons are added to the 3' and 5' ends of ISR4. do.

[0095] [Table 1]

[0096] As presented in Examples 2 and 3, the synthetic intergenic sequences presented as SEQ ID NOS: 1-6 The sequence region elements are used to express vectors in stably transformed maize and soybean plants. The reciprocity of the first transgene cassette to the second transgene cassette in the master stack demonstrated the ability to reduce the effects of

[0097] Example 2 ISR4_Stop, ISR89, ​​and ISR1 in stably transformed maize plants and ISR97 reduce interactions of transgene expression cassettes In this example, ISRs ISR4_Stop, ISR89, ​​and ISR97 are The first transfer site of the vector stack used to stably transform maize plants When inserted between a transgene expression cassette and a second transgene expression cassette, Demonstrate the ability to reduce interactions of the expression cassettes.

[0098] To assess the ability of ISRs to reduce transgene expression cassette interactions, The plant was transformed into a rhocolate plant containing two transgene expression cassettes in opposite orientations, with the ISR A binary plant transformation vector stack between two transgene expression cassettes. Two control vector stacks were also transformed into corn plants. and tested.

[0099] One control vector stack (Figure 1A, enhancer-free control) contained T-Ta.Hs p17-1:1:1 3'UTR (SEQ ID NO: 15), operably linked thereto on the 5' side The resulting neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:3, a coding sequence for IFN-γ (SEQ ID NO: 14), operably linked to IO at the 5' end; s.Act1-1:1:19 intron (SEQ ID NO: 13), which can act on the 5' side The leader L-Ta.Lhcb1:1 (SEQ ID NO: 12) was linked to the operably linked to it was a promoter called P-Os.Act1:67 (SEQ ID NO: 11 ) containing the first transgene expression cassette. A second transgene expression cassette, cloned in the opposite orientation relative to T-Os 3'UTR (SEQ ID NO: 20) of Mth-1:1:1, operably linked thereto on the 5' side The resulting GUS-1, GOI-Ec.uidA+St.LS1.nno:1, was a coding sequence (SEQ ID NO: 19) operably linked thereto at the 5' end to I-Zm.D naK:1 intron (SEQ ID NO: 18), operably linked thereto at the 5' side -Zm.39486-1:1:1 leader (SEQ ID NO: 17), which acts on the 5' side A seed-specific promoter, P-Zm.39486-1:1:1, was operably linked to the A control vector stack without enhancer was also included (row 16). It also contains an additional transgene expression cassette that was used to select for transformed cells using genomic DNA selection. I was.

[0100] The other control vector stack (Figure 1B, enhancer-containing control) was T-Ta.Hs p17-1:1:1 3'UTR (SEQ ID NO: 15), operably linked thereto on the 5' side The resulting neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:3, a coding sequence for IFN-γ (SEQ ID NO: 14), operably linked to IO at the 5' end; s.Act1-1:1:19 intron (SEQ ID NO: 13), which can act on the 5' side The leader L-Ta.Lhcb1:1 (SEQ ID NO: 12) was linked to the operably linked to it was a promoter called P-Os.Act1:67 (SEQ ID NO: 11 ), operably linked thereto at the 5' end, the cauliflower mosaic virus 35S promoter E-CaMV.35 contains tandem repeats of specific enhancer regions derived from the S.2xA1-B3-1:1:1, a potent enhancer (SEQ ID NO: 10), The first transgene expression cassette contained a transgene expression cassette that was reciprocal to the first transgene expression cassette. The second transgene expression cassette, cloned in the opposite orientation, is driven by a seed-specific promoter. The control contained the same transgene expression cassette as described above. The vector stack was also used to select for transformed cells using glyphosate selection. An additional transgene expression cassette was also included.

[0101] As illustrated in FIG. 1C, a first transgene expression cassette and a second transgene expression cassette are To assay the effectiveness of ISR in reducing interactions between sets, 4_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 used as enhancers. - Control vector stack with first transgene expression cassette and second transgene expression cassette The cassette was cloned into the Agroba vector using methods similar to those known in the art. Using the cerebellum-mediated transformation method, two control vector stacks and an ISR were Transformation of cv. LH244 maize plant cells was performed with a five-vector stack containing The transformed plant cells were induced to form whole plants.

[0102] Qualitative and quantitative GUS analysis was used to characterize selected plant organs and The activity of the expression element in the plant and tissues was evaluated. For sexual analysis, whole-mount or sectioned tissues were treated with 1 mg / mL X- G containing Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) Incubate with US staining solution at 37°C for 5 hours and decontaminate with 35% EtOH and 50% acetic acid. Under a dissecting microscope or compound microscope, the blue coloration of selected plant organs or tissues was observed. The amount of GUS expression was determined qualitatively by visual inspection for the amount of GUS expressed. For quantitative analysis of S expression, select tissues of transformed maize plants were analyzed. Total protein was extracted. 1-2 micrograms of total protein was extracted from 50 microliters of The fluorescent substrate 4-methylumbelliferyl (meth) was added at a concentration of 1 mM in the total reaction volume of the fluorogenic substrate. Incubated with β-D-glucuronide (MUG) After 1 hour of incubation at 37°C, 350 microliters of 200 ml The reaction was stopped by adding M sodium bicarbonate solution. 4-Methylumbelliferone (4-MU) is maximally fluorescent at high pH, ​​where the hydroxyl groups are ionized. The assay was stopped by the addition of basic sodium carbonate solution, while the fluorescent product 4 The amount of 4-MU formed was determined by adjusting the pH. Omega microplate reader (BMG LABTECH) (excitation 355 nm, emission The GUS activity was estimated by measuring its fluorescence using a light source (460 nm). It is provided in nanomolar units of 4-MU / hr / mg total protein.

[0103] For GUS expression levels in the R0 generation, the following tissues were sampled: leaves at the V3 stage and and roots, V7 stage leaves and roots, VT stage leaves, roots, anthers and silks, and pollination Seed embryo and seed endosperm at R3 stage after 21 days (DAP). Table 2 shows the vegetative, reproductive, and The mean GUS expression level in the stalk and seed tissues is shown, where "bdl" indicates the level of GUS expression detected. Table 3 shows the average GUS expression levels in vegetative and reproductive tissues. The control with enhancer is the first transgene expression cassette with enhancer and Corresponds to complete interaction of the second transgene expression cassette with the seed-specific promoter Therefore, it is considered to be a strong constitutive enhancer of the first transgene expression cassette. P-Zm.39486-1:1:1, a seed-specific promoter affected by The average expression level in vegetative and reproductive tissues from the GUS cassette driven by corresponds to 0 percent leakage. The leakage percentage of a vector stack containing an ISR is I Average GUS expression in vegetative and reproductive tissues of plants transformed with SR-containing constructs Divide the amount by the average GUS expression level in the vegetative and reproductive tissues of the enhancer-containing control. , determined by multiplying the result by 100.

[0104] [Table 2]

[0105] [Table 3]

[0106] As can be seen in Table 2, the enhancer control showed significantly higher phenotypes than the no-enhancer control. When the transgenic maize was transformed with α-GUS, high GUS expression was observed in all tissues of the stably transformed maize plants. This indicates that the strong enhancer in the first transgene expression cassette Shifting the seed-specific expression pattern of the second transgene expression cassette to a more constitutive expression pattern Demonstrate the changes you made.

[0107] As shown in Table 2, the strong enhancer in the first transgene expression cassette , and the interaction for the second transgene expression cassette is ISR4_Stop, ISR89 The effect was reduced when an ISR called ISR97 was inserted between the cassettes. Vegetative and reproductive tissues in vector stacks carrying Stop, ISR89, ​​and ISR97 The average GUS expression level in the tissues was much lower than that of the control vector with enhancer. The leakage percentages of ISR4_Stop, ISR89, ​​and ISR97 were respectively The percentages were 16%, 8%, and 6%, respectively, thus indicating the interaction between the two transgene expression cassettes. In comparison, ISR88 and ISR90 provided 84%, 92%, and 94% reductions, respectively. ISR86 was much more leaky (61% and 32%, respectively) and contained two transgenes. The interactions between the child expression cassettes were only reduced by 39% and 68%, respectively.

[0108] ISR4_Stop (SEQ ID NO: 1), ISR89 (SEQ ID NO: 2), and ISR97 (SEQ ID NO: Column number 5) shows the first vector in the vector stack in stably transformed maize plants. Ability to reduce interaction between one transgene expression cassette and a second transgene expression cassette He showed his strength.

[0109] Example 3 Transgene expression via ISR2 and ISR4 in stably transformed soybean plants. Reduced cassette interactions This example demonstrates that the intergenic sequence region elements ISR2 and ISR4 are The first transgene expression cassette of the vector stack used to stably transform the plant When inserted between the first transgene expression cassette and a second transgene expression cassette, Demonstrate the ability to reduce interactions between

[0110] To assess the ability of ISRs to reduce transgene expression cassette interactions, soybean The plants were constructed by culturing two transgene expression cassettes in opposite orientations, with an ISR separating these two. A binary plant transformation vector stack containing a transgene expression cassette Two control vector stacks were also transformed into soybean plants and tested. Ta.

[0111] One control vector stack (Figure 2A, enhancer-free control) was T-Mt.AC 145767v28:3 3'UTR (SEQ ID NO: 28), operable on the 5' side The concatenated coding sequence encoding GUS-2, GOI-GUS:1:2 (SEQ ID NO: No. 27), and L-Gm.Sphas1-1:1:1 operably linked thereto at the 5' end. a leader (SEQ ID NO: 26) operably linked to the 5' side thereof, P-Gm.Sph A single transfection consisting of the seed-specific promoter as1:14 (SEQ ID NO: 25) A control vector stack without an enhancer also contained the gene expression cassette. An additional transgene expression cassette was also used to select for transformed cells using biomaterial selection. It included.

[0112] The other control vector stack (Figure 2B, enhancer-containing control) was in the opposite orientation. The first transgene cassette contained TM t.AC139600v16:1 3'UTR (SEQ ID NO: 24), The operably linked neomycin-containing CR-Ec.nptII-Tn5-1:1:2 a coding sequence for phosphoryltransferase (SEQ ID NO: 23), operably linked thereto at the 5' end The leader L-Ph.DnaK-1:1:3 (SEQ ID NO: 22) was P-FMV.35 with a reconstituted and duplicated enhancer operably linked to S-enh-1:1:2, derived from the 35S promoter of Scrophulariac mosaic virus The second transgene expression cassette contained a strong promoter (SEQ ID NO: 21) was the same as the seed-specific transgene expression cassette described above. Control with enhancer The vector stack was also used for selection of transformed cells using antibiotic selection. It also contained a transgene expression cassette.

[0113] As illustrated in FIG. 2C, to assay the effectiveness of the ISRs in reducing the interaction between the first and second transgene expression cassettes, ISR2 (SEQ ID NO: 3), ISR4 (SEQ ID NO: 4), and ISR5 (SEQ ID NO: 5) were used. 4 The ISRs, ISR69 (SEQ ID NO: 6), and ISR_X (SEQ ID NO: 8), were cloned between the first and second transgene expression cassettes of the enhancer-containing control vector stack. Using Agrobacterium-mediated transformation methods similar to those known in the art, the three vector stacks containing the enhancer-less control, the enhancer-containing control, and the ISRs were transformed into cultivar A3555 soybean plant cells. The transformed plant cells were induced to form whole plants.

[0114] Qualitative and quantitative GUS analysis was performed as described above in Example 2. G The following tissues were sampled for US expression: Vn5 roots, Vn5 sink leaves, and Vn5 soybeans. R1 source leaf, R1 petiole, R1 flower, R3 immature seed, R3 pod, R5 cotyledon, yellow pod (YP) embryo, and yellow pod (YP) cotyledon.

[0115] The enhancer control was a control containing the first transgene expression cassette enhancer and the second transgene. This corresponds to the complete interaction of the gene expression cassette with the seed-specific promoter. Therefore, the expression of the first transgene is not affected by the strong constitutive enhancer of the first transgene expression cassette. It is driven by P-Gm.Sphas1:14, a seed-specific promoter influenced by The average expression level in vegetative and reproductive tissues from the GUS cassettes was 100 percent. The percent leakage of the ISR-containing construct corresponds to the percentage of the ISR-containing construct that was transformed with the ISR-containing construct. The average GUS expression levels in Vn5, R1, and R3 tissues of the transformed plants were compared. Divide by the average GUS expression level of the control Vn5, R1, and R3 tissues and multiply the result by 100. This was decided by

[0116] The average GUS expression levels of Vn5, R1, and R3 tissues are presented in Table 4 , where “nd” indicates undetermined. Average GUS expression levels in R5 and yellow pod tissues, Vn5, R1, and R3 tissues The mean expression levels of, as well as percent leakage, are presented in Table 5.

[0117] [Table 4]

[0118] [Table 5]

[0119] As can be seen in Table 4, the Vn5, R In the R1 and R3 tissues, very little GUS expression was observed. Plants transformed with the control showed high GUS expression in Vn5, R1, and R3 tissues. Similarly, as seen in Table 5, the enhancer The control transformed plants without the sensor were grown in the known seeds of P-Gm.Sphas1:14. Consistent with the specific expression pattern, high GUS expression levels were observed only in the embryos and cotyledons of the yellow pods. Very little expression was observed in R5 cotyledons, where the expression level was higher than that of R3 It can be seen that the number of seeds is slightly increased compared to immature seeds. Plants transformed with R5 cotyledons showed high levels of expression, as well as the no enhancer control. The plants with the enhancer showed an increase in yellowing of the pods, embryos, and cotyledons compared to the plants without the enhancer. The first transgene expression cassette, P-FMV.35S-enh-1:1:2 promoter, was inserted into the control The strong enhancer contained in the transgene was inserted into the second transgene expression cassette, P-Gm. It interacted with Sphas1:14 and changed its seed-specific expression to a constitutive expression pattern.

[0120] As shown in Table 5, the intergenic sequence regions ISR2, ISR4, and ISR69 indicates the first transgene expression cassette in the control construct with an enhancer, and indicates the second transgene The interactions with the expression cassette were reduced by 97%, 96%, and 95%, respectively. The ISR_X enhancer was of the first transgene expression cassette in the control construct with the second transgene expression cassette was not as effective in reducing the interaction, exhibiting a leakage of 24%. , compared with 97%, 96%, and 95% for ISR2, ISR4, and ISR69. The effect was only a 76% reduction.

[0121] ISR2 (SEQ ID NO: 3), ISR4 (SEQ ID NO: 4), and ISR69 (SEQ ID NO: 6) In a stably transformed soybean plant, the first transgene expression cassette and the second transgene expression cassette are expressed. It demonstrated the ability to reduce interactions with the transgene expression cassette.

[0122] While the principles of the present invention have been illustrated and described, arrangements of the present invention may be made without departing from such principles. It will be apparent to one skilled in the art that variations in detail and configurations may be made. We claim all modifications that come within the spirit and scope of the claims. All publications and published patent documents cited are to be read as if they were individual publications or patent issues. to the same extent as if each application were specifically and individually indicated to be incorporated by reference. This is hereby incorporated by reference.

Claims

1. 1. A recombinant DNA molecule comprising: a. a DNA sequence having at least 95 percent sequence identity to SEQ ID NO:2, wherein a polynucleotide comprising said DNA sequence reduces the effect of a first transgene expression cassette on the expression of a second transgene expression cassette in a transgenic plant; and b. A DNA sequence comprising SEQ ID NO:2; A recombinant DNA molecule as described above, comprising a DNA sequence selected from the group consisting of:

2. 2. The recombinant DNA molecule of claim 1, wherein the DNA sequence is inserted between a first expression cassette and a second expression cassette in a vector stack.

3. 2. The recombinant DNA molecule of claim 1, wherein the DNA sequence has at least 97 percent sequence identity to the DNA sequence of SEQ ID NO:2, and wherein a polynucleotide comprising the DNA sequence reduces the effect of a first transgene expression cassette on the expression of a second transgene expression cassette in a transgenic plant.

4. 2. The recombinant DNA molecule of claim 1, wherein the DNA sequence has at least 99 percent sequence identity to the DNA sequence of SEQ ID NO:2, and wherein a polynucleotide comprising the DNA sequence reduces the effect of a first transgene expression cassette on the expression of a second transgene expression cassette in a transgenic plant.

5. 2. The recombinant DNA molecule of claim 1, wherein the DNA sequence comprises SEQ ID NO:

2.

6. A transgenic plant cell comprising the recombinant DNA molecule of claim 1.

7. The transgenic plant cell of claim 6 , wherein the transgenic plant cell is a monocotyledonous plant cell.

8. The transgenic plant cell of claim 6 , wherein the transgenic plant cell is a dicotyledonous plant cell.

9. A transgenic plant, or part thereof, comprising the recombinant DNA molecule of claim 1.

10. A progeny plant of the transgenic plant according to claim 9, or a part thereof, The progeny plant, or a part thereof, wherein the progeny plant or part thereof comprises the recombinant DNA molecule.

11. A transgenic seed comprising:

10. The transgenic seed, wherein the seed comprises the recombinant DNA molecule of claim 1.

12. 1. A method of producing a commodity product, comprising:

10. The method comprising obtaining a transgenic plant or part thereof according to claim 9 and producing said commercial product therefrom.

13. 13. The method of claim 12, wherein the commodity product is selected from the group consisting of seeds, processed seeds, protein concentrates, protein isolates, starches, grains, plant parts, seed oils, biomass, fine flour, and coarse flour.

14. 1. A method for reducing interaction between a first transgene expression cassette and a second transgene expression cassette in a transgenic plant transformed with a vector stack, comprising: The method comprises: a. a first transgene expression cassette; b. a second transgene expression cassette; c) transforming a plant cell with a vector stack comprising a heterologous transfer DNA (T-DNA) comprising the recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule 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.

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