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] Incorporation of sequence listing A sequence listing in computer readable form is included in "MONS472WO_ST25. 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 No. 6,313,935, which is incorporated herein by reference in its entirety.

[0003] The present invention relates to the fields of plant molecular biology and plant genetic engineering. More specifically, the present invention The aim of this study was 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 that are useful for reducing the effects of [Background technology]

[0004] An intergenic sequence region ("ISR") is located between two or more transgene cassettes. In the case of The expression pattern of the transgene cassettes due to the interaction of expression elements between the cassettes is 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 expression of adjacent or nearby expression cassettes. For example, the cauliflower mosaic virus 35S promoter contains a cis-acting element having the Plant viral promoters such as the CaMV 35S promoter regulate 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 includes a Ca gene that drives the coding sequence of a selectable marker. Subcloning 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 leads to a more constitutive pattern of tissue-specific expression of the transgene cassette. (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 To introduce traits of environmental importance, vectors containing multiple transgene cassettes are used. Plant transformation is becoming more common. The advantage of this process is that it allows the expression of several agronomic traits. The advantage of this is that it can be used to create vector-stack plants. This is more efficient when the plant is bred together with another transgenic plant that contains additional agronomic traits. However, this allows for a more efficient and less expensive breeding process. When the set is cloned into a vector, within the vector stack, an expression cassette The expression elements from the It may affect the expression of certain genes in a particular tissue, such as expression in seeds. Expression cassettes designed to replicate in the expression vector stack are The seed-specific expression patterns were determined by the interaction between neighboring This can be achieved by changing the expression pattern of the gene to closely resemble that of a seed-specific expression cassette. Therefore, in plant biotechnology, In a method for reducing or preventing interactions between adjacent expression cassettes in a vector stack, There is a need for DNA sequences that can

[0007] Thus, the present inventors herein provide a method for the expression of vectors in transgenic plants. We disclose novel synthetic ISRs that minimize the interaction of expression cassettes within the stack. ISRs can be placed between adjacent expression cassettes in a single vector stack to This prevents interactions between the expression elements of each expression group in 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 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. 5 percent sequence identity, and (b) a sequence including any one 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 set forth in 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 The present invention includes DNA sequences that have sequence identity to the above.

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

[0011] In yet another aspect, (a) a nucleic acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1 to 6, (b) a sequence having any one of SEQ ID NOs: 1 to 6, A transgenic recombinant DNA molecule comprising a DNA sequence selected from the group consisting of Further provided herein is a transgenic plant, or a part thereof. A transgenic plant is any progeny plant that contains a recombinant DNA molecule. A transgenic plant comprising the recombinant DNA molecule, Also provided herein are seeds.

[0012] In another aspect, the present invention relates to a transgenic plant or a plant comprising a recombinant DNA molecule of the present invention. The production of commodity products, including obtaining a product or part of a product and producing a commodity product from it In one embodiment, the commodity product is a seed, a processed seed, a protein Concentrates, protein isolates, starches, grains, plant parts, seed oils, biomass, flours, and coarse flour.

[0013] In yet another aspect, the present invention relates to a transgenic plant transformed with a vector stack. The first transgene expression cassette and the second transgene expression cassette are expressed in a genic plant. The present invention provides a method for reducing interactions, 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) any of SEQ ID NOs: 1 to 6. A DNA molecule comprising a sequence selected from the group consisting of sequences comprising inserted between one transgene expression cassette and a second transgene expression cassette, Transforming a plant cell 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 one 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 the sequence SEQ ID NO:1 contains ISR4 (SEQ ID NO:4) and the nucleotide sequence 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 presented as "E-CaMV.35S" in Figures 1A-C. 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 called Act1:67.

[0025] SEQ ID NO: 12 is 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 presented as "nptII-1" in Figures 1A-C, CR-Ec.n 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, Hsp17-1:1:1.

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

[0030] SEQ ID NO: 17 is presented in Figure 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 Figures 1A-C. The process originates from the tissue-specific ST-LS1 gene (Genbank accession: X04753). Optimized synthesis of β-glucuronidase with a syntenic intron for plant expression DN of coding sequence (GUS-1:GOI-Ec.uidA+St.LS1.nno:1) It is an A sequence.

[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 presented as "P-FMV.35S" in Figures 2A-C. The DNA sequence of the promoter is .35S-enh-1:1:2.

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

[0036] SEQ ID NO:23 is presented as "nptII-2" in Figures 2A-C, CR-Ec.n 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. This is the DNA sequence of the 3'UTR, 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 GUS-2 gene, represented in Figures 2A-C as "GUS-2". 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)

[0041] SEQ ID NO: 28 is presented as "T-AC145767" in Figures 2A-C. This is the DNA sequence of the 3'UTR, AC145767v28:3. [Brief description of the drawings]

[0042] [Figure 1]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 identifiers for each genetic element as presented in the sequence brief description. A shows the transgene expression cassette configuration of a control vector stack that is an enhancer-less control. The enhancer-less control is composed of two transgene expression cassettes cloned in opposing orientations. The first transgene cassette is composed of a 3'UTR, T-Ta.Hsp17-1:1:1 (SEQ ID NO:15), operably linked to the 5' coding sequence of neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:3 (SEQ ID NO:14), operably linked to the 5' coding sequence, I-Os.Act1-1:1:19 (SEQ ID NO:13), operably linked to the 5' coding sequence, L-Ta.Lhcb1:1 (SEQ ID NO:12), and operably linked to the 5' coding sequence, P-Os.Act1:67 (SEQ ID NO:11). The second transgene cassette, cloned in the opposite orientation to the first, is composed of a 3'UTR T-Os.Mth-1:1:1 (SEQ ID NO:20), operably linked at the 5' end to a coding sequence encoding GUS-1 GOI-Ec.uidA+St.LS1.nno:1 (SEQ ID NO:19), operably linked at the 5' end to an intron I-Zm.DnaK:1 (SEQ ID NO:18), operably linked at the 5' end to a leader L-Zm.39486-1:1:1 (SEQ ID NO:17), and operably linked at the 5' end to a seed-specific promoter P-Zm.39486-1:1:1 (SEQ ID NO:16). B shows the transgene expression cassette configuration of the control vector stack, which is a control with an enhancer.The enhancer control consists of a 3'UTR, T-Ta.Hsp17-1:1:1 (SEQ ID NO:15), operably linked at the 5' end to a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:3 (SEQ ID NO:14), operably linked at the 5' end to an intron, I-Os.Act1-1:1:19 (SEQ ID NO:13), operably linked at the 5' end to a leader, L-Ta.Lhcb1:1 (SEQ ID NO:12), operably linked at the 5' end to a promoter, P-Os.Act1:67 (SEQ ID NO:11), and operably linked at the 5' end to a strong enhancer, 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, is composed of a 3'UTR T-Os.Mth-1:1:1 (SEQ ID NO:20), operably linked at the 5' end to a coding sequence encoding GUS-1 GOI-Ec.uidA+St.LS1.nno:1 (SEQ ID NO:19), operably linked at the 5' end to an intron I-Zm.DnaK:1 (SEQ ID NO:18), operably linked at the 5' end to a leader L-Zm.39486-1:1:1 (SEQ ID NO:17), and operably linked at the 5' end to a seed-specific promoter 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 expression transgene 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 on the expression of the promoter in the second expression transgene cassette, resulting in reduced expression in non-seed tissues compared to the enhancer-present control. [Diagram 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 in a single vector stack on 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 that is an enhancer-less control. The enhancer-less control (A) is composed of a 3'UTR called T-Mt.AC145767v28:3 (SEQ ID NO:28), operably linked at the 5' end to a coding sequence encoding GUS-2 called GOI-GUS:1:2 (SEQ ID NO:27), operably linked at the 5' end to a leader called L-Gm.Sphas1-1:1:1 (SEQ ID NO:26), and operably linked at the 5' end to a seed-specific promoter called P-Gm.Sphas1:14 (SEQ ID NO:25). The seed-specific promoter can drive GUS expression mainly in the seeds of soybean plants in the control without enhancer. B shows the transgene expression cassette configuration of the control vector stack, which is the control with enhancer. The control with enhancer is composed of two transgene expression cassettes in opposite orientations. The first transgene cassette is composed of the 3'UTR T-Mt.AC139600v16:1 (SEQ ID NO:24), operably linked at the 5' end to the coding sequence of neomycin phosphotransferase 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), operably linked at the 5' end to the strong promoter derived from the Scimitarium mosaic virus 35S promoter P-FMV.35S-enh-1:1:2 with a reconstituted and duplicated enhancer (SEQ ID NO:21).The second transgene cassette, cloned in the opposite direction to the first transgene cassette, is composed of a 3'UTR called T-Mt.AC145767v28:3 (SEQ ID NO:28), a coding sequence encoding GUS-2 called GOI-GUS:1:2 (SEQ ID NO:27) operably linked thereto at the 5' end, a leader called L-Gm.Sphas1-1:1:1 (SEQ ID NO:26) operably linked thereto at the 5' end, and a seed-specific promoter called P-Gm.Sphas1:14 (SEQ ID NO:25) operably linked thereto at the 5' end. The enhancer 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 Sciurus mosaic virus 35S promoter in the first transgene expression cassette, changing the expression of the second expression 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, and if the ISR is effective, it will reduce the interaction of the enhancer region of the Figwort mosaic virus 35S promoter in the first transgene expression cassette with 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 PREFERRED EMBODIMENTS

[0043] The present invention relates to novel synthetic intergenic sequence regions for use in transgenic plants. The nucleotide sequences of these novel synthetic ISRs are set forth in SEQ ID NO: These synthetic ISRs are provided as references 1-6. When inserted between a first transgene and a second transgene in a transgenic plant Correlation of expression elements in the transgene cassette to the expression of a second transgene cassette. The present invention also relates to transgenic plant cells, plants, and The present invention also provides methods for using the ISR, as well as methods for producing plants comprising 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 as understood by those of ordinary skill in the relevant art. It is to be understood according to usage.

[0045] Interaction of ISR and the first transgene expression cassette with the second transgene expression cassette for As used herein, the term "interaction" refers in certain embodiments to a vector. Transgenic plants transformed using the TASTACK 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 cis-element as-1, 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 vascular tissues in 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 was suppressed by 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 tissues. The expression profile of the control with ISR was compared with that of the test vector stack (two (composed of an enhancer-containing control with an ISR inserted between the transgene cassettes) expression profile compared to enhancer-containing controls. 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 of the test constructs were compared to 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 reduction in leakage is calculated by subtracting the percent leakage from 100 percent. It is decided.

[0049] "Intergenic sequence region" or "ISR" refers to a region of a gene that is located in an adjacent transgenic cassette. The synthesis of expression elements in the The intergenic sequence regions disclosed herein are computationally designed nucleotide sequences. 1. First transgene expression in the vector stack used to transform plant cells Reduce the interaction of the cassette with the second transgene expression cassette, thus Expression profiles of gene expression cassettes are observed individually in transgenic plants The antibodies were assayed for their ability to preserve the expression profile when cultured.

[0050] "Synthetic nucleotide sequence" or "artificial nucleotide sequence" means a nucleotide sequence that does not occur in nature. 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. The sequence shares little or no extended homology with the native sequence. Extended homology generally means 100% sequence identity over a contiguous sequence of about 25 nucleotides. It refers to extending beyond the column.

[0051] In Example 2, two vectors consisting of two transgene expression cassettes in opposite orientations were used. The vector stack was used to transform control maize plants. The stack contains the rice actin 1 promoter that drives the expression of antibiotic resistance genes. The first transgene expression cassette contained an enhancer-free control (Figure 1A), and the second transgene contained a GUS expression cassette. The second transgene expression cassette contained a seed-preferred promoter driving Maize plants transformed with this vector stack showed seed-preferential expression of GUS. The other control vector stack (enhancer-containing control) showed antibiotic resistance. operably linked to the rice actin 1 promoter driving 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 chromosomes. The GUS expression was high in the silk and seeds. wherein the enhancer of the first transgene expression cassette is The expression pattern of the expression profile of the second transgene cassette is altered to enhance the expression of the second transgene cassette. was changed from seed-dominant to constitutive.

[0052] As shown in Figure 1C, a particular computationally designed ISR was synthesized using the enhancer The IS was inserted between the first and second transgene cassettes of the control. R4_Stop (SEQ ID NO: 1), ISR89 (SEQ ID NO: 2), and ISR97 (SEQ ID NO: 5) The ISR is a first transgene expression cassette and a 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 to the expression patterns was 16 %, 8%, and 6%. Therefore, these ISRs are the first transgene expression cassette. and the second transgene expression cassette by 84%, 92%, and 94%, respectively. Decreased.

[0053] In Example 3, a similar experimental design was used in soybean to examine the efficacy of certain ISRs. The first transgene expression cassette with enhancer and the second transgene were tested. Between the expression cassettes ISR2 (SEQ ID NO: 3), ISR4 (SEQ ID NO: 4), ISR69 The (SEQ ID NO:6) insertion was the first derivative 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 expression of the first transgene expression cassette on the of the interaction of the element with 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 were Moreover, it did not result in a reduction in the interaction. Even for ISRs that were reduced in abundance, the degree of reduction varied. ) and ISR86 (SEQ ID NO: 8) had leakage of 61% and 32%, respectively, resulting in trans The interactions were reduced by 39% and 68%, respectively, in transgenic maize plants. This reduction in interactions was 84% ​​for ISR4_Stop and 92% for ISR89. , and ISR97, which was much lower than the 94%. In soybean, ISR_X (SEQ ID NO: 9) is 97% of ISR2 and 10% of ISR4. 96% and ISR69 95% ( Leakage percent, 24%). Therefore, each computationally designed ISR is unique and has a different I SR can be used in combination with different expression cassettes to generate the desired expression vector 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' ( 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 WIPO Standard ST.25 (1998), Annex 2, Tables 1 and 3.

[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 do not exist together. A DNA molecule that is composed of at least two DNA molecules that are heterologous in nature. DNA molecules that contain DNA sequences that deviate from the DNA sequence specified, DNA molecules that contain synthetic DNA sequences molecules, or incorporated into the host cell's DNA by genetic transformation or gene editing The nucleic acid may be a DNA molecule.

[0057] Any reference in this application to an "isolated DNA molecule" or equivalent term or phrase means 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, Such as intron sequences, non-translated leader sequences, promoter sequences, transcription termination sequences, etc. A nucleic acid element that is naturally found within the genomic DNA of an organism is one in which the element is native to the genome of the organism. "Isolated" refers to an element that is present within a genome and is located 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 is not naturally occurring. Unless the location in the genome is that found in Similarly, the insecticidal protein or any naturally occurring insecticidal barrier of that protein may be A nucleotide sequence encoding a polypeptide is a sequence that encodes a polypeptide that encodes a protein. The isolated nucleotide sequence is not intended to be a nucleotide sequence unless the sequence is within the DNA of the bacteria in which it is naturally found. The synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein may be For purposes of this disclosure, a sequence of the polypeptide chain will be considered isolated. Any transgenic nucleotide sequence, i.e., the gene for a plant or bacterial cell, The nucleotide sequence of the DNA to be inserted into the genome or present in an extrachromosomal vector Is it present in a plasmid or similar structure used to transform cells? , present in the genome of a plant or a bacterium, or in tissue derived from a plant or a 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 degree of sequence identity between two optimally aligned sequences. The aligned 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 Manually align with another sequence to remove any appropriate internal nucleotide insertions, deletions, or deletions. or by maximizing the number of nucleotide matches in a gapped sequence alignment. As used herein, the term "reference sequence" refers to SEQ ID NO:1. 6. The DNA sequence provided as .about.6.

[0059] As used herein, "percent sequence identity" or "percent identity" or or "% identity" refers to the percentage of identity multiplied by 100. The "percentage of identity" for optimally aligned sequences is calculated based on the optimal alignment. The number of nucleotide matches in the reference sequence is calculated based on the total number of nucleotides in the reference sequence, e.g., the total length of the reference sequence. This is 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 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 identity, 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 having at least about 100 percent identity. In certain embodiments, a given percent identity to any of SEQ ID NOs: 1-6 is The sequence has the general function of any of SEQ ID NOs: 1 to 6, i.e., Expression of a first transgene expression cassette in a genetically modified plant and expression of a second transgene cassette In certain embodiments, the sequences Sequences having a given percent identity to any of numbers 1-6 are transgenic. The expression of the first transgene expression cassette in the cloned plant is related to the expression of the second transgene cassette. The compound has the activity of any one of SEQ ID NOs: 1 to 6 in reducing the effect on the immune system.

[0060] Regulatory elements Promoters, leaders (aka 5'UTR), enhancers, introns, 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 The term refers to a DNA molecule that has gene-regulating activity. The term "regulatory 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. The ability of promoters, leaders, and endogenous genes to affect the expression of genes in plants. Regulatory elements such as enhancers, introns, and 3'UTRs can be engineered into genetically engineered plants. This is useful for modifying the phenotype of

[0061] Regulatory elements can affect gene expression patterns, e.g., their positive and / or negative effects, For example, constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological , cell cycle, and / or chemical responsiveness, and any combination thereof, as well as As used herein, a genetic disorder 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 synthesis 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 functions to regulate the expression of an operably linked transcribable DNA molecule. As used herein, the term "promoter" is used as a regulatory element. The term generally refers to the process by which RNA polymerase II and other proteins (e.g., A promoter is a DNA molecule that is involved in the recognition and binding of transcription factors (e.g., trans-acting transcription factors). The gene may first be 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: 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 of a gene (5 The nucleotide sequence was isolated from the UTR and the nucleotide sequence was aligned between 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 the heterologous or their native promoters. Leaders useful in demonstrating the present invention include those having SEQ ID NO: These include numbers 12, 17, 22, and 26.

[0065] As used herein, the term "intron" refers to an intron that is isolated or identified from a gene. can be spliced ​​out during pre-translational messenger RNA (mRNA) processing. Introns may be generally defined as regions of a DNA molecule that are omitted. It may be a synthetically produced or engineered DNA element. The vector may 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 be used as a primer. The construct may include an intron, which may 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, a "3' transcription terminator," "3' untranslated region," or "3 The term 'UTR' is used during transcription to the untranslated region in the 3' part of the mRNA molecule. The 3' untranslated region of an mRNA molecule is a region of DNA that is specifically cleaved and 3' polyadenylated. The 3'UTR can be generated by poly(A) tailing (also known as a poly(A) tail). and 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" refers to a The term cis-acting regulatory elements (also known as cis elements) refers to operably An aspect of the overall expression pattern of a transcribable DNA molecule that is linked to (but usually does not exist alone) Unlike promoters, enhancer elements confer a transcription factor (which is insufficient to drive transcription). The nucleotide sequence usually contains a transcription start site (TSS) or a TATA box, or an equivalent D The promoter or promoter fragment does not include the NA sequence. Naturally contains 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 that has a composition different from that of a first DNA molecule. Refers to a similar, but not identical, second DNA molecule. For example, One variant of the ISR has a slightly different sequence composition but is identical to the ISR from which it is derived. The first transgene expression cassette in the transgenic plant was expressed in the same manner as SR. The variant maintains the ability to reduce the effect of 2 on the expression of the transgene cassette. A shorter or truncated version of the first DNA molecule, or a sequence of the first DNA molecule. Modified versions, e.g., those with different restriction enzyme sites and / or internal deletions, substitutions, or A "variant" may also refer to a version of one or more of a reference sequence. ISRs that have nucleotide sequences that contain multiple nucleotide substitutions, deletions, or insertions are also wherein the derived intergenic sequence region elements are incorporated into the transgenic plant. Influence of a first transgene expression cassette on the expression of a second transgene cassette in In the present invention, The polynucleotide sequences provided as SEQ ID NOs: 1-6 were used to determine the composition of the original ISR. While similar but not identical to the DNA sequence, they share a common general function, i.e., transduction. Expression of a first transgene expression cassette in a genetically modified plant and expression of a second transgene cassette Create variants that still maintain the same or similar ability to reduce the impact on the In certain embodiments, a variant of any of SEQ ID NOs: 1 to 6 may be The expression cassette of the first transgene in the transgenic plant is expressed by the expression cassette of the second transgene. The present invention relates to a method for reducing the effect of any one of SEQ ID NOs: 1 to 6 on the expression of ribozymes. 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 is included within the scope of the present invention.

[0069] In certain examples, 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 copies of any one of SEQ ID NO: 1 to 6. at least about 100 contiguous nucleotides, at least about 150 contiguous nucleotides at least about 200 contiguous nucleotides, at least about 250 contiguous nucleotides at least about 300 contiguous nucleotides, at least about 350 contiguous nucleotides at least about 400 contiguous nucleotides, at least about 450 contiguous nucleotides at least about 500 contiguous nucleotides, at least about 550 contiguous nucleotides at least about 600 contiguous nucleotides, at least about 650 contiguous nucleotides at least about 700 contiguous nucleotides, at least about 750 contiguous nucleotides at least about 800 contiguous nucleotides, at least about 850 contiguous nucleotides at least about 900 contiguous nucleotides, at least about 950 contiguous 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 At least about 1700 contiguous nucleotides, at least about 1800 contiguous nucleotides, 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 nucleotide, at least about 2700 contiguous nucleotides, at least about 2800 contiguous nucleotides, at least about 2900 contiguous nucleotides, at least about 3000 contiguous nucleotides In certain embodiments, the sequence may include 10 or 15 consecutive nucleotides. Any of the fragments in row numbers 1 to 6 is used to express the first introduced gene in a transgenic plant. With regard to 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] Constructs 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, at least one DNA molecule can bind to another It includes DNA molecules that are functionally linked, i.e., operably linked, 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" refers to 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 to construct a vector stack Depending on the cloning or synthesis method used to create the From the octide to about hundreds, or thousands, or more nucleotides As used herein, the term "sequence" refers to a sequence of a sequence of a DNA molecule, which may be a nucleotide sequence. In this case, an "expression cassette" refers to 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 that contains the molecule.

[0071] As used herein, the term "operably linked" refers to a first DNA segment. The first DNA molecule binds to a second DNA molecule, and the first DNA molecule affects the function of the second DNA molecule. 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 in contact with the target gene in a cell. If the promoter regulates the transcription of a DNA molecule that is not transcribable, then the promoter will act on the DNA molecule that is not transcribable. For example, a leader can be used to affect the transcription or translation of a DNA sequence. A DNA sequence is operably linked if it can be expressed in a manner that is compatible with the DNA sequence.

[0072] A transcribable DNA molecule is transformed into a functional mRNA molecule that is translated and expressed as a protein. Methods for assembling and introducing constructs into cells in a manner that allows the constructs to be transcribed are known in the art. For the practice of the present invention, methods for preparing and using the 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 control system The control vector is included.

[0073] A variety of regulatory elements, including any of the regulatory elements provided herein. Any such regulatory element may be included in the construct. Such combinations may be designed or adapted to produce the desired accommodation. In one embodiment, the construct of the present invention is 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 coupled to a heterologous promoter, e.g., cauliflower 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. It refers to any DNA molecule that can be transcribed into a nucleic acid sequence, including a protein coding sequence. 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, different plants, DNA molecules from different organisms, or synthetic DNA molecules, e.g. Artificial, synthetic or synthetically produced DNA molecules or transgenes containing antisense messages can include, but are not limited to, DNA molecules encoding other modified versions of the Exemplary transcribable DNA molecules for incorporation into the constructs of the invention include, for example, the DNA DNA molecules or genes originating from a species other than the species into which the molecule is to be incorporated, or from the same species The source or the same species, but developed using genetic engineering methods rather than classical breeding techniques. Thus, genes that are integrated into the recipient cell are included.

[0076] A "transgene" is a gene that is distinct from a host cell at least with respect to its location in the host cell genome. A species of transcribable DNA molecule, and / or the current or any past generation of cells. It 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, such as a gene for conferring herbicide resistance in plants. or a gene capable of providing plant pest resistance in a plant. Contains genes that

[0077] A regulatory element, such as a promoter, is a transcriptionally competent D gene that is heterologous to the regulatory element. 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 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 in nature 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 molecule, a DNA molecule that contains a DNA sequence that deviates from the DNA sequence found in nature, synthetic A DNA molecule containing the DNA sequence, or a DNA molecule that has been genetically transformed or edited into a host cell It may also be a DNA molecule integrated into the DNA of a 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 the transcription factor. Alternatively, for example, the transcribable DNA molecule can be a transcription factor that induces the expression of a particular The gene or protein may be designed to ultimately result in reduced expression of the gene or protein. In one embodiment, this is achieved by using a transcribable DNA molecule oriented in the antisense direction. Those of skill 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 in conjunction 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 control plant, tissue, or cell is screened in some manner. The term "generic DNA molecule" 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 code for 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 comprising the elements.

[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 bacteria, fungus, organism, or plant, including any cell, tissue, or organ. or plant, or the progeny of a bacterium, fungus, or plant. and cells include 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 construct or vector stack has been introduced. Integrating the introduced DNA molecule into the genomic DNA of a recipient cell, tissue, organ, or organism The introduced DNA molecule may be integrated and passed on to subsequent generations. A "transgenic" or "transformed" cell or organism also includes the Progeny, and any offspring resulting from breeding programs using such transgenic organisms as parents in crosses. and may include 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 produce the introduced DNA. It is also possible to make the A molecule not be passed on to the next generation. 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. In carrying out the present invention, the method includes the steps of transforming a host cell with a β-glucosamine derivative and obtaining the transformed host cell. for transforming a plant cell 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, silicon carbide fiber stirring, 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 algae, a fungal cell, The host cell may be a fungal, bacterial, or insect cell. 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 article contains a recombinant DNA molecule of the present invention and is thus transgenic.

[0087] Transgenic plants of the invention may be self-pollinated to produce plants (homozygous for the recombinant DNA molecule). The present invention also provides a method for producing a homozygous transgenic plant 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 Such homozygous and heterozygous transgenic plants can also be provided. Both are referred to herein as "progeny plants." Progeny plants are plants that are the product of the original transgenic A transgenic plant is a plant that is derived from 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 agronomic interest, i.e. Thus, 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 in that they contain the gene(s) of interest and that they are capable of carrying out the process 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 the analysis of transformed plants. For example, methods for plant analysis include Southern or Northern blots, PCR-based approaches, biochemical analysis, phenotypic screening methods, field evaluations, and immunodiagnostic assays. The expression level of a transcribable DNA molecule can be determined by a method including, but not limited to, TaqM. an® (Applied Biosystems, Foster City, CA)) can be measured using the reagents and methods 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 and manufacturers of Wave Technologies (Madison, WI) 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, Parts of the plant of the present invention include, but are not limited to, seeds, endosperm, ovules, and pollen. The present invention also relates to a method for the production of a cell that is capable of reproduction, comprising the steps of: Also included and provided 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 a transgenic plant or part thereof comprising a recombinant DNA molecule of the present invention. Commercial products produced from the sequences are also provided. 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 a seed, a plant cell, or a part of a plant; Commodity products include processed seeds, grains, plant parts, and meal. Commercial products of the invention include, but are not limited to, those corresponding to recombinant DNA molecules of the invention. Determine the content or source of a commercial product. Detection of one or more of these DNAs in a sample may be used to identify the 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 set forth herein. Examples are provided by way of illustration only and are not intended to limit the invention, unless expressly stated otherwise. Those skilled in the art will appreciate that the techniques disclosed in the following examples are well suited to the practice of the present invention. It is understood that this represents a technique discovered by the inventors to work well. However, those of ordinary skill 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 can be obtained. All matter set forth or shown in the accompanying drawings shall be interpreted in an illustrative and not a limiting sense. This section shall be interpreted as follows: EXAMPLES

[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 could 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 correspondence between the first and second transgene cassettes. To identify synthetic ISRs that reduce interactions, we used stably transformed maize and Assays were performed in soybean plants.

[0094] A specific designed and tested ISR is presented in Table 1. ISR4_Stop is 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 expressed in stably transformed maize and soybean plants. The reciprocal relationship of a first transgene cassette to a second transgene cassette in a transstack. The results showed 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 embodiment, 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, The ability to reduce interactions of the daughter expression cassettes is demonstrated.

[0098] To evaluate the ability of ISRs to reduce transgene expression cassette interactions, The locosi plants were grown to contain two transgene expression cassettes in opposite orientations, and the ISRs were 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) was T-Ta.Hs p17-1:1:1 3'UTR (SEQ ID NO:15), operably linked thereto on the 5' side The neomycin phosphotransferase CR-Ec.nptII-Tn5-1:1:3 14, a coding sequence for IFN-γ-aspartase (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 A leader called L-Ta.Lhcb1:1 (SEQ ID NO: 12) linked to the 5' end of the operably linked to the promoter P-Os.Act1:67 (SEQ ID NO: 11) The first transgene expression cassette contained the first transgene expression cassette A second transgene expression cassette, cloned in the opposite orientation 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 was coded as GOI-Ec.uidA+St.LS1.nno:1. 19, a coding sequence for the 5′-terminal end of the I-Zm.D naK: an intron named 1 (SEQ ID NO: 18), operably linked thereto at the 5' side to L - 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 no. 16). It also contains an additional transgene expression cassette that was used to select 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 neomycin phosphotransferase CR-Ec.nptII-Tn5-1:1:3 14, a coding sequence for IFN-γ-aspartase (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 A leader called L-Ta.Lhcb1:1 (SEQ ID NO: 12) linked to the 5' end of the operably linked to the promoter P-Os.Act1:67 (SEQ ID NO: 11) operably linked thereto at the 5' end, E-CaMV.35 contains tandem repeats of a specific enhancer region derived from the S.2xA1-B3-1:1:1, a strong enhancer (SEQ ID NO: 10), The first transgene expression cassette contained a reciprocal The second transgene expression cassette, cloned in the opposite orientation to the GFP gene, is driven by a seed-specific promoter. The control had the same transgene expression cassette as above, containing the enhancer. The vector stack was also used to select for transformed cells using glyphosate selection. An additional transgene expression cassette was also included.

[0101] As shown 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) are ISRs that act as enhancers. - Control vector stack with first transgene expression cassette and second transgene expression cassette The cassette was cloned into the Agroba vector similar to methods known in the art. Using the Citrate-mediated transformation method, two control vector stacks and an ISR were LH244 maize plant cells were transformed 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 in the tissues was evaluated. For sexual analysis, whole mount or sectioned tissues were incubated 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 deactivate with 35% EtOH and 50% acetic acid. Under a dissecting 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 in the leaves. For quantitative analysis of S expression, we used 100% confidence intervals from selected tissues of transformed maize plants. 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 flask. 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 then determined using a FLUOstar® 4-MU quantification system. Omega microplate reader (BMG LABTECH) (excitation 355 nm, emission The GUS activity was estimated by measuring its fluorescence using a light source (460 nm). Provided in nanomolar units of 4-MU / hr / mg total protein.

[0103] The following tissues were sampled for GUS expression levels in the R0 generation: leaves at the V3 stage and and roots, V7 leaves and roots, VT 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 seed tissue and in the stalk is shown, where "bdl" indicates the average GUS expression level in the seed tissue. Table 3 shows that the average GUS expression levels in vegetative and reproductive tissues were below the normal range. The enhancer control is the first transgene expression cassette with enhancer and Corresponding to the 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 0 percent leakage. The leakage percentage of a vector stack containing an ISR is Average GUS expression in vegetative and reproductive tissues of plants transformed with SR-containing constructs Divide the amount of GUS expression by the average amount of GUS expression in 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 IL-1 expression levels compared to the no enhancer control. When the chromosomes were transformed with β-GUS, high GUS expression was observed in all tissues of 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 , and was reduced when 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 results were 16%, 8%, and 6%, respectively, and therefore the interaction between the two transgene expression cassettes was unclear. In comparison, ISR88 and ISR90 provided 84%, 92%, and 94% reductions in ISR86 was much more leaky (61% and 32%, respectively) and contained two transgenes. It only reduced the interactions between the child expression cassettes 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 chromosome 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 illustrates how the intergenic sequence region elements ISR2 and ISR4 are expressed in soybean plants. The first transgene expression cassette of the vector stack used to stably transform When inserted between the first transgene expression cassette and a second transgene expression cassette, The present study demonstrates the ability to reduce interactions between

[0110] To evaluate the ability of ISRs to reduce transgene expression cassette interactions, soybean The plants were grown in a medium containing two transgene expression cassettes in opposite orientations, with an ISR separating these two. A binary plant transformation vector stack containing a transgene expression cassette between Two control vector stacks were also transformed and tested into soybean plants. 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, designated GOI-GUS:1:2 (SEQ ID NO: No. 27), and L-Gm.Sphas1-1:1:1 operably linked thereto at the 5' end. operably linked thereto at the 5' side; A single transfection consisting of the seed-specific promoter as1:14 (SEQ ID NO:25) A control vector stack without enhancer also contained the gene expression cassette. Additional transgene expression cassettes were also used to select transformed cells using biomaterial selection. It included.

[0112] The other control vector stack (Figure 2B, enhancer-containing control) was The first transgene cassette contained TM t.AC139600v16:1 3'UTR (SEQ ID NO: 24), The operably linked neomycin filament, CR-Ec.nptII-Tn5-1:1:2, a coding sequence for a 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 having 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) that encodes was the same as the seed-specific transgene expression cassette described above. Control with enhancer The vector stack was also used to select transformed cells using antibiotic selection. The plasmid also contained a transgene expression cassette.

[0113] As illustrated in FIG. 2C, a first transgene expression cassette and a second transgene expression cassette are To assay the effectiveness of ISR in reducing interactions between sets, 2 (SEQ ID NO: 3), ISR4 (SEQ ID NO: 2), ISR69 (SEQ ID NO: 6), and ISR_ An ISR designated X (SEQ ID NO: 8) was added to the first vector of the enhancer-containing control vector stack. The gene expression cassette was cloned between the first and second transgene expression cassettes. Using Agrobacterium-mediated transformation methods similar to those known in the art. The three vectors were: an enhancer-less control, an enhancer-containing control, and an ISR-containing vector. The soybean plant cells of cultivar A3555 were transformed with the -stack. The 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, Vn5 soybeans, and Vn5 roots. 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 expression cassette enhancer. This corresponds to the complete interaction of the gene expression cassette with the seed-specific promoter. Therefore, the expression of the first transgene was not affected by the strong constitutive enhancer of the first transgene expression cassette. The gene was driven by P-Gm.Sphas1:14, a seed-specific promoter influenced by The average expression levels in vegetative and reproductive tissues from the GUS cassettes were 100 percent. The percent leakage of the ISR-containing construct corresponds to the percentage of leakage of the ISR-containing construct. The average GUS expression levels in Vn5, R1, and R3 tissues of the transformed plants were compared. Divide the mean GUS expression levels in the Vn5, R1, and R3 tissues from the control group and multiply the results 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” The mean GUS expression levels in R5 and yellow pod tissues, Vn5, R1, and R3 tissues The average 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 Very little GUS expression was observed in the R1 and R3 tissues. Plants transformed with the control showed high GUS expression in Vn5, R1, and R3 tissues. The observed expression levels indicate a constitutive expression pattern. Plants transformed with the no-sense control had known seeds of P-Gm.Sphas1:14. Consistent with the specific expression pattern, high GUS expression was observed only in the embryos and cotyledons of yellow pods. Very little expression was observed in R5 cotyledons, where the expression level was higher than that of R3 cotyledons. It can be seen that the amount of ripe ginger in the transformed seeds is slightly increased compared to the amount in the immature seeds. Plants transformed with the R5 cotyledon showed high levels of expression, as well as the no enhancer control. The plants with the enhancer showed an increase in yellow pods, embryos and cotyledons compared to the plants with the enhancer. The first transgene expression cassette, P-FMV.35S-enh-1:1:2 promoter The strong enhancer contained in the second transgene expression cassette, P-Gm. It interacted with Sphas1:14 and shifted its seed-specific expression to a constitutive expression pattern.

[0120] As shown in Table 5, the intergenic sequence regions ISR2, ISR4, and ISR69 of the first transgene expression cassette in the control configuration with enhancer, It was possible to reduce interactions with the expression cassette by 97%, 96%, and 95%, respectively. The ISR_X enhancer was of the first transgene expression cassette in the control configuration with and without the second transgene expression cassette was less effective at 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 transgene expression cassettes.

[0122] Although the principles of the present invention have been illustrated and described, arrangements of the present invention may be modified 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 construed as though they were individual publications or patent issues. to the same extent as if each application was specifically and individually indicated to be incorporated by reference. This is hereby incorporated by reference.

Claims

1. 1. A recombinant DNA molecule comprising: a. Has at least 85 percent sequence identity to any of SEQ ID NOs: 1-6 The sequence, b. A sequence comprising any of SEQ ID NOs: 1 to 6; The recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:

2. The DNA sequence is a first expression cassette and a second expression cassette in a vector stack.

2. The recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule is inserted between

3. The DNA sequence has at least 9 copies relative to any one of the DNA sequences of SEQ ID NOs: 1 to 6.

2. The recombinant DNA molecule of claim 1, having 0 percent sequence identity.

4. The DNA sequence has at least 9 copies relative to any one of the DNA sequences of SEQ ID NOs: 1 to 6.

2. The recombinant DNA molecule of claim 1 having 5 percent sequence identity.

5. The recombinant DNA according to claim 1, wherein the DNA sequence comprises any one of SEQ ID NOs: 1 to 6. molecule.

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

7. The transgenic plant cell of claim 5, wherein the transgenic plant cell is a monocotyledonous plant cell. Transgenic plant cells.

8. The transgenic plant cell of claim 5, wherein the transgenic plant cell is a dicotyledonous plant cell. Transgenic plant cells.

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

10. A progeny plant of the transgenic plant according to claim 8, or a part thereof, the progeny plant, or a part thereof, comprising the recombinant DNA molecule; or That part.

11. A transgenic seed, comprising:

2. A transgenic seed comprising the recombinant molecule of claim 1.

12. 1. A method of producing a commodity product, comprising: Obtaining a transgenic plant or part thereof according to claim 8 and then Producing a commodity product.

13. The commodity product may be a seed, a processed seed, a protein concentrate, a protein isolate, a starch, , grains, plant parts, seed oils, biomass, fine flours, and coarse flours according to claim 11 . method.

14. First transgene expression in transgenic plants transformed with vector stacks 1. A method for reducing interaction between a cassette and a second transgene expression cassette, comprising: The method further comprising: a. a first transgene expression cassette, b. a second transgene cassette; c. the DNA molecule is a DNA molecule including the first transgene expression cassette and the second transgene expression cassette A heterologous T- Transforming a plant cell with a vector stack containing DNA; d. Regenerating a transgenic plant from the transformed plant cell; The method comprising:

15. The DNA molecule of any one of SEQ ID NOs: 1 to 6 is the first inserted between the transgene expression cassette and the second transgene expression cassette; The method of claim 13.

Images