Novel intergene sequence regions and their uses

Abstract

The present invention provides a recombinant DNA molecule comprising a novel synthetic intergene sequence region for use in plants to reduce the interaction of the first transgene expression cassette with the second transgene cassette when inserted between the first transgene cassette and the second transgene cassette. The present invention also provides a transgenic plant, a plant cell, a plant part, and a seed comprising the novel synthetic intergene sequence region. The present invention also provides a method for reducing the interaction between transgene expression cassettes using the novel synthetic intergene sequence region.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 875,752 filed on July 18, 2019, the entirety of which is incorporated herein by reference.

[0003] Inclusion in sequence list

[0004] A file named "MONS472WO_ST25.txt" containing a computer-readable format of a sequence list was created on June 9, 2020. This file is 38,698 bytes (measured on MS-Windows®), was submitted simultaneously by electronic submission (using the U.S. Patent and Trademark Office EFS-Web Submission System), and the entirety thereof is incorporated herein by reference.

[0005] Technology field

[0006] The present invention relates to the field of plant molecular biology and plant genetic engineering. More specifically, the present invention relates to a DNA molecule useful for reducing the effect that one transgene cassette has on the expression of another transgene cassette in plants. Background Technology

[0007] An intergenic sequence region ("ISR(Intergenic Sequence Region)") is a DNA sequence that, when placed between two or more transgenic cassettes, reduces the interaction of one transgenic cassette with another transgenic cassette, thereby preventing changes in the expression pattern of the transgenic cassettes due to the interaction of expression factors between the cassettes.

[0008] Expression elements in an expression cassette, such as promoters, introns, and 3' untranslated regions (3' UTRs), contain cis-action elements that have the potential to influence the expression of adjacent or neighboring expression cassettes. For example, plant viral promoters, such as that of the cauliflower mosaic virus 35S promoter (CaMV 35S), consist of enhancer domains that can influence the transcription of nearby genes, or activating genes up to 4.3 Kb upstream or downstream from the insertion site ( Gudynaite-Savitch et al. (2009) Strategies to mitigate transgene-promoter interactions. Plant Biotechnology Journal, 7: 472-485; Benfey et al. (1990) Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. The EMBO Journal, 9:1677-1684 For example, in the case of a transgene cassette subcloned into a plant transformation vector containing a selection cassette using a CaMV 35S promoter to drive a selectable marker coding sequence, the tissue-specific expression of the transgene cassette was affected by the presence of the CaMV 35S promoter, which altered the tissue-specific expression of the transgene cassette in a more constitutive pattern ( Yoo et al. (2005) The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta, 221: 523-530 ).

[0009] Increasingly, in the field of plant biotechnology, vectors containing multiple transgenic cassettes are being used to transform plants to introduce several agronomically important traits within a single vector stack. The advantage of this process is that since several agronomic traits can be contained within a single gene locus, it enables a more efficient and less expensive breeding process when breeding vector-stacked plants with other transgenic plants containing additional agronomic traits. However, as more expression cassettes are cloned into vectors, there is a possibility that expression elements from one expression cassette may alter or influence the expression profiles of other expression cassettes within the vector stack. Expression cassettes designed to provide specific patterns of tissue expression, such as those in seeds, can alter expression as a result of interactions between expression elements of neighboring expression cassettes within the vector stack, thereby changing the seed-specific expression pattern to more closely resemble that of the neighboring cassette. This can negatively affect the intended phenotype of the seed-specific expression cassette. Therefore, there is a need in plant biotechnology for DNA sequences that can reduce or prevent interactions between adjacent and neighboring expression cassettes in a vector stack.

[0010] Accordingly, the inventors disclose herein a novel synthetic ISR that minimizes the interaction of expression cassettes in a vector stack in transgenic plants. This ISR can be placed between adjacent expression cassettes in a single vector stack to prevent the interaction between expression elements of individual cassettes, thereby maintaining the intended expression pattern and level of expression of each expression cassette within the vector stack.

[0011] Summary of the Invention

[0012] The present invention provides a novel synthetic intergene sequence region or ISR for use in plants. The present invention also provides a recombinant DNA construct comprising an ISR. The present invention also provides a transgenic plant cell, plant, and seed comprising an ISR. In one embodiment, the ISR is inserted between expression cassettes in a vector stack. The present invention also provides a method for using an ISR and a method for preparing and using a recombinant DNA construct comprising an ISR, and a transgenic plant cell, plant, and seed comprising an ISR.

[0013] Thus, in one embodiment, the present invention provides a recombinant DNA molecule comprising (a) a sequence having at least 85% sequence identity with any of SEQ ID NOs 1 to 6; and (b) a DNA sequence selected from the group consisting of sequences including any of SEQ ID NOs 1 to 6. In a specific embodiment, the recombinant DNA molecule comprises a DNA sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the DNA sequence of any of SEQ ID NOs 1 to 6.

[0014] In another embodiment, the present invention provides a transgenic plant cell comprising a recombinant DNA molecule comprising (a) a sequence having at least 85% sequence identity with any of SEQ NOs 1 to 6; and (b) a DNA sequence selected from the group consisting of sequences including any of SEQ NOs 1 to 6. In a given embodiment, the transgenic plant cell is a monocotyledonous plant cell. In another embodiment, the transgenic plant cell is a monocotyledonous plant cell or a dicot plant cell.

[0015] In another aspect, the present invention further provides a transgenic plant or a part thereof comprising a recombinant DNA molecule comprising (a) a sequence having at least 85% sequence identity with any of SEQ NOs 1 to 6; and (b) a DNA sequence selected from the group consisting of sequences comprising any of SEQ NOs 1 to 6. In a specific embodiment, the transgenic plant is any generation of offspring plants comprising the recombinant DNA molecule. The present invention also provides transgenic seeds comprising the recombinant DNA molecule that produce such a transgenic plant when grown.

[0016] In another aspect, the present invention provides a method for producing a commodity product comprising the steps of obtaining a transgenic plant or a part thereof containing a recombinant DNA molecule of the present invention and producing a commodity product therefrom. In one embodiment, the commodity product is a seed, processed seed, concentrated protein, isolated protein, starch, grain, plant part, seed oil, biomass, wheat flour, and meal.

[0017] In another aspect, the present invention provides a method for reducing the interaction between a first transgene expression cassette and a second transgene expression cassette within a transgenic plant transformed by a vector stack, the method comprising: (a) a first transgene cassette; (b) a second transgene cassette: (c) a DNA molecule comprising (i) a sequence having at least 85% sequence identity with any of SEQ ID NOs 1 to 6; and (ii) a sequence selected from the group consisting of any of SEQ ID NOs 1 to 6; wherein the DNA molecule comprises a recombinant DNA molecule including a DNA molecule inserted between the first transgene expression cassette and the second transgene expression cassette; and (d) regenerating the transgenic plant from the transgenic plant cell. In a given embodiment, the vector stack comprises more than two expression cassettes. In a further embodiment, a DNA molecule of any of sequence numbers 1 to 6 is inserted between each of the expression cassettes in the vector stack.

[0018] A brief explanation of the sequence

[0019] Sequence No. 1 is the DNA sequence of the intergene sequence region ISR4_stop, which includes ISR4 (sequence No. 4) and three stop codons at both the 5' end and the 3' end.

[0020] Sequence number 2 is the DNA sequence of the intergene sequence region ISR89.

[0021] Sequence number 3 is the DNA sequence of the intergene sequence region ISR2.

[0022] Sequence number 4 is the DNA sequence of the intergene sequence region ISR4.

[0023] Sequence number 5 is the DNA sequence of the intergene sequence region ISR97.

[0024] Sequence number 6 is the DNA sequence of the intergene sequence region ISR69.

[0025] Sequence number 7 is the DNA sequence of the intergene sequence region ISR88.

[0026] Sequence number 8 is the DNA sequence of the intergene sequence region ISR86.

[0027] Sequence number 9 is the DNA sequence of the intergene sequence region ISR_X.

[0028] Sequence No. 10 is the DNA sequence of the enhancer presented in FIGS. 1a to 1c as "E-CaMV.35S", E-CaMV.35S.2xA1-B3-1:1:1.

[0029] Sequence No. 11 is the DNA sequence of the promoter presented in FIGS. 1a to 1c as "P-Os.Act1", P-Os.Act1:67.

[0030] Sequence No. 12 is the DNA sequence of the leader or 5' UTR, L-Ta.Lhcb1:1, presented in FIGS. 1a to 1c as "L-Ta.Lhcb1".

[0031] Sequence number 13 is the DNA sequence of the intron presented in FIGS. 1a to 1c as "I-Os.Act1", I-Os.Act1-1:1:19.

[0032] Sequence No. 14 is a DNA sequence encoding neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:3, presented in FIGS. 1a to 1c as "nptII-1".

[0033] Sequence number 15 is the DNA sequence of the 3' UTR presented in FIGS. 1a to 1c as "T-Ta.Hsp17", T-Ta.Hsp17-1:1:1.

[0034] Sequence number 16 is "P-Zm.39486", the DNA sequence of the promoter presented in FIGS. 1a to 1c, P-Zm.39486-1:1:1.

[0035] Sequence number 17 is the DNA sequence of the leader or 5' UTR presented in FIGS. 1a to 1c as "L-Zm.39486", L-Zm.39486-1:1:1.

[0036] Sequence number 18 is the DNA sequence of the intron presented in FIGS. 1a to 1c as "I-Zm.DnaK", I-Zm.DnaK:1.

[0037] Sequence No. 19 is the DNA sequence of a synthetic coding sequence optimized for plant expression of β-glucuronidase (GUS-1: GOI-Ec.uidA+St.LS1.nno:1) having processable introns derived from the potato photo-induced tissue-specific ST-LS1 gene (Gene Bank accession No.: X04753) presented in FIGS. 1a to 1c as "GUS-1".

[0038] Sequence number 20 is the DNA sequence of the 3' UTR presented in FIGS. 1a to 1c as "T-Os.Mth", T-Os.Mth-1:1:1.

[0039] Sequence number 21 is the DNA sequence of the promoter presented in FIGS. 2a to 2c as "P-FMV.35S", P-FMV.35S-enh-1:1:2.

[0040] Sequence No. 22 is the DNA sequence of the leader or 5' UTR, L-Ph.DnaK-1:1:3, presented in FIGS. 2a to 2c as "L-Ph.DnaK".

[0041] Sequence No. 23 is a DNA sequence encoding neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:2, presented in FIGS. 2a to 2c as "nptII-2".

[0042] Sequence number 24 is the DNA sequence of the 3' UTR, T-Mt.AC139600v16:1, presented in FIGS. 2a to 2c as "T-AC139600".

[0043] Sequence number 25 is the DNA sequence of the promoter presented in FIGS. 2a to 2c as "P-Gm.Sphas", P-Gm.Sphas1:14.

[0044] Sequence number 26 is the DNA sequence of the leader or 5' UTR, L-Gm.Sphas1-1:1:1, presented in FIGS. 2a to 2c as "L-Gm.Sphas".

[0045] Sequence No. 27 is the DNA sequence of a synthetic coding sequence for β-glucuronidase (GUS-2: GOI-GUS:1:2) having processable introns derived from the potato photo-induced tissue-specific ST-LS1 gene (Genetic Bank Accession No.: X04753) presented in FIGS. 2a to 2c as "GUS-2".

[0046] Sequence number 28 is the DNA sequence of the 3' UTR, T-Mt.AC145767v28:3, presented in FIGS. 2a to 2c as "T- AC145767". Brief explanation of the drawing

[0047] FIGS. 1a through 1c are diagrammatic representations of vector stacks used to analyze the effectiveness of synthetic intergene sequence regions ("ISRs") in reducing the interaction between two transgenic expression cassettes in a single vector stack regarding their mutual expression in stably transformed maize plants. Reference numbers in the figures indicate corresponding sequence identifiers for each genetic element, as presented in the brief description of the sequences. FIG. 1a shows the transgenic expression cassette configuration for a control vector stack, which is a control without an enhancer. The control without an enhancer consists of two transgenic expression cassettes cloned in different orientations. The first transgene cassette consists of P-Os.Act1:67 (Sequence No. 11), 5' operably linked to a promoter and reader; L-Ta.Lhcb1:1 (Sequence No. 12), 5' operably linked to an intron; I-Os.Act1-1:1:19 (Sequence No. 13), 5' operably linked to a coding sequence for neomycin phosphotransferase; CR-Ec.nptII-Tn5-1:1:3 (Sequence No. 14), 5' operably linked to a 3' UTR; and T-Ta.Hsp17-1:1:1 (Sequence No. 15). The second transgene cassette, cloned in a different direction for the first transgene cassette, consists of P-Zm.39486-1:1:1 (SEQ No. 16), 5' operably linked to a seed-specific promoter and a reader; L-Zm.39486-1:1:1 (SEQ No. 17), 5' operably linked to an intron; I-Zm.DnaK:1 (SEQ No. 18), 5' operably linked to a coding sequence encoding GUS-1; GOI-Ec.uidA+St.LS1.nno:1 (SEQ No. 19), 5' operably linked to a 3' UTR; and T-Os.Mth-1:1:1 (SEQ No. 20). Figure 1b shows the transgene expression cassette configuration for a control vector stack, which is a control with an enhancer.The control group having an enhancer consists of E-CaMV.35S.2xA1-B3-1:1:1 (SEQ No. 10), which includes a tandem repeat of a specific enhancer region derived from the cauliflower mosaic virus 35S promoter, which is 5' operably linked to the promoter, P-Os.Act1:67 (SEQ No. 11), which is 5' operably linked to the leader, L-Ta.Lhcb1:1 (SEQ No. 12), which is 5' operably linked to the intron, I-Os.Act1-1:1:19 (SEQ No. 13), which is 5' operably linked to the coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:3 (SEQ No. 14), which is 5' operably linked to the 3' UTR, and T-Ta.Hsp17-1:1:1 (SEQ No. 15). The second transgene cassette cloned in a different direction from the first transgene cassette consists of P-Zm.39486-1:1:1 (Sequence No. 16), 5' operably linked to a seed-specific promoter and a reader; L-Zm.39486-1:1:1 (Sequence No. 17), 5' operably linked to an intron; I-Zm.DnaK:1 (Sequence No. 18), 5' operably linked to a coding sequence encoding GUS-1; GOI-Ec.uidA+St.LS1.nno:1 (Sequence No. 19), 5' operably linked to a 3' UTR; and T-Os.Mth-1:1:1 (Sequence No. 20). The control group with the enhancer in FIG. 1a lacks an ISR between the first transgene expression cassette and the second transgene expression cassette. As a result, the enhancer from the first transgene expression cassette interacts with the seed-specific promoter in the second transgene expression cassette and alters its expression, thereby changing the expression of the second transgene expression cassette from seed-specific to constitutively. In FIG. 1c, the ISR is cloned between the first transgene expression cassette and the second transgene expression cassette of the control group having the enhancer.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 transgene expression cassette, thereby reducing expression in non-seed tissues compared to the control group with the enhancer. FIGS. 2a through 2c are diagrammatic representations of vector stacks used to analyze the effectiveness of an ISR in reducing the interaction between two transgenic expression cassettes in a single vector stack regarding their mutual expression in stably transformed soybean plants. Reference numbers in the figures indicate corresponding sequence identifiers for each genetic element, as presented in the brief descriptions of the sequences. FIG. 2a shows the configuration of a transgenic expression cassette for a control vector stack, which is a control without an enhancer. The control group without an enhancer (Fig. 2a) consists of a seed-specific promoter, P-Gm.Sphas1:14 (SEQ No. 25) 5' operably linked to a leader, L-Gm.Sphas1-1:1:1 (SEQ No. 26) 5' operably linked to a coding sequence encoding GUS-2, GOI-GUS:1:2 (SEQ No. 27) 5' operably linked to a 3' UTR, and T-Mt.AC145767v28:3 (SEQ No. 28). The seed-specific promoter can primarily drive GUS expression in soybean seeds in the control group without an enhancer. Fig. 2b shows the configuration of the transgene expression cassette for the control vector stack, which is a control group with an enhancer. The control group with an enhancer consists of two transgene expression cassettes in different orientations. The first transgene cassette consists of a strong promoter derived from the Scrophularia mosaic virus 35S promoter having a rearranged and duplicated enhancer, P-FMV.35S-enh-1:1:2 (SEQ No. 21) 5' operably linked to a reader, L-Ph.DnaK-1:1:3 (SEQ No. 22) 5' operably linked to a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:2 (SEQ No. 23) 5' operably linked to a 3' UTR, and T-Mt.AC139600v16:1 (SEQ No. 24).The second transgene cassette cloned in the other direction for the first transgene cassette consists of P-Gm.Sphas1:14 (SEQ No. 25), 5' operably linked to a seed-specific promoter and a reader; L-Gm.Sphas1-1:1:1 (SEQ No. 26), 5' operably linked to a coding sequence encoding GUS-2; GOI-GUS:1:2 (SEQ No. 27), 5' operably linked to a 3' UTR; and T-Mt.AC145767v28:3 (SEQ No. 28). The control group with the enhancer lacks an ISR between the first transgene expression cassette and the second transgene expression cassette. As a result, seed-specific promoter expression in the second transgene expression cassette is influenced by the enhancer region of the Scrophularia mosaic virus 35S promoter in the first transgene expression cassette, thereby constitutively altering the expression of the second transgene expression cassette from seed-specific. In Fig. 2c, the ISR is cloned between the first transgene expression cassette and the second transgene expression cassette of the control group with the enhancer. If the ISR is effective, it will reduce the interaction between the enhancer region of the Scrophularia mosaic virus 35S promoter in the first transgene expression cassette and the promoter in the second transgene expression cassette, thereby reducing expression in non-seed tissues compared to the control group with the enhancer. Specific details for implementing the invention

[0048] The present invention provides a novel synthetic intergene sequence region or ISR for use in transgenic plants. The nucleotide sequences of this novel synthetic ISR are provided as SEQ ID NOs. 1 to 6. This synthetic ISR reduces the interaction of expression factors in the first transgenic expression cassette with the expression of the second transgenic cassette in a transgenic plant when inserted between a first transgenic cassette and a second transgenic cassette. The present invention also provides transgenic plant cells, plants, and seeds comprising the ISR. The present invention also provides a method for using the ISR and for preparing and using a recombinant DNA molecule comprising the ISR.

[0049] The following definitions and methods are provided to better limit the invention and to guide those skilled in the art in practicing the invention. Unless otherwise noted, terms should be understood by those skilled in the art according to their conventional usage.

[0050] Interaction between ISR and the first transposable expression cassette and the second transposable expression cassette

[0051] As used herein, the term “interaction” refers to the effect of one or more elements in a first transgene expression cassette on the expression pattern of a second transgene expression cassette when provided adjacent to each other in a transgenic plant transformed using a vector stack in a given embodiment.

[0052] The regulatory elements within each transgenic expression cassette consist of various cis elements coupled by transacting factors that bring about the transcription of the transgenic gene. For example, plant promoters consist of cis elements essential for the initiation and efficiency of transcription. Furthermore, plant promoters are typically composed of other cis element motifs capable of regulating transcription in response to specific stimuli, such as stress (ABRE and AB14), pathogens (W Box), or light (GT1 motif). Other cis elements may provide tissue-specific or tissue-preferred expression ( Porto et al. (2014) Plant Promoters: An Approach of Structure and Function. Mol. Biotechnology 56: 38-49For example, the cauliflower mosaic virus 35S promoter contains an enhancer region composed of two domains. The downstream domain, Domain A, confers expression primarily in the roots. The cis element within the 22-base pair region of Domain A, as-1, is primarily responsible for this expression. The upstream domain, Domain B, confers expression in most cell types of leaves and stems, as well as in the vascular tissues of the roots ( Benfey et al. (1990) Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. The EMBO Journal, 9:1677-1684 ).

[0053] When two transgenic expression cassettes are adjacent to each other in the plant genome, there is a possibility that the expression elements of one transgenic expression cassette may alter the expression of the other transgenic expression cassette. The "interaction" between one transgenic expression cassette and an adjacent transgenic expression cassette in transgenic plants is demonstrated in Examples 2 and 3 by a control group having an enhancer.

[0054] "Leakage" is a term used to describe the level of change in average expression in tissues caused by the interaction of expression elements from the first expression cassette with the expression profile of the second expression cassette. Leakage is determined by comparing the expression profile of the control with the enhancer to the expression profile of the test vector stack containing the ISR (consisting of a control with an enhancer containing an ISR inserted between two transgene cassettes). The leakage of the control without the enhancer is 100% compared to the control with the enhancer. The leakage of the construct containing the ISR is determined by dividing the average GUS expression in non-target tissues of the test construct by the average GUS expression in non-target tissues of the control with the enhancer construct and multiplying by 100. The percentage reduction in leakage is determined by subtracting the percentage leakage from 100%.

[0055] An “intergene sequence region” or “ISR” is a synthetic nucleotide sequence designed to minimize the interaction of expression elements in neighboring transgenic cassettes for each other’s expression. The intergene sequence regions disclosed herein were computer-designed and analyzed for their ability to preserve the expression profiles of each transgenic expression cassette as observed individually in transgenic plants by reducing the interaction of the first transgenic expression cassette with the second transgenic expression cassette in a vector stack used to transgenic plant cells.

[0056] "Synthetic nucleotide sequence" or "artificial nucleotide sequence" is a nucleotide sequence that is not known to occur in nature or does not occur naturally. The intergene sequence region element of the present invention comprises a synthetic nucleotide sequence. Preferably, the synthetic nucleotide sequence shares little homology with the natural sequence or does not share extended homology. In this context, extended homology generally refers to 100% sequence identity extending beyond about 25 nucleotides of adjacent sequences.

[0057] In Example 2, control maize plants were transformed using two vector stacks consisting of two transgenic expression cassettes in different orientations. One control vector stack contained a first transgenic expression cassette containing a single rice actin promoter (control without enhancer, see FIG. 1a) that promotes the expression of the antibiotic resistance gene, and a second transgenic expression cassette containing a seed-preferred promoter that promotes GUS expression. Maize plants transformed by this vector stack exhibited seed-preferred expression of GUS. The other control vector stack (control with enhancer) contained a first transgenic expression cassette containing a strong enhancer derived from CaMV 35S operably linked to a single rice actin promoter that promotes the expression of the antibiotic resistance gene, and a second transgenic expression cassette containing a seed-preferred promoter that promotes GUS expression. Corn plants transformed by the control group with the enhancer exhibited high levels of GUS expression in roots, leaves, anthers, silks, and seeds. As such, in the control group with the enhancer, the first transgenic expression cassette enhancer modified the expression pattern of the expression profile of the second transgenic expression cassette, thereby constitutively altering the expression of the second transgenic expression cassette from seed-preference.

[0058] A predetermined computer-designed ISR was inserted between the first and second transgene cassettes of the control group having an enhancer, as demonstrated in FIG. 1c. When ISR ISR4_stop (SEQ No. 1), ISR89 (SEQ No. 2), and ISR97 (SEQ No. 5) were inserted between the first and second transgene expression cassettes, the percentage leakage in the interaction between the expression pattern of the first transgene expression cassette and the expression pattern of the second transgene expression cassette was 16%, 8%, and 6%, respectively. Thus, this ISR reduced the interaction between the first and second transgene expression cassettes by 84%, 92%, and 94%, respectively.

[0059] In Example 3, a similar experimental design was used to test the efficacy of a specified ISR in soybeans. The insertion of ISR2 (SEQ No. 3), ISR4 (SEQ No. 4), and ISR69 (SEQ No. 6) between the first transgene expression cassette and the second transgene expression cassette of the control group with the enhancer reduced the effect of the expression pattern of the first transgene expression cassette on the expression pattern of the second transgene expression cassette by only 3%, 4%, and 5% leakage, respectively. This reduced the interaction of the expression elements in the first transgene expression cassette on the expression pattern of the second transgene expression cassette by 97%, 96%, and 95%, respectively.

[0060] As demonstrated in the examples, not all computer-designed intergene sequence regions were efficient in reducing interactions. Additionally, even the ISRs that did reduce interactions did so to varying degrees. For example, ISR88 (Sequence No. 7) and ISR86 (Sequence No. 8) reduced interactions by 39% and 68%, respectively, in transgenic maize plants with only 61% and 32% leakage, respectively. This reduction in interactions was significantly less compared to 84% for ISR4_stop, 92% for ISR89, ​​and 94% for ISR97. Similarly, in transgenic soybeans, ISR_X (Sequence No. 9) reduced interactions by only 76% (percent leakage, 24%), compared to 97% for ISR2, 96% for ISR4, and 95% for ISR69. As such, each computer-designed ISR is unique, and different ISRs can be used with different expression cassettes to achieve desired expression profiles for one or more genes of interest.

[0061] DNA molecule

[0062] As used herein, the terms “DNA” or “DNA molecule” refer to a double-stranded DNA molecule of genomic or synthetic origin read from the 5’ (upstream) end to the 3’ (downstream) end, i.e., a polymer of deoxyribonucleotide bases or DNA molecules. As used herein, the term “DNA sequence” refers to a sequence of nucleotides of a DNA molecule. The nomenclature used herein corresponds to that of Title 37 of § 1.822 of the United States Code of Federal Regulations and is listed in the tables of Table 1 and Table 3 of Appendix 2 of WIPO Standard ST.25 (1998).

[0063] As used herein, “heterogeneous molecule” is a molecule comprising a combination of DNA molecules that do not occur together in nature without human intervention. For example, a heterogeneous molecule may be a DNA molecule composed of at least two DNA molecules that are heterogeneous with respect to each other, a DNA molecule comprising a DNA sequence that deviates from a DNA sequence existing in nature, a DNA molecule comprising a synthetic DNA sequence, or a DNA molecule introduced into the DNA of a host cell by genetic transformation or gene editing.

[0064] References to "isolated DNA molecules" or equivalent terms or phrases herein are intended to mean that a DNA molecule exists alone or in combination with other compositions but is not found in its natural environment. For example, nucleic acid elements such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, and others found naturally within the DNA of an organism's genome are not considered "isolated" insofar as the element is within the organism's genome and is located at a position within the genome found in nature. However, each of these elements and sub-parts of these elements will be considered "isolated" within the scope of this disclosure insofar as the element is not within the organism's genome and is located at a position within the genome found in nature. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein will be an isolated nucleotide sequence insofar as the sequence encoding the protein is not found within the DNA of bacteria found in nature. A synthetic nucleotide sequence encoding an amino acid sequence of a naturally occurring insecticidal protein will be considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transformed nucleotide sequence, i.e., a nucleotide sequence of DNA that is inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, will be considered to be an isolated nucleotide sequence, whether it is present in a plasmid or similar structure used to transform the cell within the genome of a plant or bacterial cell, or present in detectable amounts in a biological sample or raw material product derived from a tissue, offspring, plant or bacterial cell.

[0065] As used herein, the term “sequence identity” refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. Optimal sequence alignment is generated by manually aligning two sequences, e.g., a reference sequence and another sequence, to maximize the number of nucleotide matches in sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term “reference sequence” refers to the DNA sequences provided as Sequence Nos. 1 through 6.

[0066] As used herein, the term “percent sequence identity” or “percent identity” or “% identity” is the identity fraction multiplied by 100. The “identity fraction” for a reference sequence and an optimally aligned sequence is the number of nucleotide matches in the optimal alignment divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides over the entire length of the reference sequence. Accordingly, one embodiment of the present invention provides a DNA molecule comprising a sequence having at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 100% identity with respect to the reference sequence provided herein as SEQ ID NOs 1 to 6. In a given embodiment, a sequence having a given percentage identity with any of sequences 1 to 6 retains the general function of any of sequences 1 to 6, that is, exhibits the same or similar ability to reduce the influence of the first transgene expression cassette on the expression of the second transgene cassette in a transgenic plant. In a given embodiment, a sequence having a given percentage identity with any of sequences 1 to 6 has the activity of any of sequences 1 to 6 in relation to reducing the influence of the first transgene expression cassette on the expression of the second transgene cassette in a transgenic plant.

[0067] Control factors

[0068] Regulatory elements such as promoters, leaders (also known as 5' UTRs), enhancers, introns, and transcription termination regions (or 3' UTRs) play an integral role in the total expression of genes in living cells. As used herein, the term “regulatory element” refers to a DNA molecule having gene-regulatory activity. As used herein, the term “gene-regulatory activity” refers to the ability to influence the expression of an operably linked transcriptional DNA molecule, for example, by influencing the transcription and / or translation of the operably linked transcriptional DNA molecule. Regulatory elements such as promoters, leaders, enhancers, introns, and 3' UTRs acting in plants are useful for modifying plant phenotypes through genetic manipulation.

[0069] Regulatory factors may be identified by quantitative or qualitative indications as well as positive and / or negative effects, such as their gene expression patterns, e.g., constitutive expression or expression that is temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle and / or chemically reactive, and any combination thereof. As used herein, “gene expression pattern” is any pattern of transcription of a DNA molecule operably linked to a transcribed RNA molecule. The transcribed RNA molecule may be translated to produce a protein molecule or may provide antisense or other regulatory RNA molecules such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), and others.

[0070] As used herein, the term “protein expression” is any pattern of translation of a transcribed RNA molecule into a protein molecule. Protein expression can be identified by its temporal, spatial, developmental, or morphological qualities, as well as by quantitative or qualitative indications.

[0071] Promoters are useful as regulatory elements for regulating the expression of operantly linked transcriptional DNA molecules. As used herein, the term “promoter” generally refers to a DNA molecule involved in the recognition and binding of other proteins, such as RNA polymerase II and trans-acting transcription factors, to initiate transcription. Promoters can be initially isolated from the 5’ untranslated region (5’ UTR) of a genomic copy of a gene. Alternatively, the promoter may be a synthetically produced or engineered DNA molecule. The promoter may also be a chimeric. A chimeric promoter is produced through the fusion of two or more heterogeneous DNA molecules. Promoters useful for demonstrating the present invention include promoter elements provided as SEQ ID NO. 11, SEQ ID NO. 16, SEQ ID NO. 21, and SEQ ID NO. 25.

[0072] As used herein, the term “leader” refers to a DNA molecule isolated from the untranslated 5’ region (5’ UTR) of a gene and generally defined as a nucleotide segment between the transcription start site (TSS) and the protein-coding sequence start site. Alternatively, the leader may be a synthetically manufactured or engineered DNA element. The leader may be used as a 5’ regulatory element to regulate the expression of an operantly linked transcription-capable DNA molecule. The leader molecule may be used with a heterogeneous promoter or its natural promoter. Leaders useful for demonstrating the present invention include SEQ NO. 12, SEQ NO. 17, SEQ NO. 22, and SEQ NO. 26.

[0073] As used herein, the term “intron” refers to a DNA molecule that can be isolated from or identified from a gene and can generally be defined as a region sliced ​​during messenger RNA (mRNA) processing prior to translation. Alternatively, an intron may be a synthetically produced or engineered DNA element. An intron may contain an enhancer element that brings about the transcription of an operably linked gene. An intron may be used as a regulatory element to regulate the expression of an operably linked transcriptional DNA molecule. A composition may contain an intron, and the intron may or may not be heterogeneous with respect to the transcriptional DNA molecule. Introns useful for demonstrating the invention are presented as SEQ ID NO. 13 and SEQ ID NO. 18.

[0074] As used herein, the terms “3’ transcription termination molecule,” “3’ untranslated region,” or “3’ UTR” refer to a DNA molecule used during transcription in the untranslated region of the 3’ portion of an mRNA molecule. The 3’ untranslated region of an mRNA molecule may be generated by specific cleavage and 3’ polyadenylation, also known as a polyA tail. The 3’ UTR may be operably linked to a transcription-capable DNA molecule and located downstream thereof, and may contain polyadenylation signals and other regulatory signals that may affect transcription, mRNA processing, or gene expression. The polyA tail is thought to act in mRNA stability and the initiation of translation.

[0075] As used herein, the terms “enhancer” or “enhancer element” refer to a cis-action regulatory element, also known as a cis-element, of an operably linked transcriptional DNA molecule that confers a mode of the overall expression pattern but is usually insufficient on its own to induce transcription. Unlike a promoter, an enhancer element does not usually contain a transcription start site (TSS) or a TATA box or an equivalent DNA sequence. A promoter or promoter fragment may naturally contain one or more enhancer elements that influence the transcription of the operably linked DNA sequence. An enhancer element may also be fused to a promoter to produce a chimeric promoter cis element that confers a mode of the overall regulation of gene expression.

[0076] As used herein, the term “variant” refers to a second DNA molecule that is similar in composition to but not identical to the first DNA molecule. For example, one variant of the ISR disclosed herein will have a slightly different sequence composition but will retain the ability to reduce the effect of the first transgene expression cassette on the expression of the second transgene cassette in transgenic plants in the same way as the ISR from which it originated. The variant may be a shorter or truncated version of the first DNA molecule, such as having different restriction enzyme sites and / or internal deletions, substitutions, or insertions, or an altered version of the first DNA molecule. The “variant” may also include an ISR having a nucleotide sequence comprising one or more nucleotide substitutions, deletions, or insertions of the reference sequence, and the derivative intergene sequence region element has a greater or lesser ability or an equivalent ability to reduce the effect of the first transgene expression cassette on the expression of the second transgene cassette in transgenic plants. In the present invention, the polynucleotide sequences provided as SEQ NOs. 1 to 6 may be used to produce variants that are similar but not identical in composition to the DNA sequence of the original ISR while still retaining the same or similar ability to reduce the influence of the first transgene expression cassette on the expression of the second transgene cassette in transgenic plants. In a given embodiment, a variant of any of SEQ NOs. 1 to 6 has the activity of any of SEQ NOs. 1 to 6 in relation to reducing the influence of the first transgene expression cassette on the expression of the second transgene cassette in transgenic plants. The preparation of such variants of the present invention is entirely within the general art of the art in the sight of the present disclosure and is included within the scope of the present invention.

[0077] In a given example, a variant of ISR may be a fragment of any of sequence numbers 1 to 6. The fragments of SEQ NOs 1 to 6 comprise at least about 50 adjacent nucleotides, at least about 100 adjacent nucleotides, at least about 150 adjacent nucleotides, at least about 200 adjacent nucleotides, at least about 250 adjacent nucleotides, at least about 300 adjacent nucleotides, at least about 350 adjacent nucleotides, at least about 400 adjacent nucleotides, at least about 450 adjacent nucleotides, at least about 500 adjacent nucleotides, at least about 550 adjacent nucleotides, at least about 600 adjacent nucleotides, at least about 650 adjacent nucleotides, at least about 700 adjacent nucleotides, at least about 750 adjacent nucleotides, at least about 800 adjacent nucleotides, at least about 850 adjacent nucleotides, at least Approximately 900 adjacent nucleotides, at least approximately 950 adjacent nucleotides, at least approximately 1000 adjacent nucleotides, at least approximately 1100 adjacent nucleotides, at least approximately 1200 adjacent nucleotides, at least approximately 1300 adjacent nucleotides, at least approximately 1400 adjacent nucleotides, at least approximately 1500 adjacent nucleotides, at least approximately 1600 adjacent nucleotides, at least approximately 1700 adjacent nucleotides, at least approximately 1800 adjacent nucleotides, at least approximately 1900 adjacent nucleotides, at least approximately 2000 adjacent nucleotides, at least approximately 2100 adjacent nucleotides, at least approximately 2200 adjacent nucleotides, at least approximately 2300 adjacent nucleotides, at least approximately 2400 adjacent nucleotides, at least approximately 2500 adjacent nucleotide,It may include at least about 2,600 adjacent nucleotides, at least about 2,700 adjacent nucleotides, at least about 2,800 adjacent nucleotides, at least about 2,900 adjacent nucleotides, at least about 3,000 adjacent nucleotides, or more. In a given embodiment, a fragment of any of SEQ NOs 1 to 6 has the activity of any of SEQ NOs 1 to 6 in relation to reducing the effect of the first transgene expression cassette on the expression of the second transgene cassette in a transgenic plant.

[0078] Works

[0079] As used herein, the term “composition” means any recombinant DNA molecule, such as a plasmid, cosmid, virus, phage, or linear or circular DNA or RNA molecule derived from any source capable of genome integration or autonomous replication, comprising at least one DNA molecule linked to another DNA molecule in a functionally operable manner, i.e., operablely linked DNA molecule. As used herein, the term “vector” means any composition that can be used for the purpose of transformation, i.e., introduction of heterologous DNA or RNA into a host cell. A “vector stack” is a vector consisting of two or more cassettes stacked together for transformation. The two or more transgenic expression cassettes in a vector stack are separated by fragments of DNA sequences ranging from approximately 10 nucleotides to approximately several hundred nucleotides, or several thousand nucleotides, or as little as greater than these, depending on the method of cloning or synthesis used to construct the vector stack. As used herein, “expression cassette” refers to a DNA molecule comprising one or more regulatory elements, typically at least a promoter and at least a transcriptional DNA molecule operably linked to a 3’ UTR.

[0080] As used herein, the term “operably linked” refers to a first DNA molecule linked to a second DNA molecule, wherein the first DNA molecule and the second DNA molecule are arranged such that the first DNA molecule influences the function of the second DNA molecule. The two DNA molecules may or may not be part of a single adjacent DNA molecule, and may or may not be adjacent. For example, if a promoter regulates the transcription of a DNA molecule of interest that is transcribable in a cell, the promoter is operably linked to the transcribable DNA molecule. A leader is operably linked to a DNA sequence, for example, when it can influence the transcription or translation of a DNA sequence.

[0081] Methods for assembling and introducing constructs into cells in such a manner that a transcription-capable DNA molecule is translated as a protein and transcribed into a functional mRNA molecule that is expressed are known in the art. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are fully known to those skilled in the art. Conventional vectors successful for the expression of nucleic acids in higher plants are fully known in the art, and Agrobacterium tumopaciens ( Agrobacterium tumefaciens It includes a vector derived from the Ti plasmid of ) and a pCaMVCN-carrying control vector.

[0082] Various regulatory elements may be included in the composition, including those provided herein. Any such regulatory elements may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce a desired regulatory feature. In one embodiment, the composition of the present invention comprises at least one regulatory element operably linked to a transcribable DNA molecule operably linked to a 3' UTR.

[0083] The composition of the present invention may include any promoter or reader provided herein or known in the art. For example, the promoter of the present invention may be operably linked to a heterogeneous untranslated 5' reader, such as one derived from the heat shock protein gene. Alternatively, the reader of the present invention may be operably linked to a heterogeneous promoter, such as the cauliflower mosaic virus 35S transcriptome promoter.

[0084] Transcriptional DNA molecules

[0085] As used herein, the term “transcribable DNA molecule” refers to any DNA molecule that can be transcribed into an RNA molecule, including, by non-limiting example, having a protein-coding sequence and preparing an RNA molecule having a sequence useful for gene repression. The types of DNA molecules may 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, such as DNA molecules containing an antisense message of a gene, or DNA molecules encoding an artificial, synthetic, or otherwise modified version of a transposable gene. Exemplary transcriptionable DNA molecules for incorporation into the composition of the present invention include, for example, DNA molecules or genes from a species other than the species into which the DNA molecule is incorporated, or genes originating from or present in the same species but incorporated into recipient cells by genetic engineering methods rather than traditional breeding techniques.

[0086] "Transposable gene" refers to a transcribable DNA molecule that is heterogeneous to the host cell with respect to at least its location in the host cell genome and / or transcriptionable DNA molecule, which is artificially incorporated into the host cell genome in a current or any prior cell generation. In certain embodiments, the transposable gene includes a gene of agricultural interest, such as a gene capable of providing herbicide resistance in a plant, or a gene capable of providing plant pest resistance in a plant.

[0087] A regulatory element, such as a promoter, can be operably linked to a transcriptional DNA molecule that is heterogeneous with respect to the regulatory element. As used herein, the term “heterogeneity” refers to a combination of two or more DNA molecules where such combination is not usually found in nature. For example, the two DNA molecules may be of different species and / or, or the two DNA molecules may be of different genes, for example, different genes from the same species or the same gene from different species. The regulatory element is heterogeneous with respect to the transcriptional DNA molecule to which it is operably linked when such combination is not usually found in nature, that is, when the transcriptional DNA molecule operably linked to the regulatory element does not occur in nature.

[0088] As used herein, “recombinant DNA molecule” is a DNA molecule comprising a combination of DNA molecules that do not occur together in nature without human intervention. For example, a recombinant DNA molecule may be a DNA molecule composed of at least two DNA molecules that are heterogeneous with respect to each other, a DNA molecule comprising a DNA sequence different from a DNA sequence existing in nature, a DNA molecule comprising a synthetic DNA sequence, or a DNA molecule incorporated into the DNA of a host cell by genetic transformation or gene editing.

[0089] A transcriptional DNA molecule can generally be any DNA molecule for which the expression of a transcript is desired. Such expression of the transcript can lead to the translation of the generated mRNA molecule and, consequently, protein expression. Alternatively, for example, a transcriptional DNA molecule can be designed to ultimately cause a reduction in the expression of a specific gene or protein. In one embodiment, this can be achieved using a transcriptional DNA molecule oriented in an antisense direction. Those skilled in the art are familiar with using such antisense techniques. Any gene can be negatively regulated in this way, and in one embodiment, a transcriptional DNA molecule can be designed for the repression of a specific gene through the expression of dsRNA, siRNA, or miRNA molecules.

[0090] Selectable markers

[0091] 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 any transcribable DNA molecule whose expression or lack thereof in a transgenic plant, tissue, or cell may be screened or scored in some manner. Selectable marker genes for use in the practice of the present invention, and associated selection and screening techniques are known in the art and include, but are not limited to, transcribable DNA molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins conferring antibiotic resistance, and proteins conferring herbicide tolerance. Examples of selectable marker transgenes are provided as SEQ ID NO. 18 and SEQ ID NO. 26.

[0092] Cell transformation

[0093] The present invention also relates to a method for producing transformed cells and plants comprising one or more regulatory elements operably linked to a transcription-capable DNA molecule.

[0094] The term “transformation” refers to the introduction of DNA molecules into a recipient host. As used herein, the term “host” refers to a bacterium, fungus, or plant, including any cell, tissue, organ, or offspring of the bacterium, fungus, or plant. Plant tissues and cells of particular interest include protoplasts, calluses, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0095] As used herein, the term “transformed” refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct or vector stack, has been introduced. The introduced DNA molecule may be incorporated into the genomic DNA of a recipient cell, tissue, organ, or organism so that the introduced DNA molecule is inherited by subsequent offspring. “Transformed” or “transformed” cells or organisms may also include offspring produced from a breeding program that uses such transformed organisms as parents in the offspring and mating of cells or organisms and exhibits an altered phenotype resulting from the presence of the foreign DNA molecule. The introduced DNA molecule may also be temporarily introduced into recipient cells so that the introduced DNA molecule is not inherited by subsequent offspring. The term “transformed” refers to bacteria, fungi, or plants containing one or more heterogeneous DNA molecules.

[0096] There are many methods fully known to those skilled in the art for introducing DNA molecules into plant cells. The process generally includes the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining the transformed host cell. In the practice of the present invention, methods and materials for transforming plant cells by introducing a plant construct into the plant genome may include any fully known and proven methods. Suitable methods include, among others, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., by PEG-mediated transformation, dehydration / inhibition-mediated DNA influx, electroporation, agitation by silicon carbide fibers, and acceleration of DNA-coated particles), and gene editing (e.g., CRISPR-Cas systems), but are not limited to these.

[0097] The host cell may be any cell or organism, such as a plant cell, an algal cell, an algae, a fungal cell, a fungus, a bacterial cell, or an insect cell. In specific embodiments, the host cell and the transformed cell may include cells from a crop.

[0098] Transgenic plants can subsequently be regenerated from the transgenic plant cells of the present invention. Seeds can be produced from these transgenic plants using conventional breeding techniques or self-pollination. These seeds and the offspring plants produced by growing from these seeds will contain the recombinant DNA molecules of the present invention and will therefore be transgenic.

[0099] The transgenic plants of the present invention may be self-pollinated to provide seeds for the homozygous transgenic plants of the present invention (homozygous to the recombinant DNA molecule), or crossed with non-transgenic plants or different transgenic plants to provide seeds for the heterozygous transgenic plants of the present invention (heterozygous to the recombinant DNA molecule). Both such homozygous plants and heterozygous transgenic plants are referred to herein as "offspring plants." Offspring plants are transgenic plants derived from the original transgenic plants and containing the recombinant DNA molecule of the present invention. Seeds produced using the transgenic plants of the present invention may be harvested and used to grow a generation of transgenic plants containing the crop of the present invention and expressing a gene of agronomic interest, i.e., offspring plants of the present invention. Descriptions of breeding methods commonly used for different crops may be found in one of several reference books, for example, Allard, Principles of Plant Breeding , John Wiley& Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Improvement , Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant Breeding Perspectives , Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses , 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development , Theory and Technique (Vol. 1) and Crop Species Soybean See (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0100] Transformed plants may be analyzed for the presence of genes or genes of interest and for expression levels and / or profiles conferred by the regulatory elements of the present invention. Those skilled in the art are aware of many methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blot or Northern blot, PCR-based approaches, biochemical analyses, phenotypic screening methods, validation tests, and immunodiagnostic assays. The expression of transcriptional DNA molecules may be measured using TaqMan® (Applied Biosystems, Foster City, California) reagents and methods as described by the manufacturer, and PCR cycle counts may be determined using the TaqMan® Testing Matrix. Alternatively, Invader® (Third Wave Technologies, Wisconsin) reagents and methods as described by the manufacturer may be used to evaluate transgenic expression.

[0101] The present invention also provides a plant part of the present invention. The plant part includes, but is not limited to, leaves, stems, roots, tubers, seeds, endosperms, ovules, and pollen. The plant part of the present invention may be viable, non-viable, regenerative, and / or non-regenerative. The present invention also includes and provides a transformed plant cell comprising a DNA molecule of the present invention. The transformed or transformed plant cell of the present invention includes a regenerative plant cell and / or a non-regenerative plant cell.

[0102] The present invention also provides a raw material product prepared from a transgenic plant or a part thereof containing the recombinant DNA molecule of the present invention. The raw material product of the present invention contains a detectable amount of DNA containing a DNA sequence selected from the group consisting of SEQ ID NOs 1 to 6. As used herein, "raw material product" refers to any composition or product made of a material derived from a transgenic plant, seed, plant cell, or plant part containing the recombinant DNA molecule of the present invention. The raw material product includes, but is not limited to, processed seeds, grains, plant parts, and coarse flour. The raw material product of the present invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecule of the present invention. One or more detections of this DNA in a sample may be used to determine the content or source of the raw material product. Any standard method of detection for DNA molecules, including the detection method disclosed herein, may be used.

[0103] The present invention may be more easily understood through reference to the following examples, which are provided in an illustrative manner unless otherwise specified and are not intended to be a limitation of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function fully in the practice of the present invention. However, those skilled in the art should understand that, in light of the present disclosure, many modifications may be made to the specific embodiments disclosed, and similar or similar results may still be obtained without departing from the spirit or scope of the invention, and therefore all objects described or illustrated in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

[0104] Examples

[0105] Example 1

[0106] Design, synthesis, and cloning of intergene sequence region elements

[0107] Synthetic intergene sequence region elements ("ISRs") were designed by computer using algorithmic methods. Each ISR was designed not to contain any possible Open Reading Frame (ORF) that would inappropriately produce unwanted proteins after insertion into the plant genome. Furthermore, many ISRs were designed to contain stop codons at the 5' and 3' ends of the ISR, arranged in such a way that they provide stop codons in all six reading frames.

[0108] Once designed, the ISR was chemically synthesized and cloned between the transgene expression cassettes in a heterogeneous vector stack. Well over 100 synthetic intergene sequence region elements were designed and analyzed in stably transformed maize and soybean plants to identify synthetic ISRs that reduced the interaction between the first transgene cassette and the second transgene cassette.

[0109] The designed and tested ISRs are presented in Table 1. ISR4_stop is a variant of ISR4, where the stop codon is attached to the 3' and 5' ends of ISR4.

[0110]

[0111] The synthetic intergene sequence region elements presented as sequence numbers 1 to 6 demonstrated the ability to reduce the interaction of the first transgene cassette with the second transgene cassette in a vector stack in stably transformed corn and soybean plants as presented in Examples 2 and 3.

[0112] Example 2

[0113] Reduction of transgenic expression cassette interactions by ISR4_cessation, ISR89, ​​and ISR97 in stably transformed maize plants

[0114] This example demonstrates the ability of ISR ISR4_stop, ISR89, ​​and ISR97 to reduce transgene expression cassette interactions when inserted between the first transgene expression cassette and the second transgene expression cassette of a vector stack used to stably transform corn plants.

[0115] Maize plants were transformed by a binary plant transformation vector stack containing an ISR and two transgene expression cassettes oriented differently between the two transgene expression cassettes to evaluate the ability of the ISR to reduce transgene expression cassette interactions. Two control vector stacks were also transformed into maize plants and tested.

[0116] One control vector stack (Fig. 1a, control without enhancer) included a first transgene expression cassette comprising P-Os.Act1:67 (Sequence No. 11) 5' operably linked to a promoter and a leader, L-Ta.Lhcb1:1 (Sequence No. 12) 5' operably linked to an intron, I-Os.Act1-1:1:19 (Sequence No. 13) 5' operably linked to a coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:3 (Sequence No. 14) 5' operably linked to a 3' UTR, and T-Ta.Hsp17-1:1:1 (Sequence No. 15). The second transgene expression cassette, cloned with a different orientation to the first transgene expression cassette, included P-Zm.39486-1:1:1 (SEQ No. 16), 5' operably linked to the seed-specific promoter and reader; L-Zm.39486-1:1:1 (SEQ No. 17), 5' operably linked to the intron; I-Zm.DnaK:1 (SEQ No. 18), 5' operably linked to the coding sequence encoding GUS-1; GOI-Ec.uidA+St.LS1.nno:1 (SEQ No. 19), 5' operably linked to the 3' UTR; and T-Os.Mth-1:1:1 (SEQ No. 20). The control group without the enhancer vector stack also included an additional transgene expression cassette used for the selection of transformed cells using glyphosate selection.

[0117] Another control vector stack (Fig. 1b, control with enhancer) comprises E-CaMV.35S.2xA1-B3-1:1:1 (SEQ No. 10), containing a tandem repeat of a specific enhancer region derived from the cauliflower mosaic virus 35S promoter, 5' operably linked to a strong enhancer and promoter; P-Os.Act1:67 (SEQ No. 11), 5' operably linked to a reader; L-Ta.Lhcb1:1 (SEQ No. 12), 5' operably linked to an intron; I-Os.Act1-1:1:19 (SEQ No. 13), 5' operably linked to a coding sequence for neomycin phosphotransferase; and CR-Ec.nptII-Tn5-1:1:3 (SEQ No. 14), 5' operably linked to a 3' UTR. It included a first transgene expression cassette containing T-Ta.Hsp17-1:1:1 (sequence number 15). A second transgene expression cassette cloned with a different orientation to the first transgene expression cassette contained a seed-specific promoter and was the same transgene expression cassette as described above. The control group with the enhancer vector stack also included an additional transgene expression cassette used for the selection of transformed cells using glyphosate selection.

[0118] To analyze the effectiveness of ISRs in reducing the interaction between the first transgene expression cassette and the second transgene expression cassette, ISRs ISR4_stop (SEQ No. 1), ISR89 (SEQ No. 2), ISR97 (SEQ No. 5), ISR88 (SEQ No. 7), and ISR86 (SEQ No. 8) were cloned between the first transgene expression cassette and the second transgene expression cassette of the control group having an enhancer vector stack as illustrated in FIG. 1c. LH244 maize plant cells were transformed using an Agrobacterium-mediated transformation method similar to those known in the art, with two control vector stacks and five vector stacks containing ISRs. The transformed plant cells were induced to form whole plants.

[0119] Qualitative and quantitative GUS analyses were used to evaluate the activity of expressed elements in selected plant organs and tissues of transgenic plants. For the qualitative analysis of GUS expression by histochemical staining, whole-tissue specimens or sectioned tissues were incubated with a GUS staining solution containing 1 mg / mL of X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) at 37°C for 5 hours and destained with 35% EtOH and 50% acetic acid. GUS expression was qualitatively determined by visual examination of selected plant organs or tissues for excision or blue staining under a compound microscope. For the quantitative analysis of GUS expression by enzymatic assay, total protein was extracted from selected tissues of transgenic maize plants. 1 to 2 micrograms of total protein were incubated with 4-methylumbelliferyl-β-D-glucuronide (MUG), a fluorescent substrate, at a concentration of 1 mM in a total reaction volume of 50 microliters. After incubation at 37°C for 1 hour, the reaction was stopped by adding 350 microliters of 200 mM sodium bicarbonate solution. The reaction product, 4-methylumbelliferone (4-MU), exhibits maximum fluorescence at high pH, ​​where the hydroxyl group is ionized. The addition of the basic sodium carbonate solution simultaneously stops the assay and adjusts the pH to quantify the fluorescent product 4-MU. The amount of 4-MU formed was estimated by measuring fluorescence using a FLUOstar Omega Microplate Reader (BMG LABTECH) (excitation at 355 nm, emission at 460 nm). GUS activity values ​​are provided in nmole of 4-MU / hour / mg total protein.

[0120] The following tissues were sampled for GUS expression in the R0 generation: V3 stage leaves and roots at 21 days after pollination (DAP); V7 stage leaves and roots; VT stage leaves, roots, anthers, and silks; and R3 stage seed embryos and seed endosperms. Table 2 shows the average GUS expression in plant growth, reproductive, and seed tissues, where "bdl" indicates that GUS expression is lower than the detection level. Table 3 shows the average GUS expression in plant growth and reproductive tissues. The control group with the enhancer is considered to represent the total interaction between the first transgene expression cassette enhancer and the seed-specific promoter of the second transgene expression cassette. Therefore, the average plant growth and reproductive tissue expression from the GUS cassette driven by P-Zm.39486-1:1:1, a seed-specific promoter influenced by the strong constitutive enhancer of the first transgene expression cassette, exhibits 100% leakage. The percentage leakage of a vector stack containing an ISR was determined by dividing the average GUS expression in the plant growth and reproductive tissues of plants transformed by the construct containing the ISR by the average GUS expression in the plant growth and reproductive tissues of a control group with an enhancer, and multiplying the result by 100.

[0121]

[0122]

[0123] As can be seen in Table 2, the control group with the enhancer demonstrated high GUS expression in all tissues of stably transgenic maize plants compared to the control group without the enhancer. This demonstrates that the strong enhancer in the first transgene expression cassette transformed the seed-specific expression pattern of the second transgene expression cassette into a more constitutive expression pattern.

[0124] As shown in Table 2, the interaction of strong enhancers from the first transgene expression cassette to the second transgene expression cassette was reduced when ISR ISR4_stop, ISR89, ​​and ISR97 were inserted between the cassettes. The average GUS expression in plant growth and germ tissues in vector stacks containing ISR4_stop, ISR89, ​​and ISR97 was significantly lower than that of the control group with the enhancer vector. The percentage leakage of ISR4_stop, ISR89, ​​and ISR97 was 16%, 8%, and 6%, respectively, providing a reduction of 84%, 92%, and 94% in the interaction between the two transgene expression cassettes, respectively. In comparison, ISR88 and ISR86 were much more leakage (61% and 32%, respectively), but reduced the interaction between the two transgene expression cassettes by only 39% and 68%, respectively.

[0125] ISR4_stop (sequence number 1), ISR89 (sequence number 2), and ISR97 (sequence number 5) demonstrated the ability to reduce the interaction between the first transgenic expression cassette and the second transgenic expression cassette in a vector stack in stably transformed maize plants.

[0126] Example 3

[0127] Reduction of transgenic expression cassette interactions by ISR2 and ISR4 in stably transformed soybean plants

[0128] This example demonstrates the ability of intergene sequence region elements, ISR2 and ISR4, to reduce transgene expression cassette interactions when inserted between the first transgene expression cassette and the second transgene expression cassette of a vector stack used to stably transform soybean plants.

[0129] Soybean plants were transformed by a binary plant transformation vector stack containing two transgene expression cassettes with an ISR and different orientations between the two transgene expression cassettes to evaluate the ability of the ISR to reduce transgene expression cassette interactions. Two control vector stacks were also transformed into soybean plants and tested.

[0130] One control vector stack (Fig. 2a, control without enhancer) contained a single transgene expression cassette consisting of a seed-specific promoter, P-Gm.Sphas1:14 (SEQ No. 25) 5' operably linked to a leader, L-Gm.Sphas1-1:1:1 (SEQ No. 26) 5' operably linked to a coding sequence encoding GUS-2, GOI-GUS:1:2 (SEQ No. 27) 5' operably linked to a 3' UTR, and T-Mt.AC145767v28:3 (SEQ No. 28). The control without enhancer vector stack also contained an additional transgene expression cassette used for the selection of cells transformed using antibiotic selection.

[0131] Another control vector stack (Fig. 2b, control with enhancer) contained two transgene expression cassettes with different orientations. The first transgene cassette contained a strong promoter derived from the Scrophularia mosaic virus 35S promoter with rearranged and duplicated enhancers, P-FMV.35S-enh-1:1:2 (SEQ No. 21) 5' operably linked to the leader, L-Ph.DnaK-1:1:3 (SEQ No. 22) 5' operably linked to the coding sequence for neomycin phosphotransferase, CR-Ec.nptII-Tn5-1:1:2 (SEQ No. 23) 5' operably linked to the 3' UTR, and T-Mt.AC139600v16:1 (SEQ No. 24). The second transgene expression cassette was identical to the seed-specific transgene expression cassette described above. The control group with the enhancer vector stack also included an additional transgene expression cassette used for the selection of transformed cells using antibiotic selection.

[0132] To analyze the effectiveness of ISRs in reducing the interaction between the first transgene expression cassette and the second transgene expression cassette, ISRs ISR2 (SEQ No. 3), ISR4 (SEQ No. 2), ISR69 (SEQ No. 6), and ISR_X (SEQ No. 8) were cloned between the first transgene expression cassette and the second transgene expression cassette of a control group having an enhancer vector stack as illustrated in FIG. 2c. Soybean plant cells of variety A3555 were transformed using an Agrobacterium-mediated transformation method similar to those known in the art by a control group without an enhancer, a control group with an enhancer, and a three-vector stack containing ISRs. The transformed plant cells were induced to form whole plants.

[0133] Qualitative and quantitative GUS analyses were performed as previously described in Example 2. The following tissues were sampled for GUS expression in the R0 generation: Vn5 root, Vn5 submerged leaf, Vn5 basal leaf, R1 basal leaf, R1 petiole, R1 flower, R3 immature seed, R3 pod, R5 cotyledon, yellow pod (YP) embryo, and yellow pod (YP) cotyledon.

[0134] The control group with the enhancer is considered to represent the total interaction between the first transgene expression cassette enhancer and the seed-specific promoter of the second transgene expression cassette. Therefore, the average plant growth and reproductive tissue expression from the GUS cassette driven by P-Gm.Sphas1:14, a seed-specific promoter influenced by the strong constitutive enhancer of the first transgene expression cassette, exhibits 100% leakage. The percentage leakage of the compost containing ISR was determined by dividing the average GUS expression in the Vn5, R1, and R3 tissues of plants transformed by the compost containing ISR by the average GUS expression in the Vn5, R1, and R3 tissues of the control group with the enhancer, and multiplying the result by 100.

[0135] The average GUS expression of Vn5, R1, and R3 tissues is presented in Table 4, where "nd" indicates that it is not determined. The average GUS expression of R5 and yellow pod tissues, the average expression of Vn5, R1, and R3 tissues, and the percentage leakage are presented in Table 5.

[0136]

[0137]

[0138] As shown in Table 4, very low GUS expression is observed in Vn5, R1, and R3 tissues in plants transformed by the control without the enhancer. Plants transformed by the control with the enhancer demonstrate a constitutive expression pattern, with high GUS expression observed in Vn5, R1, and R3 tissues. Similarly, as shown in Table 5, plants transformed by the control without the enhancer exhibit only high GUS expression in yellow pod embryos and cotyledons, consistent with the known seed-specific expression pattern of P-Gm.Sphas1:14. Very low expression is observed in R5 cotyledons, where expression appears slightly increased compared to R3 immature seeds. Plants transformed by the control with the enhancer show high expression levels in R5 cotyledons and an increase in yellow pod embryos and cotyledons compared to plants transformed by the control without the enhancer. Thus, the strong enhancer contained in the P-FMV.35S-enh-1:1:2 promoter of the first transgenic expression cassette of the control group having the enhancer interacted with the seed-specific expression of P-Gm.Sphas1:14 of the second transgenic expression cassette and altered it into a constitutive expression pattern.

[0139] As demonstrated in Table 5, the intergene sequence regions ISR2, ISR4, and ISR69 were able to reduce the interaction of the first transgene expression cassette to the second transgene expression cassette of the control with the enhancer configuration by 97%, 96%, and 95%, respectively (leaking out by only 3%, 4%, and 5%). ISR_X was not as effective in reducing the interaction of the first transgene expression cassette to the second transgene expression cassette of the control with the enhancer configuration and exhibited 24% leakage. ISR_X reduced the interaction by 76% compared to 97%, 96%, and 95% for only ISR2, ISR4, and ISR69.

[0140] ISR2 (sequence number 3), ISR4 (sequence number 4), and ISR69 (sequence number 6) demonstrated the ability to reduce the interaction between the first transgenic expression cassette and the second transgenic expression cassette in stably transformed soybean plants.

[0141] Although the principles of the present invention are illustrated and described, it should be clear to those skilled in the art that the arrangement and details of the present invention may be modified without departing from such principles. The inventors claim all modifications within the spirit and scope of the claims. All publications and published patent documents cited herein are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

Claims

Claim 1 A recombinant DNA molecule comprising a DNA sequence consisting of SEQ ID NO. 1 or SEQ ID NO.

4. Claim 2 In claim 1, the DNA sequence is a recombinant DNA molecule inserted between the first expression cassette and the second expression cassette in a vector stack. Claim 3 A transformed plant cell comprising the recombinant DNA molecule of claim 1. Claim 4 In paragraph 3, the transformed plant cell is a transformed plant cell that is a monocotyledonous plant cell. Claim 5 In paragraph 3, the transformed plant cell is a transformed plant cell that is a dicotyledonous plant cell. Claim 6 A transgenic plant or a part thereof comprising the recombinant DNA molecule of claim 1. Claim 7 A progeny plant or part thereof of the transgenic plant of paragraph 6, wherein the progeny plant or part thereof comprises a recombinant DNA molecule. Claim 8 As a transgenic seed, the seed is a transgenic seed comprising the recombinant molecule of claim 1. Claim 9 A method for manufacturing a commodity product, comprising the steps of obtaining a transgenic plant or a part thereof according to claim 6 and manufacturing a commodity product therefrom. Claim 10 A method according to claim 9, wherein the raw material product is selected from the group consisting of seeds, processed seeds, concentrated protein, isolated protein, starch, grains, plant parts, seed oil, biomass, wheat flour, and coarse meal. Claim 11 A method for reducing the interaction between a first transgene expression cassette and a second transgene expression cassette within a transgenic plant transformed by a vector stack, the method comprising the steps of: a. transforming a plant cell by a vector stack comprising: a first transgene expression cassette; b. a second transgene cassette; c. a recombinant DNA molecule of claim 1, wherein the DNA molecule comprises a heterogeneous transfer DNA (T-DNA) comprising a recombinant DNA molecule inserted between the first transgene expression cassette and the second transgene expression cassette; and d. regenerating a transgenic plant from the transformed plant cell. Claim 12 In claim 11, the DNA molecule of sequence number 1 or sequence number 4 is inserted within the vector stack between the first transgene expression cassette and the second transgene expression cassette. Claim 13 delete Claim 14 delete Claim 15 delete

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