Plant regulatory elements and uses thereof

Abstract

The present invention provides recombinant DNA molecules and constructs for modulating gene expression in plants, as well as their nucleotide sequences. The invention also provides transgenic plants, plant cells, plant parts, and seeds comprising recombinant DNA molecules operably linked to heterologous transcribable DNA molecules, as well as methods of their use.

Description

[0001] This application is a divisional application of the invention patent application with application date of August 2, 2019, application number 201980042761.4, and invention name “Plant regulatory elements and their uses”.

[0002] Citation of Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 714,228, filed on August 3, 2018, which is incorporated herein by reference in its entirety.

[0004] Incorporation of Sequence Listing

[0005] The sequence list contained in the file named "38-21-62691-0001_Seqlist_ST25.txt" is 31,060 bytes (as Microsoft ), created on July 2, 2019, and submitted electronically together and incorporated herein by reference. Technical Field

[0006] The present invention relates to the fields of plant molecular biology and plant genetic engineering. More specifically, the present invention relates to DNA molecules for regulating gene expression in plants. Background Art

[0007] Regulatory elements are genetic elements that regulate gene activity by regulating the transcription of an operably linked transcribable DNA molecule. Such elements may include promoters, leader sequences, introns, and 3' untranslated regions, and are used in the fields of plant molecular biology and plant genetic engineering. Summary of the invention

[0008] The present invention provides novel synthetic gene regulatory elements for plants. The present invention also provides recombinant DNA molecule constructs comprising the regulatory elements. The present invention also provides transgenic plant cells, plants and seeds comprising the regulatory elements. In one embodiment, the regulatory elements are operably connected to a transcribable DNA molecule. In certain embodiments, the transcribable DNA molecule can be heterologous relative to the regulatory sequence. Therefore, in specific embodiments, the regulatory element sequence provided by the present invention can be defined as being operably connected to a heterologous transcribable DNA molecule. The present invention also provides methods for using regulatory elements and preparing and using recombinant DNA molecules comprising the regulatory elements and transgenic plant cells, plants and seeds comprising the regulatory elements operably connected to transcribable DNA molecules.

[0009] Thus, in one aspect, the present invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85% sequence identity to any one of SEQ ID NOs: 1-19 and SEQ ID NO: 26; (b) a sequence comprising any one of SEQ ID NOs: 1-19 and SEQ ID NO: 26; and (c) a fragment of any one of SEQ ID NOs: 1-19 and SEQ ID NO: 26, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable DNA molecule. "Heterologous transcribable DNA molecule" means that the transcribable DNA molecule is heterologous with respect to the polynucleotide sequence to which it is operably linked. 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 one of SEQ ID NO: 1-19 and SEQ ID NO: 26. In a specific embodiment, the DNA sequence comprises a regulatory element. In some embodiments, the regulatory element comprises a promoter. In yet other embodiments, the regulatory element comprises an intron. In yet other embodiments, the regulatory element comprises a 3′UTR. In yet other embodiments, the heterologous transcribable DNA molecule comprises a gene of agronomic 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. In yet other embodiments, the heterologous transcribable DNA molecule comprises a sequence encoding a small RNA such as a dsRNA, miRNA or siRNA. In still other embodiments, the present invention provides a construct comprising a recombinant DNA molecule as provided herein.

[0010] In another aspect, provided herein is a transgenic plant cell comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85% sequence identity to any one of SEQ ID NO: 1-19 and SEQ ID NO: 26; (b) a sequence comprising any one of SEQ ID NO: 1-19 and SEQ ID NO: 26; and (c) a fragment of any one of SEQ ID NO: 1-19 and SEQ ID NO: 26, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA molecule. In certain embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[0011] In yet another aspect, the present invention also provides a transgenic plant or part thereof, which comprises a recombinant DNA molecule containing a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-19 and SEQ ID NO: 26; b) a sequence comprising any one of SEQ ID NO: 1-19 and SEQ ID NO: 26; and c) a fragment of any one of SEQ ID NO: 1-19 and SEQ ID NO: 26, wherein the fragment has gene regulatory activity; wherein the sequence is operably linked to a heterologous transcribable DNA molecule. In a specific embodiment, the transgenic plant is a progeny plant of any generation comprising the recombinant DNA molecule. The present invention also provides a transgenic seed comprising the recombinant DNA molecule, which produces such a transgenic plant when grown.

[0012] In another aspect, the present invention provides a method for producing a commodity, the method comprising obtaining a transgenic plant or part thereof containing the recombinant DNA molecule of the present invention, and producing a commodity from the transgenic plant or part thereof. In one embodiment, the commodity is seed, processed seed, protein concentrate, protein isolate, starch, grain, plant part, seed oil, biomass, flour and meal.

[0013] In yet another aspect, the present invention provides a method for producing a transgenic plant comprising a recombinant DNA molecule of the present invention, the method comprising transforming a plant cell with the recombinant DNA molecule of the present invention to produce a transformed plant cell, and regenerating a transgenic plant from the transformed plant cell.

[0014] Sequence Description

[0015] SEQ ID NO: 1 is the DNA sequence of a synthetic regulatory expression element set (EXP) EXP-Zm.GSP850, which comprises a synthetic promoter (P-Zm.GSP850.nno:4) operably linked 5' to a synthetic leader sequence (L-Zm.GSP850.nno:3).

[0016] SEQ ID NO:2 is the DNA sequence of the synthetic promoter P-Zm.GSP850.nno:4.

[0017] SEQ ID NO:3 is the DNA sequence of the synthetic leader sequence L-Zm.GSP850.nno:3.

[0018] SEQ ID NO:4 is the DNA sequence of the synthetic EXP EXP-Zm.GSP850.nno+Zm.GSI153.nno:2, which comprises a synthetic promoter (P-Zm.GSP850.nno:4), which is 5′ operably linked to a synthetic leader sequence (L-Zm.GSP850.nno:3), and the leader sequence is 5′ operably linked to a synthetic intron (I-Zm.GSI153.nno:1).

[0019] SEQ ID NO:5 is the DNA sequence of synthetic intron I-Zm.GSI153.nno:1.

[0020] SEQ ID NO:6 is the DNA sequence of the synthetic EXP EXP-Zm.GSP990, comprising a synthetic promoter (P-Zm.GSP990.nno:2) operably linked 5' to a synthetic leader sequence (L-Zm.GSP990.nno:1).

[0021] SEQ ID NO:7 is the DNA sequence of the synthetic promoter P-Zm.GSP990.nno:2.

[0022] SEQ ID NO:8 is the DNA sequence of the synthetic leader sequence L-Zm.GSP990.nno:1.

[0023] SEQ ID NO:9 is the DNA sequence of synthetic EXP EXP-Zm.GSP990.nno+Zm.GSI197.nno:2, which comprises a synthetic promoter (P-Zm.GSP990.nno:2), which is 5′ operably linked to a synthetic leader sequence (L-Zm.GSP990.nno:1), and the synthetic leader sequence is 5′ operably linked to a synthetic intron (I-Zm.GSI197.nno:1).

[0024] SEQ ID NO:10 is the DNA sequence of synthetic intron I-Zm.GSI197.nno:1.

[0025] SEQ ID NO:11 is the DNA sequence of the synthetic EXP EXP-Zm.GSP850.nno+Zm.GSI140.nno:1, which comprises a synthetic promoter (P-Zm.GSP850.nno:4), which is 5′ operably linked to a synthetic leader sequence (L-Zm.GSP850.nno:3), and the synthetic leader sequence is 5′ operably linked to a synthetic intron (I-Zm.GSI140.nno:1).

[0026] SEQ ID NO:12 is the DNA sequence of synthetic intron I-Zm.GSI140.nno:1.

[0027] SEQ ID NO: 13 is the DNA sequence of the synthetic 3'UTR T-Zm.GST9.nno:2.

[0028] SEQ ID NO:14 is the DNA sequence of the synthetic 3'UTR T-Zm.GST18.nno:2.

[0029] SEQ ID NO:15 is the DNA sequence of synthetic EXP EXP-Zm.GSP850.nno+Zm.DnaK:1, which comprises a synthetic promoter (P-Zm.GSP850.nno:4), which is 5′ operably linked to a synthetic leader sequence (L-Zm.GSP850.nno:3), and the synthetic leader sequence is 5′ operably linked to an intron (I-Zm.DnaK:1).

[0030] SEQ ID NO:16 is the DNA sequence of synthetic EXP EXP-Zm.GSP990.nno+Zm.DnaK:1, which comprises a synthetic promoter (P-Zm.GSP990.nno:2), which is 5′ operably linked to a synthetic leader sequence (L-Zm.GSP990.nno:1), and the synthetic leader sequence is 5′ operably linked to an intron (I-Zm.DnaK:1).

[0031] SEQ ID NO: 17 is the DNA sequence of the synthetic enhancer E-Zm.GSP850, which is derived from the synthetic promoter P-Zm.GSP850.nno:4.

[0032] SEQ ID NO: 18 is the DNA sequence of the synthetic enhancer E-Zm.GSP990, which is derived from the synthetic promoter P-Zm.GSP990.nno:2.

[0033] SEQ ID NO: 19 is the DNA sequence of the 3'UTR T-Sb.Nltp4-1:1:2, which is derived from the NLTP4 (non-specific lipid transfer protein 4) gene from Sorghum bicolor.

[0034] SEQ ID NO: 20 is a synthetic coding sequence optimized for plant expression of β-glucuronidase (GUS) with a processable intron derived from the potato light-inducible, tissue-specific ST-LS1 gene (Genbank Accession No.: X04753).

[0035] SEQ ID NO:21 is the DNA sequence of EXP EXP-CaMV.35S, which comprises a 35S promoter and a leader sequence derived from cauliflower mosaic virus.

[0036] SEQ ID NO:22 is the DNA sequence of intron I-Zm.DnaK:1, which is derived from the heat shock protein 70 (Hsp70) gene (DnaK) from maize (Zeamays).

[0037] SEQ ID NO:23 is the DNA sequence of 3'UTR T-Os.LTP:1, which is derived from the lipid transfer protein-like gene (LTP) from rice (Oryza sativa).

[0038] SEQ ID NO: 24 is the coding sequence of β-glucuronidase (GUS) with a processable intron derived from the potato light-inducible, tissue-specific ST-LS1 gene (Genbank Accession No.: X04753).

[0039] SEQ ID NO:25 is The coding sequence of the luciferase fluorescent protein (Promega, Madison, WI 53711) Nluc was engineered by directed evolution of luciferase from deep-sea shrimp (Oplophorus gacilirostris).

[0040] SEQ ID NO:26 is the DNA sequence of the synthetic 3'UTR T-Zm.GST43.nno:1. DETAILED DESCRIPTION

[0041] The present invention provides synthetic regulatory elements with gene regulatory activity in plants. The nucleotide sequences of these synthetic regulatory elements are provided as SEQ ID NOs: 1-18 and SEQ ID NO: 26. These synthetic regulatory elements can affect the expression of transcribable DNA molecules operably linked in plant tissues, and thus regulate the gene expression of transgenics operably linked in transgenic plants. The present invention also provides novel endogenous regulatory elements with gene regulatory activity in plants, and is provided as SEQ ID NO: 19. The present invention also provides methods for modifying, producing and using recombinant DNA molecules containing the provided synthetic and endogenous regulatory elements. The present invention also provides compositions comprising transgenic plant cells, plants, plant parts and seeds containing the recombinant DNA molecules of the present invention, as well as methods for preparing and using the compositions.

[0042] The following definitions and methods are provided to better define the present invention and guide those of ordinary skill in the art to practice the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those of ordinary skill in the relevant art.

[0043] DNA molecules

[0044] As used herein, the term "DNA" or "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e., a polymer or DNA molecule of deoxynucleotide bases read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to the nomenclature of Title 37 of Section 1.822 of the U.S. Code of Federal Regulations, and is listed in the tables in Tables 1 and 3 of Appendix 2 of WIPO Standard ST.25 (1998).

[0045] As used herein, a "recombinant DNA molecule" is a DNA molecule that contains a combination of DNA molecules that would not naturally exist together without human intervention. For example, a recombinant DNA molecule can be a DNA molecule composed of at least two DNA molecules that are relatively heterologous to each other, a DNA molecule that contains a DNA sequence that deviates from a DNA sequence that occurs in nature, a DNA molecule that contains a synthetic DNA sequence or DNA molecule that has been incorporated into the DNA of a host cell by genetic transformation or gene editing.

[0046] As used herein, a "synthetic nucleotide sequence" or "artificial nucleotide sequence" is a nucleotide sequence that is known not to exist in nature or does not exist naturally. The gene regulatory element of the present invention comprises a synthetic nucleotide sequence. Preferably, the synthetic nucleotide sequence shares little or no extended homology with the natural sequence. In this context, extended homology generally refers to 100% sequence identity extending over a continuous sequence of about 25 nucleotides.

[0047] In the present application, reference to "isolated DNA molecules" or equivalent terms or phrases is intended to mean that the DNA molecule is a DNA molecule that exists alone or in combination with other compositions but is not in its natural environment. For example, nucleic acid elements naturally present in the DNA of the genome of an organism, such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc. are not considered to be "isolated", as long as the elements are in the genome of the organism and in the position of the genome in which they naturally occur. However, each of these elements and sub-portions of these elements will be "isolated" within the scope of the present disclosure, as long as the elements are not in the genome of the organism and are not in the position of the genome in which they naturally occur. Similarly, the nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of the protein will be an isolated nucleotide sequence, as long as the nucleotide sequence is not in the DNA of a bacterium in which the sequence encoding the protein is naturally present. For the purposes of the present disclosure, a synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered to be isolated. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA that is inserted into the genome of a cell of a plant or bacterium or that is present in an extrachromosomal vector, whether it is present in a plasmid or similar construct used to transform cells, in the genome of a plant or bacterium, or in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacterium, is considered an isolated nucleotide sequence.

[0048] 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. An optimal sequence alignment is generated by manually aligning two sequences, such as a reference sequence and another sequence, to maximize the number of nucleotide matches in the sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term "reference sequence" refers to the DNA sequences provided as SEQ ID NOs: 1-19 and SEQ ID NO: 26.

[0049] As used herein, the term "percent sequence identity" or "percent identity" or "% identity" is the identity score multiplied by 100. The "identity score" for a sequence optimally aligned to a reference sequence is the number of nucleotide matches in the optimal alignment divided by the total number of nucleotides in the reference sequence, e.g., the total number of nucleotides throughout the entire length of the reference sequence. Thus, one embodiment of the 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 to a reference sequence when optimally aligned with the reference sequence provided herein as SEQ ID NOs: 1-19 and SEQ ID NO: 26. A DNA molecule having a percentage sequence identity to a reference molecule can exhibit the activity of the reference sequence.

[0050] Regulatory elements

[0051] Regulatory elements, such as promoters, leader sequences (also known as 5'UTRs), enhancers, introns, and transcription termination regions (or 3'UTRs) play an indispensable role in the overall expression of genes in living cells. As used herein, the term "regulatory element" refers to a DNA molecule with gene regulatory activity. As used herein, the term "gene regulatory activity" refers to the ability to affect the expression of an operably linked transcribable DNA molecule, for example, by affecting the transcription and / or translation of the operably linked transcribable DNA molecule. Regulatory elements, such as promoters, leader sequences, enhancers, introns, and 3'UTRs play a role in plants to modify plant phenotypes through genetic engineering.

[0052] As used herein, "regulatory expression element group" or "EXP" sequence can refer to a group of operably linked regulatory elements, such as enhancers, promoters, leader sequences and introns. For example, the regulatory expression element group can consist of, for example, a promoter operably linked 5' to a leader sequence. EXPs used to implement the present invention include SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16.

[0053] Regulatory elements can be characterized by their gene expression patterns, for example, positive and / or negative effects, such as constitutive expression or time, space, development, organization, environment, physiology, pathology, cell cycle and / or chemical responsiveness expression, and any combination thereof and by quantitative or qualitative indication. As used herein, "gene expression pattern" is any pattern of RNA molecules transcribed by operably connected DNA molecules. The RNA molecules transcribed can be translated to produce protein molecules, or antisense or other regulatory RNA molecules can be provided, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA) etc.

[0054] As used herein, the term "protein expression" is any pattern of translation of transcribed RNA molecules into protein molecules. Protein expression can be characterized by its temporal, spatial, developmental or morphological properties, as well as quantitative or qualitative indications.

[0055] Promoter can be used as a regulatory element for regulating the expression of operably connected transcribable DNA molecules. As used herein, the term "promoter" generally refers to a DNA molecule that participates in recognition and binding RNA polymerase II and other proteins (such as trans-acting transcription factors) to initiate transcription. The promoter can be initially separated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. Alternatively, the promoter can be a synthetically produced or manipulated DNA molecule. The promoter can also be chimeric. Chimeric promoters are produced by the fusion of two or more heterologous DNA molecules. Promoters for implementing the present invention include promoter elements or fragments or variants thereof provided with SEQ ID NO:2 and 7 or included in any one of SEQ ID NO:1,4,6,9,11,15 and 16. In a specific embodiment of the present invention, the claimed DNA molecules as described herein and any variants or derivatives thereof are further defined as comprising promoter activity, i.e., being able to act as promoters in host cells such as transgenic plants. In still other specific embodiments, the fragment may be defined as exhibiting the promoter activity possessed by the initiator promoter molecule from which it is derived, or the fragment may comprise a "minimal promoter" that provides a basal level of transcription and consists of a TATA box or an equivalent DNA sequence for recognizing and binding the RNA polymerase II complex to initiate transcription.

[0056] In one embodiment, a fragment of an EXP sequence or promoter sequence disclosed herein is provided. As described above, a promoter fragment may contain promoter activity and may be used alone or in combination with other promoters and promoter fragments, such as for constructing a chimeric promoter, or in combination with other expression elements and expression element fragments. In a specific embodiment, a fragment of a promoter is provided, the fragment comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900 or at least about 1000 or longer continuous nucleotides of a DNA molecule having promoter activity as disclosed herein. Methods for generating such fragments from a starting promoter molecule are well known in the art.

[0057] In other embodiments, a fragment of an enhancer or intron sequence disclosed herein is provided. An enhancer or intron fragment may include the activity of the basic molecule derived therefrom, and may be used alone or in combination with other regulatory elements, including promoters, leader sequences, other enhancers, other introns or their fragments. In a specific embodiment, an enhancer or intron fragment is provided, the fragment comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900 or at least about 1000 or longer continuous nucleotides of a DNA molecule having an enhancer or intron activity as disclosed herein. The method for producing such a fragment from a starting molecule is well known in the art.

[0058] In other embodiments, a fragment of a 3'UTR sequence disclosed herein is provided. The 3'UTR fragment may include the activity of the basic 3'UTR molecule derived therefrom, and may be used alone or in combination with other regulatory elements, including promoters, leader sequences, introns or their fragments. In a specific embodiment, a fragment of an intron is provided, the fragment comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900 or at least about 1000 or longer continuous nucleotides of a DNA molecule having 3'UTR activity as disclosed herein. The method of producing such a fragment from an initial 3'UTR molecule is well known in the art.

[0059] Compositions derived from any of the promoter elements provided as SEQ ID NOs: 2 and 7 or contained within any of SEQ ID NOs: 1, 4, 6, 9, 11, 15, and 16, such as internal or 5' deletions, for example, can be generated using methods known in the art to improve or alter expression, including by removing elements that have a positive or negative effect on expression; duplication of elements that have a positive or negative effect on expression; and / or duplication or removal of elements that have a tissue-specific or cell-specific effect on expression. Compositions derived from any of the promoter elements provided as SEQ ID NOs: 2 and 7 or contained within any of SEQ ID NOs: 1, 4, 6, 9, 11, 15, and 16, comprising a 3' deletion in which the TATA box element or its equivalent and downstream sequences are removed, can be used, for example, to prepare enhancer elements. Further deletions can be made to remove any element that has a positive or negative; tissue-specific; cell-specific or time-specific (such as but not limited to circadian) effect on expression. Any of the promoter elements provided as SEQ ID NOs: 2 and 7 or contained within any of SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16, and fragments or enhancers derived therefrom, can be used to prepare chimeric transcriptional regulatory element compositions.

[0060] According to the present invention, the presence of known promoter elements, i.e. DNA sequence characteristics, such as TATA boxes and other known transcription factor binding site motifs, of promoters or promoter fragments can be analyzed. The identification of such known promoter elements can be used by those skilled in the art to design promoter variants with expression patterns similar to the original promoter.

[0061] As used herein, the term "leader sequence" refers to a DNA molecule isolated from the non-translated 5' region (5'UTR) of a gene, and is generally defined as a nucleotide segment between a transcription start site (TSS) and a protein coding sequence start site. Alternatively, the leader sequence can be a synthetically produced or manipulated DNA element. The leader sequence can be used as a 5' regulatory element for regulating the expression of an operably linked transcribable DNA molecule. The leader sequence molecule can be used with a heterologous promoter or with its native promoter. The leader sequence for implementing the present invention includes SEQ ID NO: 3 and 8; or any one of the leader sequence elements or its fragments or variants contained in any one of SEQ ID NO: 1, 4, 6, 9, 11, 15 and 16. In a specific embodiment, such DNA sequences can be defined as being able to act as leader sequences in host cells, including, for example, transgenic plant cells. In one embodiment, such sequences are decoded as comprising leader sequence activity.

[0062] The leader sequence (also referred to as 5'UTR) presented in any of SEQ ID NOs: 3 and 8 or the leader sequence elements contained within any of SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16 may consist of a regulatory element or may adopt a secondary structure that may affect the transcription or translation of an operably linked transcribable DNA molecule. According to the present invention, the leader sequence presented in any of SEQ ID NOs: 3 and 8 or the leader sequence elements contained within any of SEQ ID NOs: 1, 4, 6, 9, 11, 15 and 16 may be used to prepare a chimeric regulatory element that affects the transcription or translation of an operably linked transcribable DNA molecule.

[0063] As used herein, the term "intron" refers to a DNA molecule that can be isolated or identified from a gene, and can generally be defined as a region spliced ​​out during the processing of the messenger RNA (mRNA) prior to translation. Alternatively, an intron can be a synthetically produced or manipulated DNA element. An intron can contain an enhancer element that affects the transcription of an operably linked gene. An intron can be used as a regulatory element for regulating the expression of an operably linked transcribable DNA molecule. A construct can include an intron, and the intron may be heterologous or may not be heterologous relative to the transcribable DNA molecule. Examples of introns in this area include rice actin introns and corn HSP70 introns.

[0064] In plants, relative to constructs lacking introns, some introns are included in gene constructs to cause increased accumulation of mRNA and protein. This effect has been referred to as "intron-mediated enhancement" (IME) of gene expression. In corn genes (e.g. tubA1, Adh1, Sh1 and Ubil), rice genes (e.g. tpi) and dicot genes (e.g. dicot genes from petunia (e.g. rbcS), potato (e.g. st-ls1) and from Arabidopsis thaliana (e.g. ubq3 and pat1)), introns known to stimulate expression in plants have been identified. It has been shown that deletions or mutations in intron splicing sites reduce gene expression, which suggests that IME may require splicing. However, IME in dicots has been shown by point mutations in the splicing sites from Arabidopsis thaliana pat1 gene. It has been shown that repeatedly using the same intron in a plant exhibits disadvantages. In those cases, it is necessary to have a collection of basic control elements to construct appropriate recombinant DNA elements. Exemplary introns useful in practicing the present invention are presented in SEQ ID NOs: 5, 10, and 12.

[0065] As used herein, the term "3' transcription termination molecule", "3' untranslated region" or "3'UTR" refers to a DNA molecule used when transcribing into the untranslated region of the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule can be produced by specific cleavage and 3' polyadenylation (also known as a polyA tail). The 3'UTR can be operably linked to and located downstream of the transcribable DNA molecule, and can include a polyadenylation signal and other regulatory signals that can affect transcription, mRNA processing or gene expression. The polyA tail is believed to play a role in mRNA stability and translation initiation. Examples of 3' transcription termination molecules in the art are the nopaline synthase 3' region, the wheat hsp17 3' region, the pea rubisco small subunit 3' region, the cotton E6 3' region, and the coixin 3'UTR.

[0066] 3'UTRs are often found to be useful for the recombinant expression of specific DNA molecules. A weak 3'UTR may produce read-through, which may affect the expression of DNA molecules located in adjacent expression cassettes. Appropriate control of transcription termination can prevent read-through into DNA sequences located downstream (e.g., other expression cassettes), and can further allow efficient recycling of RNA polymerase to improve gene expression. Efficient termination of transcription (releasing RNA polymerase II from DNA) is a prerequisite for transcription reinitiation and therefore directly affects overall transcript levels. After transcription termination, mature mRNA is released from the synthesis site and the template is transported to the cytoplasm. Eukaryotic mRNA accumulates in the form of poly (A) in vivo, making it difficult to detect transcription termination sites by conventional methods. However, it is difficult to predict functional and efficient 3'UTRs by bioinformatics methods because there is no conserved DNA sequence that allows easy prediction of effective 3'UTRs.

[0067] From an implementation perspective, it is generally beneficial for the 3′UTR used in the expression cassette to have the following properties. First, the 3′UTR should be able to efficiently and effectively terminate transcription of the transgene and prevent the transcript from reading through into any adjacent DNA sequence that may consist of another expression cassette, such as in the case of multiple expression cassettes residing in one transfer DNA (T-DNA) or adjacent chromosomal DNA into which the T-DNA has been inserted. Second, the 3′UTR should not cause a reduction in transcriptional activity conferred by the promoter, leader sequence, enhancer, and intron used to drive expression of the DNA molecule. Finally, in plant biotechnology, the 3′UTR is often used to initiate amplification reactions of reverse transcribed RNA extracted from transformed plants and is used to: (1) evaluate the transcriptional activity or expression of the expression cassette when integrated into the plant chromosome; (2) evaluate the number of copies inserted into the plant DNA; and (3) evaluate the zygosity of the resulting seeds after breeding. The 3′UTR is also used in amplification reactions of DNA extracted from transformed plants to characterize the integrity of the inserted cassette. The 3′UTR used to implement the present invention is presented in SEQ ID NOs: 13, 14, 19, and 26.

[0068] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting regulatory element, also known as a cis-element, which confers aspects of the overall expression pattern, but is often insufficient to drive transcription of an operably linked transcribable DNA molecule alone. Unlike promoters, enhancer elements often do not contain a transcription start site (TSS) or a TATA box or equivalent DNA sequence. A promoter or promoter fragment may naturally contain one or more enhancer elements that affect the transcription of an operably linked DNA sequence. Enhancer elements may also be fused to a promoter to produce a chimeric promoter cis-element, which confers aspects of the overall regulation of gene expression.

[0069] It is believed that many promoter enhancer elements bind DNA binding proteins and / or affect DNA topology, thereby generating a local conformation that selectively allows or restricts RNA polymerase to approach the DNA template or promotes the selective opening of the double helix at the transcription start site. Enhancer elements can play a role in binding transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors can interact with more than one enhancer domain with different affinities. Enhancer elements can be identified by a variety of techniques, including deletion analysis, i.e., one or more nucleotides are deleted from the 5′ end or inside of the promoter; DNA binding protein analysis of in vivo genomic footprints using DNase I footprints, methylation interference, electrophoretic mobility shift assays, polymerase chain reaction (PCR) mediated by ligation and other conventional assays or by using known cis-element motifs or enhancer elements as target sequences or target motifs and conventional DNA sequence comparison methods (such as BLAST) DNA sequence similarity analysis. The fine structure of the enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods known in the art. Enhancer elements can be obtained by chemical synthesis or by separation from regulatory elements containing such elements, and they can be synthesized together with additional flanking nucleotides containing available restriction enzyme sites to facilitate subsequence manipulation. Therefore, the present invention encompasses the design, construction and use of enhancer elements for regulating the expression of operably linked transcribable DNA molecules according to the methods disclosed herein. Exemplary enhancers for practicing the present invention are presented in SEQ ID NOs: 17 and 18.

[0070] As used herein, the term "chimeric" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein neither the first DNA molecule nor the second DNA molecule usually exists in this configuration, i.e., fused to each other. Therefore, a chimeric DNA molecule is a new DNA molecule that does not normally exist in nature in other ways. As used herein, the term "chimeric promoter" refers to a promoter produced by the manipulation of such a DNA molecule. A chimeric promoter can combine two or more DNA fragments, for example, a fusion of a promoter and an enhancer element. Therefore, the present invention encompasses the design, construction, and use of chimeric promoters for regulating the expression of operably linked transcribable DNA molecules according to the methods disclosed herein.

[0071] Chimeric regulatory elements can be designed to include various constituent elements that can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation of independent clones, modular assembly of PCR products during amplification, or direct chemical synthesis of regulatory elements, as well as other methods known in the art. The resulting various chimeric regulatory elements can be composed of the same constituent elements or variants of the same constituent elements, but these constituent elements differ in one or more DNA sequences, which contain one or more connecting DNA sequences that allow the components to be operably linked. In the present invention, the DNA sequences provided as SEQ ID NOs: 1-19 and SEQ ID NO: 26 can provide regulatory element reference sequences, wherein the constituent elements containing the reference sequences can be linked by methods known in the art, and can contain substitutions, deletions and / or insertions of one or more nucleotides or mutations naturally occurring in bacterial and plant cell transformation.

[0072] As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, which is similar but not identical to the first DNA molecule in composition, and wherein the second DNA molecule still retains the general function of the first DNA molecule, i.e., the same or similar expression pattern, for example, by more or less equivalent transcriptional activity. The variant can be a shorter or truncated version of the first DNA molecule or an altered version of the sequence of the first DNA molecule, such as a variant with different restriction enzyme sites and / or internal deletions, substitutions or insertions. "Variants" can also encompass regulatory elements having a nucleotide sequence comprising substitutions, deletions or insertions of one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equivalent transcriptional activity or translational activity than the corresponding parent regulatory molecule. Regulatory element "variants" will also encompass variants produced by naturally occurring mutations in bacterial and plant cell transformation. In the present invention, the polynucleotide sequences provided with SEQ ID NOs: 1-19 and SEQ ID NO: 26 can be used to generate variants similar but not identical in composition to the DNA sequence of the initial regulatory element, while the variants still retain the general function of the initial regulatory element, i.e., the same or similar expression pattern. The generation of such variations of the present invention is well within the skill of one of ordinary skill in the art in light of the present disclosure and are encompassed within the scope of the present invention.

[0073] The efficacy of the modifications, duplications or deletions described herein on the desired expression of a particular transgene can be tested empirically in stable and transient plant assays, such as those described in the working examples herein, to verify results which can vary depending on the changes made and the target of the changes in the starting DNA molecule.

[0074] Construct

[0075] As used herein, the term "construct" 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 genomic integration or autonomous replication, comprising DNA molecules, wherein at least one DNA molecule has been linked to another DNA molecule in a functionally operable manner, i.e., operably linked. As used herein, the term "vector" means any construct that can be used for the purpose of transformation (i.e., the introduction of heterologous DNA or RNA into a host cell). The construct typically comprises one or more expression cassettes. As used herein, "expression cassette" refers to a DNA molecule that comprises at least a transcribable DNA molecule that is operably linked to one or more regulatory elements (usually at least a promoter and 3'UTR).

[0076] As used herein, the term "operably connected" refers to a first DNA molecule that is connected to a second DNA molecule, wherein the first DNA molecule and the second DNA molecule are arranged so that the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may be a part of a single continuous DNA molecule or may not be a part of a single continuous DNA molecule, and may be adjacent or may not be adjacent. For example, if the promoter regulates the transcription of the interested transcribable DNA molecule in the cell, the promoter is operably connected to the transcribable DNA molecule. For example, when the leader sequence can affect the transcription or translation of a DNA sequence, the leader sequence is operably connected to the DNA sequence.

[0077] In one embodiment, the construct of the present invention can be provided as a double tumor-inducing (Ti) plasmid border construct having a right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) region of a Ti plasmid isolated from Agrobacterium tumefaciens, the region comprising T-DNA, the T-DNA together with the transfer molecule provided by the Agrobacterium tumefaciens cell, allows the T-DNA to be integrated into the genome of the plant cell (see, e.g., U.S. Pat. No. 6,603,061). The construct may also comprise a plasmid backbone DNA segment that provides replication function and antibiotic selection in bacterial cells, e.g., an Escherichia coli replication origin (such as ori322), a broad host range replication origin (such as oriV or oriRi), and a coding region for a selectable marker (such as Spec / Strp), the coding region encoding a Tn7 aminoglycoside adenyltransferase (aadA) or gentamicin (Gm, Gent) selectable marker gene that confers resistance to spectinomycin or streptomycin. For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, or LBA4404, however other strains known to those skilled in the art of plant transformation may also function in the present invention.

[0078] Methods for assembling constructs and introducing them into cells in such a way that transcribable DNA molecules are transcribed into functional mRNA molecules that are translated and expressed as proteins are known in the art. Conventional compositions and methods for preparing and using constructs and host cells for practicing the present invention are well known to those skilled in the art. Typical vectors for expressing nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN transfer control vector.

[0079] Various regulatory elements may be included in the construct, including any regulatory element provided herein. Any such regulatory element may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce desirable regulatory characteristics. In one embodiment, the construct of the present invention comprises at least one regulatory element operably linked to a transcribable DNA molecule, which is operably linked to a 3'UTR.

[0080] Constructs of the present invention may comprise any promoter or leader sequence provided herein or known in the art. For example, a promoter of the present invention may be operably linked to a heterologous non-translated 5' leader sequence, such as a 5' leader sequence derived from a heat shock protein gene. Alternatively, a leader sequence of the present invention may be operably linked to a heterologous promoter, such as a cauliflower mosaic virus 35S transcript promoter.

[0081] The expression cassette may also include a transit peptide coding sequence encoding a peptide for subcellular targeting of an operably linked protein, particularly targeting chloroplasts, leucoplasts or other plastid organelles; mitochondria; peroxisomes; vacuoles; or peptides of extracellular locations. Many chloroplast localization proteins are expressed by nuclear genes as precursors and targeted to chloroplasts by chloroplast transit peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those chloroplast proteins associated with ribulose-1,5-bisphosphate carboxylase small subunit (SSU), ferredoxin, ferredoxin oxidoreductase, light-harvesting complex protein I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS). Chloroplast transit peptides are described in, for example, U.S. Patent No. 7,193,133. It has been demonstrated that non-chloroplast proteins can be targeted to chloroplasts by expression of heterologous CTPs operably linked to transgenic genes encoding non-chloroplast proteins.

[0082] Transcriptable DNA molecules

[0083] As used herein, the term "transcribable DNA molecule" refers to any DNA molecule that can be transcribed into an RNA molecule, including but not limited to DNA molecules having protein coding sequences and DNA molecules that produce RNA molecules having sequences for gene suppression. The types of DNA molecules may include but are not limited to DNA molecules from the same plant, DNA molecules from another plant, DNA molecules from different organisms, or synthetic DNA molecules, such as DNA molecules containing antisense information for a gene or DNA molecules encoding artificial, synthetic, or otherwise modified transgenic types. Exemplary transcribable DNA molecules for incorporation into the constructs of the present invention include, for example, DNA molecules or genes from species other than the species into which the DNA molecule is incorporated, or genes derived from the same species or present in the same species but incorporated into recipient cells by genetic engineering methods rather than classical breeding techniques.

[0084] "Transgene" refers to a transcribable DNA molecule that is heterologous to the host cell, at least with respect to its location in the host cell genome, and / or that was artificially incorporated into the genome of the host cell in the current or any previous generation of the host cell.

[0085] Regulatory elements, such as promoters of the present invention, can be operably linked to transcribable DNA molecules that are heterologous to the regulatory elements. As used herein, the term "heterologous" refers to a combination of two or more DNA molecules, but this combination does not usually exist in nature. For example, the two DNA molecules can be derived from different species, and / or the two DNA molecules can be derived from different genes, for example, different genes from the same species or the same gene from different species. Therefore, if this combination does not normally exist in nature, that is, the transcribable DNA molecule does not naturally occur to be operably linked to the regulatory element, then the regulatory element is heterologous to the transcribable DNA molecule that is operably linked.

[0086] The transcribable DNA molecule can be generally any DNA molecule that needs to express a transcript. The expression of this transcript can make the translation of the mRNA molecule obtained, and therefore make protein expression. Alternatively, for example, a transcribable DNA molecule can be designed to eventually cause the expression of a specific gene or protein to decrease. In one embodiment, this can be achieved by using a transcribable DNA molecule directed in an antisense direction. Those of ordinary skill in the art are familiar with using this antisense technology. Any gene can be negatively regulated in this way, and in one embodiment, a transcribable DNA molecule can be designed to suppress a specific gene by expressing dsRNA, siRNA or miRNA molecules.

[0087] Thus, one embodiment of the present invention is a recombinant DNA molecule comprising a regulatory element of the present invention, such as the regulatory elements provided in SEQ ID NOs: 1-19 and SEQ ID NO: 26, operably linked to a heterologous transcribable DNA molecule so as to regulate transcription of the transcribable DNA molecule at a desired level or in a desired pattern when the construct is integrated into the genome of a transgenic plant cell. In one embodiment, the transcribable DNA molecule comprises a protein coding region of a gene, and in another embodiment, the transcribable DNA molecule comprises an antisense region of a gene.

[0088] Genes of interest in agronomy

[0089] The transcribable DNA molecule can be the interested gene of agronomy.As used herein, term " the interested gene of agronomy " refers to when expressing in specific plant tissue, cell or cell type, the transcribable DNA molecule of ideal characteristic is given.The product of the interested gene of agronomy can work in plant, to cause the impact on plant morphology, physiology, growth, development, yield, cereal composition, nutritional profile, disease or pest resistance and / or environment or chemical tolerance, or can serve as pesticide in the diet of the insect that feeds on plant.In one embodiment of the invention, regulating and controlling element of the present invention is mixed in the construct so that the regulating and controlling element is operably connected to the transcribable DNA molecule as the interested gene of agronomy.In the transgenic plant containing this construct, the expression of the interested gene of agronomy can give useful agronomy traits. For example, beneficial agronomic traits may include, but are not limited to, herbicide tolerance, insect control, improved yield, disease resistance, pathogen resistance, improved plant growth and development, improved starch content, improved oil content, improved fatty acid content, improved protein content, improved fruit ripening, enhanced animal and human nutrition, biopolymer production, environmental stress resistance, pharmaceutical peptides, improved processing quality, improved flavor, hybrid seed production utility, improved fiber production, and desirable biofuel production.

[0090] Non-limiting examples of genes of agronomic interest known in the art include genes for herbicide resistance (U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increased yield (U.S. Pat. Nos. USRE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; and 5,7 16,837), insect control (U.S. Patent Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,61 5; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658; 5,880,275; 5,763,245; and 5,763,241), fungal disease resistance (U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,96 2), virus resistance (U.S. Patent Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (U.S. Patent No. 6,228,992), bacterial disease resistance (U.S. Patent No. 5,516,671), plant growth and development (U.S. Patent Nos. 6,723,897 and 6,518,488), starch yield (U.S. Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), improved oil yield (U.S. Patent Nos. 6,444,876; 6,426,447;and 6,380,462), high oil yield (U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), improved fatty acid content (U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,46 1; and 6,459,018), high protein yield (U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Pat. Nos. USRE37,543; 6,228,623; and 5,958,74 5 and 6,946,588), resistance to environmental stress (U.S. Pat. No. 6,072,103), pharmaceutical peptides and secretory peptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing properties (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No. 6,531,648), low raffinose (U.S. Pat. No. 6,166,292), industrial enzyme production (U.S. Pat. No. 6,080,560), and improved digestibility (U.S. Pat. No. 6,531,648). No. 5,543,576), improved flavor (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,229,114), hybrid seed yield (U.S. Patent No. 5,689,041), fiber yield (U.S. Patent Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720), and biofuel yield (U.S. Patent No. 5,998,700).

[0091] Alternatively, the gene of agronomic interest can affect the above-mentioned plant characteristics or phenotypes by encoding an RNA molecule that causes targeted regulation of gene expression of an endogenous gene, for example, by antisense (see, e.g., U.S. Pat. No. 5,107,065); inhibitory RNA ("RNAi", including regulation of gene expression by miRNA-mediated, siRNA-mediated, trans-acting siRNA-mediated, and staged sRNA-mediated mechanisms, for example, as described in published applications U.S. 2006 / 0200878 and U.S. 2008 / 0066206 and U.S. Patent Application No. 11 / 974,469); or co-suppression-mediated mechanisms. The RNA can also be a catalytic RNA molecule (e.g., a ribozyme or riboswitch; see, e.g., U.S. 2006 / 0200878) that is engineered to cleave a desired endogenous mRNA product. Methods for constructing a construct and introducing it into a cell in such a way that a transcribable DNA molecule is transcribed into a molecule capable of causing gene inhibition are known in the art.

[0092] Selectable markers

[0093] Selectable marker transgenics can also be used together with the regulatory elements of the present invention. As used herein, the term "selectable marker transgenics" refers to any transcribable DNA molecules that can be screened or scored in a certain way to express or lack in transgenic plants, tissues or cells. Selectable marker genes for implementing the present invention and their related selection and screening techniques are known in the art, and include but are not limited to transcribable DNA molecules encoding beta-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic resistance, and proteins that confer herbicide tolerance. Examples of selectable marker transgenics are provided with SEQ ID NOs: 20 and 24.

[0094] Cell transformation

[0095] The present invention also relates to methods of producing transformed cells and plants comprising one or more regulatory elements operably linked to a transcribable DNA molecule.

[0096] The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" refers to bacteria, fungi or plants, including any cell, tissue, organ or progeny of bacteria, fungi or plants. Specific plant tissues and cells of interest include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos and pollen.

[0097] As used herein, the term "transformation" refers to a cell, tissue, organ or organism into which a foreign DNA molecule (such as a construct) has been introduced. The introduced DNA molecule can be integrated into the genomic DNA of the recipient cell, tissue, organ or organism so that the introduced DNA molecule is inherited by subsequent progeny. A "transgenic" or "transformed" cell or organism can also include progeny of the cell or organism, as well as progeny produced from a breeding program that uses such a transgenic organism as a parent in hybridization and exhibits a phenotype that is altered by the presence of the foreign DNA molecule. The introduced DNA molecule can also be transiently introduced into the recipient cell so that the introduced DNA molecule is not inherited by subsequent progeny. The term "transgenic" refers to a bacterium, fungus or plant containing one or more heterologous DNA molecules.

[0098] There are many methods well known to those skilled in the art for introducing DNA molecules into plant cells. The process generally comprises the following steps: selecting a suitable host cell, transforming the host cell using a vector, and obtaining the transformed host cell. In the implementation of the present invention, the methods and materials for transforming plant cells by introducing plant constructs into the plant genome may include any well-known and proven methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., by PEG-mediated transformation, drying / inhibition-mediated DNA uptake, electroporation, acceleration using silicon carbide fiber stirring and DNA-coated particles), gene editing (e.g., CRISPR-Cas systems), etc.

[0099] The host cell can 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 can include cells from a crop plant.

[0100] Transgenic plants can then be regenerated from the transgenic plant cells of the present invention. Seeds can be produced from such transgenic plants using conventional breeding techniques or self-pollination. Such seeds and progeny plants grown from such seeds will contain the recombinant DNA molecules of the present invention, and therefore they will be transgenic.

[0101] Transgenic plant of the present invention can be self-pollinated to provide the seed of homozygous transgenic plant of the present invention (homozygote of recombinant DNA molecule) or hybridized with non-transgenic plant or different transgenic plant to provide the seed of heterozygous transgenic plant of the present invention (heterozygote of recombinant DNA molecule). Such homozygous transgenic plant and heterozygous transgenic plant are all referred to as "progeny plant" in this article. Progeny plant is the transgenic plant derived from the initial transgenic plant, and comprises the recombinant DNA molecule of the present invention. The seed produced by transgenic plant of the present invention can be harvested, and used for planting the generations of transgenic plant, i.e., progeny plant of the present invention, which comprises construct of the present invention and expresses the gene of interest of agronomy. Descriptions of breeding methods commonly used for different crops can be found in one of several reference books, see, for example, Allard, Principles of Plant Breeding, John Wiley and 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 (eds.), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd ed., Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0102] The transformed plants can be analyzed for the presence of the gene or genes of interest and the expression level and / or distribution conferred by the regulatory elements of the present invention. Those skilled in the art are aware of many methods that can be used to analyze transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blot or Northern blot, PCR-based methods, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays. Expression of transcribable DNA molecules was measured using ELISA (Applied Biosystems, Foster City, CA) reagents and methods described by the manufacturer and using The test matrix determines the number of PCR cycles. Alternatively, (Third Wave Technologies, Madison, WI) reagents and methods as described by the manufacturer can be used to assess transgene expression.

[0103] The present invention also provides parts of the plant of the present invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperms, ovules and pollen. Plant parts of the present invention can be viable, non-viable, regenerative and / or non-regenerative. The present invention also includes and provides transformed plant cells containing the DNA molecules of the present invention. Transformed plant cells or transgenic plant cells of the present invention include regenerative and / or non-regenerative plant cells.

[0104] The present invention also provides a commodity produced by a transgenic plant or part thereof containing the recombinant DNA molecule of the present invention. The commodity of the present invention comprises a detectable amount of DNA, and the DNA comprises a DNA sequence selected from the group consisting of SEQ ID NO: 1-19 and SEQ ID NO: 26. As used herein, "commodity" refers to any composition or product composed of materials derived from a transgenic plant, seed, plant cell or plant part containing the recombinant DNA molecule of the present invention. Commodities include but are not limited to processed seeds, grains, plant parts and meal. The commodity 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 such DNA in a sample can be used to determine the content or source of the commodity. Any standard DNA molecular detection method can be used, including the detection method disclosed herein.

[0105] The present invention can be more easily understood by reference to the following examples, which are provided in an illustrative manner and are not intended to limit the present invention unless specified. It will be appreciated by those skilled in the art that the technology disclosed in the following examples represents the technology found by the inventors to work well in the practice of the present invention. However, according to the present disclosure, it will be appreciated by those skilled in the art that many changes can be made in the specific embodiments disclosed and still obtaining similar or similar results without departing from the spirit and scope of the present invention, so all materials listed should be interpreted as illustrative, rather than limiting.

[0106] Example

[0107] Example 1

[0108] Design, synthesis and cloning of synthetic regulatory elements

[0109] Novel synthetic transcriptional regulatory elements are synthetic expression elements designed by algorithmic methods. These computationally designed regulatory elements are chemically synthesized and cloned to prepare synthetic regulatory expression element sets (EXPs). More than 1,000 synthetic regulatory elements were designed and assayed in corn protoplasts and stably transformed corn plants to identify those synthetic regulatory elements that provide desired properties (such as protein expression levels and expression patterns). The synthetic elements of the present invention provide various modes of constitutive expression, which are used to drive the expression of many different coding sequences and interfere with RNAs of interest in agronomy.

[0110] The designed synthetic transcriptional regulatory elements do not have extended homology to any known nucleic acid sequence found in nature, but affect the transcription of an operably linked coding sequence in the same manner as naturally occurring promoters, leader sequences, introns, and 3'UTRs. The synthetic EXPs and their corresponding synthetic promoters, leader sequences and introns, as well as the synthetic 3'UTR are presented in Table 1. The synthetic EXPs were cloned into a binary plant transformation vector using methods known in the art, operably linked to a β-glucuronidase (GUS) coding sequence, and the expression level and pattern in stably transformed maize plants were evaluated.

[0111] Analysis of regulatory element TSS and intron / exon splicing junctions can be performed using transformed plant tissues. Briefly, plants are transformed using a plant expression vector comprising a cloned DNA fragment operably linked to a heterologous transcribable DNA molecule. Next, the DNA sequence of the generated mRNA transcripts is analyzed using the 5′ RACE system version 2.0 for rapid amplification of cDNA ends (Invitrogen, Carlsbad, California 92008) to confirm regulatory element TSS and intron / exon splicing junctions. The synthetic 3′ UTR is characterized with respect to its effect on gene expression and proper termination of transcripts.

[0112] In addition to synthetic expression elements, provided herein is a novel endogenous 3'UTR T-Sb.Nltp4-1:1:2 derived from the Sorghum bicolor nonspecific lipid transfer protein 4 gene and provided as SEQ ID NO: 19. T-Sb.Nltp4-1:1:2 was characterized in a similar manner to the synthetic 3'UTR.

[0113] Table 1. Synthetic transcriptional regulatory expression element set, promoter, leader sequence, intron and 3′UTR.

[0114]

[0115]

[0116] Example 2

[0117] Analysis of regulatory elements driving GUS synthesis in maize leaf protoplasts

[0118] Vectors, particularly expression vectors containing test regulatory elements driving expression of a beta-glucuronidase (GUS) transgene, are used to transform corn leaf protoplasts. The resulting transformed corn leaf protoplasts are analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0119] Expression vectors containing synthetic expression elements were used to transform corn protoplasts derived from leaf tissue. The expression levels and patterns of these synthetic expression element vectors in corn protoplasts were compared with those of expression elements known in the art. Separate experiments were performed to evaluate the activity of EXP (EXP-Zm.GSP850 (SEQ ID NO: 1) and EXP-Zm.GSP990 (SEQ ID NO: 6)); introns (I-Zm.GSI153.nno: 1 (SEQ ID NO: 5) and I-Zm.GSI197.nno: 1 (SEQ ID NO: 10)); and 3′UTR (T-Zm.GST9.nno: 2 (SEQ ID NO: 13) and T-Zm.GST18.nno: 2 (SEQ ID NO: 14)). The expression element was cloned into an expression vector and operably linked to the GUS coding sequence GOI-Ec.uidA+St.LS1:1:1 (SEQ ID NO:24) containing a processable intron. A control expression vector contained different configurations of known expression elements, which varied depending on the type of element being evaluated (EXP, intron or 3'UTR). A plasmid for co-transformation of protoplasts and data normalization was also constructed using methods known in the art. It contained a transgene cassette consisting of EXP EXP-CaMV.35S (SEQ ID NO:21) operably linked 5' to the coding sequence The coding sequence for the luciferase fluorescent protein (Promega, Madison, WI 53711), referred to herein as Nluc (SEQ ID NO: 25), was operably linked 5' to the 3' UTR T-Os.LTP: 1 (SEQ ID NO: 23).

[0120] Corn leaf protoplasts were transformed using a PEG-based transformation method similar to methods known in the art. Protoplast cells were transformed into a ninety-six (96) well format. Twelve (12) micrograms of test vector DNA or control vector DNA and six (6) micrograms of Vector DNA was used to transform 3.2 × 10 5Protoplasts. After transformation, the protoplasts were incubated in the dark at 25 ° C for sixteen to twenty hours. After incubation, the protoplasts were lysed, and the lysate was used to measure luciferase and GUS expression. In order to lyse the cells, the cells in the plate were precipitated by centrifugation, washed, resuspended in a smaller volume, and transferred to a strip well tube. The tube was centrifuged again, and the supernatant was aspirated, leaving the protoplast cell pellet. The cell pellet was resuspended in QB buffer (100mM KPO4, pH 7.8; 1mM EDTA; 1% Triton X-100; 10% glycerol; 1mM DTT). The cells were lysed by vigorous pipetting of the cells several times, vortexing the tubes and incubating the tubes on ice for five minutes. The lysate was then centrifuged to precipitate cell debris. The resulting lysate was then transferred to a clean plate.

[0121] Use QB buffer Luciferase activity was determined using luciferase assay substrate (Promega, Madison, WI 53711). The luciferase assay substrate / QB solution was mixed together in a white ninety-six (96) well plate. Fluorescence was measured using a plate reader (BMG LABTECH Inc., Cary, NC 27513).

[0122] GUS activity is assayed using the fluorescent substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of fifty (50) microliters. The reaction product, 4-methylumbelliferyl (4-MU), is maximally fluorescent at high pH, ​​where the hydroxyl group is ionized. The assay is terminated by the simultaneous addition of an alkaline solution of sodium carbonate, and the pH is adjusted to quantify the fluorescent product. An aliquot of the lysate is mixed with an aliquot of MUG dissolved in QB buffer and incubated at 37°C. A small aliquot of the lysate / MUG reaction mixture is removed and added to the stop buffer at three different time points: (1) immediately after the mixed lysate / MUG reaction as "time zero minutes"; (2) twenty minutes; and (3) sixty minutes. Use Fluorescence was measured by a plate reader (BMG LABTECH Inc., Cary, NC 27513) with excitation at 355 nm and emission at 460 nm. Expression levels were expressed as "nMMUG hydrolyzed" derived from an in-plate standard curve.

[0123] For each plate, each construct was transformed in four (4) to eight (8) wells. An aliquot was taken from each transformation for MUG assay and "nM MUG hydrolysis" was derived from an in-plate standard curve. An aliquot was also taken from each transformation for Read( RLU). The average nM MUG hydrolysis / for each construct was calculated relative to the EXP-CaMV.35S / I-Zm.DnaK:1 / T-Os.LTP:1 construct set as 100%. RLU normalization.

[0124] Analysis of GUS expression in maize leaf protoplasts driven by the synthetic EXP EXP-Zm.GSP850.

[0125] Maize leaf protoplast cells were transformed with expression vectors constructed using methods known in the art and containing expression elements driving GUS expression. Two test expression vectors contained transgenic cassettes containing synthetic EXP EXP-Zm.GSP850 (SEQ ID NO: 1). Synthetic EXP-Zm.GSP850 consisted of a synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO: 2), which was 5'operably linked to a synthetic leader sequence L-Zm.GSP850.nno:3 (SEQ ID NO: 3). The first test vector contained EXP-Zm.GSP850, which was 5'operably linked to a coding sequence encoding GUS (SEQ ID NO: 24), which contained a processable intron, which was 5'operably linked to a 3'UTR T-Os.LTP: 1 (SEQ ID NO: 23). The second transgene cassette comprises EXP-Zm.GSP850, which is 5' operably linked to intron I-Zm.DnaK:1 (SEQ ID NO:22), which is 5' operably linked to a GUS coding sequence, which is 5' operably linked to the 3' UTR T-Os.LTP:1.

[0126] Three control expression vectors were also constructed and used to transform corn leaf protoplasts. The first control expression vector contained a promoterless transgenic cassette and consisted of intron I-Zm.DnaK:1, which was 5'operably linked to a GUS coding sequence, which was 5'operably linked to a 3'UTR T-Os.LTP:1. The second control vector contained an intronless transgenic cassette and consisted of EXP EXP-CaMV.35S (SEQ ID NO:21), which was 5'operably linked to a GUS coding sequence, which was 5'operably linked to a 3'UTR T-Os.LTP:1. The third control vector comprises a transgene cassette containing EXP EXP-CaMV.35S, wherein EXP is 5′ operably linked to intron I-Zm.DnaK:1, which is 5′ operably linked to a GUS coding sequence, which is 5′ operably linked to a 3′ UTR T-Os.LTP:1.

[0127] All five vectors were used to transform corn leaf protoplasts. Transformation and lysis of protoplast cells were performed as described herein. Luciferase and GUS expression were measured as described herein. Table 2 shows the average GUS expression measured and is expressed as a percentage of expression relative to the third control expression vector containing EXP-CaMV.35S and I-Zm.DnaK:1 that drives GUS.

[0128] Table 2. Average percentage of GUS expression in maize leaf protoplasts transformed with test and control vectors.

[0129] Tablet No. Promoter Intron average value SD Repetitions 39 No promoter I-Zm.DnaK:1 0.1 0.337 6 54 No promoter I-Zm.DnaK:1 0.4 0.315 8 95 No promoter I-Zm.DnaK:1 0.1 0.534 8 103 No promoter I-Zm.DnaK:1 0.5 0.149 8 39 EXP-CaMV.35S No introns 48.3 2.018 6 54 EXP-CaMV.35S No introns 46.5 3.949 8 95 EXP-CaMV.35S No introns 46.8 4.369 8 103 EXP-CaMV.35S No introns 40 3.333 8 39 EXP-CaMV.35S I-Zm.DnaK:1 100 5.117 6 54 EXP-CaMV.35S I-Zm.DnaK:1 100 6.465 8 95 EXP-CaMV.35S I-Zm.DnaK:1 100 18.603 8 103 EXP-CaMV.35S I-Zm.DnaK:1 100 5.164 8 95 EXP-Zm.GSP850 No introns 14 1.844 8 103 EXP-Zm.GSP850 No introns 8.4 0.336 8 39 EXP-Zm.GSP850 I-Zm.DnaK:1 19 0.732 4 54 EXP-Zm.GSP850 I-Zm.DnaK:1 22 1.954 8

[0130] As can be seen in Table 2 above, EXP-Zm.GSP850 (SEQ ID NO: 1) was able to drive GUS transgene expression in maize leaf protoplasts when compared to maize leaf protoplast cells transformed with a promoterless construct.

[0131] Analysis of GUS expression in maize leaf protoplasts driven by the synthetic EXP EXP-Zm.GSP990.

[0132] Maize leaf protoplast cells were transformed with an expression vector constructed to contain an expression element driving GUS expression. The test expression vector contained a transgenic cassette containing a synthetic EXP EXP-Zm.GSP990 (SEQ ID NO: 6) operably linked 5' to intron I-Zm.DnaK: 1 (SEQ ID NO: 22), which was operably linked 5' to a coding sequence encoding GUS (SEQ ID NO: 20), which was operably linked 5' to 3' UTR T-Os.LTP: 1. Synthetic EXP-Zm.GSP990 (SEQ ID NO: 6) consisted of a synthetic promoter P-Zm.GSP990.nno: 2 (SEQ ID NO: 7), which was operably linked 5' to a synthetic leader sequence L-Zm.GSP990.nno: 1 (SEQ ID NO: 8). Three control expression vectors were also transformed into maize leaf protoplasts and constructed as described above. Table 3 shows the average percentage of expression relative to the third control expression vector containing EXP-CaMV.35S and I-Zm.DnaK:1 driving GUS.

[0133] Table 3. Average percentage of GUS expression in maize leaf protoplasts transformed with test and control vectors.

[0134] Tablet No. Promoter Intron average value SD Repetitions 71 No promoter I-Zm.DnaK:1 0.3 0.125 6 108 No promoter I-Zm.DnaK:1 -0.6 1.211 8 71 EXP-CaMV.35S No introns 45.1 7.791 6 108 EXP-CaMV.35S No introns 39.9 2.636 8 71 EXP-CaMV.35S I-Zm.DnaK:1 100 13.591 6 108 EXP-CaMV.35S I-Zm.DnaK:1 100 7.425 8 71 EXP-Zm.GSP990 I-Zm.DnaK:1 52.3 13.658 4 108 EXP-Zm.GSP990 I-Zm.DnaK:1 45.2 5.468 8

[0135] As can be seen in Table 3, EXP-Zm.GSP990 (SEQ ID NO: 6) was able to drive GUS transgene expression in maize leaf protoplasts when compared to maize leaf protoplast cells transformed with a promoterless construct.

[0136] Analysis of enhanced GUS expression by synthetic intron I-Zm.GSI153.nno:1

[0137] Maize leaf protoplast cells were transformed with expression vectors constructed to contain expression elements driving GUS expression. The test expression vector was used to determine the enhancement of GUS expression by the synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO:5) driven by EXP-CaMV.35. The transgenic cassette contained EXP EXP-CaMV.35, which was 5′operably linked to the synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO:5), which was 5′operably linked to the coding sequence encoding GUS (SEQ ID NO:24), which was 5′operably linked to the 3′UTR T-Os.LTP:1. Two control expression vectors were also constructed and used to transform maize leaf protoplasts. The first control expression vector comprises an intronless transgene cassette and consists of EXP EXP-CaMV.35S, which is 5'operably linked to a GUS coding sequence, which is 5'operably linked to the 3'UTR T-Os.LTP: 1. The second control vector comprises a transgene cassette, which comprises EXP EXP-CaMV.35S, which is 5'operably linked to an intron I-Zm.DnaK: 1, which is 5'operably linked to a GUS coding sequence, which is 5'operably linked to the 3'UTR T-Os.LTP: 1. Table 4 shows the average percentage of expression relative to the second control expression vector comprising both EXP-CaMV.35S and I-Zm.DnaK: 1 driving GUS.

[0138] Table 4. Average percentage of GUS expression in maize leaf protoplasts transformed with test and control vectors.

[0139] Tablet No. Promoter Intron average value SD Repetitions 10 EXP-CaMV.35S No introns 52.8 8.428 6 13 EXP-CaMV.35S No introns 44.4 4.586 8 10 EXP-CaMV.35S I-Zm.DnaK:1 100 16.646 6 13 EXP-CaMV.35S I-Zm.DnaK:1 100 13.123 8 10 EXP-CaMV.35S I-Zm.GSI153.nno:1 83 5.601 4 13 EXP-CaMV.35S I-Zm.GSI153.nno:1 83.6 7.596 8

[0140] As seen in Table 4, the synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO:5) enhanced the expression of the GUS transgene driven by EXP-CaMV.35S in maize leaf protoplasts when compared to the intronless control expression vector.

[0141] Analysis of enhanced GUS expression by synthetic intron I-Zm.GSI197.nno:1

[0142] Maize leaf protoplast cells were transformed with expression vectors constructed to contain expression elements driving GUS expression. The test expression vectors were used to determine the enhancement of GUS expression by the synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO:10) driven by EXP-CaMV.35. The transgenic cassette contained EXP EXP-CaMV.35, which was 5′operably linked to the synthetic intron I-Zm.GSI197.nno:1, which was 5′operably linked to the coding sequence encoding GUS (SEQ ID NO:24), which was 5′operably linked to the 3′UTR T-Os.LTP:1. Three control expression vectors were also constructed and used to transform maize leaf protoplasts. The first control expression vector comprises a promoterless transgene cassette and consists of intron I-Zm.DnaK:1, which is 5'operably linked to a GUS coding sequence, which is 5'operably linked to a 3'UTR T-Os.LTP: 1. The second control vector comprises an intronless transgene cassette and consists of EXP EXP-CaMV.35S, which is 5'operably linked to a GUS coding sequence, which is 5'operably linked to a 3'UTR T-Os.LTP: 1. The third control vector comprises a transgene cassette, which comprises EXP EXP-CaMV.35S, which is 5'operably linked to an intron I-Zm.DnaK:1, which is 5'operably linked to a GUS coding sequence, which is 5'operably linked to a 3'UTR T-Os.LTP: 1. Table 5 shows the average percentage of expression relative to a third control vector containing both EXP-CaMV.35S and I-Zm.DnaK:1 driving GUS.

[0143] Table 5. Average percentage of GUS expression in maize leaf protoplasts transformed with test and control vectors.

[0144] Tablet No. Promoter Intron average value SD Repetitions 125 No promoter I-Zm.DnaK:1 1.1 0.317 6 146 No promoter I-Zm.DnaK:1 0.6 0.101 8 125 EXP-CaMV.35S No introns 43.8 4.081 6 146 EXP-CaMV.35S No introns 41 3.666 8 125 EXP-CaMV.35S I-Zm.DnaK:1 100 11.287 6 146 EXP-CaMV.35S I-Zm.DnaK:1 100 8.506 8 125 EXP-CaMV.35S I-Zm.GSI197.nno:1 150 12.451 4 146 EXP-CaMV.35S I-Zm.GSI197.nno:1 109.8 8.001 8

[0145] As seen in Table 5, the synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10) enhanced expression of the GUS transgene driven by EXP-CaMV.35S in corn leaf protoplasts when compared to the intronless control expression vector. The enhancement of expression was greater than that conferred by intron I-Zm.DnaK:1 when compared to a third control expression vector comprising EXP EXP-CaMV.35S operably linked 5' to intron I-Zm.DnaK:1.

[0146] Analysis of the enhanced GUS expression by the synthetic 3′UTRs T-Zm.GST9.nno:2 and T-Zm.GST18.nno:2.

[0147] Maize leaf protoplast cells were transformed using expression vectors constructed to contain expression elements driving GUS expression. Two test vectors contained transgenic cassettes for analysis of enhanced GUS expression conferred by 3′UTR T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14), and consisted of EXP-CaMV.35S, the EXP being 5′operably linked to intron I-Zm.DnaK: 1, the intron being 5′operably linked to a coding sequence encoding GUS (SEQ ID NO: 24), the coding sequence being 5′operably linked to 3′UTR T-Zm.GST9.nno:2 (SEQ ID NO: 13) or 3′UTR T-Zm.GST18.nno:2 (SEQ ID NO: 14). As described above, three control expression vectors were also constructed and used to transform maize leaf protoplasts. Table 6 shows the average percentage of expression relative to a third control expression vector containing both EXP-CaMV.35S and I-Zm.DnaK:1 driving GUS.

[0148] Table 6. Average percentage of GUS expression in maize leaf protoplasts transformed with test and control vectors.

[0149]

[0150]

[0151] As seen in Table 6, 3'UTRs T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) enhanced GUS expression relative to the control in corn leaf protoplasts.

[0152] Example 3

[0153] In stably transformed LH244 maize plants, the synthetic EXP

[0154] Analysis of GUS expression driven by EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1

[0155] Vectors, particularly plant expression vectors containing test regulatory elements driving expression of a beta-glucuronidase (GUS) transgene, are used to transform corn plants. The resulting plants are analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0156] Corn plants were transformed using a plant GUS expression construct. The regulatory elements were cloned into a basic plant expression vector using standard methods known in the art. The resulting plant expression vector contains the left border region of Agrobacterium tumefaciens (B-AGRtu. left border); a first transgenic selection box for selecting transformed plant cells that confer resistance to the herbicide glyphosate; a second transgenic box for evaluating the activity of a synthetic regulatory element, the synthetic regulatory element comprising a synthetic EXP EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) or EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11), the synthetic EXP will be 5′operably linked to a synthetic coding sequence encoding β-glucuronidase designed for expression in plant cells (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:11). NO:20), the synthetic coding sequence contains a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (Genbank Accession No.: X04753), the processable intron is 5'operably linked to the 3' termination region, T-Sb.Nltp4-1:1:2 (SEQ ID NO:19); and the right border region of Agrobacterium tumefaciens (B-AGRtu.right border). The synthetic EXP EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) consists of the synthetic promoter P-Zm.GSP850.nno:4 (SEQID NO:2), which is 5′ operably linked to the synthetic leader sequence L-Zm.GSP850.nno:3 (SEQ ID NO:3), and the synthetic leader sequence is 5′ operably linked to the synthetic intron I-Zm.GSI153.nno:1 (SEQ ID NO:5). The synthetic EXP EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) consists of the synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO:2), which is 5′ operably linked to the synthetic leader sequence L-Zm.GSP850.nno:3 (SEQ ID NO:3), and the synthetic leader sequence is 5′ operably linked to the synthetic intron I-Zm.GSI140.nno:1 (SEQ ID NO:12).

[0157] As is well known in the art, the binary transformation vector constructs described above were used to transform maize variety LH244 plant cells by Agrobacterium-mediated transformation. The resulting transformed plant cells were induced to form complete maize plants.

[0158] Use qualitative and quantitative GUS analysis to assess the expression element activity in selected plant organs and tissues in transformed plants. In order to qualitatively analyze GUS expression by histochemical staining, whole specimen embedding or sliced ​​tissue was incubated together with the GUS staining solution containing 1mg / mL X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronic acid) at 37°C for 5 hours, and decolorized with 35% EtOH and 50% acetic acid. Qualitatively determine the expression of GUS by visual inspection of blue coloration of selected plant organs or tissues under a dissecting microscope or a compound microscope.

[0159] In order to quantitatively analyze GUS expression by enzymatic assay, total protein was extracted from the selected tissues of the transformed corn plants. One to two micrograms of total protein were incubated with the fluorescent substrate 4-methylumbelliferyl-β-D-glucuronic acid (MUG) at a concentration of 1mM in a 50 microliter total reaction volume. After incubation at 37°C for 1 hour, the reaction was terminated by adding 350 microliters of 200mM sodium bicarbonate solution. The reaction product 4-methylumbelliferyl (4-MU) is the maximum fluorescence at high pH, ​​where the hydroxyl group is ionized. Alkaline sodium carbonate solution was added to terminate the assay at the same time, and pH was adjusted for quantitative fluorescent product 4-MU. The fluorescence of 4-MU was estimated by using FLUOstar Omega microplate reader (BMG LABTECH) (excitation at 355nm, emission at 460nm) to measure its formation amount. GUS activity values ​​are provided in nanomoles of 4-MU / hour / mg total protein.

[0160] The following tissues were sampled for GUS expression in the R0 generation: leaves and roots at the V4 stage; leaves and roots at the V7 stage; leaves, roots and flowers / anthers at the VT stage; cobs / silks at the R1 stage; and seed embryos and seed endosperms at the R3 stage 21 days after pollination (DAP). Table 7 shows the average quantitative GUS expression values ​​for each synthetic EXP.

[0161] Table 7. Average quantitative GUS expression in stably transformed LH244 variety maize plants driven by the synthetic EXPs EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1.

[0162]

[0163] As seen in Table 7, the synthetic GSP850 promoter and leader sequences (P-Zm.GSP850.nno:4 (SEQ ID NO:2) and L-Zm.GSP850.nno:3 (SEQ ID NO:3)) drive constitutive expression of GUS in stably transformed LH244 variety maize plants. Molecular analysis of transcription start sites showed that the TSS of the GSP850 promoter and leader sequences are identical. The synthetic introns I-Zm.GSI153.nno:1 (SEQ ID NO:5) and I-Zm.GSI140.nno:1 (SEQ ID NO:12) had different effects on expression in the different tissues sampled. Molecular analysis of intron splice sites showed that the processing of the synthetic introns is consistent. The overall enhancement of GUS expression was higher in most tissue samples of plants containing I-Zm.GSI153.nno:1 (SEQ ID NO:5), except that the GUS expression levels of leaves at the V4 stage, roots at the V7 stage, and R3 seed embryos were relatively similar. Relative to I-Zm.GSI140.nno:1 (SEQ ID NO:12), the enhancement of expression conferred by I-Zm.GSI153.nno:1 (SEQ ID NO:5) was about 7.5 times higher in V4 roots, about 7.7 times higher in VT leaves, about 6.0 times higher in VT roots, about 4.3 times higher in VT flowers / anthers, about 3.2 times higher in R1 cobs / silks, and about 2.3 times higher in R3 seed endosperm.

[0164] Example 4

[0165] In stably transformed maize plants of variety 01DKD2, the synthetic EXP

[0166] EXP-Zm.GSP850.nno+Zm.DnaK:1、

[0167] Analysis of GUS expression driven by EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1

[0168] Vectors, particularly plant expression vectors containing test regulatory elements driving expression of a beta-glucuronidase (GUS) transgene, are used to transform corn plants. The resulting plants are analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0169] Transformation of maize plants using the plant GUS expression construct Regulatory elements were cloned into basic plant expression vectors using standard methods known in the art. The resulting plant expression vector contains the left border region of Agrobacterium tumefaciens (B-AGRtu.left border); a first transgenic selection box for selecting transformed plant cells that confer resistance to the herbicide glyphosate; a second transgenic box for evaluating the activity of a regulatory element, the regulatory element comprising a synthetic EXP EXP-Zm.GSP850.nno+Zm.DnaK:1 (SEQ ID NO:15), EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) or EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11), the synthetic EXP is 5′ operably linked to a synthetic coding sequence encoding β-glucuronidase designed for expression in plant cells (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:12). NO:20), the synthetic coding sequence contains a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (Genbank Accession No.: X04753), which is 5'operably linked to the 3' terminator region T-Sb.Nltp4-1:1:2 (SEQ ID NO:19); and the right border region of Agrobacterium tumefaciens (B-AGRtu. right border). Synthetic EXP EXP-Zm.GSP850.nno+Zm.DnaK:1 (SEQ ID NO:15) consists of a synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO:2), which is 5'operably linked to a synthetic leader sequence L-Zm.GSP850.nno:3 (SEQ ID NO:3), which is 5'operably linked to intron I-Zm.DnaK:1 (SEQ ID NO:22). The synthesis of EXP EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 (SEQ ID NO:4) and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 (SEQ ID NO:11) was as described in Example 3.

[0170] As is well known in the art, the binary transformation vector construct described above by Agrobacterium-mediated transformation was used to transform corn variety 01DKD2 plant cells. The resulting transformed plant cells were induced to form complete corn plants. Qualitative and quantitative GUS expression was determined as described in Example 3. Table 8 shows the average quantitative GUS expression values ​​for each synthetic EXP.

[0171] Table 8. Average quantitative GUS expression driven by synthetic EXPs EXP-Zm.GSP850.nno+Zm.DnaK:1, EXP-Zm.GSP850.nno+Zm.GSI153.nno:2 and EXP-Zm.GSP850.nno+Zm.GSI140.nno:1 in stably transformed 01DKD2 variety maize plants.

[0172]

[0173]

[0174] As seen in Table 8, the synthetic GSP850 promoter and leader sequences (P-Zm.GSP850.nno:4 (SEQ ID NO:2) and L-Zm.GSP850.nno:3 (SEQ ID NO:3)) drove constitutive expression of GUS in stably transformed LH244 variety maize plants. Synthetic introns I-Zm.GSI153.nno:1 (SEQ ID NO:5) and I-Zm.GSI140.nno:1 (SEQ ID NO:12) enhanced expression relative to intron I-Zm.DnaK:1 (SEQ ID NO:22) in all tissues assayed.

[0175] Example 5

[0176] In stably transformed maize plants of variety 01DKD2, the synthetic EXP

[0177] Analysis of GUS expression driven by EXP-Zm.GSP990.nno+Zm.DnaK:1 and EXP-Zm.GSP990.nno+Zm.GSI197.nno:2

[0178] Vectors, particularly plant expression vectors containing test regulatory elements driving expression of a beta-glucuronidase (GUS) transgene, are used to transform corn plants. The resulting plants are analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0179] Corn plants were transformed using a plant GUS expression construct. The regulatory elements were cloned into a basic plant expression vector using standard methods known in the art. The resulting plant expression vector contains the left border region of Agrobacterium tumefaciens (B-AGRtu. left border); a first transgenic selection box for selecting transformed plant cells that confer resistance to the herbicide glyphosate; a second transgenic box for evaluating the activity of the regulatory element, the regulatory element comprising a synthetic EXP EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO:16) or EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQID NO:9), the synthetic EXP will be 5′operably linked to a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20) encoding β-glucuronidase designed for expression in plant cells, the synthetic coding sequence containing a light-induced tissue-specific ST-LS1 gene derived from potato (Gen bank accession number: X04753) of the processable intron, the processable intron will be 5'operably linked to the 3' termination region T-Sb.Nltp4-1:1:2 (SEQ ID NO: 19); and the right border region of Agrobacterium tumefaciens (B-AGRtu. right border). Synthetic EXP EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 (SEQ ID NO: 9) consists of a synthetic promoter P-Zm.GSP990.nno:2 (SEQ ID NO: 7), the synthetic promoter will be 5'operably linked to a synthetic leader sequence L-Zm.GSP990.nno:1 (SEQ ID NO: 8), and the synthetic leader sequence will be 5'operably linked to a synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10). The synthetic EXP EXP-Zm.GSP990.nno+Zm.DnaK:1 (SEQ ID NO:16) consists of the synthetic promoter P-Zm.GSP990.nno:2 (SEQ ID NO:7), which is 5′ operably linked to the synthetic leader sequence L-Zm.GSP990.nno:1 (SEQ ID NO:8), and the synthetic leader sequence is 5′ operably linked to the intron I-Zm.DnaK:1 (SEQ ID NO:22).

[0180] As is well known in the art, the binary transformation vector construct described above by Agrobacterium-mediated transformation was used to transform corn variety 01DKD2 plant cells. The resulting transformed plant cells were induced to form complete corn plants. Qualitative and quantitative GUS expression was determined as described in previous Example 3. Table 9 shows the average quantitative GUS expression values ​​for each synthetic EXP, where "ND" means not determined.

[0181] Table 9. Average quantitative GUS expression driven by synthetic EXPs EXP-Zm.GSP990.nno+Zm.DnaK:1 and EXP-Zm.GSP990.nno+Zm.GSI197.nno:2 in stably transformed 01DKD2 variety maize plants.

[0182]

[0183]

[0184] It can be seen that the synthetic GSP990 promoter and leader sequence (P-Zm.GSP990.nno:2 (SEQ ID NO:7) and L-Zm.GSP990.nno:1 (SEQ ID NO:8)) drive the expression of GUS. Molecular analysis of the transcription start site showed that the TSS of the GSP990 promoter and leader sequence were consistent. Relative to intron I-Zm.DnaK:1 (SEQ ID NO:22), the synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO:10) weakened expression in some tissues while enhancing expression in other tissues. For example, GUS expression in leaves was weakened at the V4, V7 and VT stages. Relative to I-Zm.DnaK:1, GUS expression was slightly enhanced in the roots of V7 and VT. The flower / anther expression of I-Zm.GSI197.nno:1 (SEQ ID NO: 10) was enhanced by about 2.7 fold relative to I-Zm.DnaK:1. The difference in expression conferred by I-Zm.GSI197.nno:1 (SEQ ID NO: 10) relative to I-Zm.DnaK:1 can be very useful in situations where lower leaf expression and higher flower / anther expression are desired. Molecular analysis of intron splice sites indicated that the synthetic intron I-Zm.GSI197.nno:1 (SEQ ID NO: 10) is processed consistently.

[0185] Example 6

[0186] Analysis of the effects of synthetic 3′UTRs T-Zm.GST9.nno:2, T-Zm.GST18.nno:2, and T-Zm.GST43.nno:1 and native T-Sb.Ntlp4-1:1:2 on GUS expression in stably transformed 01DKD2 maize plants

[0187] Vectors, particularly plant expression vectors containing test regulatory elements driving expression of a beta-glucuronidase (GUS) transgene, are used to transform corn plants. The resulting plants are analyzed for GUS protein expression to assess the effect of the selected regulatory elements on expression.

[0188] Corn plants were transformed using a plant GUS expression construct. The regulatory elements were cloned into a basic plant expression vector using standard methods known in the art. The resulting plant expression vector contains the left border region of Agrobacterium tumefaciens (B-AGRtu. left border); a first transgenic selection box for selecting transformed plant cells that confer resistance to the herbicide glyphosate; a second transgenic box for evaluating the activity of a 3′UTR regulatory element, the regulatory element comprising EXP EXP-CaMV.35S (SEQ ID NO: 21), the EXP being 5′ operably linked to intron I-Zm.DnaK: 1 (SEQ ID NO: 22), the intron being 5′ operably linked to a synthetic coding sequence encoding β-glucuronidase designed for expression in plant cells (GUS, GOI-Ec.uidA+St.LS1.nno: 1, SEQ ID NO:20), the synthetic coding sequence contains a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (Genbank Accession No.: X04753), which is 5'operably linked to the 3' termination region; and the right border region of Agrobacterium tumefaciens (B-AGRtu.right border). Three test expression vectors contain 3'UTRs T-Zm.GST9.nno:2 (SEQ ID NO:13), T-Zm.GST18.nno:2 (SEQ ID NO:14), or T-Zm.GST43.nno:1 (SEQ ID NO:26) operably linked to the GUS coding sequence. Another test expression vector contains the native 3'UTR T-Sb.Nltp4-1:1:2 (SEQ ID NO:19) operably linked to the GUS coding sequence and is used to compare the expression between the native 3'UTR and the synthetic 3'UTR.

[0189] As is well known in the art, the binary transformation vector construct described above was used to transform maize variety 01DKD2 plant cells by Agrobacterium-mediated transformation. The resulting transformed plant cells were induced to form complete maize plants.

[0190] Qualitative and quantitative GUS analysis was used to evaluate the activity of the expression element in the V4 leaf and root tissues of the transformed plants and was performed as described in Example 3 above. The effect of the synthetic 3'UTR was evaluated by comparing the effect of the 3'UTR T-Sb.Nltp4-1:1:2 (SEQ ID NO:19) on expression. The resulting transcripts were analyzed to determine whether proper termination had occurred and that there was no read-through of the transcripts. One method of evaluating whether there was read-through was by using amplification primers corresponding to a portion of the T-DNA border sequence (3' to 3'UTR) to amplify the transcript cDNA. The average GUS expression of plants transformed with four constructs comprising T-Zm.GST9.nno:2 (SEQ ID NO:13), T-Zm.GST18.nno:2 (SEQ ID NO:14), T-Zm.GST43.nno:1 (SEQ ID NO:26) and T-Sb.Nltp4-1:1:2 (SEQ ID NO:19) is provided in Table 10.

[0191] Table 10. Average quantitative GUS expression in stably transformed 01DKD2 variety maize plants from constructs containing three different 3'UTRs.

[0192]

[0193] As seen in Table 10, both T-Zm.GST9.nno:2 (SEQ ID NO: 13) and T-Zm.GST18.nno:2 (SEQ ID NO: 14) enhanced GUS expression driven by EXP-CaMV.35S operably linked to I-Zm.DnaK: 1 relative to 3′UTR T-Sb.Nltp4-1: 1:2. GUS expression in plants containing T-Zm.GST9.nno:2 (SEQ ID NO: 13) was higher than that in plants containing T-Zm.GST18.nno:2. T-Zm.GST43.nno: 1 (SEQ ID NO: 26) enhanced GUS expression in V4 roots but attenuated expression in V4 leaves relative to T-Sb.Nltp4-1: 1:2. Analysis of GUS transcripts for all four constructs indicated that the transcripts were properly terminated and there was no evidence of read-through in the resulting GUS transcripts. 3'UTR T-Zm.GST9.nno:2 (SEQ ID NO: 13), T-Zm.GST18.nno:2 (SEQ ID NO: 14) and T-Zm.GST43.nno:1 (SEQ ID NO: 26) operate in a similar manner to the native 3'UTR and demonstrate regulation of GUS expression relative to the native 3'UTR T-Sb.Nlt p4-1: 1: 2. All four synthetic 3'UTRs and the additional native 3'UTR T-Sb.Nlt p4-1: 1: 2 provide a range of expression values ​​that can be used to fine-tune expression in stably transformed corn plants.

[0194] Example 7

[0195] Enhancer elements derived from regulatory elements

[0196] Enhancers are derived from promoter elements presented in SEQ ID NOs: 2 and 7. Enhancer elements may consist of one or more cis-regulatory elements that, when 5' or 3' operably linked to a promoter element in cooperation with 5' or 3' operably linked to another enhancer element, can enhance or modulate the expression level of a transcribable DNA molecule, or provide for expression of a transcribable DNA molecule in a specific cell type or plant organ in cooperation with a specific time point of development or circadian rhythm. Enhancers are prepared by removing the TATA box or a functionally similar element and any downstream sequences from the promoter, which allows initiation of transcription from the promoters presented in SEQ ID NOs: 2 and 7 or fragments thereof.

[0197] The TATA box in plant promoters is not as highly conserved as the TATA box in some other eukaryotic organisms. Therefore, in order to define a fragment as an enhancer, the transcription start site (TSS) of the gene must first be determined, where the 5'UTR is transcribed first. An enhancer derived from the synthetic promoter P-Zm.GSP850.nno:4 (SEQ ID NO:2) can include nucleotides 1 to 418 of SEQ ID NO:2, thereby generating the synthetic enhancer E-Zm.GSP850 (SEQ ID NO:17). An enhancer derived from the synthetic promoter P-Zm.GSP990.nno:2 (SEQ ID NO:7) can include nucleotides 1 to 416 of SEQ ID NO:7, thereby generating the synthetic enhancer E-Zm.GSP990 (SEQ ID NO:18). An enhancer derived from a promoter can include fragments of SEQ ID NOs:17 and 18, or repeats of SEQ ID NOs:17 and 18 or fragments of each of them. The effectiveness of synthetic enhancers derived from synthetic promoters was empirically determined by constructing chimeric transcriptional regulatory elements comprising fragments derived from the synthetic promoters P-Zm.GSP850.nno:4 (SEQ ID NO:2) or P-Zm.GSP990.nno:2 (SEQ ID NO:7), which were operably linked to the promoter and leader sequence and used to drive expression of transcribable DNA molecules (such as GUS) in stable or transient plant assays.

[0198] Further refinement of the enhancer element may be required and validated empirically. In addition, the position of the enhancer element relative to other elements within the chimeric transcriptional regulatory element is also empirically determined, as the order of each element within the chimeric transcriptional regulatory element can impart different effects depending on the relative position of each element. Some promoter elements will have multiple TATA boxes or TATA box-like elements and potentially multiple transcription start sites. In those cases, it may be necessary to first determine the location of the first TSS and then begin designing the enhancer using the first TSS to prevent potential transcription initiation from occurring in the putative enhancer element.

[0199] Enhancer elements derived from promoter elements presented in SEQ ID NOs: 2 and 7 are cloned using methods known in the art to be operably linked 5' or within the promoter element, or to be operably linked 5' or 3' to another enhancer element, which is operably linked to the promoter. Alternatively, enhancer elements can be cloned using methods known in the art to provide larger enhancer elements, which are composed of two or more enhancer copies and cloned using methods known in the art to be operably linked 5' or 3' to the promoter element or to be operably linked 5' or 3' to another enhancer element, which is operably linked to the promoter to create a chimeric transcriptional regulatory element. Enhancer elements derived from promoters (derived from genes from organisms of multiple genera) can be operably linked to enhancers derived from synthetic promoters.

[0200] A GUS expressing plant transformation vector can be constructed using methods known in the art similar to the constructs described in Example 3, wherein the resulting plant expression vector contains the left border region of Agrobacterium tumefaciens (B-AGRtu.left border); a first transgenic selection cassette for selecting transformed plant cells that confer resistance to the herbicide glyphosate; and a second transgenic cassette for testing enhancer elements, which consists of an enhancer element that is 5′ or 3′ operably linked to a promoter element or is 5′ or 3′ operably linked to an additional enhancer element, which in turn is operably linked to a promoter, which is 5′ operably linked to a leader sequence element, which is 5′ operably linked to an intron element, which is operably linked to a coding sequence for β-glucuronidase (GOI-Ec.uidA+St.LS1.nno:1, SEQ ID NO:20), the coding sequence contains a processable intron derived from the potato light-induced tissue-specific ST-LS1 gene (Genbank accession number: X04753), the processable intron is operably linked to the 3' terminator region from the rice lipid transfer protein-like gene (T-Os.LTP:1, SEQ ID NO:23); and the right border region of Agrobacterium tumefaciens (B-AGRtu.right border). The resulting plasmid is used to transform corn plants or other monocotyledonous plants by the above method. Alternatively, protoplast cells derived from corn or other monocotyledonous plants are transformed using methods known in the art for transient assays

[0201] GUS expression driven by a regulatory element comprising one or more enhancers is evaluated in a stable or transient plant assay to determine the effect of the enhancer element on the expression of a transcribable DNA molecule. Modification of one or more enhancer elements or duplication of one or more enhancer elements can be performed based on empirical experiments and the resulting gene expression regulation observed using each regulatory element combination. Changing the relative position of one or more enhancers in the resulting regulatory or chimeric regulatory element may affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element and is empirically determined to determine the optimal enhancer for the desired transgenic expression profile in corn plants or other genus plants.

[0202] Having illustrated and described the principles of the invention, it will be apparent to those skilled in the art that modifications in arrangement and detail may be made to the invention without departing from such principles. We claim protection for 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 if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to SEQ ID NO: 14; b) a sequence comprising SEQ ID NO: 14; and c) a fragment of SEQ ID NO: 14, wherein the fragment has gene regulatory activity; Wherein the DNA sequence is operably linked to a heterologous transcribable DNA molecule.

2. The recombinant DNA molecule of claim 1, wherein the sequence comprises at least 90% sequence identity to the DNA sequence of SEQ ID NO:

14.

3. The recombinant DNA molecule of claim 1, wherein the sequence comprises at least 95% sequence identity to the DNA sequence of SEQ ID NO:

14.

4. The DNA sequence of claim 1, wherein the DNA sequence comprises gene regulatory activity.

5. The recombinant DNA molecule of claim 1, wherein the heterologous transcribable DNA molecule comprises a gene of agronomic interest.

6. The recombinant DNA molecule of claim 5, wherein the gene of agronomic interest confers herbicide tolerance in plants.

7. The recombinant DNA molecule of claim 5, wherein the gene of agronomic interest confers pest resistance in plants.

8. The recombinant DNA molecule of claim 1, wherein the heterologous transcribable DNA molecule encodes a dsRNA, a miRNA, or a siRNA.

9. A transgenic plant cell comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity to SEQ ID NO: 14; b) a sequence comprising SEQ ID NO: 14; and c) a fragment of SEQ ID NO: 14, wherein the fragment has gene regulatory activity; Wherein the DNA sequence is operably linked to a heterologous transcribable DNA molecule.

10. The transgenic plant cell of claim 9, wherein the transgenic plant cell is a monocotyledonous plant cell.

11. The transgenic plant cell of claim 9, wherein the transgenic plant cell is a dicotyledonous plant cell.

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

13. A progeny plant or part thereof of the transgenic plant of claim 12, wherein said progeny plant or part thereof comprises said recombinant DNA molecule.

14. A transgenic seed, wherein the seed comprises the recombinant DNA molecule of claim 1.

15. A method for producing a commodity, comprising obtaining the transgenic plant or a part thereof according to claim 12, and producing a commodity from the transgenic plant or a part thereof.

16. The method of claim 15, wherein the commodity is a seed, processed seed, protein concentrate, protein isolate, starch, grain, plant part, seed oil, biomass, flour or meal.

17. A method for expressing a transcribable DNA molecule, comprising obtaining the transgenic plant according to claim 12, and culturing the plant, wherein the transcribable DNA is expressed.

Images