Plant regulatory elements and uses thereof

Abstract

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

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 375,114, filed on September 9, 2022, which is hereby incorporated by reference in its entirety.

[0003] Incorporation of Sequence Listing

[0004] The sequence listing contained in the file named "AGOE009WO_ST26.xml" (56.2 KB, measured in Microsoft), created on August 29, 2023, and submitted here by electronic submission, is hereby incorporated by reference. and is incorporated herein by reference. Technical Field

[0005] The present invention relates to plant molecular biology and plant genetic engineering, and DNA molecules for regulating gene expression in plants. Background Art

[0006] Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of an operably linked transcribable polynucleotide molecule. Such elements include promoters, leader sequences, introns, and 3' untranslated regions, and can be used in the fields of plant molecular biology and plant genetic engineering. Summary of the Invention

[0008] The present invention provides novel gene regulatory elements for plants. The present invention also provides DNA constructs comprising the regulatory elements. The present invention also provides transgenic plant cells, plants, and seeds comprising the regulatory elements. Sequences operably linked to a transcribable polynucleotide molecule can be provided. In one embodiment, the transcribable polynucleotide molecule can be heterologous with respect to the regulatory sequences provided herein. Thus, in certain embodiments, the regulatory element sequences provided by the present invention can be defined as being operably linked to a heterologous transcribable polynucleotide molecule. The present invention also provides methods of preparing and using the regulatory elements, DNA constructs comprising the regulatory elements, and transgenic plant cells, plants, and seeds comprising the regulatory elements operably linked to a transcribable polynucleotide molecule.

[0009] Accordingly, on the one hand, the present invention provides a DNA molecule comprising a DNA sequence selected from the following: a) a sequence having at least about 85% sequence identity with any one of SEQ ID NOs: 1-24; b) a sequence comprising any one of SEQ ID NOs: 1-24; c) a fragment of a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1-24, wherein the fragment has gene regulatory activity; d) a fragment of any one of SEQ ID NOs: 1-24, wherein the fragment has gene regulatory activity; and e) a combination thereof; wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule. In some embodiments, the DNA molecule is active as a promoter. In further embodiments, the DNA molecule further comprises a heterologous regulatory element. In certain embodiments, the DNA molecule has at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity with the DNA sequence of any one of SEQ ID NOs: 1-24. In certain embodiments of the DNA molecule, the DNA sequence comprises a regulatory element. In some embodiments, the regulatory element comprises a promoter. In certain embodiments, the heterologous transcribable polynucleotide molecule comprises an agronomic gene, such as a gene capable of providing increased yield in a plant, a gene capable of providing increased root growth in a plant, a gene capable of providing increased drought tolerance in a plant or a gene capable of providing increased starch content in a plant.

[0010] On the other hand, the present invention provides a construct comprising at least one copy of the DNA molecule provided herein and an operably linked transcribable agronomic gene. In some embodiments, the construct comprises, in the 5'-3' direction: (a) at least one copy of the DNA molecule; (b) an operably linked transcribable agronomic gene; and (c) a gene termination sequence. In further embodiments, the transcribable agronomic gene comprises an open reading frame encoding a polypeptide.

[0011] The present invention also provides a transgenic plant cell comprising the heterologous DNA construct provided by the present invention, said DNA construct comprising the sequence of any one of SEQ ID NOs: 1-24 or a fragment or variant thereof, wherein said sequence is operably linked to a heterologous transcribable polynucleotide molecule. For example, in a further embodiment, the transgenic plant cell may comprise a sequence selected from: a) a sequence having at least about 85% sequence identity with any one of SEQ ID NOs: 1-24; b) a sequence comprising any one of SEQ ID NOs: 1-24; c) a fragment of a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1-24, wherein said fragment has gene regulatory activity; d) a fragment of any one of SEQ ID NOs: 1-24, wherein said fragment has gene regulatory activity; and e) a combination thereof; wherein said sequence is operably linked to a heterologous transcribable polynucleotide 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. In a particular embodiment, the transgenic plant cell is a cassava plant cell.

[0012] The present invention further provides a transgenic plant or a part thereof comprising the DNA molecule provided herein, said DNA molecule comprising a DNA sequence selected from: a) a sequence having at least about 85% sequence identity with any one of SEQ ID NOs: 1-24; b) a sequence comprising any one of SEQ ID NOs: 1-24; c) a fragment of a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1-24, wherein said fragment has gene regulatory activity; d) a fragment of any one of SEQ ID NOs: 1-24, wherein said fragment has gene regulatory activity; and e) a combination thereof; wherein said sequence is operably linked to a second heterologous transcribable polynucleotide molecule. In a particular embodiment, the transgenic plant can be any generation of progeny plants comprising the DNA molecule relative to the starting transgenic plant comprising the DNA molecule. Transgenic seeds comprising the DNA molecule according to the present invention are also further provided.

[0013] In another aspect, the present invention provides a method for producing a commodity, comprising obtaining a transgenic plant or a part thereof according to the present invention and producing a commodity therefrom. In one embodiment, the commodity of the present invention is a protein concentrate, a protein isolate, a grain, a starch, a seed, a grit, a flour, a biomass or a seed oil. In another aspect, the present invention provides a commodity produced by the above method. For example, in one embodiment, the present invention provides a commodity comprising a DNA molecule provided herein, the DNA molecule comprising a DNA sequence selected from the following: a) a sequence having at least about 85% sequence identity with any one of SEQ ID NOs: 1-24; b) a sequence comprising any one of SEQ ID NOs: 1-24; c) a fragment of a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1-24, wherein the fragment has gene regulatory activity; d) a fragment of any one of SEQ ID NOs: 1-24, wherein the fragment has gene regulatory activity; and e) a combination thereof; wherein the sequence is operably linked to a heterologous transcribable polynucleotide molecule.

[0014] In yet another aspect, the present invention provides a method for expressing a transcribable polynucleotide molecule, which comprises obtaining a transgenic plant according to the present invention, for example, a plant comprising a DNA molecule as described herein, and cultivating a plant in which the transcribable polynucleotide in the DNA molecule is expressed.

[0015] In this specification and the claims, unless the context otherwise requires, the word "comprise" and its variants, such as "comprises" and "comprising", shall be understood to mean including the stated component, step and / or value or a combination thereof, but not excluding any other component, step and / or value or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Summary showing the approximate promoter activities of 10 promoters in source leaves, stems and storage roots. The relative gene expression of different transcripts (normalized to MeGAPDH) was determined and the data was used to infer the approximate activities of the promoter elements controlling their expression. Source leaves (afternoon) fully exposed, stem segments below the first branch point, and storage root material from the two thickest storage roots of each plant were sampled using field-grown cassava plants.

[0017] Figure 2A1-2F6 shows representative GUS staining patterns from at least three events: pAtCAB1::GUS, pStLS1::GUS, pAtRBCS3B::GUS, pMeGBSS1, pStSSS3, and pStSTP1 promoter-reporter plants. For pAtCAB1::GUS, A1) source leaf (inset = close-up); B1) sink leaf; C1) new leaf; D1) petiole cross-section; E1) upper stem cross-section; F1) lower stem cross-section; G1) storage root cross-section; H1) fibrous root; I1) GUS expression levels (%) of four pAtCAB1::GUS lines relative to three pCaMV35S::GUS lines. For pStLS1::GUS, A2) source leaf; B2) sink leaf; C2) petiole cross-section; D2) upper stem cross-section; E2) storage root cross-section; H2) fibrous root. For pAtRBCS3B::GUS, A3) source leaf; B3) sink leaf; C3) petiole cross-section; D3) upper stem cross-section; E3) storage root cross-section; H3) fibrous root. For pMeGBSS1::GUS, A4) source leaf; B4) sink leaf; C4) petiole cross-section; D4) upper stem cross-section; E4) storage root cross-section; H4) fibrous root. For pStSSS3::GUS, A5) source leaf; B5) sink leaf; C5) petiole cross-section; D5) upper stem cross-section; E5) storage root cross-section; H4) fibrous root. For pStSTP1::GUS, A6) source leaf; B6) sink leaf; C6) petiole cross-section; D6) upper stem cross-section; E6) storage root cross-section; H6) fibrous root. Plants were grown in the field at the experimental station of National Chung Hsing University, Taiwan, or in the greenhouse in Erlangen, Germany. Tissues from approximately 3-month-old cassava plants were used.

[0018] Figure 3 A-3I shows representative GUS staining patterns of three pMePsbr::GUS promoter-reporter lines. A) source leaf; B) sink leaf; C) new leaf; D) petiole cross-section; E) upper stem cross-section; F) lower stem cross-section; G) storage root cross-section; H) fibrous root; I) GUS expression levels (%) of three pPsbR::GUS lines relative to three pCaMV35S::GUS lines. Bars represent means with standard deviation (n = 4).

[0019] Figure 4 A-4H shows representative GUS staining patterns of four pAtSUC2::GUS promoter-reporter lines. A) source leaf (inset = close-up); B) sink leaf (inset = close-up); C) new leaf; D) petiole cross-section; E) upper stem cross-section; F) lower stem cross-section; G) storage root cross-section; H) fibrous root (inset = root tip).

[0020] Figure 5A-5H shows the representative GUS staining patterns of four pCmGolS1 promoter-reporter gene lines. A) Source leaf (inset = close-up); B) Sink leaf (inset = close-up); C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root (inset = close-up); H) Fibrous root (inset = root tip).

[0021] Figure 6 A-6H shows the representative GUS staining patterns of four pCoYMV promoter-reporter gene lines. A) Source leaf (inset = close-up); B) Sink leaf (inset = close-up); C) New leaf; D) Cross-section of petiole (inset = close-up); E) Cross-section of upper stem (inset = close-up); F) Cross-section of lower stem (inset = close-up); G) Cross-section of storage root (inset = close-up); H) Fibrous root (inset = root tip).

[0022] Figure 7 A-7H shows the representative GUS staining patterns of at least four pMeSWEET1-like promoter-reporter gene lines. A) Source leaf; B) Sink leaf; C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem (inset = close-up); F) Cross-section of lower stem (inset = close-up); G) Cross-section of storage root (inset = close-up); H) Fibrous root (inset = developing lateral root).

[0023] Figure 8 A-8H shows the representative GUS staining patterns of at least four pMeSUS1 promoter-reporter gene lines. A) Source leaf (inset = close-up); B) Sink leaf (inset = close-up); C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root; H) Fibrous root; I) GUS expression levels (%) of three pMeSUS1::GUS lines relative to three pCaMV35S::GUS lines. Bars represent means with standard deviations (n = 4).

[0024] Figure 9 A-9H shows the representative GUS staining patterns of four pStPatatin class I promoter-reporter gene lines. A) Source leaf; B) Sink leaf; C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root; H) Fibrous root; I) GUS expression levels (%) of three pStPatatin:GUS lines relative to three pCaMV35S::GUS lines. Bars represent means with standard deviations (n = 4).

[0025] Figure 10A-10H shows representative GUS staining patterns of at least four pStB33 promoter-reporter gene lines. A) Source leaf; B) Sink leaf; C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root; H) Fibrous root (insert = root tip); I) GUS expression levels (%) of three pStB33::GUS lines relative to three pCaMV35S::GUS lines. Bars represent mean values with standard deviation (n = 4).

[0026] Figure 11 A-11H shows representative GUS staining patterns of four pStGBSS1 promoter-reporter gene lines. A) Source leaf; B) Sink leaf; C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root; H) Fibrous root (insert = root tip); I) GUS expression levels (%) of three pStGBSS1:GUS lines relative to three pCaMV35S::GUS lines. Bars represent mean values with standard deviation (n = 4).

[0027] Figure 12 A-12H shows representative GUS staining patterns of four pMeGPT promoter-reporter gene lines. A) Source leaf; B) Sink leaf (insert = close-up); C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root; H) Fibrous root; I) GUS expression levels (%) of three pMeGPT::GUS lines relative to three pCaMV35S::GUS lines. Bars represent mean values with standard deviation (n = 4).

[0028] Figure 13 A-13H shows representative GUS staining patterns of the pManes.14g071100 promoter-reporter gene line. A) Source leaf; B) Sink leaf; C) Shoot tip; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root; H) Fibrous root.

[0029] Figure 14 A-14H shows representative GUS staining patterns of at least four pIbSRD1 promoter-reporter gene lines. A) Source leaf; B) Sink leaf; C) New leaf; D) Cross-section of petiole; E) Cross-section of upper stem; F) Cross-section of lower stem; G) Cross-section of storage root (insert = close-up); H) Fibrous root.

[0030] Figure 15A-15G shows the sampling positions and tissue descriptions of cassava plants. Different organs and organ sections were sampled from three-month-old cassava 60444 plants grown in the greenhouse and counterstained with 10% toluidine blue solution. The numbered circles in the left panel indicate the approximate sampling positions of each sample for GUS staining. The samples were taken from cassava plants transformed with the promoter::uidA construct and their tissue-specific staining patterns were analyzed to show their respective promoter activities. Note the brown color of the developing (sink) leaves in circle A, which is easily distinguishable from the green color of the fully expanded source leaves in circle B. A) New / sink leaves, B) Source leaves, C) Cross-section of petiole, D) Cross-section of upper stem, E) Cross-section of lower stem, F) Cross-section of storage root, G) Fibrous root. Abbreviations: Collenchyma (Col), Cork cambium (CC), Major vein (MaV), Mesophyll (Mes), Minor vein (MiV), Parenchyma (Par), Periderm (Per), Phelloderm (Phe), Phloem parenchyma (PPar), Phloem (Phl), Pith parenchyma (PiPar), Pith (Pi), Protoxylem (PX), Sclerenchyma (Scl), Vascular cambium (VC), Vascular ray (VR), Xylem fiber (XF), Xylem parenchyma (XPar), Xylem vessel (XV).

[0031] Figure 16 A-16H shows the representative GUS staining patterns of three pCaMV35S promoter-reporter gene lines. A) Source leaves, B) Sink leaves, C) Cross-section of petiole, D) Cross-section of upper stem, E) Cross-section of storage root, F) Fibrous root.

[0032] Figure 17 A-17F shows the representative GUS staining patterns of four pStFBPase cyt promoter-reporter gene lines. A) Source leaves, B) Sink leaves, C) Cross-section of petiole, D) Cross-section of upper stem, E) Cross-section of storage root, F) Fibrous root.

[0033] Figure 18 A-18F shows the representative GUS staining patterns of four pDjDIO3 promoter-reporter gene lines. A) Source leaves, B) Sink leaves, C) Cross-section of petiole, D) Cross-section of upper stem, E) Cross-section of storage root, F) Fibrous root.

[0034] Brief Description of the Sequences

[0035] SEQ ID NO:1 is the promoter sequence of the bidirectional sugar transporter SWEET1 gene (MeSWEET1) from Manihot esculenta.

[0036] SEQ ID NO:2 is the promoter sequence of the glucose-6-phosphate / phosphate transporter gene (MeGPT) from Manihot esculenta.

[0037] SEQ ID NO:3 is the promoter sequence of the cassava photosystem II subunit R gene (MePsbR).

[0038] SEQ ID NO:4 is the promoter sequence of the cassava NADH-ubiquinone reductase complex 1 MLRQ subunit (B12D) gene (Manes.14g071100).

[0039] SEQ ID NO:5 is the promoter sequence of the cassava sucrose synthase 1 gene (MeSUS1).

[0040] SEQ ID NO:6 is the promoter sequence of the Arabidopsis thaliana chlorophyll A / B binding protein gene (AtCAB1), At1g29930.

[0041] SEQ ID NO:7 is the promoter sequence that controls the transcription of a single viral mRNA encoding the entire Comellina yellow mottle virus (CoYMV) genome.

[0042] SEQ ID NO:8 is the promoter sequence of the potato (Solanum tuberosum) cytoplasmic fructose-1,6-bisphosphatase gene (StFBPase cyt )

[0043] SEQ ID NO:9 is the promoter sequence of the Dioscorea japonica dioscorine 3 subunit gene (DjDio3).

[0044] SEQ ID NO:10 is the promoter sequence of the Arabidopsis thaliana fructose bisphosphate aldolase 2 gene (AtFBA2), AT4G38970.

[0045] SEQ ID NO:11 is the promoter sequence of the melon (Cucumis melo) galactinol synthase 1 gene (CmGolS1).

[0046] SEQ ID NO:12 is the promoter sequence of the Arabidopsis thaliana glyceraldehyde 3-phosphate dehydrogenase subunit A gene (AtGAPA), AT3G26650.

[0047] SEQ ID NO:13 is the promoter sequence of the potato granule-bound starch synthase 1 gene (StGBSS1).

[0048] SEQ ID NO:14 is the promoter sequence of the cassava granule-bound starch synthase 1 gene (MeGBSS1).

[0049] SEQ ID NO:15 is the promoter sequence of the potato leaf-specific 1 gene (StLS1), X04753.1.

[0050] SEQ ID NO:16 is the promoter sequence of the sweet potato (Ipomoea batatas) Mads-Box protein SRD1 gene (IbSRD1).

[0051] SEQ ID NO:17 is the promoter sequence of the potato PATATIN class 1 gene (StPat).

[0052] SEQ ID NO:18 is the promoter sequence of the Arabidopsis thaliana ribulose bisphosphate carboxylase small subunit 1A gene (AtRBCS1A), AT1G67090.

[0053] SEQ ID NO:19 is the promoter sequence of the tomato (Solanum lycopersicum) ribulose bisphosphate carboxylase small subunit 2 gene (SlRBCS2), X66069.1.

[0054] SEQ ID NO:20 is the promoter sequence of the Arabidopsis thaliana ribulose bisphosphate carboxylase small subunit 3 gene (AtRBCS3B), At5g38410.

[0055] SEQ ID NO:21 is the promoter sequence of the potato soluble starch synthase 3 gene (StSSS3).

[0056] SEQ ID NO:22 is the promoter sequence of the potato starch phosphorylase 1 gene (StSTP1), X73684.1.

[0057] SEQ ID NO:23 is the promoter sequence of the potato B33 gene (StB33).

[0058] SEQ ID NO:24 is the promoter sequence of the Arabidopsis thaliana sucrose-proton symporter 2 gene (AtSUC2). Detailed Description of the Invention

[0060] There is an urgent need to increase agricultural production in sub-Saharan Africa to combat hunger and malnutrition. According to the latest report of the Food and Agriculture Organization of the United Nations (FAO), 239 million people in sub-Saharan Africa are chronically hungry; and at least 399 million people are moderately food insecure. In addition, in recent years, food insecurity in sub-Saharan Africa has deteriorated mainly due to climate change, conflicts, and economic slowdown.

[0061] The starchy crop cassava is a key staple food in sub-Saharan Africa, providing food for millions of people. Due to the limited amount of arable land available for farming in this region, increasing cassava yield and / or nutritional content through plant genetic engineering represents an important approach to support food security in sub-Saharan Africa, especially for small-scale farmers with limited agricultural input resources such as industrial fertilizers. The successful implementation of many plant genetic engineering concepts depends on the availability of correct spatio-temporal expression tools; however, fully characterized cassava promoters are scarce in the public domain. Additionally, yield and other beneficial traits can be polygenic, depending on the interaction of many genes. The flux through biochemical pathways is usually coordinated with the flux of competing pathways, and thus, effective metabolic engineering will be achieved only by controlling multiple genes of the same or interconnected pathways. Therefore, a large number of new fully characterized promoters are needed to support cassava biotechnology methods, especially for applications that require the expression of multiple genes in different tissues or subcellular compartments. In particular, autotrophic tissue-specific promoter sequences, heterotrophic tissue (such as phloem or storage parenchyma) - specific promoters, or promoters with very cell-specific expression patterns will be valuable.

[0062] Accordingly, the present disclosure provides polynucleotide molecules having beneficial gene regulatory activity from plant species. The present invention provides the design, construction, and use of these polynucleotide molecules. Nucleotide sequences of these polynucleotide molecules are provided herein, such as SEQ ID NO: 1 - 24. These polynucleotide molecules are capable of affecting the expression of an operably linked transcribable polynucleotide molecule in a plant tissue and thus selectively regulating gene expression or the activity of an encoded gene product in a transgenic plant. The present invention also provides methods for modifying, producing, and using them. The present invention also provides compositions, transformed host cells, transgenic plants, and seeds containing a promoter and / or other disclosed nucleotide sequences, as well as methods for preparing and using them.

[0063] The following definitions and methods are provided to better define the present invention and guide one of ordinary skill in the art in practicing the present invention. Unless otherwise indicated, terms shall be understood according to the ordinary usage of one of ordinary skill in the relevant art.

[0064] DNA molecule

[0065] As used herein, the term "DNA" or "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin read from the 5' (upstream) end to the 3' (downstream) end, i.e., a polymer of deoxyribonucleotide bases or polynucleotide molecules. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The terms used herein correspond to those in 37 C.F.R. § 1.822 and are as shown in the tables of Appendix 2, Tables 1 and 3 of WIPO Standard ST.25 (1998).

[0066] As used herein, the term "isolated DNA molecule" refers to a DNA molecule that is at least partially separated from other molecules that are normally associated with it in its original or native state. In one embodiment, the term "isolated" refers to a DNA molecule that is at least partially separated from some of the nucleic acids that flank the DNA molecule in its original or native state. Thus, a DNA molecule that is fused to regulatory or coding sequences that are not normally associated with it (e.g., as a result of recombinant techniques) is considered isolated herein. Such a molecule is considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution together with other DNA molecules because they are not in their original state.

[0067] As used herein, a "recombinant DNA molecule" is a DNA molecule that contains a combination of DNA molecules that do not naturally occur together without human intervention. For example, a recombinant DNA molecule can be a DNA molecule composed of at least two DNA molecules that are heterologous to each other, a DNA molecule that contains a DNA sequence different from a naturally occurring DNA sequence, or a DNA molecule that has been integrated into the DNA of a host cell by genetic transformation or gene editing.

[0068] The polynucleotides of the present invention can be synthetic nucleotide sequences. A "synthetic nucleotide sequence" is a nucleotide sequence that is not known to exist in nature or is not naturally occurring. In some embodiments, the polynucleotide has little or no extended homology to a natural sequence. As used herein, extended homology generally refers to 100% sequence identity of a continuous sequence extending more than about 25 nucleotides.

[0069] Any number of methods well known to those skilled in the art can be used to isolate and manipulate the DNA molecules or fragments thereof disclosed in the present invention. For example, PCR (polymerase chain reaction) technology can be used to amplify a specific starting DNA molecule and / or generate variants of the original molecule. DNA molecules or fragments thereof can also be obtained by other techniques, such as directly synthesizing the fragment by chemical methods, which is typically accomplished using an automated oligonucleotide synthesizer.

[0070] As used herein, the term "sequence identity" refers to the degree of identity between two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences. The optimal sequence alignment is generated by manually aligning two sequences (e.g., a reference sequence and another sequence) to maximize the number of nucleotides that match in the sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term "reference sequence" refers to the sequences provided as the polynucleotide sequences of SEQ ID NO: 1-24.

[0071] As used herein, the term "percent sequence identity" or "percent identity" or "% identity" is the identity score multiplied by 100. The "identity score" of a sequence optimally aligned with 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 over the full length of the entire reference sequence. Thus, one embodiment of the invention is a DNA molecule comprising a sequence that has at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with the reference sequence when optimally aligned with the reference sequence provided herein as SEQ ID NO: 1-24. In certain embodiments, such sequences may be defined as having gene regulatory activity or the activity of the reference sequence.

[0072] Regulatory element

[0073] A regulatory element is a DNA molecule having gene regulatory activity, i.e., a DNA molecule having the ability to affect the transcription and / or translation of an operably linked transcribable polynucleotide molecule. Thus, the term "gene regulatory activity" refers to the ability to affect the expression pattern of an operably linked transcribable polynucleotide molecule by affecting the transcription and / or translation of the operably linked transcribable polynucleotide molecule. As used herein, a transcriptional regulatory sequence can comprise an operably linked expression element such as an enhancer, a promoter, a leader sequence such as a 5'-untranslated region or a portion thereof, an intron, a 3'-untranslated region or a portion thereof, a terminator, a transcriptional termination region (or 3'UTR) or a chromatin control element that functions in plants, and can thus be used to genetically engineer the modification of plant phenotypes. Thus, a transcriptional regulatory sequence can comprise, for example, a 5' of a promoter operably linked to a leader sequence, the 5' of which is in turn operably linked to an intron sequence. The leader sequence and the intron can positively affect the transcription of an operably linked transcribable polynucleotide molecule and the translation of the resulting transcribed RNA. The preprocessed RNA molecule comprises a leader sequence and an intron, which can affect the post-transcriptional processing of the transcribed RNA and / or the export of the transcribed RNA molecule from the nucleus to the cytoplasm. After the post-transcriptional processing of the transcribed RNA molecule, the leader sequence can remain as part of the final messenger RNA and can positively affect the translation of the messenger RNA molecule.

[0074] Regulatory elements such as promoters, enhancers, leader sequences such as 5'-untranslated regions or portions thereof, introns, 3'-untranslated regions or portions thereof, transcriptional termination regions (or 3'UTRs) or chromatin control elements are DNA molecules having gene regulatory activity and play important roles in the overall expression of genes in living cells. The term "regulatory element" refers to a DNA molecule having gene regulatory activity, i.e., a DNA molecule having the ability to affect the transcription and / or translation of an operably linked transcribable polynucleotide molecule. Thus, isolated regulatory elements such as promoters and leader sequences that function in plants can be used to genetically engineer the modification of plant phenotypes.

[0075] It is recognized that the polynucleotides of the present invention can comprise multiple regulatory elements such as promoters and enhancers. It is also recognized that some genetic regulatory elements act in concert with other genetic regulatory elements to control the regulation of an operably linked gene of interest. In addition, it is recognized that some genetic regulatory elements such as promoters or enhancers can be separated from the transcriptional region of the gene of interest by DNA of 1, 2, 3 or more kilobases.

[0076] The present invention also provides methods for controlling gene expression. "Controlling gene expression" refers to controlling the expression of an RNA transcript and can further encompass the translation of the transcript, or even the activity or function of the encoded protein. Controlling gene expression can include affecting one or more of RNA transcription, processing, turnover and / or translation.

[0077] The genetic regulatory elements disclosed herein can be implemented as regulatory sequences to control gene expression in a "desired manner". The desired manner of gene expression can be temporal, spatial, or any combination thereof in a target organism, including but not limited to constitutive expression, tissue-preferred expression, and organ-preferred expression. The desired manner of gene expression can also be expression in response to biotic stresses (such as fungal, bacterial, and viral pathogens, insects, herbivores, etc.) and / or abiotic stresses (such as wounding, drought, cold, heat, high nutrient levels, low nutrient levels, metals, light, herbicides, and other synthetic chemicals, etc.).

[0078] Regulatory elements can be characterized by their expression pattern effects (qualitative and / or quantitative), such as positive or negative effects and / or constitutive or other effects, such as their temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemical responsiveness expression patterns, and any combination thereof, as well as by quantitative or qualitative representations. A promoter can be used as a regulatory element to regulate the expression of an operably linked transcribable polynucleotide molecule.

[0079] As used herein, "gene expression pattern" is any pattern of transcription of an operably linked DNA molecule into a transcribed RNA molecule. The transcribed RNA molecule can be translated to produce a protein molecule or can provide an antisense or other regulatory RNA molecule, such as dsRNA, tRNA, rRNA, miRNA, etc.

[0080] As used herein, the term "protein expression" is any pattern of translation of a transcribed RNA molecule into a protein molecule. Protein expression can be characterized by its temporal, spatial, developmental, or morphological characteristics and by quantitative or qualitative representations.

[0081] Promoter

[0082] A regulatory element, such as a promoter of the present invention, can be operably linked to a transcribable DNA molecule that is heterologous to the regulatory element. As used herein, the term "heterologous" refers to such a combination when the combination of two or more DNA molecules does not normally 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, such as different genes from the same species or the same gene from different species. Thus, if the combination does not normally exist in nature, i.e., the transcribable DNA molecule is not naturally operably linked to the regulatory element, the regulatory element is heterologous with respect to the operably linked transcribable DNA molecule.

[0083] A transcribable DNA molecule can generally be any DNA molecule for which expression of a transcript is desired. Such expression of the transcript can lead to translation of the resulting mRNA molecule and thus to protein expression. Alternatively, for example, a transcribable DNA molecule can be designed to ultimately result in decreased expression of a particular gene or protein. In one embodiment, this can be achieved by using a transcribable DNA molecule oriented in the antisense direction. Those of ordinary skill in the art are familiar with the use of such antisense technology. Any gene can be negatively regulated in this manner, and in one embodiment, a transcribable DNA molecule can be designed to inhibit a particular gene through the expression of dsRNA, siRNA, or miRNA molecules.

[0084] In some embodiments, the present disclosure provides polynucleotides containing a promoter and / or enhancer. A "promoter" refers to a nucleotide sequence capable of controlling the expression of an operably linked coding sequence or other sequence encoding an RNA that need not be translated into a protein. Thus, a polynucleotide can include proximal promoter elements as well as more distal upstream elements, the latter generally referred to as enhancers. An "enhancer" refers to a DNA sequence that can stimulate promoter activity and can be a native element of the promoter or a heterologous element inserted to enhance promoter level or tissue specificity. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental conditions. It is also recognized that since the exact boundaries of regulatory sequences are not fully defined in most cases, some variant nucleic acid fragments may have the same or similar promoter activity.

[0085] A promoter that causes a gene to be expressed in most cell types of an organism and at most times is generally referred to as a "constitutive promoter". Expression of a gene in most cell types of an organism is herein referred to at most as "constitutive gene expression" or "constitutive expression".

[0086] In some embodiments, the regulatory element is an expression-enhancing intron. An "expression-enhancing intron" or "enhancing intron" is an intron that can cause an increase in the expression of a gene operably linked thereto. Although the mechanism by which the present invention relies on a specific biology is not known, it is believed that the expression-enhancing introns of the present invention enhance expression through intron-mediated enhancement (IME). It is recognized that naturally occurring introns that enhance expression through IME are typically found within 1 Kb of the transcription start site of their native gene (see, Rose et al. (2008) Plant Cell 20:543-551). Such introns are typically the first intron, whether the first intron is in the 5'UTR or the coding sequence, and are required to be in the transcription region. When introns that enhance expression solely through IME are inserted into the non-transcribed region of a gene, such as a promoter, they do not enhance gene expression. That is, they do not function as transcriptional enhancers. Unless otherwise stated or obvious from the context, the expression-enhancing introns of the present invention can enhance gene expression when found in the transcription region of a gene, but do not enhance gene expression when they occur in a non-transcribed region such as a promoter.

[0087] In some embodiments, the promoter is a plant promoter. A "plant promoter" is a promoter that can initiate transcription in a plant cell, regardless of whether its source is a plant cell. For example, it is well known that Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses, and bacteria such as Agrobacterium and Bradyrhizobium, as well as synthetic promoters that can initiate transcription in a plant cell. Plant promoters can be constitutive promoters, non-constitutive promoters, inducible promoters, repressible promoters, tissue-specific promoters (e.g., root-specific promoters, stem-specific promoters, leaf-specific promoters), tissue-preferred promoters (e.g., root-preferred promoters, stem-preferred promoters, leaf-preferred promoters), cell type-specific or preferred promoters (e.g., meristematic cell-specific / preferred promoters), or many other types. In some embodiments, the variant polynucleotides or fragments described herein include additional known cis-acting sequences to drive the transcription of a gene to express in a desired manner.

[0088] In some embodiments, the promoter is a constitutive promoter. A "constitutive promoter" is a promoter that is active under most conditions and / or at most developmental stages. There are several advantages to using a constitutive promoter in an expression vector used in plant biotechnology, such as: high-level production of a protein for selection of transgenic cells or plants; high-level expression of a reporter protein or scorable marker, allowing easy detection and quantification; high-level production of a transcription factor as part of a regulatory transcription system; production of a compound that requires ubiquitous activity in a plant; and production of a compound required at all stages of plant development. By way of illustration, constitutive promoters can include the CaMV 19S promoter, the CaMV 35S promoter (U.S. Patent Nos. 5,352,605; 5,530,196 and 5,858,742), opine promoters, ubiquitin promoters, actin promoters, alcohol dehydrogenase promoters, and the like. In some embodiments, a synthetic promoter prepared as described herein is used to drive the expression of a heterologous sequence, while the CaMV 35S promoter is used to drive the expression of a second sequence.

[0089] In some embodiments, the promoter is a non-constitutive promoter. A "non-constitutive promoter" is a promoter that is active under certain conditions, in certain types of cells, and / or at certain developmental stages. For example, tissue-specific, tissue-preferred, cell-type-specific, cell-type-preferred, inducible promoters, and promoters under developmental control are non-constitutive promoters. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues such as stems, leaves, roots, or seeds.

[0090] In some embodiments, the promoter is an inducible or repressible promoter. An "inducible" or "repressible" promoter is a promoter that is under the control of chemical or environmental factors. Examples of environmental conditions that can affect the transcription of an inducible promoter include cold, heat, drought, the presence of certain chemicals, or light.

[0091] In some embodiments, the promoter is a tissue-specific promoter. A "tissue-specific" promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of a gene, tissue-specific expression is the result of several interacting levels of gene regulation. Thus, in the art, it is sometimes preferred to use promoters from homologous or closely related plant species to achieve efficient and reliable expression of a transgene in a specific tissue. This is one of the main reasons for the large number of tissue-specific promoters isolated from specific plants and tissues found in the scientific and patent literature. Non-limiting examples of known tissue-specific promoters can include the β-amylase gene or the hordein gene promoter of barley (for seed gene expression), the tomato pz7 and pz130 gene promoters (for ovary gene expression), the tobacco RD2 gene promoter (for root gene expression), the banana TRX promoter and the melon actin promoter (for fruit gene expression), and embryo-specific promoters, such as those associated with the amino acid permease gene (AAP1), oleate 12-hydroxylase: desaturase gene from Lesquerella (LFAH12), 2S2 albumin gene (2S2), fatty acid elongase gene (FAE1) or leafy cotyledon gene (LEC2).

[0092] In some embodiments, the promoter is a tissue-preferred promoter. A "tissue-preferred" promoter is a promoter that primarily initiates transcription in certain tissues, but not necessarily exclusively or only in certain tissues.

[0093] In some embodiments, the promoter is a cell-type-specific promoter. A "cell-type-specific" promoter is a promoter that drives expression primarily in certain cell types in one or more organs, such as vascular cells, stem cells in roots, leaves, and stems.

[0094] In some embodiments, the promoter is a cell-type-preferred promoter. A "cell-type-preferred" promoter is a promoter that primarily drives most of the expression, but not necessarily exclusively or only in certain cell types in one or more organs, such as vascular cells, stem cells in roots, leaves, and stems.

[0095] In some embodiments, the promoter is a root-specific promoter. A "root-specific" promoter is a promoter that initiates transcription only in root tissue.

[0096] In some embodiments, the promoter is a root-preferred promoter. A "root-preferred" promoter is a promoter that primarily initiates transcription in root tissue, but not necessarily exclusively or only in root tissue.

[0097] In some embodiments, the present invention provides methods for obtaining inbred plants comprising a polynucleotide sequence. As used herein, the term "inbred line" or "inbred plant" is used in the context of the present invention. This also includes any single-gene transformation of such inbred lines. The phrase "monoallelically transformed plant" as used herein refers to those plants developed by a plant breeding technique called backcrossing, in which substantially all of the desired morphological and physiological characteristics of the inbred line are restored except for the single allele transferred into the inbred line by the backcrossing technique.

[0098] The term "promoter" or a molecule having "promoter activity" as used herein generally refers to a DNA molecule that participates in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A promoter can initially be isolated from the 5' untranslated region (5' UTR) of a genomic copy of a gene. Alternatively, a promoter can be a DNA molecule that is synthetically produced or engineered. A promoter can also be chimeric, i.e., a promoter produced by fusing two or more heterologous DNA molecules. Promoters that can be used to practice the present invention include SEQ ID NO: 1-24, or fragments, variants, functional fragments or variants thereof, or combinations thereof. In specific embodiments of the present invention, such molecules and any variants or derivatives thereof as described herein are further defined as comprising promoter activity, i.e., being capable of acting as a promoter in a host cell, such as a transgenic plant. In further specific embodiments, a fragment can be defined as exhibiting the promoter activity of the starting promoter molecule from which it is derived, or a fragment can comprise a "minimal promoter" that provides a basal level of transcription and contains a TATA box or equivalent sequence for the recognition and binding of the RNA polymerase II complex for initiating transcription.

[0099] In one embodiment, fragments of the promoter sequences disclosed herein are provided. The promoter fragments can comprise promoter activity as described above and can be used alone or in combination with other promoters and promoter fragments, such as for constructing chimeric promoters. In specific embodiments, promoter fragments are provided that comprise at least about 50, 95, 150, 250, 500, 750, 1000, 1250, 1500, 1750 or at least about 2000 consecutive nucleotides, or longer, of any one of SEQ ID NO: 1-24 or of a polynucleotide molecule having promoter activity as disclosed herein. Fragments of SEQ ID NO: 1-24 can have the activity of the reference promoter sequence.

[0100] Compositions derived from any of the promoters shown in SEQ ID NO: 1-24 (e.g., internal or 5' deletions) 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; duplicating elements that have a positive or negative effect on expression; and / or duplicating or removing elements that have a tissue- or cell-specific effect on expression. Compositions derived from any of the promoters shown in SEQ ID NO: 1-24 can be used, for example, to prepare enhancer elements, said promoters comprising 3' deletions where the TATA box element or its equivalent sequence and downstream sequences are removed. Further deletions can be made to remove any element that has a positive or negative; tissue-specific; cell-specific; or timing-specific (e.g., but not limited to circadian rhythm) effect on expression. Any of the promoters shown in SEQ ID NO: 1-24 and their derived fragments or enhancers can be used to prepare chimeric transcriptional regulatory element compositions, which consist of any of the promoters shown in SEQ ID NO: 1-24 and their derived fragments or enhancers operably linked to other enhancers and promoters. The efficacy of the modifications, duplications, or deletions described herein on the desired expression aspects of a particular transgene can be empirically tested in stable and transient plant assays, such as those described in the working examples herein, to verify the results, which can vary depending on the changes made and the goals of the starting molecule modification.

[0101] As used herein, the term "leader sequence" refers to a DNA molecule isolated from the untranslated 5' region (5' UTR) of the genomic copy of a gene and is generally defined as the nucleotide segment between the transcription start site (TSS) and the start site of the protein-coding sequence. Alternatively, the leader sequence can be a synthetically produced or engineered DNA element. The leader sequence can be used as a 5' regulatory element to regulate the expression of an operably linked transcribable polynucleotide molecule. The leader molecule can be used with a heterologous promoter or its native promoter. Thus, the promoter molecules of the present invention can be operably linked to their native leader sequence or can be operably linked to a heterologous leader sequence. Leader sequences known in the art can be used to practice the present invention. The leader sequence (5' UTR) can consist of regulatory elements or can adopt a secondary structure that affects the transcription or translation of the transgene. Leader sequences known in the art can be used according to the present invention to prepare chimeric regulatory elements that affect the transcription or translation of the transgene. In addition, leader sequences can be used to prepare chimeric leader sequences that affect the transcription or translation of the transgene.

[0102] The introduction of foreign genes into new plant hosts does not always result in high expression of the input genes. In addition, when dealing with complex traits, it is sometimes necessary to regulate several genes with spatially or temporally different expression patterns. Introns can mainly provide such regulation. However, the repeated use of the same intron in one plant has shown disadvantages. In those cases, a collection of basic control elements for constructing suitable recombinant DNA elements is needed.

[0103] According to the present invention, a promoter or a promoter fragment can be analyzed for the presence of known promoter elements, i.e., DNA sequence features such as the TATA box and other known transcription factor binding site motifs. A person skilled in the art can use the identification of such known promoter elements to design promoter variants having a similar expression pattern to the original promoter.

[0104] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting transcriptional regulatory element, also referred to as a cis-element, which confers aspects of the overall expression pattern of an operably linked polynucleotide sequence, but which generally is not sufficient alone to drive transcription. Unlike a promoter, an enhancer element generally does not include a transcription start site (TSS) or a TATA box or equivalent sequence. A promoter may naturally contain one or more enhancer elements that affect the transcription of an operably linked polynucleotide sequence. An isolated enhancer element also may be fused to a promoter to produce a chimeric promoter cis-element that confers aspects of the overall regulation of gene expression. A promoter or promoter fragment may contain one or more enhancer elements that affect the transcription of an operably linked gene. Many promoter enhancer elements are thought to bind DNA-binding proteins and / or affect DNA topology, creating a local conformation that selectively allows or restricts RNA polymerase access to the DNA template or promotes selective opening of the double helix at the transcription start site. Enhancer elements can be used to bind 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, in which one or more nucleotides are deleted from the 5'-end or internally from a promoter; DNA-binding protein analysis using DNaseI footprinting, methylation interference, electrophoretic mobility shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or DNA sequence similarity analysis using a known cis-element motif or enhancer element as a target sequence or target motif, in which conventional DNA sequence comparison methods such as BLAST are used. The fine structure of an enhancer domain can be further investigated by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Enhancer elements can be obtained by chemical synthesis or by isolation from regulatory elements that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation. Accordingly, the present invention encompasses the design, construction, and use of enhancer elements according to the methods for regulating the expression of an operably linked transcribable polynucleotide molecule disclosed herein. Enhancer sequences derived from CaMV also can be used (e.g., U.S. Patent Nos. 5,164,316; 5,196,525; 5,322,938; 5,530,196; 5,352,605; 5,359,142; and 5,858,742).

[0105] In plants, inclusion of some introns in a gene construct results in increased mRNA and protein accumulation relative to constructs lacking introns.

[0106] This effect is known as "intron-mediated enhancement" (IME) of gene expression (Mascarenhas et al., (1990) Plant Mol. Biol. 15:913-920). Introns that stimulate expression in plants have been identified in maize genes such as tubA1, Adh1, Sh1, Ubi1 (Jeon et al. (2000) Plant Physiol. 123:1005-1014; Callis et al. (1987) Genes Dev. 1:1183-1200; Vasil et al. (1989) Plant Physiol. 91:1575-1579; Christiansen et al. (1992) Plant Mol. Biol. 18:675-689) and rice genes such as salt, tpi: McElroy et al., Plant Cell 2:163-171 (1990); Xu et al., Plant Physiol. 106:459-467 (1994)). Similarly, introns from dicotyledonous plant genes such as from petunia (e.g., rbcS), potato (e.g., st-ls1) and Arabidopsis (e.g., ubq3 and pat1) have been found to increase the rate of gene expression (Dean et al. (1989) Plant Cell 1:201-208; Leon et al. (1991) Plant Physiol. 95:968-972; Norris et al. (1993) Plant Mol Biol 21:895-906; Rose and Last (1997) Plant J. 11:455-464). It has been shown that deletions or mutations within intron splice sites reduce gene expression, indicating that IME may require splicing (Mascarenhas et al. (1990) Plant Mol Biol. 15:913-920; Clancy and Hannah (2002) Plant Physiol. 130:918-929). However, it has been shown by point mutations within the splice site of the pat1 gene from Arabidopsis that some IME in dicotyledons do not require splicing itself (Rose and Beliakoff (2000) Plant Physiol. 122:535-542).

[0107] Enhancing gene expression through introns is not a universal phenomenon because the insertion of some introns into recombinant expression cassettes fails to enhance expression (e.g., introns from dicotyledonous plant genes (rbcS gene from pea, phaseolin gene from bean, and stls-1 gene from potato) and introns from maize genes (adh1 gene, the ninth intron, hsp81 gene, the first intron)) (Chee et al. (1986) Gene 41:47-57; Kuhlemeier et al. (1988) Mol Gen Genet 212:405-411; Mascarenhas et al. (1990) Plant Mol. Biol. 15:913-920; Sinibaldi and Mettler (1992) In WE Cohn, K Moldave, eds, Progress in Nucleic Acid Research and Molecular Biology, Vol 42. Academic Press, New York, pp 229-257; Vancanneyt et al. 1990 Mol. Gen. Genet. 220:245-250). Thus, not every intron can be used to manipulate the gene expression level of non-endogenous or endogenous genes in transgenic plants. To increase the expression rate of a given gene, it is unknown in the prior art what characteristics or specific sequence features must be present in the intron sequence, and thus it is impossible to predict from the prior art whether a given plant intron (when used heterologously) will result in enhanced expression at the DNA level or the transcript level (IME).

[0108] As used herein, the term "chimeric" refers to a single DNA molecule produced by fusing a first DNA molecule with a second DNA molecule, where neither the first nor the second DNA molecule is normally found in that configuration, i.e., fused to each other. Thus, the chimeric DNA molecule is a new DNA molecule not normally found in nature. As used herein, the term "chimeric promoter" refers to a promoter produced by such manipulation of DNA molecules. A chimeric promoter can combine two or more DNA fragments; an example is the fusion of a promoter with an enhancer element. Thus, chimeric promoters designed, constructed, and used according to the methods disclosed herein for regulating the expression of operably linked transcribable polynucleotide molecules are encompassed by this disclosure.

[0109] As used herein, the term "variant" refers to a second DNA molecule that is similar but not identical to the first DNA molecule in composition, but the second DNA molecule still maintains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern. The variant can be a shorter or truncated form of the first DNA molecule and / or an altered form of the first DNA molecule sequence, such as a form with different restriction enzyme sites and / or internal deletions, substitutions and / or insertions. "Variant" can also encompass regulatory elements having a nucleotide sequence that replaces, deletes and / or inserts one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equivalent transcription or translation activity than the corresponding parent regulatory molecule. Regulatory element "variants" also encompass variants produced by naturally occurring mutations in bacterial and plant cell transformation. In the present invention, polynucleotide sequences such as those provided by SEQ ID NO: 1-24 can be used to produce variants that are similar but not identical to the polynucleotide sequences of the original regulatory elements in composition, while still maintaining the general functionality of the original regulatory elements, i.e., the same or similar expression pattern. According to the present disclosure, the generation of these variants of the present invention is completely within the ordinary skill of the art, and is encompassed within the scope of the present invention. Chimeric regulatory element "variants" contain the same constituent elements as the reference sequence, but the constituent elements comprising the chimeric regulatory element can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, 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 chimeric regulatory element "variant" can be composed of the same constituent elements as the reference sequence or variants thereof, but differs in one or more sequences containing one or more connection sequences that allow the constituent parts to be operably linked. In the present invention, the polynucleotide sequences provided by SEQ ID NO: 1-24 provide reference sequences, wherein the constituent elements comprising the reference sequence can be linked by methods known in the art, and can contain substitutions, deletions and / or insertions of one or more nucleotides or mutations that occur naturally in bacterial and plant cell transformation.

[0110] Construct

[0111] As used herein, the term "construct" refers to any recombinant polynucleotide molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or a linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule derived from any source, which is capable of genome integration or autonomous replication, comprising a polynucleotide molecule, wherein one or more polynucleotide molecules are linked in a functionally operable manner, i.e., operably linked. As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. The term includes expression cassettes isolated from any of the aforementioned molecules.

[0112] Expression cassette

[0113] The polynucleotides of the present invention can be provided in an expression cassette for expressing a target gene in a target plant or other organism or host cell. It is recognized that the polynucleotides of the present invention and expression cassettes containing them can be used for expression in human and non-human host cells, including but not limited to host cells from plants, animals, fungi, and algae. In one embodiment of the present invention, the host cell is a human host cell or a host cell line that cannot differentiate into a human.

[0114] The expression cassette can include 5′ and 3′ regulatory sequences operably linked to the target gene to be expressed. "Operably linked" refers to a functional connection between two or more elements. For example, an operable connection between one or more genetic regulatory elements and a target gene is a functional connection between the target gene and one or more genetic regulatory elements that allow the expression of the target gene. The operably linked elements can be contiguous or non-contiguous. The cassette can additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene can be provided on multiple expression cassettes. Such expression cassettes have multiple restriction sites and / or recombination sites for inserting polynucleotides under the transcriptional regulation of a regulatory region. The expression cassette can additionally contain a selectable marker gene. The expression cassette can include a transcriptional and translational initiation region (i.e., a promoter), the polynucleotide to be expressed, and a transcriptional and translational termination region (i.e., a terminator) that is functional in a plant or other organism or host cell in the transcriptional 5′-3′ direction. The regulatory regions (i.e., the promoter, transcriptional regulatory region, and translational termination region) and / or the polynucleotide to be expressed can be native / to the host cell or similar to each other. In some embodiments, the promoter can be provided by the polynucleotides of the present invention.

[0115] If desired, the polynucleotide can be optimized to increase expression in the transformed plant. That is, the polynucleotide can be synthesized using plant-preferred codons to improve expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of the use of host-preferred codons. Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

[0116] Additional sequence modifications that enhance gene expression in a cellular host are known. These sequence modifications include the elimination of coding polyadenylation signals, exon-intron splice site signals, transposable element-like repeats, and other such well-characterized sequences that may be detrimental to gene expression. The G-C content of the sequence can be adjusted to an average level for a given cellular host, where the level is calculated by reference to known genes expressed in that host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0117] Expression cassettes can additionally contain 5' leader sequences. Such leader sequences can function to enhance translation. Translational leader sequences are known in the art and include: picornavirus leader sequences such as the EMCV leader sequence (encephalomyocarditis virus 5' non-coding region) (Elroy-Stein et al., (1989) PNAS USA 86:6126-6130); potyvirus leader sequences such as the TEV leader sequence (tobacco etch virus) (Gallie et al., (1995) Gene 165(2):233-238), MDMV leader sequence (maize dwarf mosaic virus) (Virology 154:9-20), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al., (1991) Nature 353:90-94); the untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al., (1987) Nature 325:622-625); the tobacco mosaic virus (TMV) leader sequence (Gallie et al. (1989), in Molecular Biology of RNA, Cech ed. (Liss, New York), pp. 237-256); and the maize chlorotic mottle virus leader sequence (MCMV) (Lommel et al., (1991) Virology 81:382-385). See also Della-Cioppa et al. (1987), Plant Physiol. 84:965-968. In preparing expression cassettes, a variety of DNA fragments can be manipulated so that the DNA sequences are in the appropriate orientation and, as needed, in the proper reading frame. For this purpose, linkers or adaptors can be used to join the DNA fragments or other manipulations can be included to provide convenient restriction sites, remove superfluous DNA, eliminate restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and replacement, such as transitions and transversions, can be involved.

[0118] The expression cassette may also include a selectable marker gene for selecting transformed cells. The selectable marker gene is used to select transformed cells or tissues. The selectable marker gene can be for positive or negative selection. For positive selection, an exogenous gene is provided to plant cells to enable them to utilize substrates present in the medium that otherwise cannot be used, such as mannose or xylose (e.g., see U.S. Patent Nos. 5,767,378; 5,994,629). However, more typically, negative selection is used because it is more efficient, using a selection agent such as a herbicide or antibiotic that kills or inhibits the growth of untransformed plant cells and reduces the likelihood of chimeras. Non-limiting exemplary marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds, such as glufosinate, bromoxynil, imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine, benzonitrile, and 2,4-dichlorophenoxyacetic acid (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng. 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan fluorescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol. 129:913-42), and yellow fluorescent protein (PhiYFP from Evrogen TM, see Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, generally see Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) PNAS 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) PNAS 86:5400-5404; Fuerst et al. (1989) PNAS 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) PNAS 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) PNAS 89:3952-3956; Baim et al. (1991) PNAS 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillen and-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) PNAS 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724; Bourouis et al., EMBO J. 2(7):1099-1104 (1983) White et al., Nucl Acids Res 18:1062 (1990), Spencer et al., Theon Appl Genet 79:625-631 (1990), U.S. Patent Nos. 5,034,322; 6,174,724; 6,255,560; 4,795,855; 5,378,824 and 6,107,549. These disclosures are incorporated herein by reference.

[0119] The above list of selectable marker genes is not restrictive. Any selectable marker gene can be used in the present invention. There are many methods for obtaining plant transformation vectors and transforming plants. See, for example, An, G. et al. (1986) Plant Pysiol., 81: 301-305; Fry, J., et al. (1987) Plant Cell Rep. 6: 321-325; Block, M. (1988) Theor. Appl Genet. 76: 767-774; Hinchee, et al. (1990) Stadler. Genet. Symp. 203212.203-212; Cousins, et al. (1991) Aust. J. Plant Physiol. 18: 481-494; Chee, P.P. and Slightom, J.L. (1992) Gene 118: 255-260; Christou, et al. (1992) Trends. Biotechnol. 10: 239-246; D'Halluin, et al. (1992) Bio / Technol. 10: 309-314; Dhir, et al. (1992) Plant Physiol. 99: 81-88; Casas et al. (1993) PNAS 90: 11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P: 119-124; Davies, et al. (1993) Plant Cell Rep. 12: 180-183; Dong, J.A. and Mchughen, A. (1993) Plant Sci. 91: 139-148; Franklin, C.I. and Trieu, T.N. (1993) Plant. Physiol. 102: 167; Golovkin, et al. (1993) Plant Sci. 90: 41-52; Guo Chin Sci. Bull. 38: 2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N.M. and Park, W.D. (1994) Crit. Rev. Plant. Sci. 13: 219-239; Barcelo, et al. (1994) Plant J. 5: 583-592; Becker, et al. (1994) Plant J. 5: 299-307; Borkowska et al. (1994) Acta.Physiol Plant. 16:225 - 230; Christou, P. (1994) Agro.Food.Ind.Hi Tech. 5:17 - 27; Eapen et al. (1994) Plant Cell Rep. 13:582 - 586; Hartman, et al. (1994) Bio - Technology 12:919 - 923; Ritala, et al. (1994) Plant.Mol.Biol. 24:317 - 325 and Wan, Y.C. and Lemaux, P.G. (1994) Plant Physiol. 104:37 - 48.

[0120] As used herein, the term "operably linked" refers to the joining of a first molecule to a second molecule, wherein the molecules are arranged such that the first molecule affects the function of the second molecule. The two molecules can be or not be part of a single continuous molecule and can be or not be adjacent. For example, if a promoter regulates the transcription of a target transcribable polynucleotide molecule in a cell, the promoter is operably linked to the transcribable polynucleotide molecule. For example, a leader sequence is operably linked to a coding sequence when it is capable of acting as a leader sequence for the polypeptide encoded by the coding sequence.

[0121] For the transformation of plants and plant cells, the nucleotide sequences of the present invention are inserted into any vector known in the art suitable for the expression of nucleotide sequences in plants or plant cells using standard techniques. The choice of vector depends on the preferred transformation technique and the target plant species to be transformed.

[0122] Methodologies for constructing plant expression cassettes and introducing foreign nucleic acids into plants are well known in the art and have been described previously. For example, tumor-inducing (Ti) plasmid vectors can be used to introduce foreign DNA into plants. There are numerous patents controlling Agrobacterium-mediated transformation and specific DNA delivery plasmids designed for Agrobacterium - for example, U.S. Patent No. 4,536,475, EP0265556, EP0270822, WO8504899, WO8603516, U.S. Patent No. 5,591,616, EP0604662, EP0672752, WO8603776, WO9209696, WO9419930, WO9967357, U.S. Patent No. 4,399,216, WO8303259, U.S. Patent No. 5,731,179, EP068730, WO9516031, U.S. Patent No. 5,693,512, 6,051,757 and EP904362A1. Agrobacterium-mediated plant transformation involves first placing a DNA fragment cloned on a plasmid into live Agrobacterium cells, which are then used to transform individual plant cells. Thus, Agrobacterium-mediated plant transformation is an indirect method of plant transformation. Agrobacterium-mediated plant transformation methods involving the use of vectors without T-DNA are also well known to those skilled in the art and can be used in the present invention. See, for example, U.S. Patent No. 7,250,554, which uses P-DNA instead of T-DNA in the transformation vector. Agrobacterium tumefaciens is a naturally occurring bacterium that is capable of inserting its DNA (genetic information) into plants, causing a type of damage to the plant called crown gall. Transgenic plants formed using Agrobacterium transformation methods typically contain a single gene on one chromosome, although multiple copies are possible. Such transgenic plants can be referred to as hemizygous for the added gene.

[0123] Other methods for delivering exogenous DNA or other exogenous nucleic acids include PEG-mediated protoplast transformation, electroporation, whisker microinjection, and biolistic or particle bombardment for direct DNA uptake. These methods are known in the art. (U.S. Patent No. 5,405,765 to Vasilet al.; Bilang et al. (1991) Gene 100:247-250; Scheid et al., (1991) Mol. Gen. Genet. 228:104-112; Guerche et al., (1987) Plant Science 52:111-116; Neuhause et al., (1987) Theor. Appl Genet. 75:30-36; Klein et al., (1987) Nature 327:70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227:1229-1231; DeBlock et al., (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988); Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989); M.E. Fromm et al., Nature, 319, 791 (1986); H.J. Jones et al., Plant Mol. Biol., 13, 501 (1989) and H.Yang et al., Plant Cell Reports, 7, 421 (1988); UMizuno et al., 2004; Petolino et al., 2000; U.S. Patent No. 5,302,523; and U.S. Patent Application Publication No. 20040197909; Kaepler et al., 1992; Raloff, 1990; Wang, 1995; U.S. Patent Nos. 5,204,253; 5,015,580; 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318; 5,641,664; 5,736,369 and 5,736,369; International Patent Application Publication Nos. WO2002 / 038779 and WO / 2009 / 117555; Lu et al., (Plant Cell Reports, 2008, 27:273 - 278); Watson et al., Recombinant DNA, Scientific American Books (1992); Hinchee et al., Bio / Tech. 6:915 - 922 (1988); McCabe et al., Bio / Tech. 6:923 - 926 (1988); Toriyama et al., Bio / Tech. 6:1072 - 1074 (1988); Fromm et al., Bio / Tech. 8:833 - 839 (1990); Mullins et al., Bio / Tech. 8:833 - 839 (1990); Hiei et al., Plant Molecular Biology 35:205 - 218 (1997); Ishida et al., Nature Biotechnology 14:745 - 750 (1996); Zhang et al., Molecular Biotechnology 8:223 - 231 (1997); Ku et al., Nature Biotechnology 17:76 - 80 (1999); and Raineri et al., Bio / Tech.8:33-38(1990), each of which is incorporated herein by reference in its entirety). The transformation method depends on the plant cells to be transformed, the stability of the vector used, the expression level of the gene product, and other parameters. Specific methods for transforming certain plant species (such as maize, rice, wheat, barley, soybean) are described in U.S. Patent Nos. 4,940,838, 5,464,763, 5,149,645, 5,501,967, 6,265,638, 4,693,976, 5,635,381, 5,731,179, 5,693,512, 6,162,965, 5,693,512, 5,981,840, 6,420,630, 6,919,494, 6,329,571, 6,215,051, 6,369,298, 5,169,770, 5,376,543, 5,416,011, 5,569,834, 5,824,877, 5,959,179, 5,563,055, and 5,968,830, each of which is incorporated herein by reference in its entirety.

[0124] Other suitable methods for introducing a nucleotide sequence into a plant cell and subsequently inserting it into the plant genome include microinjection as described in Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described in Riggs et al. (1986) PNAS 83:5602-5606, Agrobacterium-mediated transformation as described in Townsend et al., U.S. Patent No. 5,563,055, Zhao et al., U.S. Patent No. 5,981,840, Yukou et al., WO 94 / 000977, and Hideaki et al., WO 95 / 06722, direct gene transfer as described in Paszkowski et al. (1984) EMBO J. 3:2717-2722, and ballistic particle acceleration such as described below: Sanford et al., U.S. Patent No. 4,945,050; Tomes et al., U.S. Patent No. 5,879,918; Tomes et al., U.S. Patent No. 5,886,244; Bidney et al., U.S. Patent No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00 / 28058). See also Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al.(1998) Theor. Appl. Genet. 96: 319 - 324 (soybean); Datta et al. (1990) Biotechnology 8: 736 - 740 (rice); Klein et al. (1988) PNAS 85: 4305 - 4309 (maize); Klein et al. (1988) Biotechnology 6: 559 - 563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et al., U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer - Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91: 440 - 444 (maize); Fromm et al. (1990) Biotechnology 8: 833 - 839 (maize); Hooykaas - Van Slogteren et al. (1984) Nature (London) 311: 763 - 764; Bowen et al., U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) PNAS 84: 5345 - 5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197 - 209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415 - 418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560 - 566 (whisker - mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495 - 1505 (electroporation); Li et al.(1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium); all of these are incorporated herein by reference.

[0125] The polynucleotides of the present invention can be introduced into plants by contacting the plants with a virus or viral nucleic acid. Generally, these methods involve incorporating the polynucleotide constructs of the present invention into viral DNA or RNA molecules. In addition, it is recognized that the promoters of the present invention also encompass promoters for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing the proteins encoded therein (including viral DNA or RNA molecules) are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931; which are incorporated herein by reference.

[0126] In some embodiments, the polynucleotides of the present invention can be introduced into plants using sexual hybridization between two lines, followed by repeated backcrossing between the hybrid progeny and one of the parents until a plant with the desired characteristics is obtained. However, this method is limited to plants that can be sexually hybridized, and genes other than the desired gene will be transferred.

[0127] Recombinant DNA technology allows plant researchers to circumvent these limitations by enabling plant geneticists to identify and clone specific genes for desired traits (such as resistance to insect pests) and to introduce these genes into already useful plant varieties. Once an exogenous gene has been introduced into a plant, that plant can be used in conventional plant breeding programs (such as pedigree breeding, single-seed descent breeding programs, reciprocal recurrent selection) to produce progeny that also contain the target gene.

[0128] In some embodiments, homologous recombination can be used to introduce genes in a site-directed manner. Homologous recombination allows site-specific modification of endogenous genes, and thus can correct genetic or acquired mutations, and / or can engineer new alterations into the genome. Homologous recombination and site-directed integration in plants are discussed, for example, in U.S. Patent Nos. 5,451,513; 5,501,967, and 5,527,695.

[0129] The transformed cells can be cultivated into plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be cultivated, pollinated with the same transformed line or a different line, and the resulting hybrids with constitutive expression of the desired phenotypic characteristics can be identified. Two or more generations can be cultivated to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and the seeds can then be harvested to ensure that the expression of the desired phenotypic characteristics has been achieved.

[0130] In this way, the present invention provides transformed seeds (also referred to as "transgenic seeds") in which the polynucleotide constructs of the present invention (such as the expression cassettes of the present invention) are stably incorporated into their genomes.

[0131] In certain embodiments, the nucleic acid molecules and polynucleotide constructs of the present invention can be provided to plants using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, directly introducing the sequence or its variants and fragments into the plant or introducing the transcript into the plant. These methods include, for example, microinjection, electroporation, or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al. (1994) PNAS 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107: 775-784, Sheen, J. 2002. A transient expression assay using maize mesophyll protoplasts. Sheen, J. 2001. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 2001 December; 127: 1466-1475, Anderson et al., U.S. Patent No. 7,645,919 B2, all of which are incorporated herein by reference. Alternatively, polynucleotides can be transiently transformed into plants using techniques known in the art.

[0132] In one embodiment, the constructs of the invention can be provided as a binary Ti plasmid border DNA construct having the right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of a Ti plasmid isolated from Agrobacterium tumefaciens containing T-DNA, which together with the transfer molecule provided by an Agrobacterium tumefaciens cell allows integration of the T-DNA into the genome of a plant cell (e.g., see U.S. Patent No. 6,603,061). The construct may also contain a plasmid backbone DNA segment that provides replication function and antibiotic selection in bacterial cells, such as an Escherichia coli origin of replication like ori322, a broad host origin of replication like oriV or oriRi, and a selectable marker encoding a Tn7 aminoglycoside adenylyltransferase (aadA) that confers spectinomycin or streptomycin resistance (e.g., Spec / Strp), or the coding region of a gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host strain is typically Agrobacterium tumefaciens ABI, C58 or LBA4404; however, other strains known to those skilled in the art of plant transformation can also function in the present invention.

[0133] Methods are known in the art for assembling and introducing constructs into cells in such a way that the transcribable polynucleotide molecule is transcribed into a functional mRNA molecule, which is translated and expressed as a protein product. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art, e.g., see Molecular Cloning: A Laboratory Manual, 3 rdedition Volumes 1, 2, and 3 (2000) J. Sambrook, D. W. Russell and N. Irwin, Cold Spring Harbor Laboratory Press. Methods for preparing recombinant vectors particularly suitable for plant transformation include, but are not limited to, those described in U.S. Patent Nos. 4,971,908; 4,940,835; 4,769,061; and 4,757,011 in their entirety. Vectors of these types have also been reviewed in the scientific literature (e.g., see Rodriguez et al., Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, (1988) and Glick et al., Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, FL. (1993)). Typical vectors for expressing nucleic acids in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers et al., Methods in Enzymology 153:253-277 (1987)). Other recombinant vectors that can be used for plant transformation, including the pCaMVCN transfer control vector, have also been described in the scientific literature (e.g., see Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824-5828 (1985)).

[0134] A variety of regulatory elements can be included in the construct, including any of the regulatory elements provided herein. Any such regulatory element can be provided in combination with other regulatory elements, such as any combination of the sequences shown in SEQ ID NOs: 1-24. Such combinations can be designed or modified to produce the desired regulatory characteristics. In one embodiment, the construct of the present invention comprises at least one regulatory element operably linked to at least one transcribable polynucleotide molecule, which is operably linked to at least one 3' UTR.

[0135] The constructs of the present invention may include any promoter or leader sequence provided herein or known in the art. For example, the promoter of the present invention may be operably linked to a heterologous untranslated 5' leader sequence, such as a leader sequence from a heat shock protein gene (see, e.g., U.S. Patent Nos. 5,659,122 and 5,362,865). Alternatively, the leader sequence of the present invention may be operably linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcriptional promoter (see, U.S. Patent No. 5,352,605).

[0136] As used herein, the term "intron" refers to a DNA molecule that can be isolated or identified from the genomic copy of a gene and is generally defined as the region spliced out during pre-mRNA processing prior to translation. Alternatively, an intron may be a synthetically produced or engineered DNA element. An intron may contain enhancer elements that affect the transcription of an operably linked gene. Introns can be used as regulatory elements to modulate the expression of an operably linked transcribable polynucleotide molecule. A DNA construct may contain an intron, and the intron may or may not be heterologous to the transcribable polynucleotide molecule sequence. Examples of introns in the art include the rice actin intron (U.S. Patent No. 5,641,876) and the maize HSP70 intron (U.S. Patent No. 5,859,347). In addition, when modifying intron / exon boundary sequences, it is preferred to avoid using the nucleotide sequence AT or nucleotide A just before the 5' end of the splice site (GT), and the nucleotide G or nucleotide sequence TG just after the 3' end of the splice site (AG) to eliminate the possibility of forming an unwanted start codon during messenger RNA processing into the final transcript. Thus, the sequences around the 5' or 3' end splice sites of an intron can be modified in this manner.

[0137] As used herein, the term "3' transcription termination molecule" or "3' UTR" refers to a DNA molecule that is used to generate the 3' untranslated region (3' UTR) of an mRNA molecule during transcription. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation (also known as the polyA tail). The 3' UTR can be operably linked to a transcribable polynucleotide molecule and be located downstream thereof, and can include polynucleotides that provide polyadenylation signals and other regulatory signals capable of affecting transcription, mRNA processing, or gene expression. The PolyA tail is thought 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 (see Fraley, et al., Proc. Natl. Acad. Sci. USA, 80:4803-4807 (1983)); the 3' region of wheat hsp17; the 3' region of pea ribulose bisphosphate carboxylase-oxygenase small subunit; the cotton E6 3' region (U.S. Patent 6,096,950); the 3' region disclosed in WO 0011200A2; and the coixin 3' UTR (U.S. Patent No. 6,635,806).

[0138] Transcribable polynucleotide molecule

[0139] As used herein, the term "transcribable polynucleotide molecule" refers to any DNA molecule capable of being transcribed into an RNA molecule, including but not limited to molecules having protein-coding sequences and molecules that produce RNA molecules having sequences for gene silencing. The types of DNA molecules can 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 gene antisense information, or artificial, synthetic, or other modified forms of DNA molecules encoding transgenes. Exemplary transcribable DNA molecules for incorporation into the constructs of the present invention can include, for example, DNA molecules or genes from species other than the species into which the DNA molecule is incorporated, or genes that are derived from or present in the same species but are incorporated into recipient cells by genetic engineering methods rather than classical breeding techniques.

[0140] "Transgene" refers to a transcribable polynucleotide molecule that is heterologous to the host cell (at least its location in the genome) and / or a transcribable polynucleotide molecule that has been artificially introduced into the genome of the host cell in the current or any previous generation of cells.

[0141] The promoter of the present invention, such as SEQ ID NO: 1-24, can be operably linked to a transcribable polynucleotide molecule that is heterologous to the promoter molecule. As used herein, the term "heterologous" refers to such a combination when the combination of two or more polynucleotide molecules does not normally exist in nature. For example, the two molecules can be from different species and / or the two molecules can be from different genes, such as different genes from the same species or the same gene from different species. Additionally, the two molecules can be from separate locations within the same gene, where such a combination of molecules does not normally exist in nature. If the combination does not normally exist in nature, i.e., the transcribable polynucleotide molecule is not a naturally occurring combination operably linked to the promoter molecule, then the promoter is heterologous with respect to the operably linked transcribable polynucleotide molecule.

[0142] As used herein, the term "overexpression" refers to an increase in the level of expression of a transcribable polynucleotide molecule or protein in a plant, plant cell, or plant tissue compared to its expression in a wild-type plant, cell, or tissue at any developmental or temporal stage of the gene. Overexpression can occur in plant cells that normally lack expression of the target transcribable polynucleotide molecule. Overexpression can also occur in plant cells in which the transcribable polynucleotide molecule or a functionally equivalent molecule is normally endogenously expressed, but such endogenous expression is at a lower level compared to overexpression. Thus, overexpression results in a greater production of the polypeptide in the plant, cell, or tissue than the endogenous production or "overproduction".

[0143] In certain embodiments, the expression or overexpression of the transcribable polynucleotide molecules disclosed herein can directly or indirectly affect enhanced traits or altered phenotypes. In some cases, this can be achieved, for example, by effectively increasing yield by expressing one or more genes with spatio-temporal precision. In certain exemplary embodiments, the protein produced by the transcribable polynucleotide molecule can result in an increase in the starch content in the plant.

[0144] The transcribable polynucleotide molecule can generally be any DNA molecule that requires the expression of an RNA transcript. Such expression of the RNA transcript can lead to the translation of the resulting mRNA molecule and thus to protein expression. Alternatively, for example, the transcribable polynucleotide molecule can be designed to ultimately result in a decrease in the expression of a specific gene or protein. In one embodiment, this can be achieved by using a transcribable polynucleotide molecule oriented in the antisense direction. Those of ordinary skill in the art are familiar with the use of such antisense technology. Briefly, when the antisense transcribable polynucleotide molecule is transcribed, the RNA product hybridizes to the complementary RNA molecule within the cell and sequesters it. This double-stranded RNA molecule cannot be translated into a protein by the cell's translation machinery and is degraded in the cell. Any gene can be negatively regulated in this manner.

[0145] Accordingly, one embodiment of the present invention is a regulatory element of the present invention, such as the regulatory elements provided in SEQ ID NO: 1-24, which is operably linked to a transcribable polynucleotide molecule such that when the construct is integrated into the genome of a plant cell, it regulates the transcription of the transcribable polynucleotide molecule at a desired level or in a desired pattern. In one embodiment, the transcribable polynucleotide molecule comprises the protein-coding region of a gene, and the promoter affects the transcription of the RNA molecule that is translated and expressed as a protein product.

[0146] In another embodiment, the transcribable polynucleotide molecule comprises the antisense region of a gene, and the promoter affects the transcription of an antisense RNA molecule, double-stranded RNA, or other similar inhibitory RNA molecule in order to inhibit the expression of a target specific RNA molecule in a target host cell.

[0147] Agronomic target gene

[0148] The transcribable polynucleotide molecule may be an agronomic target gene. As used herein, the term "agronomic target gene" refers to a transcribable polynucleotide molecule that confers a desired trait when expressed in a particular plant tissue, cell, or cell type, such as traits related to plant morphology, physiology, growth, development, yield, grain composition, product, nutritional profile, disease or pest resistance, environmental or chemical tolerance, and / or that can act as an insecticide in the diet of pests that feed on plants. In one embodiment of the present invention, the regulatory element of the present invention is incorporated into a construct such that the regulatory element is operably linked to a transcribable DNA molecule that is an agronomic target gene.

[0149] Agronomic target genes reflect commercial markets and interests in crop development. The crops and markets of interest change, and as developing countries open up world markets, new crops and technologies will also emerge. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increases, the selection of genes for transformation will change accordingly.

[0150] In the transgenic plants of the present invention, the expression of an agronomic target gene can confer beneficial agronomic traits. Beneficial agronomic traits can include, for example but not limited to, genes encoding important or desired traits of agronomy, herbicide tolerance or resistance, insect control, insect resistance, altered yield, disease resistance, pathogen resistance, altered plant growth and development, altered starch content, grain characteristics, modified oil content, modified fatty acid content, altered protein content, altered fruit ripening, abiotic stress tolerance, enhanced animal and human nutrition, biopolymer production, environmental stress resistance, pharmaceutical peptides, sterility, improved processing quality, improved flavor, hybrid seed production utility, improved fiber production, commercial product production, and biofuel production.

[0151] Examples of agronomic target genes can include, but are not limited to, genes encoding proteins important for agronomy, such as yield proteins, stress-resistant proteins, development control proteins, tissue differentiation proteins, meristem proteins, environmental response proteins, senescence proteins, hormone response proteins, insect resistance proteins, abscission proteins, source proteins, sink proteins, flower inhibition proteins, seed proteins, herbicide-resistant proteins, disease-resistant proteins, fatty acid biosynthetic enzymes, tocopherol biosynthetic enzymes, amino acid biosynthetic enzymes, insecticidal proteins or genes for any other reagent (e.g., antisense or RNAi molecules targeting specific genes for inhibition). The products of agronomic target genes can act within plants to affect plant physiology or metabolism.

[0152] In certain embodiments, the promoter sequences disclosed herein as SEQ ID NOs: 1-24 can be used to express transgenes that improve photosynthesis and sucrose production, sucrose export, drought resistance, and pest resistance. For example, the promoter pMePsbr (SEQ ID NO: 3) or pAtCAB1 (SEQ ID NO: 6) and other autotrophic tissue-specific promoters are particularly useful for driving the expression of these genes. Examples of agronomic target genes involved in improving photosynthesis and yield can alter non-photochemical quenching (NPQ), reduce photorespiration, or improve Calvin cycle efficiency, and include, but are not limited to, violaxanthin de-epoxidase (VDE), zeaxanthin epoxidase (ZEP), and photosystem II subunit S (PsbS) that alter the NPQ response in plants (Kromdijk et al. (2016)); and knockout of genes for glycolate dehydrogenase, malate synthase, and glycolate / glycerate transporter that introduce a photorespiration bypass (Soute et al. (2019)); and the cyanobacterial bifunctional enzyme fructose-1,6-bisphosphatase / sedoheptulose-1,7-bisphosphatase or sedoheptulose-1,7-bisphosphatase, and the red algal gene cytochrome C6 (Lopez-Calcagno et al. (2020)). Examples of agronomic target genes involved in improving sucrose production and yield by altering sugar metabolism include Escherichia coli pyrophosphatase (PPase) or sequences for RNAi that target leaf ADP-glucose pyrophosphorylase (AGPase) to improve sucrose production and export (Jonik et al. (2012)). Examples of agronomic target genes involved in drought resistance include the brassinosteroid receptor family gene BRL3, which has been shown to improve drought resistance without negatively affecting yield, as demonstrated by (Fàbregas et al., 2018). Examples of agronomic target genes involved in pest resistance include, but are not limited to, DNA polymerase δ subunit 1 (MePOLD1), a gene that has been shown by Lim et al. (2022) to mediate resistance against ACMV.

[0153] In certain embodiments, the promoter sequences disclosed herein as SEQ ID NOs: 1-24 can be used to express transgenes that improve sucrose and nitrogen partitioning in cassava. For example, the promoters pMeSUS1 (SEQ ID NO:5) and pMeSWEET1-like (SEQ ID NO:1), as well as other phloem / phloem parenchyma-specific promoters, are particularly useful for driving the expression of agronomic target genes involved in enhancing the expression of sugar transporters and amino acid transporters that support phloem loading or phloem unloading of sugars and amino acids. Examples of agronomic target genes involved in the expression of sugar transporters and amino acid transporters include, but are not limited to, Sucrose Transporter (SUT / SUC)-family proteins, Sucrose Unloading Efflux Transporter (SWEET)-family proteins, UMAMIT-family proteins, and Amino Acid Permease (AAP)-family proteins. Examples of agronomic target genes that can be used to improve uptake strength by enhancing sucrose consumption include, but are not limited to, Sucrose Synthase (SUS)-family proteins. Examples of agronomic target genes that can block sugar transporters and alter sugar distribution in plants include, but are not limited to, FLOWERING LOCUS T (FT)-related genes, such as SELF PRUNING 6A (SP6A) genes, which have been shown to be able to block SWEET transporters (Abelenda et al. (2019)).

[0154] In certain embodiments, the promoter sequences disclosed herein as SEQ ID NOs: 1-24 can be used to express transgenes that improve starch yield, storage root size, starch quality, nutritional quality, pest resistance, or post-harvest physiological deterioration. For example, the promoter pMeGPT (SEQ ID NO: 2) and other storage root-specific promoters are particularly useful for driving the expression of agronomic target genes involved in increasing starch yield, storage root size, starch quality, nutritional quality, pest resistance, or post-harvest physiological deterioration. Examples of agronomic target genes involved in altering sugar and starch metabolism in storage roots include, but are not limited to, genes that alter cytosolic sugar-converting enzymes such as sucrose synthase (SUS) genes, fructokinase (FRK) genes, UDP-glucose pyrophosphorylase (UGP) genes, phosphoglucose isomerase (PGI) genes, and phosphoglucomutase (PGM) genes. Examples of agronomic target genes involved in the import of starch precursor metabolites include, but are not limited to, glucose-6-phosphate transporters (GPTs) or nucleotide transporter family proteins, which have been shown by Zhang et al. (2008) and Jonik et al. (2012) to increase starch concentration. Examples of agronomic target genes involved in amyloplast starch formation include, but are not limited to, phosphoglucomutase genes, ADP-glucose pyrophosphorylase (AGPase) genes, soluble starch synthase genes, and granule-bound starch synthase genes. Examples of agronomic target genes involved in starch granule targeting, starch granule remodeling, or starch degradation include, but are not limited to, α- and β-amylase genes, debranching enzyme genes (DBEs), starch phosphorylation genes, early starvation (ESV) genes, proteins targeted to starch (PTST) genes, myosin-like chloroplast proteins (MRCs), or myosin-binding filament protein 1 (MFP1). Examples of agronomic target genes involved in altering hormonal regulation include, but are not limited to, cytokinin synthesis genes such as isopentenyl transferase genes (IPTs), CYP735A genes, or loner (LOG) genes. Examples of agronomic target genes that alter auxin synthesis genes include, but are not limited to, YUCCA genes. Agronomic target genes involved in vitamin formation or mineral acquisition include, but are not limited to, genes involved in β-carotene synthesis such as phytoene synthase (PTS) genes (Welsch et al. (2010)), or genes involved in vitamin b6 synthesis such as PDX1 and PDX2 (Li et al. (2015)), or genes involved in mineral accumulation such as vacuolar iron transporter (VIT) genes, iron transporter (IRT) genes, and ferritin (FER) genes (Narayanan et al. (2019)). The promoter sequences described herein can also be used to drive genes involved in root pathogen resistance or genes that reduce post-harvest deterioration.Examples of genes for reducing post - harvest degradation include, but are not limited to, redox - protective genes such as peroxidase (PER) genes, glutathione reductase (GSR) genes or ascorbate peroxidase (APX) genes (Vanderschuren et al. (2014)).

[0155] In certain embodiments, the promoter sequences disclosed herein as SEQ ID NO: 1 - 24 can be used to drive the expression of transgenes that improve storage root size by altering vascular cambium activity. For example, the promoter pManes.14g071100 (SEQ ID NO: 4) and other storage root cambium - specific promoters can be used to drive the expression of transgenes that improve storage root size by altering vascular cambium activity. Examples of agronomic purpose genes that participate in altering hormone activity or genes that alter vascular cambium cell division or cell differentiation include, but are not limited to, the ANTINTEGUMENTA gene or genes involved in xylem cell formation such as genes of the WUSCHEL - RELATED - HOMEOBOX gene family, the MONOPTEROUS gene, and the BREDIPEDICELLUS / KNAT genes. Auxin biosynthesis genes such as YUCCA, auxin response factor family genes, cytokinin biosynthesis genes such as LONELY GUY (LOG) genes, and cytokinin response genes such as LATERAL ORGAN BOUNDARY DOMAIN (LBD) genes are other examples of agronomic purpose genes that can be used in conjunction with the promoter sequences disclosed herein.

[0156] Alternatively, an agronomic target gene can affect the above-described plant traits 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. Patent No. 5,107,065); inhibitory RNA (“RNAi”, including regulation of gene expression via miRNA-, siRNA-, trans-acting siRNA- and phased sRNA-mediated mechanisms, as described, for example, in published applications US2006 / 0200878 and US2008 / 0066206 and U.S. Patent Application 11 / 974,469); or co-suppression-mediated mechanisms. The RNA can also be a catalytic RNA molecule engineered to cleave a desired endogenous mRNA product (e.g., a ribozyme or riboswitch; see, e.g., US2006 / 0200878). Thus, any transcribable polynucleotide molecule encoding a transcriptional RNA molecule that affects an important agronomic phenotype or morphological change can be used to practice the present invention. Methods for constructing constructs and introducing them into cells in such a way that the transcribable polynucleotide molecule is transcribed into a molecule capable of causing gene silencing are known in the art. For example, post-transcriptional gene silencing of gene expression in plant cells using constructs with transcribable polynucleotide molecules in antisense orientation is disclosed in U.S. Patent Nos. 5,107,065 and 5,759,829, and post-transcriptional gene silencing of gene expression in plants using constructs with transcribable polynucleotide molecules in sense orientation is disclosed in U.S. Patent Nos. 5,283,184 and 5,231,020. Expression of a transcribable polynucleotide in a plant cell can also be used to inhibit plant pests that feed on the plant cell, such as compositions isolated from coleopteran pests (U.S. Patent Publication No. US20070124836) and compositions isolated from nematode pests (U.S. Patent Publication No. US20070250947). Plant pests include, but are not limited to, arthropod pests, nematode pests, and fungal or microbial pests. Exemplary transcribable polynucleotide molecules for incorporation into the constructs of the present invention include, for example, DNA molecules or genes from species other than the target species, or genes derived from or present in the same species, but incorporated into the recipient cell by genetic engineering methods rather than classical breeding or propagation techniques. The types of polynucleotide molecules can include, but are not limited to, polynucleotide molecules already present in the plant cell, polynucleotide molecules from another plant, polynucleotide molecules from a different organism, or externally produced polynucleotide molecules, such as polynucleotide molecules containing gene antisense information, or artificial, synthetic or other modified forms of polynucleotide molecules encoding a transgene.

[0157] The target genes can include, for example, those genes involved in information, such as zinc fingers, those genes involved in communication, such as kinases, and those genes involved in housekeeping, such as heat shock proteins; those genes involved in oil, starch, carbohydrate or nutrient metabolism; genes encoding enzymes and other proteins from plants and other sources (including prokaryotes and other eukaryotes).

[0158] Examples of agronomic target genes known in the art include those for herbicide resistance (U.S. Patent 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. Patent Nos. RE38,446; 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 5,716,837), 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; 6,177,615; 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. Patent 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 6,506,962), 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 production (U.S. Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oil production (U.S. Patent Nos. 6,444,876; 6,426,447 and 6,380,462), high oil production (U.S. Patent No. 6,495,739;those of 5,608,149; 6,483,008 and 6,476,295), modified fatty acid content (U.S. Patent 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; 6,489,461 and 6,459,018), high protein production (U.S. Patent No. 6,380,466), fruit ripening (U.S. Patent No. 5,512,466), enhanced animal and human nutrition (U.S. Patent Nos. 6,723,837; 6,653,530; 6,541,259; 5,985,605 and 6,171,640), biopolymers (U.S. Patent No. RE37,543; 6,228,623 and 5,958,745 and 6,946,588), environmental stress resistance (U.S. Patent No. 6,072,103), pharmaceutical peptides and secreted peptides (U.S. Patent Nos. 6,812,379; 6,774,283; 6,140,075 and 6,080,560), improved processing traits (U.S. Patent No. 6,476,295), improved digestibility (U.S. Patent No. 6,531,648), raffinose - low (U.S. Patent No. 6,166,292), industrial enzyme production (U.S. Patent No. 5,543,576), improved flavor (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,229,114), hybrid seed production (U.S. Patent No. 5,689,041), fiber production (U.S. Patent Nos. 6,576,818; 6,271,443; 5,981,834 and 5,869,720) and biofuel production (U.S. Patent No. 5,998,700).;

[0159] Alternatively, an agronomic target gene 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. Patent No. 5,107,065); inhibitory RNA (“RNAi”, including regulation of gene expression by miRNA -, siRNA -, trans - acting siRNA - and phased sRNA - mediated mechanisms, e.g., as described in published applications U.S. 2006 / 0200878 and U.S. 2008 / 0066206 and U.S. Patent Application Serial No. 11 / 974,469); or co - suppression - mediated mechanisms. The RNA can also be a catalytic RNA molecule (e.g., ribozyme or riboswitch; see, e.g., U.S. 2006 / 0200878), which is engineered to cleave a desired endogenous mRNA product. Methods are known in the art for constructing constructs and introducing them into cells in a manner that will transcribe the DNA molecule into a molecule capable of causing gene silencing.

[0160] Selectable marker

[0161] The selectable marker transgene can also be used with the regulatory elements of the present invention. As used herein, the term "selectable marker transgene" refers to any transcribable DNA molecule whose expression or lack of expression in a transgenic plant, tissue or cell can be screened or scored in some way. As used herein, the term "marker" refers to any transcribable polynucleotide molecule whose expression or lack of expression can be screened or scored in some way. Marker genes useful in practicing the present invention include, but are not limited to, those encoding β-glucuronidase (GUS as described in U.S. Patent No. 5,599,670), green fluorescent protein and its variants (GFP as described in U.S. Patents Nos. 5,491,084 and 6,146,826), proteins conferring antibiotic resistance or proteins conferring herbicide tolerance.

[0162] The term "selectable marker" also includes genes encoding a secreted marker, the secretion of which can be detected as a means of identifying or selecting transformed cells. Examples include markers encoding a secreted antigen that can be recognized by antibody interaction, or even a secreted enzyme whose activity can be assayed. Selectable secreted marker proteins can be classified into several categories, including small, diffusible proteins that can be detected (e.g., by ELISA), small active enzymes that can be detected in the extracellular solution (e.g., α-amylase, β-lactamase, phosphinothricin transferase), or proteins that are inserted or restricted in the cell wall (e.g., proteins containing a leader sequence, such as those found in an extended expression unit or tobacco pathogenesis-related proteins, also known as tobacco PR-S). Other possible selectable marker genes will be apparent to those skilled in the art and are included in the present invention.

[0163] A "selectable marker" refers to a sequence that can be used to distinguish between transformed and untransformed genes. A reporter gene is a test sequence whose expression can be quantified. A reporter gene can serve as a marker for a transgene. In some embodiments, the transgene of the present invention comprises at least one reporter gene. As used herein, a "reporter molecule" or "reporter gene" refers to a nucleic acid molecule encoding a detectable marker. Reporter genes can be, for example, luciferase (e.g., firefly luciferase or Renilla luciferase), β-galactosidase, chloramphenicol acetyltransferase (CAT), or fluorescent proteins (e.g., green fluorescent protein (GFP), red fluorescent protein (DsRed), yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein, or variants thereof, including enhanced variants such as enhanced GFP (eGFP)). Reporter genes can be detected by reporter gene assays. Reporter gene assays can measure the level of reporter gene expression or activity by a number of methods, including, for example, measuring the level of reporter gene mRNA, the level of reporter protein, or the amount of reporter protein activity. Reporter molecule assays are known in the art or are otherwise disclosed herein.

[0164] Cell transformation

[0165] The present invention also relates to methods for generating transformed cells and plants, said transformed cells and plants comprising a promoter operably linked to a transcribable polynucleotide molecule.

[0166] The term "transformation" refers to the introduction of nucleic acid into a recipient host. As used herein, the term "host" refers to a bacterium, fungus or plant, including any cell, tissue, organ or progeny of a bacterium, fungus or plant. Particularly interesting plant tissues and cells include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos and pollen.

[0167] As used herein, the term "transformed" refers to a cell, tissue, organ or organism into which an exogenous polynucleotide molecule (e.g., a construct) has been introduced. The introduced polynucleotide molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced polynucleotide molecule is inherited by subsequent progeny. A "transgenic" or "transformed" cell or organism may also include the progeny of such cell or organism, as well as progeny produced by a breeding program that uses such transgenic organism as a hybrid parent and exhibits a phenotypic change due to the presence of the exogenous polynucleotide molecule. The term "transgenic" refers to a bacterium, fungus or plant that contains one or more heterologous polynucleotide molecules. As used herein, the terms "heterologous DNA sequence", "exogenous DNA fragment" or "heterologous nucleic acid", "transgene", "exogenous polynucleotide" each refer to a sequence that is exogenous (e.g., non-native) to a particular host cell, or a sequence that has been modified from its original form and / or locus if from the same source or species; a sequence that is heterologous to the host cell at least in terms of its position in the genome; a promoter that is not the natural promoter of the polynucleotide to which it is operably linked, and / or has been artificially incorporated into the genome of the host cell in the current or any previous generation of the cell.

[0168] There are many methods for introducing polynucleotide molecules into plant cells. The methods generally include the steps of selecting a suitable host cell, transforming the host cell with a recombinant vector and obtaining the transformed host cell. Suitable methods include bacterial infection (e.g., Agrobacterium), binary bacterial artificial chromosome vectors, direct delivery of DNA (e.g., by PEG-mediated transformation, desiccation / inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers and acceleration of DNA-coated particles, etc. (reviewed in Potrykus, et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:205 (1991))), gene editing (e.g., CRISPR-Cas system), etc.

[0169] Techniques for introducing DNA molecules into cells are well known to those skilled in the art. In the practice of the present invention, methods and materials for transforming plant cells by introducing a plant DNA construct into the plant genome may include any well-known and proven methods. Any transformation method can be used to transform a host cell with one or more promoters and / or constructs of the present invention. The host cell can be any cell or organism, such as a plant cell, an algal cell, an alga, a fungal cell, a fungus, a bacterial cell, or an insect cell. Preferred hosts and transformed cells include cells from plants, Aspergillus, yeast, insects, bacteria, and algae. In a specific embodiment, the host cell and the transformed cell may include cells from a crop plant.

[0170] In various embodiments, the methods described herein may involve introducing a polynucleotide construct into a plant. "Introducing" means presenting the polynucleotide construct to the plant in such a way that the construct enters the interior of a plant cell. The methods of the present invention do not depend on a particular method for introducing the polynucleotide construct into the plant, as long as the polynucleotide construct enters the interior of at least one cell of the plant. Methods for introducing a polynucleotide construct into a plant are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0171] "Stable transformation" means that the polynucleotide construct introduced into the plant integrates into the genome of the plant and can be inherited by its progeny. "Transient transformation" means that the polynucleotide construct introduced into the plant does not integrate into the genome of the plant.

[0172] Transgenic plants can subsequently be regenerated from the transgenic plant cells of the present invention. Using conventional breeding techniques or self-pollination, seeds can be produced from the transgenic plant. Such seeds and the progeny plants grown from such seeds will contain the recombinant DNA molecule of the present invention and will thus be transgenic.

[0173] Regenerated transgenic plants can self-pollinate to provide homozygous transgenic plants (homozygous for the recombinant DNA molecule). Alternatively, pollen obtained from the regenerated transgenic plants can be crossed with non-transgenic plants to provide seeds of heterozygous transgenic plants (heterozygous for the recombinant DNA molecule), preferably inbred lines of species of agronomic importance. Such homozygous and heterozygous transgenic plants are both referred to herein as "progeny plants". Progeny plants are transgenic plants derived from the original transgenic plants and containing the recombinant DNA molecule of the present invention. Seeds produced using the transgenic plants of the present invention can be harvested and used to grow generations of transgenic plants, i.e., the progeny plants of the present invention, which contain the construct of the present invention and express the agronomic purpose gene. Descriptions of breeding methods commonly used for different traits and crops can be found in one of several reference books, see, for example, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of crop improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of variety development, Theory and Technique, (Vol.1) and Crop Species Soybean (Vol 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987). Conversely, pollen from non-transgenic plants can be used to pollinate the regenerated transgenic plants.

[0174] The presence of the gene of interest in the transformed plants and the expression level and / or profile conferred by the regulatory elements of the present invention can be analyzed. Those skilled in the art know a variety of methods that can be used to analyze transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blotting or northern blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field evaluations, and immunoassay diagnostics. The expression of the transcribable polynucleotide molecule can be determined using the methods described by the manufacturer (Applied Biosystems, Foster City, CA) reagents and methods are used for measurement and TestingMatrix to determine the PCR cycle time. Alternatively, the (Third Wave Technologies, Madison, WI) reagents and methods can be used for transgene expression.

[0175] Seeds of the plants of the present invention can be harvested from fertile transgenic plants and used to grow progeny of the transformed plants of the present invention, including hybrid plant lines containing the construct of the present invention and expressing an agronomic gene of interest.

[0176] The present invention also provides plant parts of the present invention. Plant parts include but are not limited to leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. The plant parts of the present invention can be active, inactive, renewable, and / or non-renewable. The present invention also includes and provides transformed plant cells containing the DNA molecule of the present invention. The transformed or transgenic plant cells of the present invention include renewable and / or non-renewable plant cells. The present invention also includes and provides transformed plant cells containing the nucleic acid molecule of the present invention.

[0177] Transgenic plants can transmit the transgenic polynucleotide molecule to their progeny. The progeny include any renewable plant part or seed that contains the transgene derived from the ancestral plant. The transgenic plants are preferably homozygous for the transformed polynucleotide molecule and transmit this sequence to all progeny through sexual reproduction. The progeny can be grown from seeds produced by the transgenic plants. These additional plants can then self-pollinate to produce true plant breeding lines. The progeny of these plants are evaluated for gene expression and the like. Gene expression can be detected by several commonly used methods, such as western blotting, northern blotting, immunoprecipitation, and ELISA.

[0178] Commodity

[0179] The present invention provides commodities comprising the DNA molecules according to the present invention. As used herein, "commodity" refers to any composition or product composed of materials derived from plants, seeds, plant cells or plant parts comprising the DNA molecules of the present invention. Commodities can be sold to consumers and can be either active or inactive. Inactive commodities include, but are not limited to, inactive seeds and grains; processed seeds, seed parts and plant parts; dehydrated plant tissues, frozen plant tissues and processed plant tissues; seeds and plant parts, oils, meal, flour, flakes, bran, fiber, milk, cheese, paper, cream, wine and any other food for human consumption processed for use as animal feed for land and / or aquatic animals; and biomass and fuel products. Active commodities include, but are not limited to, seeds and plant cells. Thus, plants comprising the DNA molecules according to the present invention can be used to manufacture any commodity normally obtained from a plant or its parts. The commodities of the present invention contain a detectable amount of DNA corresponding to the recombinant DNA molecules of the present invention. Detection of one or more such DNAs in a sample can be used to determine the content or origin of the commodity. Any standard method for detecting DNA molecules can be used, including the detection methods disclosed herein.

[0180] plant

[0181] The nucleic acid molecules and polynucleotide constructs of the present invention can be used to transform any plant species, including but not limited to monocotyledonous and dicotyledonous plants. Examples of target plant species include but are not limited to Arabidopsis thaliana, chili peppers (Capsicum spp.; e.g., Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens, etc.), cowpea (Vigna unguiculata), tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), eggplant (Solanum melongena), petunia (Petunia spp., e.g., Petunia x hybrida or Petunia hybrida), maize or corn (Zea mays), Brassica spp. (e.g., Brassica napus, B. rapa, B. juncea), especially Brassica species that can be used as a source of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., Pennisetum glaucum, Panicum miliaceum, Setaria italica, Setaria viridis, Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.)) avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), switchgrass (Panicum virgatum), algae (e.g., Chlamydomonas reinhardtii, Botryococcus braunii, Chlorella spp., Dunaliella tertiolecta, Gracilaria spp.), oats, barley, vegetables, ornamental plants, and conifers. The nucleic acid molecules and polynucleotide constructs of the invention can also be used for the transformation of any algal species.

[0182] As used herein, the term "plant" refers to any living organism belonging to the plant kingdom (i.e., any genus / species within the plant kingdom). In some embodiments, the plant is a tree, herb, shrub, grass, vine, fern, moss, or green alga. The plant can be monocotyledonous (monocots) or dicotyledonous (dicots). Examples of specific plants that can contain the polynucleotides of the present invention include, but are not limited to, Arabidopsis, Brachypodium, switchgrass, maize, potato, rose, apple tree, sunflower, wheat, rice, banana, plantain, tomato, opo, pumpkin, zucchini, lettuce, Chinese cabbage, oak, guzmania, geranium, hibiscus, clematis, poinsettias, sugarcane, taro, duck weed, pine tree, Kentucky bluegrass, zoysiagrass, coconut tree, cauliflower, cavalo, collards, yardlong bean, kale, rutabaga, mustard, rape and other Brassica leafy vegetable crops, bulb vegetables (e.g., garlic, leek, onion (dry bulb onion, scallions, long onions), chive), citrus fruits (e.g., grapefruit, lemon, lime, orange, tangerine, citrus hybrids, pomelo), melon vegetables (e.g., gherkin, citron melon, edible gourd, West Indian cucumber, melon (including hybrids and / or cultivars of cucumber melons), watermelon, cantaloupe and other melon vegetable crops), fruit vegetables (including eggplant, gooseberry, pepino, pepper, tomato, tree tomato), grape, leafy vegetables (e.g., romaine lettuce), root / tuber and bulb vegetables (e.g., potato, yam, cassava, taro), tree nuts (almond, walnut, pistachio and pecan), berries (e.g., tomato, barberry, currants, elderberry, gooseberry, honeysuckle, mayapples, nannyberries, Oregon grape, sea buckthorn, hackberry, bearberry, blueberry, strawberry, sea grape, blackberry, Japanese apricot, loganberries, raspberry, beautyberry, roughberry and wineberry), cereal crops (e.g., maize, rice, wheat, barley, sorghum, millet, oats, rye, triticale, buckwheat, fonio, quinoa, oil palm), cruciferous plants and leguminous plants, pome fruits (e.g., apple, pear), stone fruits (e.g., coffee, date, mango, olive, coconut, oil palm, pistachio, almond, apricot, cherry, European plum, nectarine, peach and plum), vines (e.g., table grape, wine grape), fiber crops (e.g., hemp, cotton), ornamental plants, etc.

[0183] The present invention has been generally described above. The present invention will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the present invention unless otherwise specified. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, based on the present disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and still obtain similar or comparable results. Therefore, all content set forth or shown in the accompanying drawings should be construed as illustrative and not restrictive. Example

[0184] Example 1: Activity and tissue specificity of various promoters in transgenic, field-grown cassava plants.

[0185] Field trials were conducted on transgenic cassava plants expressing various combinations of metabolic activity genes that alter photosynthesis, transport, and storage metabolism at the Experimental Station of National Chung Hsing University in Taiwan. Cassava plants (genotype 60444) were transformed as previously described (Bullet al., 2009). Plants were transformed with one of seven different vectors, each containing 3 - 6 candidate promoters, each promoter driving a different detectable coding sequence. A total of 10 candidate promoter elements were tested to determine their expression profiles. Promoters were evaluated based on their specificity for autotrophic (i.e., "source" tissues) and / or heterotrophic (i.e., "sink" tissues).

[0186] Table 1. Summary of 10 promoters tested in field-grown cassava plants

[0187]

[0188] Analysis of over 400 field-grown plants generated using seven different constructs and representing 84 transgenic events was performed to determine the expression profiles of 10 candidate promoter sequences. Cassava source leaf, stem, and storage root samples were analyzed using quantitative RT-PCR, and the results for each promoter were summarized ( Figure 1)。Field trials revealed very strong source leaf expression for transcripts controlled by promoters pSlRBCS2 (SEQ ID NO:19) and pAtRBCS1A (SEQ ID NO:18), although with large variation. Their transcripts were approximately 4 - 5 times more abundant than transcripts controlled by the next strongest leaf promoter pAtCAB1 (SEQ ID NO:6). High abundance in source leaves was also observed for transcripts controlled by pAtGAPA (SEQ ID NO:12) and pStLS1 (SEQ ID NO:15). Moderate transcript abundance in source leaves was detected for pMeGBSS1 (SEQ ID NO:14) and pAtRBCS3B (SEQ ID NO:20). Low levels in source leaves were found for transcripts controlled by pStSSS3 (SEQ ID NO:21) and pAtFBA2 (SEQ ID NO:10), and no transcripts were found for pStSTP1 (SEQ ID NO:22). In heterotrophic organs, moderate to low levels were detected for transcripts controlled by pMeGBSS1 (SEQ ID NO:14), pStSSS3 (SEQ ID NO:21) and pStSTP1 (SEQ ID NO:22). Low levels were found for pAtRBCS1A (SEQ ID NO:18), and residual levels were found in heterotrophic tissues for transcripts controlled by leaf promoters pSlRBCS2 (SEQ ID NO:19), pStLS1 (SEQ ID NO:15) and pAtFBA2 (SEQ ID NO:10).

[0189] Based on the transcript abundances observed in different tissues ( Figure 1), the pSlRBCS2 and pAtRBCS1A promoters (SEQ ID NO: 19 and 18) appear to have very high activity in source leaves, although pAtRBCS1A (SEQ ID NO: 18) also appears to have low levels of activity in sink organs. The AtCAB1 (SEQ ID NO: 6) and AtGAPA (SEQ ID NO: 12) promoters are characterized by high and very specific source leaf expression, while the pAtRBCS3B (SEQ ID NO: 20) and pAtFBA2 promoters (SEQ ID NO: 20 and SEQ ID NO: 10) behave rather weakly. The StLS1 promoter (SEQ ID NO: 15) also shows weak activity in source leaves, with additional residual activity in sink organs. The MeGBSS1 (SEQ ID NO: 14), StSSS3 (SEQ ID NO: 21), and StSTP1 (SEQ ID NO: 22) promoters are expected to be specific for heterotrophic organs. However, the abundance of transcripts controlled by the MeGBSS1 (SEQ ID NO: 14) and StSSS3 (SEQ ID NO: 21) promoters is comparable among the three tissues tested. In these experiments, only pStSTP1 (SEQ ID NO: 22) appears to have specific activity for heterotrophic organs ( Figure 1 ). According to the PCR results, all three of these promoter sequences produce rather weak activity.

[0190] Example 2: Analysis of promoter activity and tissue specificity in cassava plants.

[0191] Analyze the expression patterns of 19 different promoter-GUS constructs in cassava plants. Briefly, all promoter-GUS constructs were generated using Golden Gate cloning (Engler et al. 2014). The promoters AtCAB1 (SEQ ID NO: 6), pSlRBCS2 (SEQ ID NO: 19), AtRBCS3B (SEQ ID NO: 20) and pStLS1 (SEQ ID NO: 15) were taken from the "MoClo Plant Parts Kit" (Engler et al. 2014). All other promoter elements were generated by PCR amplification or DNA synthesis. The promoters AtCAB1 (SEQ ID NO: 6), AtGAPA (SEQ ID NO: 12), AtFBA2 (SEQ ID NO: 10), AtRBCS3B (SEQ ID NO: 20), MeGBSS1 (SEQ ID NO: 14), StB33 (SEQ ID NO: 23), StFBPasecyt (SEQ ID NO: 8), StLS1 (SEQ ID NO: 15), StSSS3 (SEQ ID NO: 21) and StSTP1 (SEQ ID NO: 22) were maintained in the level 0 promoter module (GGAT-TACT). The promoters AtSUC2 (SEQ ID NO: 24), CmGolS1 (SEQ ID NO: 11), CoYMV (SEQ ID NO: 7), DjDIO3 (SEQ ID NO: 9), IbSRD1 (SEQ ID NO: 16), MeGPT (SEQ ID NO: 2), MePsbr (SEQ ID NO: 3), AtRBCS1A (SEQ ID NO: 18), MeSUS1 (SEQ ID NO: 5), MeSWEET1-like (SEQ ID NO: 1), StGBSS1 (SEQ ID NO: 13) and StPat (SEQ ID NO: 17) were maintained in the level 0 promoter + 5'UTR module (GGAT-AATG). All level 0 promoter modules (GGAT-TACT) were fused with the tobacco mosaic virus 5'UTR (pICH41402; Engler et al. (2014)), the modified β-glucuronidase coding sequence ("GUSPlus"; Broothaerts et al. (2005)), the Escherichia coli nopaline synthase 3'UTR + terminator (pICH41421; Engler et al. (2014)) and the 1-1f level acceptor (pICH47732; Engler et al. (2014)) to generate the corresponding promoter-reporter cassettes.All 0-level promoter + 5'UTR modules (GGAT - AATG) were fused with a modified β-glucuronidase coding sequence (“GUSPlus”; Broothaerts et al. (2005)), the Escherichia coli nopaline synthase 3'UTR + terminator (pICH41421; Engler et al. (2014)) and a 1-1f level receptor (pICH47732; Engler et al. (2014)) or a 1-3f level receptor (pICH47751; Engler et al. (2014)) to generate the corresponding promoter-reporter cassette. The level 1 plasmids containing the corresponding promoter-reporter cassette were transferred into the transformation vector p134GG (Mehdi et al., 2019) to generate the final level 2 transformation plasmids. Cassava plants (genotype 60444) were transformed with the promoter-reporter constructs as previously described (Bull et al., 2009). Hygromycin-resistant transformants were screened by β-glucuronidase histological staining. Plants with clear GUS staining were maintained in tissue culture and their tissue-specific expression patterns were analyzed continuously.

[0192] To analyze these promoter-GUS plants, up to 7 different tissues were sampled and stained and examined under a microscope. New leaves, developing leaves, fully developed leaves, petioles, upper stem segments, lower stem segments, storage root segments and fibrous roots ( Figure 15 ). New leaves and developing leaves are characterized by being brown and are called “sink” leaves (defined as leaves with net carbon input), while green fully expanded leaves are considered “source” leaves (defined as leaves with net carbon output).

[0193] For staining, cassava tissues were sampled into ice-cold 90% acetone solution. Leaf samples were taken with a leaf punch and cross-sections were prepared manually with a razor blade. With GUS staining buffer ((200 mM NaP pH7, 100 mM K3[Fe(CN6)], 100 mM K4[Fe(CN6)], 500 mM EDTA, 0.5% Cover these sections with gold and infiltrate them under full vacuum for 10 minutes. Remove the GUS staining buffer and replace it with fresh GUS staining solution, which contains GUS staining buffer with 0.75 mg / ml 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc; pre-dissolved in a small amount of DMSO). Infiltrate the GUS staining solution under full vacuum for 10 minutes. Incubate the infiltrated tissue overnight at a temperature of 37 °C or stop shortly after incubation (in the case of very rapid staining) (e.g., pCoYMV, pDjDIO3). After removing the GUS staining solution, add 70% ethanol to the tissue sections and incubate at a temperature of 37 °C until the tissue becomes clear. Take optical microscope images on a Zeiss Axioskop or Zeiss STEMI SV11 stereomicroscope (Zeiss, Wetzlar, Germany).

[0194] To quantify GUS expression, RNA extraction from cassava source leaves and storage roots was performed using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA). cDNA was generated from 0.2 - 1 μg of RNA using RevertAid H Minus reverse transcriptase as indicated by the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). Dilute the cDNA 1:10 and use qPCR Master Mix (Promega, Madison, WI, USA) to examine the quantification of gene expression. Mix the analytes in a 96-well plate and measure them in an AriaMx real-time PCR system (Agilent, Santa Clara, CA, USA). Using MeGAPDH (Manes.06g116400) as a reference gene, the normalized GUS expression of the promoter::GUS lines was determined by the 2 -ΔCt -ΔΔCt calculation method. The normalized GUS expression of each promoter::GUS line was calculated relative to the normalized expression of the pCaMV35S::GUS line and shown as a percentage of the relative expression of the pCaMV35S::GUS line to provide an approximate classification of the expression intensity.

[0195] Plants containing the reporter gene construct showed GUS staining in different tissues and cell types, as described in detail in the following examples. To more accurately determine these different cell types, counterstain with toluidine blue. As Figure 15 shown, "source" and "sink" leaves can be easily divided into vascular bundles and mesophyll cells ( Figure 15 A - B). In the petiole, collenchyma, sclerenchyma, phloem, protoxylem / xylem parenchyma, medullary parenchyma, and medullary cells can be distinguished from the outside to the inside (Figure 15 C). Depending on the position of the stem, the stem tissue is characterized by collenchyma, sclerenchyma, phloem, vascular cambium, and varying degrees of secondary xylem and pith tissue ( Figure 15 D-E). In particular, the lower heterotrophic stem tissue shows an increase in the level of secondary xylem tissue, which is composed of xylem fibers, water-transporting xylem vessels, and starch-storing xylem parenchyma cells ( Figure 15 E). The storage root has, from the outside to the inside, periderm tissue, phellogen, phelloderm / phloem parenchyma, phloem, vascular cambium, and xylem cells. Adjacent to the xylem vessels, the xylem tissue is mainly dominated by xylem parenchyma cells that store starch in the storage root ( Figure 15 F). The lower stem and the storage root, two heterotrophic starch-storing tissues, are generally similar, and both tissues are characterized by numerous vascular strands that ensure the connection of the assimilation and water transport systems, despite the increased distance formed by secondary xylem during secondary growth ( Figure 15 E-F).

[0196] Example 3: Analysis of GUS expression driven by selected promoters in transgenic plants from field tests.

[0197] For 6 out of 10 promoters (pAtCAB1, pStLS1 (SEQ ID NO:15), pAtRBCS3, pMeGBSS1 (SEQ ID NO:14), pStSSS3 (SEQ ID NO:21), pStSTP1 (SEQ ID NO:22)) that were included in the multi-gene construct plants and their gene expression in the field was tested (Example 1; Figure 1 ), dedicated promoter-GUS plants were also generated as described (Example 2; Figure 2 ).

[0198] In the pAtCAB1::GUS event, staining was observed in the mesophyll of source leaves, sink leaves, and new leaves ( Figure 2 A1-C1). The petiole and upper stem cross-sections showed staining in the sclerenchyma, outer parenchyma, and protoxylem regions ( Figure 2 D1-E1). The lower stem segments, storage roots, and fibrous roots showed no GUS staining at all ( Figure 2F1-H1). Thus, these results demonstrate that the promoter element of AtCAB1 (SEQ ID NO:6) can drive expression in autotrophic cassava tissues but is inactive in heterotrophic plant parts. To determine the approximate expression intensity of these promoter elements in reporter plants, the relative expression levels of different lines were measured and compared with the relative expression levels of pCaMV35S in three pCaMV35S::GUS lines. The CaMV35S promoter also has ubiquitous activity in cassava (Figure S2), and its expression intensity serves as a clear reference point. Compared with the promoter element of CaMV35S, the promoter of AtCAB1 (SEQ ID NO:6) showed approximately 25% to 45% activity respectively ( Figure 2 I1). Since pCaMV35S is a well-documented strong promoter, the promoter AtCAB1 (SEQ ID NO:6) can drive specific and reasonably strong expression in autotrophic tissues of cassava, which is consistent with Figure 1 the field expression results shown in

[0199] The promoter pStLS1 (SEQ ID NO:15) showed expected staining in source and sink leaves ( Figure 2 A2-B2). However, it also showed staining in the phloem and xylem tissues of petioles, stems, and storage roots ( Figure 2 C2-E2). Only the fibrous roots were not stained ( Figure 2 F2). This staining pattern matches the expression results ( Figure 1 ), indicating that pStLS1 (SEQ ID NO:15) is active in both source and sink tissues of cassava.

[0200] Similar to the low transcript levels observed for pAtRBCS3B (SEQ ID NO:20; Figure 1 ), a rather weak staining pattern was observed for pAtRBCS3B::GUS. Staining was observed in source and sink leaves ( Figure 2 A3-B3) and unexpectedly in the storage root cambium region ( Figure 2 E3). It seems that pAtRBCS3B (SEQ ID NO:20) is not a strongly or specifically expressed promoter that can be modified in cassava.

[0201] The promoter pMeGBSS1 (SEQ ID NO:14) was expected to be sink-specific (Koehorst-van Putten et al., 2012). However, activity was observed in both source and sink tissues. Source leaves ( Figure 2 A4) and sink leaves ( Figure 2 B4) were stained, and strong staining was observed in the phloem region of petioles ( Figure 2C4). All cell types of the stem and storage roots were stained except for the pith of the stem ( Figure 2 D4 - E4). The fibrous roots were not stained ( Figure 2 F4). Although the staining in the stem and storage roots seems stronger than in other tissues, the promoter activity inferred from the expression results ( Figure 1 ) indicates that the activity is rather equal between source and sink. In any case, the promoter is not storage root - specific in these experiments.

[0202] Contrary to the expression results of the field experiment ( Figure 1 ), indicating comparable source and sink activities for the StSSS3 promoter (SEQ ID NO: 21), pStSSS3::GUS plants showed heterotrophic tissue - specific staining. In the protoxylem of the petiole ( Figure 2 C5) and the stem ( Figure 2 D5), staining was observed in the phloem and xylem regions of the storage root ( Figure 2 E5), but not in the fibrous roots ( Figure 2 F5). This may be because the promoter used in the polygenic construct ( Figure 1 ) might be affected by adjacent promoters.

[0203] The promoter StSTP1 (SEQ ID NO: 22) showed weak but sink - specific behavior in the polygenic construct plants ( Figure 1 ). A matching staining pattern was observed in the promoter - GUS plants. The activities in source and sink leaves were confined to the vascular system ( Figure 2 A6 - B6). The petiole showed staining outside the protoxylem and sclerenchyma ( Figure 2 C6). Although the fibrous roots showed no staining except at the root tip ( Figure 2 F6), most of the staining was observed in the stem ( Figure 2 D6) and the storage root ( Figure 2 E6). Although the activity seems limited, pStSTP1 (SEQ ID NO: 22) can mediate rather sink - specific expression.

[0204] Example 4: Analysis of the expression of the promoter sequence in autotrophic tissues.

[0205] As described in the foregoing embodiments, several promoter elements tested can mediate specific expression patterns in autotrophic tissues of cassava plants (e.g., pSlRBCS2 (SEQ ID NO:19), pAtCAB1 (SEQ ID NO:6)), and some can mediate rather specific expression patterns in heterotrophic organs (e.g., pStSSS3, pStSTP1). However, none of these promoters seems to show particularly strong and specific targeting to sink tissues. Therefore, 13 additional reporter gene lines containing promoter-GUS constructs were generated, the constructs containing promoters with potential transport and heterotrophic storage tissue specificity (Table 2).

[0206] Table 2. Summary of 13 additional promoter sequences analyzed in cassava

[0207]

[0208]

[0209] Two additional promoter-GUS constructs were generated, one including the promoter StFBPase of cytosolic fructose-1,6-bisphosphatase cyt (SEQ ID NO:8), and the other including the promoter MePsbr (SEQ ID NO:3). The promoter StFBPase cyt (SEQ ID NO:8) and the promoter MePsbR (SEQ ID NO:3) were selected as candidates based on RNA transcription data. Contrary to the expected mesophyll-specific staining pattern, in addition to staining in the mesophyll of source leaves ( Figure 17 A) and sink leaves ( Figure 17 B), pStFBPase cyt (SEQ ID NO:8) showed considerable staining in the phloem and cambial regions of stems ( Figure 17 D) and storage roots ( Figure 17 E).

[0210] However, a very specific staining pattern was found for pMePsbR (SEQ ID NO:3; Figure 3 ). Here, staining was observed in the mesophyll of source leaves, sink leaves and new leaves ( Figure 3 A-C). Cross-sections of petioles showed labeling of most cell types except sclerenchyma and pith tissues ( Figure 3 D), while upper stem segments showed staining in pith parenchyma, phloem and cambial regions as well as collenchyma ( Figure 3 E). Heterotrophic lower stem segments, storage roots and fibrous roots showed no GUS staining at all ( Figure 3 F-H).

[0211] Thus, the results described herein show for the first time that the promoter MePsbR (SEQ ID NO:3) can drive the specific expression of a heterologous transcribable polynucleotide in autotrophic cassava tissues. To determine the approximate expression intensity of pMePbsbR, we determined the relative expression levels of different lines and compared them with the relative expression levels of pCaMV35 in three pCaMV35S::GUS. Compared with the promoter element of CaMV35S, the promoter MePsbR (SEQ ID NO:3) showed approximately 15% to 35% activity( Figure 3 I). Similar expression levels were obtained for pAtCAB1 (SEQ ID NO:6), and since pCaMV35S is a well-demonstrated strong promoter, the promoter MePsbR (SEQ ID NO:3) can drive specific and reasonably strong expression in autotrophic tissues of cassava.

[0212] Example 5: Analysis of promoter sequence expression in phloem tissue.

[0213] The promoter AtSUC2 (SEQ ID NO:24) was selected and expected to be phloem-specific in cassava because this promoter has been used as a phloem-specific tool in numerous studies in different species (Stadler and Sauer, (2019)). Indeed, the pAtSUC2::GUS lines showed significant staining in the minor and major leaf veins of source leaves( Figure 4 A), sink leaves( Figure 4 B), new leaves( Figure 4 C), as well as in the phloem region of petioles( Figure 4 D), upper stems( Figure 4 E), lower stems( Figure 4 F), and storage roots( Figure 4 G). The punctate GUS staining in the phloem is likely caused by the staining of phloem companion cells. The vascular systems of fibrous roots and root tips also showed GUS staining( Figure 4 H). In addition, some staining was observed in the protoxylem and xylem parenchyma regions( Figure 4 D-F). These results demonstrate that pAtSUC2 (SEQ ID NO:24) is well-suited to drive specific expression in phloem companion cells also in cassava.

[0214] Two additional phloem promoters were selected for testing, one being the melon promoter sequence driving the expression of galactinol synthase 1 (pGolS1; SEQ ID NO:11), and one being the sequence from Commelina yellow mottle virus (pCoYMV; SEQ ID NO:7). The former sequence was previously described as having specific activity for loading phloem, as GUS staining was specifically observed in the minor veins of source leaves (Haritatos et al., 2000). The latter promoter sequence was described as a promoter with high-level expression specific to phloem cells and phloem-associated cells (Medberry et al., 1992). Additionally, GUS staining was observed in phloem unloading tissues such as the tapetum (Medberry et al., 1992).

[0215] GUS staining of transgenic pCmGolS1::GUS plant lines revealed specific staining of the minor veins in cassava source leaves ( Figure 5 A), matching the results obtained in a previous publication (Haritatos et al., 2000). Most lines also showed slight patchy staining in the veins of sink leaves and new leaves ( Figure 5 B-C), staining in the protoxylem / xylem parenchyma of petioles ( Figure 5 D) and green stems ( Figure 5 E), as well as slight staining in the pith tissue of autotrophic and heterotrophic stem tissues ( Figure 5 E-F). While storage roots showed very little staining ( Figure 5 G), fibrous roots also showed slight patchy staining ( Figure 5 H). In summary, the promoter sequence used showed most activity in the minor veins of source leaves, but also some activity in non-phloem-associated tissues of cassava. Despite the activity outside the leaf, the promoter can still be a useful tool for phloem loading-centered biotechnological methods.

[0216] In contrast to the pCmGolS1::GUS plant lines that showed preferential activity in loading phloem, the pCoYMV::GUS plant lines seemed to be more specific to transporting and unloading phloem. None of the lines studied showed any staining of the vascular system in source leaves, but rather showed a wound-induced staining pattern due to staining at the cut site and punctate staining within the mesophyll ( Figure 6 A) or in fibrous roots ( Figure 6 H). In sink leaves, staining was only observed outside the vascular system, possibly representing phloem parenchyma ( Figure 6 B-C). In addition to some staining in the protoxylem and pith parenchyma ( Figure 6 D-F), in petioles ( Figure 6 D), autotrophic stems (Figure 6 E), heterotrophic stems ( Figure 6 F) and storage roots ( Figure 6 G) showed significant staining in the phloem tissue. Interestingly, as shown by the staining of the vascular strands in the following stems and storage roots, the tissues that are important for lateral transport were also stained in these promoter-reporter plants ( Figure 6 F - G). In summary, the analyzed pCoYMV sequence is rather specific for the transport and unloading phloem tissue, which is consistent with previous results showing promoter activity in vascular and reproductive tissues (Medberry et al., 1992). Although not a quantitative measure, all pCoYMV::GUS lines stained within seconds of adding the staining buffer, indicating very strong activity for the transport and unloading phloem tissue.

[0217] Promoter pMeSWEET1 - like (SEQ ID NO:1; Figure 7 ) also showed staining in the phloem region, although with lower specificity compared to pAtSUC2 (SEQ ID NO:24; Figure 4 ). The promoter element pMeSWEET1 - like (SEQ ID NO:1) was selected as a candidate based on RNA transcription data. The promoter - GUS lines containing this promoter element revealed staining in the vascular systems of source - and sink leaves ( Figure 7 A - B), as well as in the phloem and parenchyma tissues of petioles and stems ( Figure 7 D - G). The outer storage root regions containing phloem and phloem parenchyma showed significant GUS staining ( Figure 7 G). In addition, the promoter pMeSWEET1 - like (SEQ ID NO:1) showed activity in the vascular system of fibrous roots and at the root tips ( Figure 7 H). As disclosed for the first time herein, these results indicate that the promoter pMeSWEET1 - like (SEQ ID NO:1) has preferential activity in the phloem and parenchyma cells of cassava.

[0218] Based on RNA transcription data, the promoter MeSUS1 (SEQ ID NO:5) was selected as a candidate for testing as a putative phloem promoter. The pSUS1::GUS lines showed an interesting staining pattern similar to the pCoYMV promoter ( Figure 6 ). pSUS1 was active in the major leaf veins of the leaf vascular system ( Figure 8 A - B), in phloem and parenchyma cell types ( Figure 8 D - G) and in the vascular system of fibrous roots ( Figure 8 H). It showed significant staining in the vascular strands of stems and storage roots ( Figure 8(F - G), and the staining pattern in the storage roots indicates preferential activity in the phloem unloading zones and in the young xylem cells of the storage roots. Figure 8 G). This staining pattern matches the symplastic unloading pattern of cassava described previously and the metabolic gradients observed previously in storage roots.

[0219] To determine the approximate expression intensity of pMeSUS1 (SEQ ID NO:5), the relative expression levels of different lines were tested and compared with the relative expression levels of pCaMV35 determined in three pCaMV35S::GUS lines. The promoter element MeSUS1 (SEQ ID NO:5) showed approximately 5 - 20% activity compared to the promoter element of CaMV35S. Figure 8 I). Although its expression is much weaker than that of the more parenchyma - dominated promoters described in Example 6, the MeSUS1 (SEQ ID NO:5) promoter is active in many fewer cells throughout the storage root, thinning the specific signal. In summary, these results demonstrate for the first time that the promoter pMeSUS1 (SEQ ID NO:5) can be used as a promoter for applications focused on phloem transport and unloading.

[0220] Example 6: Analysis of promoter sequence expression in heterotrophic storage tissues.

[0221] Storage root - specific promoters are of particular interest for cassava. However, very few storage root - specific promoters are known in the art for cassava. A notable example is the promoter sequence (pStPat; SEQ ID NO:17) of the potato PATATIN class I promoter encoding the tuber storage protein patatin, which was previously shown to mediate this specific expression pattern in cassava. The promoter element pStPat (SEQ ID NO:17) was included in this study to confirm its tissue - specificity and activity. Additionally, the promoter elements pStB33 (SEQ ID NO:23), pStGBSS1 (SEQ ID NO:13), pDjDIO3 (SEQ ID NO:9), and pMeGPT (SEQ ID NO:2) were selected for testing. The promoters StB33 (SEQ ID NO:23), StGBSS1 (SEQ ID NO:13), and DjDIO3 (SEQ ID NO:9) were previously understood to have preferential storage organ activity in other plants. The promoter MeGPT (SEQ ID NO:2) was selected as a candidate based on RNA transcription data.

[0222] As expected, the promoter pStPat (SEQ ID NO:17) showed strong expression in storage roots and the highest specificity for storage root expression among all tested promoters. These lines showed no staining in leaves and petioles ( Figure 9 A-D), only weak staining in upper and lower stems ( Figure 9 E-F), and no staining in fibrous roots ( Figure 9 H). However, strong staining was observed in the xylem core region of storage roots ( Figure 9 G), which is mainly composed of xylem parenchyma cells. Depending on the respective line, the relative expression level of pStPat was about 40 - 160% compared to the relative expression level of pCaMV35 ( Figure 9 I). These results emphasize the storage root specificity of pStPat in cassava and confirm the high promoter activity in storage roots.

[0223] The promoter StB33 (SEQ ID NO:23), which is also part of the class I family of patatin genes, also seems to be very suitable for driving strong expression in the heterotrophic storage tissues of cassava. The pStB33::GUS lines showed staining in the minor veins of source leaves but no staining in sink leaves and petioles ( Figure 10 A-D). The upper stem tissue showed staining in collenchyma and protoxylem ( Figure 10 E), while the heterotrophic lower stem segments ( Figure 10 F) and storage roots showed strong staining in xylem and phloem parenchyma ( Figure 10 G). In addition, the vascular system and root tips of fibrous roots were stained ( Figure 10 F). Depending on the respective line, the relative expression level of pStB33 (SEQ ID NO:23) was about 20 - 80% compared to the relative expression level of pCaMV35. Thus, pStB33 (SEQ ID NO:23) is quite specific for sink tissues and has high activity in sink organs.

[0224] The pStGBSS1::GUS lines showed a staining pattern with mainly active in the phloem and xylem parenchyma cells of storage roots ( Figure 11 G). They also showed staining in the collenchyma of petioles and stems ( Figure 11 D-F), the pith parenchyma ( Figure 11 E-F), and the vascular system of fibrous roots ( Figure 11 H). In contrast to the two patatin promoters pStPat and pStB33 (SEQ ID NO:17 and 23; Fig.9 - 10), pStGBSS1 (SEQ ID NO:13) showed activity in both the source leaf and sink leaf vascular systems ( Figure 11A - B). Depending on the respective line, the relative GUS expression level caused by pStGBSS1 (SEQ ID NO: 13) is about 60 - 120% compared to the relative expression level caused by pCaMV35 ( Figure 11 I). Thus, pStGBSS1 (SEQ ID NO: 13) shows a similar silencing activity to pStPat (SEQ ID NO: 17), but seems to be less specific due to its higher activity in leaves, petioles, and stems of some cell types.

[0225] The pStGBSS1::GUS lines show a staining pattern with predominant activity in the phloem and xylem parenchyma cells of storage roots ( Figure 11 G). They also show staining in the collenchyma of petioles and stems ( Figure 11 D - F), the pith parenchyma ( Figure 11 E - F), and the vascular system of fibrous roots ( Figure 11 H). In contrast to the two patatin promoters pStPat and pStB33 (SEQ ID NO: 17 and 23; Fig. 9 - 10), pStGBSS1 (SEQ ID NO: 13) shows activity in both the source leaf and sink leaf vascular systems ( Figure 11 A - B). Depending on the respective line, the relative GUS expression level caused by pStGBSS1 (SEQ ID NO: 13) is about 60 - 120% compared to the relative expression level caused by pCaMV35 ( Figure 11 I). Thus, pStGBSS1 (SEQ ID NO: 13) shows a similar silencing activity to pStPat (SEQ ID NO: 17), but seems to be less specific due to its higher activity in leaves, petioles, and stems of some cell types.

[0226] Although the promoters pStPat (SEQ ID NO: 17), pStB33 (SEQ ID NO: 23), pStGBSS1 (SEQ ID NO: 13), and pMeGPT (SEQ ID NO: 2) all show preferential activity in heterotrophic storage tissues, the promoter of the dioscin 3 small subunit gene from Dioscorea japonica (DjDIO3) does not. Contrary to what has been previously suggested in the art, the pDjDIO3::GUS lines show a rather ubiquitous staining pattern in cassava ( Figure 18 ).

[0227] Example 7: Analysis of promoter sequence expression in cambial tissue.

[0228] To achieve transgenic intervention targeting cassava secondary growth, promoters with different activities in the vascular cambium would be useful tools. To this end, the sweet potato MADS-box transcription factor pIbSRD1 (SEQ ID NO:16) in cassava, which is the tissue specificity of promoters previously characterized in thale cress, carrot, potato, and sweet potato. In sweet potato, it was shown that SRD1 expression is auxin-responsive and the transcript is located in the primary cambium, secondary cambium, and primary phloem cells. Major promoter activity in thale cress could be demonstrated in the vascular system including periderm and endodermis, while promoter activity was strong in all cells of carrot taproot and potato tuber.

[0229] Promoter activity in cassava was similar to the results obtained in sweet potato and thale cress. In the vascular systems of source leaves, sink leaves, and new leaves ( Figure 14 A-C) and the vascular system of fibrous roots ( Figure 14 H), as well as in the protoxylem and xylem vasculature of petioles and stems ( Figure 14 D-E), significant staining was observed. In addition, strong staining was observed in the vascular cambium and phellogen of stems and storage roots ( Figure 14 E-G). Together, these results demonstrate that pIbSRD1 (SEQ ID NO:16) has specific activity in cells with meristem identity in cassava.

[0230] The promoter Manes.14g071100 (SEQ ID NO:4) was also tested based on its expected ubiquitous activity due to its expected role in mitochondrial function. However, targeted testing using the specific promoter sequence of SEQ ID NO:4 revealed a surprising but useful expression pattern, as Figure 13 shown. Four lines of this promoter::reporter gene combination were analyzed. One line showed no staining, but three lines showed very similar patterns with different intensities.

[0231] The pManes.14g071100::GUS line exhibited storage root cambium specificity as well as minor activity in shoot tips ( Figure 13 ).

[0232] Therefore, the promoter Manes.14g071100 (SEQ ID NO:4) has interesting applications for the expression of regulators of vascular cambium development due to its specificity for the storage root vascular cambium. This may lead to an increase in storage root size. This staining pattern supports the use of the promoter pManes.14g071100 (SEQ ID NO:4) as a new promoter element for developmental applications in cassava.

[0233] Example 8: Summary of promoter element specificity in cassava.

[0234] Among the leaf promoters tested, StFBPase cyt (SEQ ID NO:8), AtFBA2 (SEQ ID NO:10), AtGAPA (SEQ ID NO:12), StLS1 (SEQ ID NO:15) and AtRBCS3B (SEQ ID NO:20) showed weak and / or non-specific expression. However, the promoters AtCAB1 (SEQ ID NO:6) and MePsbR (SEQ ID NO:3) were shown to be specific and relatively strong, making them fully suitable tools for transgene expression in cassava photosynthetic tissues. In particular, the results described herein reveal the MePsbR promoter (SEQ ID NO:3) as a viable method for expressing heterologous polynucleotide molecules in the photosynthetic tissues of cassava. Although dedicated promoter-GUS lines were not established for the promoters SlRBCS2 (SEQ ID NO:19) and AtRBCS1A (SEQ ID NO:18), they showed very high activity in source leaf tissues in transcriptional studies. In addition, pSlRBCS2 (SEQ ID NO:19) also appears to be specific for this tissue.

[0235] The tested promoters AtSUC2 (SEQ ID NO:24), CmGolS1 (SEQ ID NO:11), CoYMV (SEQ ID NO:7), MeSWEET1-like (SEQ ID NO:1) and StSTP1 (SEQ ID NO:22) can be used as expression tools for phloem tissues, where some of these promoters have expression in specific phloem tissues. For example, although pAtSUC2 (SEQ ID NO:24) has specific expression along the entire phloem, the promoters pCmGolS1 (SEQ ID NO:11) or pCoYMV (SEQ ID NO:7) can target the loading or transport / unloading phloem, respectively. The promoters MeSWEET1-like (SEQ ID NO:1) and StSTP1 (SEQ ID NO:22) can be used to target the phloem of cassava, especially phloem parenchyma. The promoter MeSUS1 (SEQ ID NO:5) also has considerable phloem activity, as well as storage tissue activity, especially in cells close to the vascular cambium. These results suggest that the MeSUS1 promoter sequence (SEQ ID NO:5) can be an important tool for methods centered on increased sink demand.

[0236] Among the promoters with preferential activity in heterotrophic storage tissues, MeGBSS1 (SEQ ID NO:14) and StSSS3 (SEQ ID NO:21) appear to show low specificity or, in the case of pStSSS3 (SEQ ID NO:21), weak activity. As previously mentioned, the promoter StPat (SEQ ID NO:17) has been demonstrated to be highly active and highly storage root-specific. However, pStB33 (SEQ ID NO:23), pMeGPT (SEQ ID NO:2), and pStGBSS1 (SEQ ID NO:13) are also very strong promoters for expression in sink tissues as they are mainly active in starch storage stem and storage root tissues. They also appear to have an expression intensity comparable to that of StPat (SEQ ID NO:17). These promoters can be used in larger transgenic stacks that attempt to avoid the repetition of the same promoter sequence to avoid silencing or recombination effects.

[0237] Although pDjDIO3 (SEQ ID NO:9) may be very strong (as it shows strong GUS staining within seconds of adding the staining buffer), the promoter specificity is low. In contrast, pIbSRD1 (SEQ ID NO:16) shows a highly specific expression pattern with high activity in dividing cells. This promoter could be an important tool for more developmentally focused approaches targeting stem cells.

[0238] In summary, we have confirmed a number of tissue-specific promoter elements that allow for targeted transgenic expression in a variety of cassava tissues. In addition, various promoter elements for expressing heterologous sequences in cassava have been described for the first time (e.g., MeSWEET1 (SEQ ID NO:1), MeGPT (SEQ ID NO:2), MePsbR (SEQ ID NO:3), pManes.14g071100 (SEQ ID NO:4), and MeSUS1 (SEQ ID NO:5)).

[0239] Table 3 provides a summary of the tissue-specific promoters for each tissue. These promoter sequences will support further transgenic applications for spatial and temporal expression in cassava.

[0240] Table 3. Tissue Specificity of Promoters Disclosed herein

[0241]

[0242]

[0243] After the principles of the present invention have been described and explained, it will be apparent to those skilled in the art that the arrangements and details of the present invention can be modified without departing from those principles. We claim all modifications within the spirit and scope of the claims. All publications and patent documents cited herein are hereby incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A DNA molecule comprising a DNA sequence selected from: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-15, 17-19, and 21-24; b) a sequence comprising any one of SEQ ID NO: 1-15, 17-19, and 21-24; c) a fragment of a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-15, 17-19, and 21-24, wherein said fragment has gene regulatory activity; d) a fragment of any one of SEQ ID NO: 1-15, 17-19, and 21-24, wherein said fragment has gene regulatory activity; and e) a combination thereof, wherein said sequence is operably linked to a heterologous transcribable polynucleotide molecule.

2. The DNA molecule according to claim 1, wherein said DNA sequence is active as a promoter.

3. The DNA molecule according to claim 1, wherein said DNA molecule further comprises a heterologous regulatory element.

4. The DNA molecule according to claim 1, wherein said sequence has at least 90% sequence identity with the DNA sequence of any one of SEQ ID NO: 1-15, 17-19, and 21-24.

5. The DNA molecule according to claim 1, wherein said sequence has at least 95% sequence identity with the DNA sequence of any one of SEQ ID NO: 1-15, 17-19, and 21-24.

6. The DNA molecule according to claim 1, wherein said DNA sequence comprises gene regulatory activity.

7. The DNA molecule according to claim 1, wherein said heterologous transcribable polynucleotide molecule comprises an agronomic gene.

8. The DNA molecule according to claim 7, wherein said agronomic gene confers increased yield, increased root growth, or increased drought tolerance to a plant.

9. The DNA molecule according to claim 7, wherein said agronomic gene confers increased starch content to a plant.

10. A construct comprising at least one copy of the DNA molecule according to claim 1, and an operably linked transcribable agronomic gene.

11. The construct according to claim 10, wherein said construct comprises, in the 5′-3′ direction: (a) at least one copy of said DNA molecule; (b) an operably linked transcribable agronomic gene; and (c) a gene termination sequence.

12. The construct according to claim 10, wherein said transcribable agronomic gene comprises an open reading frame encoding a polypeptide.

13. A transgenic plant cell comprising a heterologous DNA molecule comprising a sequence selected from: a) a sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-15, 17-19, and 21-24; b) a sequence comprising any one of SEQ ID NO: 1-15, 17-19, and 21-24; c) a fragment of a sequence having at least 85% sequence identity to any one of SEQ ID NO: 1-15, 17-19, and 21-24, wherein said fragment has gene regulatory activity; d) a fragment of any one of SEQ ID NO: 1-15, 17-19, and 21-24, wherein said fragment has gene regulatory activity; and e) a combination thereof, wherein said sequence is operably linked to a heterologous transcribable polynucleotide molecule.

14. The transgenic plant cell according to claim 13, wherein said transgenic plant cell is a monocotyledonous plant cell.

15. The transgenic plant cell according to claim 13, wherein said transgenic plant cell is a dicotyledonous plant cell.

16. A transgenic plant or a part thereof, comprising the DNA molecule according to claim 1.

17. A progeny plant or a part thereof of the transgenic plant according to claim 16, wherein said progeny plant or a part thereof comprises said DNA molecule.

18. A transgenic seed, wherein said seed comprises the DNA molecule according to claim 1.

19. A method for producing a commodity, comprising obtaining the transgenic plant or a part thereof according to claim 16 and producing a commodity therefrom.

20. The method according to claim 19, wherein said commodity is a protein concentrate, a protein isolate, a grain, a starch, a seed, a grit, a flour, a biomass, or a seed oil.

21. A commodity, comprising the DNA molecule according to claim 1.

22. The commodity according to claim 21, wherein said commodity is a protein concentrate, a protein isolate, a grain, a starch, a seed, a grit, a flour, a biomass, or a seed oil.

23. A method for expressing a transcribable polynucleotide molecule, which comprises obtaining the transgenic plant according to claim 16 and cultivating a plant in which said transcribable polynucleotide is expressed.

24. A method for expressing an agronomic gene in a plant or a plant cell, said method comprising incorporating into the plant cell a construct comprising the DNA molecule according to claim 1 operably linked to a transcribable agronomic gene, wherein said DNA molecule is capable of driving the expression of the operably linked agronomic gene in said plant cell.

25. The method according to claim 24, further comprising regenerating a transformed plant from said plant cell.

26. The method according to claim 24, wherein said plant cell is stably transformed with said construct.

27. A method for producing a transgenic plant cell, comprising introducing the DNA molecule according to claim 1 into a plant cell.

28. The method according to claim 27, wherein introducing said DNA molecule into said plant cell comprises transformation.

29. The method according to claim 28, further comprising regenerating a transgenic plant from said plant cell.

30. The method according to claim 27, wherein introducing said DNA molecule into said plant cell comprises crossing the transgenic plant according to claim 16 with another plant to produce a progeny plant comprising said plant cell.

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