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(12) Patent Application Publication (10) Pub. No.: US 2005/0010974A1 Milligan Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2005/0010974A1 Milligan Et Al US 20050010974A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0010974A1 Milligan et al. (43) Pub. Date: Jan. 13, 2005 (54) PROMOTERS FOR REGULATION OF GENE Publication Classification EXPRESSION IN PLANT ROOTS (51) Int. Cl." ............................ C12N 15/82; C12O 1/68; (76) Inventors: Stephen B Milligan, Kirkland, WA C12N 15/87; C12N 15/63; (US); Dale Skalla, Research Triangle C12N 15/85; C12N 5/10; Park, NC (US); Kay Lawton, Research C12N 15/09; CO7H 21/04 Triangle Park, NC (US) (52) U.S. Cl. ..................... 800/287; 536/23.6; 435/320.1; 435/455; 435/419; 435/468; Correspondence Address: 800/278; 435/6; 536/24.33 Randee S Schwatz Syngenta Biotechnology 3054 Cornwallis Road (57) ABSTRACT Research Triangle Park, NC 27709 (US) The present invention is directed to promoters isolated from (21) Appl. No.: 10/490,147 maize and functional equivalents thereto. The promoters of the present invention have particular utility in driving root (22) PCT Filed: Nov. 4, 2002 Specific expression of heterologous genes that impart (86) PCT No.: PCT/US02/35374 increased agronomic, horticultural and/or pesticidal charac teristics to a given promoters of the invention and trans (30) Foreign Application Priority Data formed plant tissues containing DNA molecules comprising a promoter of the invention operably linked to a heterolo Nov. 7, 2001 (US)........................................... 60337026 gous gene or genes, and Seeds thereof. US 2005/0010974 A1 Jan. 13, 2005 PROMOTERS FOR REGULATION OF GENE latory Sequences may be short regions of DNA sequence EXPRESSION IN PLANT ROOTS 6-100 base pairs that define the binding sites for trans-acting factors, Such as transcription factors. Regulatory Sequences FIELD OF THE INVENTION may also be enhancers, longer regions of DNA sequence that 0001. The present invention relates generally to the field can act from a distance from the core promoter region, of plant molecular biology and the regulation of gene Sometimes over Several kilobases from the core region. expression in plants. More Specifically, the present invention Regulatory Sequence activity may be influenced by trans relates to the regulation of gene expression in plant roots. acting factors including general transcription machinery, transcription factors and chromatin assembly factors. BACKGROUND OF THE INVENTION 0006 Frequently, it is desirable to have tissue-specific 0002 Manipulation of crop plants to alter and/or improve expression of a gene of interest in a plant. TiSSue-specific phenotypic characteristics (Such as productivity or quality) promoters promote expression exclusively in one set of requires the expression of heterologous genes in plant tis tissues without expression throughout the plant, tissue Sues. Such genetic manipulation has become possible by preferred promoters promote expression at a higher level in virtue of two discoveries: the ability to transform heterolo a Subset of tissues with Significantly less expression in the gous genetic material into a plant cell and by the existence other tissues of the plant. For example, one may desire to of promoters that are able to drive the expression of the express a value-added product only in corn Seed but not in heterologous genetic material. the remainder of the plant. Another example is the produc tion of male Sterility by tissue-specific ablation. In this case, 0003. It is advantageous to have the choice of a variety of a phytotoxic product is expressed only in the male tissue of different promoters So as to give the desired effect(s) in the the plant to ablate that specific tissue while other tissues of transgenic plant. Suitable promoters may be selected for a the flower as well as the rest of the plant remain intact. Many particular gene, construct, cell, tissue, plant or environment. aspects of agricultural biotechnology use and require tissue Promoters that are useful for plant transgene expression Specific expression. include those that are inducible, Viral, Synthetic, constitutive (Odell et al., 1985, Nature 313: 810-812; Granger & Cyr, 0007 One important example of a need for promoters is 2001, Plant Cell Repo. 20: 227-234), temporally regulated, for the expression of Selected genes in plant roots. The plant Spatially regulated, tissue-specific, and Spatio-temporally root consists of many cell types Such as epidermal, root cap, regulated (Kuhlemeier et al. 1987, Ann. Rev. Plant Physiol. columella, cortex, pericycle, vascular and root hair forming Plant Mol. Biol. 38: 221-257). Promoters from bacteria, trichoblasts, organized into tissueS or regions of the root, for fungi, viruses and plants have been used to control gene example, the root tip, root epidermis, meristematic Zone, expression in plant cells. primary root, lateral root, root hair, and vascular tissue. Promoters isolated as root-Specific or root-preferred can be 0004 Promoters consist of several regions that are nec biased towards promotion of expression in one or a few of essary for full function or the promoter. Some of these these cell types. This cell-specific activity can be useful for regions are modular, in other words they can be used in Specific applications Such as regulating meristematic activity isolation to confer promoter activity or they may be in only the meristematic cell Zone or expression of A assembled with other elements to construct new promoters. nematicidal gene in only the cell types that are contacted by The first of these promoter regions lies immediately the nematode pest. In other cases, broader cell-type speci upstream of the coding Sequence and forms the “core ficity may be desired to express genes of interest throughout promoter region' containing consensus Sequences, normally the root tissue. This may be useful in expressing an insec 20-70 base pairs immediately upstream of the coding ticidal gene to control an insect pest that feeds on plant roots, Sequence. The core promoter region contains a TATA box for instance corn rootworm (Diabrotica spp.). Broader cell and often an initiator element as well as the initiation site. type root Specificity may be accomplished with a single The precise length of the core promoter region is not fixed root-specific promoter with broad cell-type specificity or by but is usually well recognizable. Such a region is normally using two or more root-Specific or root-preferred promoters present, with Some variation, in most promoters. The base of different cell-type specificities for expression. A limited Sequences lying between the various well-characterized ele number of examples of root-preferred and root-specific ments appear to be of lesser importance. The core promoter promoters have been described. These include the RB7 region is often referred to as a minimal promoter region promoter from Nicotiana tabacum (U.S. Pat. Nos. 5,459.252 because it is functional on its own to promote a basal level and 5,750,386); the ARSK1 promoter from Arabidopsis of transcription. thaliana (Hwang and Goodman (1995) Plant J 8:37-43), the 0005 The presence of the core promoter region defines a MR7 promoter from Zea mays (U.S. Pat. No. 5,837,848), the Sequence as being a promoter: if the region is absent, the ZRP2 promoter of Zea mays (U.S. Pat. No. 5,633,363), and promoter is non-functional. The core region acts to attract the MTL promoter from Zea mays (U.S. Pat. Nos. 5,466,785 the general transcription machinery to the promoter for and 6,018,099). Many of these examples disclose promoters transcription initiation. However, the core promoter region with expression patterns confined to a limited number of is insufficient to provide fill promoter activity. A series of root tissues. Others fail to provide the root-specificity regulatory Sequences, often upstream of the core, constitute needed for expression of Selected genes. Thus, there is a the remainder of the promoter. The regulatory Sequences need in the art for isolation and characterization of new root determine expression level, the Spatial and temporal pattern promoters to obtain those of different breadth, expression of expression and, for a Subset of promoters, expression level and Specificity of cell-type expression for root-specific under inductive conditions (regulation by external factors and root-preferred expression, particularly for root-specific Such as light, temperature, chemicals and hormones). Regu expression. US 2005/0010974 A1 Jan. 13, 2005 SUMMARY OF THE INVENTION not limited to green fluorescent protein (GFP), f-glucu ronidase (GUS), and luciferase (LUC). In yet another 0008. Within the present invention, compositions and embodiment, the expression cassette comprises a Selectable methods for directing root-specific expression in transgenic marker coding Sequence including but not limited to phos plants are provided. In particular, novel nucleic acid mol phomannose isomerase (PMI), an antibiotic resistance gene ecules isolated from Zea mayS, that drive expression of Such as hygromycin, kanamycin and the like, a herbicide heterologous genes in a root-specific manner in plants, are tolerance gene Such as phosphinothricin and the like and provided. The invention is further drawn to expression bamase (bar). cassettes and vectors comprising the novel nucleic acid molecules of the invention operably linked to heterologous 0012. The present invention also provides a recombinant coding Sequences. The invention is still further drawn to vector comprising the expression cassette of the invention. transgenic plants comprising the expression cassettes of the In a preferred embodiment, the recombinant vector is a invention. The present invention also provides methods for plasmid. Specifically expressing a heterologous coding Sequence in 0013 Further, the present invention provides a transgenic transgenic plant roots, for isolating a root-specific cDNA, non-human host cell comprising the expression cassette of for isolating a nucleic acid molecule useful for directing the invention. A transgenic host cell according to this aspect root-specific expression and for isolating a root-specific of the invention is preferably a plant cell.
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