# Patent

## Title
Sucrose synthase 3 promoter from rice and uses thereof

## Abstract

Rice sucrose synthase 3 regulatory regions suitable for directing expression of a heterologous nucleic acid are described, as well as nucleic acid constructs that include these regulatory regions. Also disclosed are transgenic plants that contain such constructs and methods of producing such transgenic plants.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of, and claims the benefit of and priority under 35 U.S.C. § 120 to U.S. patent application Ser. No. 10/966,482, filed Oct. 14, 2004, which is incorporated by reference in its entirety herein.


TECHNICAL FIELD
This document provides compositions and methods involved in regulating gene expression in eukaryotic organisms (e.g., plants).
BACKGROUND
An essential element for genetic engineering of plants is the ability to express genes using various regulatory regions. The expression pattern of a transgene, conferred by a regulatory region is critical for the timing, location, and conditions under which a transgene is expressed, as well as the intensity with which the transgene is expressed in a transgenic plant. There is continuing need for suitable regulatory regions that can facilitate transcription of sequences that are operably linked to the regulatory region
SUMMARY
This document provides compositions and methods involving regulatory regions having the ability to direct transcription in eukaryotic organisms (e.g., plants). For example, this document provides regulatory regions having the ability to direct transcription in plant ovules prior to fertilization and in seeds during early stages of development. Also provided herein are nucleic acid constructs, plant cells, and plants containing such regulatory regions; methods of producing plant cells and plants containing such regulatory regions; and methods of using such regulatory regions to express polynucleotides in plants and to alter the phenotype of plant cells. Regulatory regions that direct transcription during seed development can be used, for example, to manipulate genomic imprinting in crop plants, resulting in enhanced seed development and increased yield.
In one embodiment, an isolated nucleic acid including a regulatory region having a length of 1735 to 1890 nucleotides and 80 percent or greater sequence identity to SEQ ID NO:1 is provided, where the regulatory region directs transcription, in a plant ovule within 24 hours post-fertilization, of an operably linked heterologous polynucleotide. The sequence identity can be 85 percent or greater, 90 percent or greater, or 95 percent or greater.
In another embodiment, an isolated nucleic acid including a regulatory region having a nucleic acid sequence corresponding to SEQ ID NO:1 is provided, where the regulatory region directs transcription, in a plant ovule within 24 hours post -fertilization, of an operably linked polynucleotide.
In another embodiment, an isolated nucleic acid including a regulatory region having a length of 1735 to 1890 nucleotides and 80 percent or greater sequence identity to SEQ ID NO:1 is provided, where the regulatory region directs transcription in an unfertilized plant ovule of an operably linked heterologous polynucleotide. The sequence identity can be 85 percent or greater, 90 percent or greater, or 95 percent or greater.
In a further embodiment, an isolated nucleic acid including a regulatory region having a nucleic acid sequence corresponding to SEQ ID NO:1 is provided, where the regulatory region directs transcription in an unfertilized plant ovule of an operably linked polynucleotide.
A regulatory region can include an intron. A regulatory region can include one or more of a TATA box, a CAAT box, a GCN4 box, an endosperm box, a prolamin box, and a legumin box. A regulatory region can include all of a TATA box, a CAAT box, a GCN4 box, an endosperm box, a prolamin box, and a legumin box.
In another aspect, a nucleic acid construct including an isolated nucleic acid including a regulatory region is provided, where the nucleic acid is operably linked to a heterologous polynucleotide. The heterologous polynucleotide can include a nucleic acid sequence encoding a polypeptide. The heterologous polynucleotide can be in an antisense orientation relative to the regulatory region. The heterologous polynucleotide can be transcribed into an antisense RNA capable of inhibiting expression of a DNA methyltransferase. The heterologous polynucleotide can be transcribed into an interfering RNA. The heterologous polynucleotide can be transcribed into an interfering RNA against a DNA methyltransferase.
In another embodiment, a transgenic plant or plant cell is provided, where the plant or plant cell includes an isolated nucleic acid including a regulatory region operably linked to a heterologous polynucleotide. The plant or plant cell can be a monocot. The heterologous polynucleotide can include a nucleic acid sequence encoding a polypeptide. The heterologous polynucleotide can be in an antisense orientation relative to the regulatory region. The heterologous polynucleotide can be transcribed into an interfering RNA.
In a further embodiment, a seed from a transgenic plant is provided.
In another aspect, a method of producing a transgenic plant is provided. The method can include (a) introducing into a plant cell an isolated polynucleotide including an isolated nucleic acid including a regulatory region operably linked to a heterologous polynucleotide, and (b) growing a plant from the plant cell. The plant can be a monocot. The heterologous polynucleotide can include a nucleic acid sequence encoding a polypeptide. The heterologous polynucleotide can be in an antisense orientation relative to the regulatory region. The heterologous polynucleotide can be transcribed into an interfering RNA.
In another aspect, a transgenic plant produced by the method described above is provided.
In yet another aspect, a seed from a transgenic plant described above is provided.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.


DETAILED DESCRIPTION
This document provides isolated nucleic acids comprising regulatory regions. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, and DNA (or RNA) containing nucleic acid analogs. Polynucleotides can have any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs.
As used herein, “isolated,” when in reference to a nucleic acid, refers to a nucleic acid that is separated from other nucleic acids that are present in a genome, e.g., a plant genome, including nucleic acids that normally flank one or both sides of the nucleic acid in the genome. The term “isolated” as used herein with respect to nucleic acids also includes any non-naturally-occurring sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment) independent of other sequences. An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, a bacterium, or into the genome of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.
Regulatory Regions
A regulatory region described herein is a nucleic acid that can direct transcription of a heterologous nucleic acid in certain cell types when the regulatory region is operably linked 5′ to the heterologous nucleic acid. As used herein, “heterologous nucleic acid” refers to a nucleic acid other than the naturally occurring coding sequence to which the regulatory region was operably linked in a plant. With regard to one regulatory region provided herein, pOs530c10 (SEQ ID NO:1), a heterologous nucleic acid is a nucleic acid other than the sucrose synthase 3 coding sequence. The term “operably linked” refers to positioning of a regulatory region and a transcribable sequence in a nucleic acid so as to allow or facilitate transcription of the transcribable sequence. For example, a regulatory region is operably linked to a coding sequence when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into a protein encoded by the coding sequence.
Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and other regulatory regions that can reside within coding sequences.
For example, a plant regulatory region can include one or more of the following elements: a CAAT box, a TATA box, a GCN4 box, an endosperm box, a prolamin box, and a legumin box.
The CAAT box is a conserved nucleotide sequence involved in initiation of transcription. The CAAT box functions as a recognition and binding site for regulatory proteins called transcription factors.
The TATA box is another conserved nucleotide sequence involved in transcription initiation. The TATA box seems to be important in determining accurately the position at which transcription is initiated.
The GCN4 box, the endosperm box, and the prolamin box are three different nucleotide sequence motifs that are conserved in the regulatory regions of storage protein genes. These nucleotide sequences are thought to confer endosperm-specific gene expression.
The legumin box, also referred to as the RY repeat motif or the Sph element, is another nucleotide sequence element involved in seed-specific gene expression. The legumin box acts as both an enhancer for seed-specific gene expression and a repressor of expression in vegetative tissue.
The nucleic acid sequence set forth in FIG. 1 and SEQ ID NO:1 is an example of a regulatory region provided herein. However, a regulatory region can have a nucleotide sequence that deviates from that set forth in SEQ ID NO:1, while retaining the ability to direct expression of an operably linked nucleic acid. For example, a regulatory region having 80% or greater (e.g., 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater) sequence identity to the nucleotide sequence set forth in SEQ ID NO:1 can direct expression of an operably linked nucleic acid. The nucleic acid sequences set forth in SEQ ID NOs:2-4 are additional examples of regulatory regions provided herein.
The term “percent sequence identity” refers to the degree of identity between any given query sequence, e.g., SEQ ID NO:1, and a subject sequence. A percent identity for any subject nucleic acid relative to a query nucleic acid can be determined as follows. A query nucleic acid sequence is aligned to one or more subject nucleic acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid sequences to be carried out across their entire length (global alignment). ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For alignment of multiple nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; and gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: G, P, S, N, D, Q, E, R, K; and residue-specific gap penalties: on. The ClustalW output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site (ebi.ac.uk/clustalw).
To determine a percent identity between a query sequence and a subject sequence, ClustalW divides the number of matching nucleotides by the number of nucleotides of the shorter sequence, and multiplies the result by 100. The output is the percent identity of the subject sequence with respect to the query sequence. For example, if a query sequence and a subject sequence are each 500 bases long and have 200 contiguous bases that are identical, the subject sequence would have 40 percent sequence identity to the query sequence. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two sequence lengths. For example, if 100 bases match between a 400 nucleotide query sequence and a 500 nucleotide subject sequence, the subject sequence would have 25 percent identity to the query sequence. If the shorter sequence is less than 150 bases in length, the number of matches are divided by 150 and multiplied by 100 to obtain a percent sequence identity.
It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It is also noted that the length value will always be an integer.
A regulatory region featured herein can be made by cloning 5′ flanking sequences of a sucrose synthase 3 gene, as described in more detail below. Alternatively, a regulatory region can be made by chemical synthesis and/or polymerase chain reaction (PCR) technology. PCR refers to a procedure or technique in which target nucleic acids are amplified. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. PCR is described, for example in PCR Primer: A Laboratory Manual, Ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification. See, for example, Lewis, Genetic Engineering News 12(9):1 (1992); Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990); and Weiss, Science 254:1292 (1991). Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.
Various lengths of a regulatory region described herein can be made by similar techniques. For example, a regulatory region can be made that has a length of 1735 nucleotides to 1890 nucleotides or any length therebetween, such as a length of 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, or 1889 nucleotides.
The ability of a regulatory region to direct expression of an operably linked nucleic acid can be assayed using methods known to one having ordinary skill in the art. In particular, regulatory regions of varying lengths can be operably linked to a reporter nucleic acid and used to transiently or stably transform a cell, e.g., a plant cell. Suitable reporter nucleic acids include β-glucuronidase (GUS), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and luciferase (LUC). Expression of the gene product encoded by the reporter nucleic acid can be monitored in such transformed cells using standard techniques.
A regulatory region can influence the tissue-specificity of the transcription of an operably linked heterologous nucleic acid. When a heterologous nucleic acid is operably linked to a tissue-, organ-, or cell-specific regulatory region, transcription occurs only or predominantly in a particular tissue, organ, and cell type, respectively. For example, a regulatory region can be essentially specific to a plant ovule. An ovule is a structure in a flower that contains the female gametophyte and develops into a seed. A female gametophyte is also referred to in angiosperms as the embryo sac. The seed is a mature ovule, including the embryo, the endosperm, and the seed coat.
In some cases, a regulatory region can direct transcription primarily in a plant ovule that has not been fertilized, such as in an un-pollinated embryo sac. In some cases, a regulatory region can direct transcription in a plant ovule that has been fertilized, such as in a plant ovule starting within 24 hours post-fertilization to at least five days (e.g., six, seven, eight, nine, 10, 11, 12, 13, or 14 days) after pollination. In some cases, a regulatory region can direct transcription in endosperm tissue starting within 24 hours after fertilization to at least five days after pollination.
Nucleic Acid Constructs
Nucleic acid constructs containing nucleic acids such as those described herein are also provided. A nucleic acid construct can be a vector. A vector is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term “vector” includes cloning, transformation, and expression vectors, as well as viral vectors and integrating vectors. An expression vector is a vector that includes one or more regulatory regions. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpesviruses, cytomegalovirus, vaccinia viruses, adenoviruses, adeno-associated viruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).
A nucleic acid construct includes a regulatory region as disclosed herein. A construct can also include a heterologous nucleic acid operably linked to the regulatory region, in which case the construct can be introduced into an organism and used to direct expression of the operably linked nucleic acid. The heterologous nucleic acid can be operably linked to the regulatory region in the sense or antisense orientation. The regulatory region can be operably linked from approximately 1 to 150 nucleotides upstream of the ATG translation start codon of a heterologous nucleic acid in the sense orientation. For example, the regulatory region can be operably linked 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, or 150 nucleotides upstream of the ATG translation start codon of a heterologous nucleic acid in the sense orientation. In some cases, the regulatory region can be operably linked from approximately 151 to 500 nucleotides upstream of the ATG translation start codon of a heterologous nucleic acid in the sense orientation. In some cases, the regulatory region can be operably linked from approximately 501 to 1125 nucleotides upstream of the ATG translation start codon of a heterologous nucleic acid in the sense orientation.
In some embodiments, a heterologous nucleic acid is transcribed and translated into a polypeptide. Suitable polypeptides include, without limitation, screenable and selectable markers such as green fluorescent protein, yellow fluorescent protein, luciferase, β-glucuronidase, or neomycin phosphotransferase II. Suitable polypeptides also include polypeptides that affect growth and/or hormone production. In some embodiments a heterologous nucleic acid encodes a polypeptide involved in nutrient utilization. In other embodiments, a heterologous polynucleotide encodes a non-plant protein of pharmaceutical or industrial interest. In some cases, a heterologous nucleic acid encodes a polypeptide involved in DNA methylation, such as a cytosine DNA methyltransferase.
A nucleic acid construct may include a heterologous nucleic acid that is transcribed into an RNA useful for inhibiting expression of an endogenous gene. Suitable constructs from which such an RNA can be transcribed include antisense constructs. Antisense nucleic acid constructs can include a regulatory region of the invention operably linked, in antisense orientation, to a nucleic acid molecule that is heterologous to the regulatory element. Thus, for example, a transcription product can be similar or identical to the sense coding sequence of an endogenous polypeptide, but transcribed into an RNA that is unpolyadenylated, lacks a 5′ cap structure, or contains an unsplicable intron. Constructs containing operably linked nucleic acid molecules in sense orientation can be used to inhibit the expression of a gene. Methods of co-suppression using a full-length cDNA sequence as well as a partial cDNA sequence are known in the art. See, e.g., U.S. Pat. No. 5,231,020.
Alternatively, a heterologous nucleic acid can be transcribed into a ribozyme. See, U.S. Pat. No. 6,423,885. Heterologous nucleic acid molecules can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide. Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contain a 5′-UG-3′ nucleotide sequence. The construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Pat. No. 5,254,678. Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo. Perriman et al., Proc. Natl. Acad. Sci. USA 92(13):6175-6179 (1995); de Feyter and Gaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes in Plants,” Edited by Turner, P.C, Humana Press Inc., Totowa, N.J. RNA endoribonucleases such as the one that occurs naturally in Tetrahymena thermophila, and which have been described extensively by Cech and collaborators can be useful. See, for example, U.S. Pat. No. 4,987,071.
A nucleic acid construct also may include a heterologous nucleic acid that is transcribed into an interfering RNA. See, e.g., U.S. Pat. No. 6,753,139; U.S. Patent Publication 20040053876; and U.S. Patent Publication 20030175783. Methods for designing and preparing interfering RNAs to target an endogenous gene are known to those of skill in the art.
An RNA useful for inhibiting expression of an endogenous gene can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure. One strand of the stem portion of a double stranded RNA can comprise a sequence that is similar or identical to the sense coding sequence of an endogenous polypeptide, and that is from about 10 nucleotides to about 2,500 nucleotides in length. In some embodiments, the stem portion is similar or identical to UTR sequences 5′ of the coding sequence. In some embodiments, the stem portion is similar or identical to UTR sequences 3′ of the coding sequence. The length of the sequence that is similar or identical to the sense coding sequence, the 5′ UTR, or the 3′ UTR can be from 10 nucleotides to 50 nucleotides, from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides. In some embodiments the length of the sequence that is similar or identical to the sense coding sequence, the 5′ UTR, or the 3′ UTR can be from 25 nucleotides to 500 nucleotides, from 25 nucleotides to 300 nucleotides, from 25 nucleotides to 1,000 nucleotides, from 100 nucleotides to 2,000 nucleotides, from 300 nucleotides to 2,500 nucleotides, from 200 nucleotides to 500 nucleotides, from 1,000 nucleotides to 2,500 nucleotides, or from 200 nucleotides to 1,000 nucleotides. The other strand of the stem portion of a double stranded RNA can comprise an antisense sequence of an endogenous polypeptide, and can have a length that is shorter, the same as, or longer than the length of the corresponding sense sequence. The loop portion of a double stranded RNA can be from 10 nucleotides to 2,500 nucleotides in length, e.g., from 15 nucleotides to 100 nucleotides, from 20 nucleotides to 300 nucleotides, from 25 nucleotides to 400 nucleotides, or from 30 to 2,000 nucleotides in length. The loop portion of the RNA can include an intron. See, e.g., WO 98/53083; WO 99/32619; WO 98/36083; WO 99/53050; and U.S. patent publications 20040214330 and 20030180945. See also, U.S. Pat. Nos. 5,034,323; 6,452,067; 6,777,588; 6,573,099; and U.S. Pat. No. 6,326,527.
A suitable interfering RNA also can be constructed as described in Brummell et al., Plant J. 33:793-800 (2003).
If desired, a nucleic acid construct further can include a 3′ untranslated region (3′ UTR), which can increase stability of a transcribed sequence by providing for the addition of multiple adenylate ribonucleotides at the 3′ end of the transcribed mRNA sequence. A 3′ UTR can be, for example, the nopaline synthase (NOS) 3′ UTR. A nucleic acid construct also can contain inducible elements, intron sequences, enhancer sequences, insulator sequences, or targeting sequences other than those present in a regulatory region described herein. Regulatory regions and other nucleic acids can be incorporated into a nucleic acid construct using methods known in the art.
A nucleic acid construct may contain more than one regulatory region. In some embodiments, each regulatory region is operably linked to a heterologous nucleic acid. For example, a nucleic acid construct may contain two regulatory regions, each operably linked to a different heterologous nucleic acid. The two regulatory regions can be the same or different, and one or both of the regulatory regions in such a construct can be a regulatory region described herein.
Transgenic Plants and Cells
The vectors provided herein can be used to transform plant cells and generate transgenic plants. Thus, transgenic plants and plant cells containing the nucleic acids described herein also are provided, as are methods for making such transgenic plants and plant cells. A plant or plant cell can be transformed by having the construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid sequence with each cell division. Alternatively, the plant or plant cell also can be transiently transformed such that the construct is not integrated into its genome. Transiently transformed cells typically lose some or all of the introduced nucleic acid construct with each cell division, such that the introduced nucleic acid cannot be detected in daughter cells after sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
Typically, transgenic plant cells used in the methods described herein constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques.
As used herein, a transgenic plant also refers to progeny of an initial transgenic plant. Progeny includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F1, F2, F3, F4, F5, F6 and subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants, or seeds formed on FLBC1, F1BC2, F1BC3, and subsequent generation plants. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain plants and seeds homozygous for the nucleic acid construct.
Alternatively, transgenic plant cells can be grown in suspension culture, or tissue or organ culture. Solid and/or liquid tissue culture techniques can be used. When using solid medium, transgenic plant cells can be placed directly onto the medium or can be placed onto a filter film that is then placed in contact with the medium. When using liquid medium, transgenic plant cells can be placed onto a floatation device, e.g., a porous membrane that contacts the liquid medium. Solid medium typically is made from liquid medium by adding agar. For example, a solid medium can be Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g., kinetin.
Techniques for transforming a wide variety of higher plant species are known in the art. The polynucleotides and/or recombinant vectors described herein can be introduced into the genome of a plant host using any of a number of known methods, including electroporation, microinjection, and biolistic methods. Alternatively, polynucleotides or vectors can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Such Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well known in the art. Other gene transfer and transformation techniques include protoplast transformation through calcium or PEG, electroporation-mediated uptake of naked DNA, electroporation of plant tissues, viral vector-mediated transformation, and microprojectile bombardment (see, e.g., U.S. Pat. Nos. 5,538,880; 5,204,253; 5,591,616; and 6,329,571). If a cell or tissue culture is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures using techniques known to those skilled in the art.
The polynucleotides and vectors described herein can be used to transform a number of monocotyledonous plants and plant cell systems, including monocots such as banana, barley, date palm, field corn, garlic, millet, oat, oil palm, onion, pineapple, popcorn, rice, rye, sorghum, sudangrass, sugarcane, sweet corn, switchgrass, turf grasses, and wheat.
Thus, the methods and compositions described herein can be utilized with monocotyledonous plants such as those belonging to the orders Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Liliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. 
The methods and compositions can be used over a broad range of plant species, including species from the monocot genera Agrostis, Allium, Ananas, Andropogon, Asparagus, Avena, Cynodon, Elaeis, Eragrostis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pennisetum, Phleum, Phoenix, Poa, Saccharum, Secale, Sorghum, Triticum, Zoysia and Zea. 
A transformed cell, callus, tissue, or plant can be identified and isolated by selecting or screening the engineered plant material for particular traits or activities, e.g., those encoded by marker genes or antibiotic resistance genes. Such screening and selection methodologies are well known to those having ordinary skill in the art. In addition, physical and biochemical methods can be used to identify transformants. These include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, S1 RNase protection, primer-extension, quantitative real-time PCR, or reverse transcriptase PCR (RT-PCR) amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are well known. After a polynucleotide is stably incorporated into a transgenic plant, it can be introduced into other plants using, for example, standard breeding techniques.
A regulatory region disclosed herein can be used to express any of a number of heterologous nucleic acids of interest in a plant. For example, a regulatory region disclosed herein can be used to express a polypeptide or an interfering RNA. In some cases, a regulatory region disclosed herein can be used to express a cytosine DNA methyltransferase in female gametophyte cells of a plant. In some cases, a regulatory region disclosed herein can be used to express an interfering RNA that inhibits transcription of an endogenous cytosine DNA methyltransferase in female gametophyte cells of a plant. Expression of such a polypeptide or interfering RNA can affect the phenotype of a plant (e.g., a transgenic plant) when expressed in the plant, e.g., at the appropriate time(s), in the appropriate tissue(s), or at the appropriate expression levels. Thus, transgenic plants (or plant cells) can have an altered phenotype as compared to a corresponding control plant (or plant cell) that either lacks the transgene or does not express the transgene. A corresponding control plant can be a corresponding wild-type plant, a corresponding plant that is not transgenic but otherwise is of the same genetic background as the transgenic plant of interest, or a corresponding plant of the same genetic background in which expression of the transgene is suppressed, inhibited, or not induced (e.g., where expression is under the control of an inducible promoter). A plant can be said “not to express” a transgene when the plant exhibits less than 10% (e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) of the amount of the polypeptide, mRNA encoding the polypeptide, or transcript of the transgene exhibited by the plant of interest. Expression can be evaluated using methods including, for example, quantitative real-time PCR, RT-PCR, Northern blots, S1 RNase protection, primer extensions, Western blots, protein gel electrophoresis, immunoprecipitation, enzyme-linked immunoassays, microarray technology, and mass spectrometry. It should be noted that if a transgene is expressed under the control of a tissue-specific or broadly expressing promoter, expression can be evaluated in a selected tissue or in the entire plant. Similarly, if a transgene is expressed at a particular time, e.g., at a particular time during development or upon induction, expression can be evaluated selectively during a desired time period.
Use of a regulatory region provided herein to inhibit transcription of an endogenous cytosine DNA methyltransferase in female gametophyte cells of a plant can, after pollination, lead to the formation of seeds having an increased weight compared to the weight of seeds from a corresponding control plant. In some embodiments, use of the methods and compositions described herein to express a cytosine DNA methyltransferase in female gametophyte cells of a plant can, after pollination, lead to the formation of seeds having a decreased weight compared to the weight of seeds from a corresponding control plant.
Seeds of transgenic plants describe herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture. Packaging material such as paper and cloth are well known in the art. Such a bag of seed preferably has a package label accompanying the bag, e.g., a tag or label secured to the packaging material, a label printed on the packaging material, or a label inserted within the bag. The package label may indicate the seed contained therein incorporates transgenes that provide increased seed weight.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
Isolation of a 5′-Flanking Region of a Sucrose Synthase Gene
Rice gene expression profiles (Lan et al., Plant Mol. Biol. 54(4):471-87 (2004)) were analyzed to identify genes that were highly expressed in pistil five days after pollination and that were not expressed in un-pollinated pistil. Sucrose synthase 3 was identified as such a gene. The expression level of sucrose synthase 3 in pistil five days after pollination was about 28-fold higher than the expression level of sucrose synthase 3 in unpollinated pistil.
The sequence of the sucrose synthase 3 expressed sequence tag (EST) used by Lan et al. to construct a cDNA microarray was retrieved from the website of the National Center for Gene Research, Chinese Academy of Sciences (ncgr.ac.cn/EST.html). The sucrose synthase 3 EST sequence was then compared to sequences in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST), and a corresponding complementary DNA (cDNA) sequence was identified. The cDNA sequence was then compared to cDNA clones in the database of full-length cDNA clones from japonica rice (Knowledge-based Oryza Molecular biological Encyclopedia: KOME; cdna01.dna.affrc.gojp/cDNA/) to identify a predicted full-length cDNA sequence. The predicted full-length cDNA sequence was used to perform a BLAST search and retrieve the corresponding genomic DNA sequence. By aligning the genomic DNA sequence with the coding sequence, the ATG start codon and the 5′-flanking region were identified. Approximately two kilobases of the 5′-flanking region were isolated. This sequence, designated pOs530c10 (SEQ ID NO:1), was cloned into an expression vector such that it was operably linked to a Histone-Yellow Fluorescent Protein (YFP) expression cassette that had previously been tested using a 35S promoter.
Example 2
Analysis of pOs530c10 Activity
Rice of the Kitaake cultivar was transformed with an expression vector containing a Histone-YFP coding sequence under the transcriptional control of pOs530c10. Ten putative transformants were selected and screened for the presence of the transgene using the polymerase chain reaction (PCR) with gene specific primers. The transgene was present in all ten transformants.
Whole ovules were collected from four of the transgenic plants. Approximately ten pre-fertilization ovules were dissected from the pistil of each plant, flash frozen in liquid nitrogen, and pooled for RNA extraction. Post-fertilization ovules were collected separately and processed in a similar manner. RNA samples were extracted from pre- and post-fertilization ovules and analyzed using reverse transcription PCR (RT-PCR). Plants in which expression of the Histone-YFP fusion protein was detected by RT-PCR were analyzed further using confocal microscopy. At least four ovules were dissected from each plant that was positive for Histone-YFP expression according to the RT-PCR assay. Isolated post-fertilization (one to two days after pollination) and pre-fertilization ovules were analyzed for Histone-YFP expression using confocal microscopy with different light channels. Ovules were examined using a YFP channel, a chlorophyll channel, and a bright field.
Microscopy analysis indicated that pOs530c10 was active as a promoter in early stages of seed development. YFP expression was observed in five out of five transformed plants analyzed. YFP expression was observed in seeds as early as 24 hours after pollination. These results suggest that pOs530c10 is active as a promoter in early stages of endosperm development, about one to two days after pollination. In addition, results from other experiments indicated that pOs530c10 was active as a promoter in endosperm at 14 days after pollination.
It has been reported that a promoter may be transcriptionally active at least 24 hours before fluorescence from an operably linked Green Fluorescent Protein (GFP) reporter polypeptide can be visualized. Based on the microscopy data using YFP, pOs530c10 is active immediately after fertilization.
Pre-fertilization ovules arising from plants transformed with the expression vector containing the Histone-YFP coding sequence under the transcriptional control of pOs530c10 were also analyzed. Microscopy analysis carried out as described above indicated that YFP expression also occurred in the embryo sac prior to pollination.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

## Claims

1. An isolated nucleic acid comprising a regulatory region comprising the nucleotide sequence set forth in SEQ ID NO:1, wherein said regulatory region directs transcription, in a plant ovule within 24 hours post-fertilization, of an operably linked heterologous polynucleotide.

2. The nucleic acid of claim 1 wherein said regulatory region comprises an intron.

3. An isolated nucleic acid comprising a regulatory region comprising the nucleotide sequence set forth in SEQ ID NO:1, wherein said regulatory region directs transcription in an unfertilized plant ovule of an operably linked heterologous polynucleotide.

4. The nucleic acid of claim 3 wherein said regulatory region comprises an intron.

5. A nucleic acid construct comprising the nucleic acid of claim 1 operably linked to a heterologous polynucleotide.

6. The nucleic acid construct of claim 5 wherein said heterologous polynucleotide comprises a nucleic acid sequence encoding a polypeptide.

7. The nucleic acid construct of claim 5 wherein said heterologous polynucleotide is in an antisense orientation relative to said regulatory region.

8. The nucleic acid construct of claim 5 wherein said heterologous polynucleotide is transcribed into an antisense RNA capable of inhibiting expression of a DNA methyltransferase.

9. The nucleic acid construct of claim 5 wherein said heterologous polynucleotide is transcribed into an interfering RNA.

10. The nucleic acid construct of claim 5 wherein said heterologous polynucleotide is transcribed into an interfering RNA that inhibits transcription of a DNA methyltransferase.

11. A transgenic plant or plant cell wherein said plant or plant cell comprises the nucleic acid of claim 1 operably linked to a heterologous polynucleotide.

12. The transgenic plant or plant cell of claim 11 wherein said heterologous polynucleotide comprises a nucleic acid sequence encoding a polypeptide.

13. The transgenic plant or plant cell of claim 11 wherein said heterologous polynucleotide is in an antisense orientation relative to said regulatory region.

14. The transgenic plant or plant cell of claim 11 wherein said heterologous polynucleotide is transcribed into an interfering RNA.

15. A method of producing a transgenic plant, said method comprising (a) introducing into a plant cell an isolated polynucleotide comprising the nucleic acid of claim 1 operably linked to a heterologous polynucleotide, and (b) growing a plant from said plant cell.

16. The method of claim 15 wherein said heterologous polynucleotide comprises a nucleic acid sequence encoding a polypeptide.

17. The method of claim 15 wherein said heterologous polynucleotide is in an antisense orientation relative to said regulatory region.

18. The method of claim 15 wherein said heterologous polynucleotide is transcribed into an interfering RNA.