CA2325819C - Seed-coat promoters, genes and gene products - Google Patents

Abstract

The present invention is directed to isolated genomic sequences that are differentially expressed within seed-coat tissues. These DNAs are expressed within the inner integument, thick, or thin, walled parenchyma , endothelium, palisade, hourglass, or stellate parenchyma cells of the seed-coat. Furthermore, this invention relates to promoter regions obtained from genomic sequences that are differentially expressed in seed-coat tissues, and their use for directing seed-coat specific expression of genes of interest within transformed plant cells or plants. Exemplified promoters include those obtained from the differential screening of a seed-coat library and cryptic promoters obtained using T-DNA tagging using a promoterless marker gene.

Description

SEED-COAT PROMOTERS, GENES AND GENE PRODUCTS
Field of Invention This invention relates to seed-coat promoters, genes and proteins encoded by these genes. More specifically, it relates to genes and promoters that are. developmentally regulated and expressed, or activated, within tissues comprising the seed-coat, and tissues directly associated with the seed-coat, of plants. Furthermore, this invention also relates to proteins encoded by genes expressed within these tissues are their localization within, or onto, the seed-coat.

Background and Prio:r Art Bacteria from ffie genus Agrobacterium have the ability to transfer specific segments of DNA (T-DNA) to plant cells, where they stably integrate into the nuclear chromosomes. Analyses of plants harbouring the T-DNA have revealed that this genetic element may be integrated at numerous locations, and can occasionally be found within genes. One strategy which may be exploited to identify integration events within genes is to transform plant cells with specially designed T-DNA vectors which contain a reporter gene, devoid of cis-acting transcriptional and translational expression signals (i.e.
promoterless), located at the end of the T-DNA. Upon integration, the initiation codon of the promoterless gene (reporter gene) will be juxtaposed to plant sequences. The consequence of T-DNA insertion adjacent to, and downstream of, gene promoter elements may be the activation of reporter gene expression. The resulting hybrid genes, referred to as T-DNA-mediated gene fusions, consist of unknown and thus uncharacterized plant promoters residing at their natural location within the chromosome, and the coding sequence of a marker gene located on the inserted T-DNA (Fobert et al., 1991, Plant Mol. Biol. 17, 837-851).

It has generally been assumed that activation of promoterless or enhancerless marker genes result from T-DNA insertions within or immediately adjacent to genes. The recent isolation of several T-DNA insertional mutants (Koncz et al., 1992, Plant Mol. Biol. 20, 963-976; reviewed in Feldmann, 1991, Plant J. 1, 71-82; Van Lijsebettens et al., 1991, Plant Sci. 80, 27-37;
Walden et al., 1991, Plant J. 1: 281-288; Yanofsky et al., 1990, Nature 346, 35-39), shows that this is the case for at least some insertions. However, other possibilities exist. One of these is that integration of the T-DNA activates silent regulatory sequences that are not associated with genes. Lindsey et al.

(1993, Transgenic Res. 2, 33-47) referred to such sequences as "pseudo-promoters" and suggested that they may be responsible for activating marker genes in some transgenic lines.

Inactive regulatory sequences that are buried in the genome but with the capability of being fuinctional when positioned adjacent to genes have been described in a variety of organisms, where they have been called "cryptic promoters" (Al-Shawi et al., 1991, Mol. Cell. Biol. 11, 4207-4216; Fourel et al., 1992, Mol. Cell. Biol. 12, 5336-5344; Irniger et al., 1992, Nucleic Acids Res. 20, 4733-4739; Takahashi et al., 1991, Jpn J. Cancer Res. 82, 1239-1244). Cryptic promoters can be found in the introns of genes, such as those encoding for yeast actin (Irniger et al., 1992, Nucleic Acids Res. 20, 4733-4739), and a mamma;lian melanoma-associated antigen (Takahashi et al., 1991, Jpn J. Cancer Res. 82, 1239-1244). It has been suggested that the cryptic promoter of the yeast actin gene may be a relict of a promoter that was at one time active but lost function once the coding region was assimilated into the exon-intron structure of the present-day gene (Irniger et al., 1992, Nucleic Acids Res. 20, 4733-4739). A cryptic promoter has also been found in an untranslated region of the second exon of the woodchuck N-myc proto-oncogene (Fourel et al., 1992, Mol. Cell. Biol. 12, 5336-5344). This cryptic promoter is responsible for activation of a N-myc2, a functional processed gene which arose from ret:roposition of N-nryc transcript (Fourel et al., 1992, Mol.

Cell. Biol. 12, 5336-5344). These types of regulatory sequences have not yet been isolated from plants.

Weber et al. (1995, Plant Cell 7:1835-1846) disclose a cDNA sequence of a seed-coat associated invertase. However, all of the cDNA's characterized were found to be expressed in tissues other than the seed-coat, including anthers, cotyledon, stem and root. Furthermore, no promoter was isolated, characterized, or disclosed.
Described herein is the occurrence of seed-coat genes and promoters that have been obtained as a result of differential screening of seed-coat genomic libraries, or generated by tagging with a promoterless GUS (P-glucuronidase) T-DNA vector, or by identification of genes that are highly expressed in the seed-coat or associated tissues. Expression analysis of these DNA's reveal that they are spatially and developmentally regulated in seed coats. Prior to this work, promoters, as well as genes specifically expressed in seed coat tissues had riot been isolated or reported. Furthermore, proteins encoded by genes that are expressed within seed-coat, or associated with seed-coat tissues, are also disclosed.

Summary of Inventicin This invention relates to seed-coat promoters and genes. More specifically, it relates to genes and promoters that are developmentally regulated and expressed, or activated, within tissues comprising the seed-coat of plants, and tissues directly associated with the seed-coat, of plants.
Furthermore, this invention also relates to proteins encoded by genes expressed within these tissues ar.id their localization within, or onto, the seed-coat..

A transgenic tobacco plant, T218, contained a 4.7 kb EcoRI fragment containing the 2.2 kb promoterless GUS-nos gene and 2.5 kb of 5' flanking tobacco DNA. Deletion of the region approximately between 2.5 and 1.0 kb of the 5' flanking region did not alter GUS expression, as compared to the entire 4.7 kb GUS fusion. A further deletion to 0.5 kb of the 5' flanking site resulted in complete loss of GtJS activity. Thus the region between 1.0 and 0.5 of the 5' flanking region of the tobacco DNA contains the elements essential to gene activation. This region is contained within a Xbal - SnaBI restriction site fragment of the flanking tobacco DNA. Furthermore, other promoters have been identified that are differentially expressed within the seed-coats of plants, and that are capable oi= driving expression of heterologous genes that are operatively linked tliereto.

Thus according to the present invention there is provided an isolated genomic DNA molecule, differentially expressed in seed coat tissues.
Furthermore, this genomic DNA molecule is differentially expressed within the outer integument of the seed coat, the inner integument of the seed coat, the thick walled parenchyma of the seed coat, the thin walled parenchyma of the seed coat, the endothelium of the seed coat, the hourglass cells of the seed coat, the palisade of the seed coat, the stellate parenchyma of the seed coat, or the membranous endocarp, or a combination thereof.

This invention is also directed to a seed-coat promoter obtained from the genomic DNA molecule as described above. Also considered within the scope of the present invention is a cryptic seed coat promoter. Furthermore, this invention is directed to a seed coat promoter, as described above, that controls the differential expression of a gene associated therewith, within the outer integument of the seecl coat, the inner integument of the seed coat, the thick walled parenchyma of the seed coat, the thin walled parenchyma of the seed coat, the endothelium of the seed coat, the hourglass cells of the seed coat, the palisacle of the seed coat, or the stellate parenchyma the seed coat, membranous endocarp, or a combination thereof This invention also relates to an isolated genomic DNA characterized by the restriction map selected from the group consisting of Figure 12 (a), Figure 12 (b), Figure 12 (c) and Figure 12 (d).

According to the present invention, there is also provided an isolated seed-coat promoter. Furthermore, this seed coat promoter may be obtained from angiosperms. More specifically, this seed-coat promoter is obtained from the group consisting of tobacco or soybean.

This invention is also directed to a cloning vector comprising a gene encoding a protein and an isolated seed-coat promoter, wherein the gene is under the control of the seed-coat promoter. Furthermore, this invention includes a plant cell which has been transformed with such a vector.

This invention also provides for a transgenic plant containing a seed-coat promoter, operatiively linked to a gene encoding a protein.

The present invention is also directed to a seed-coat promoter comprising at least 10 contiguous nucleotides of nucleotides 1-2526 of SEQ ID
NO:7, or an analogue of the sequence defined by nucleotides 1-2526 of SEQ ID
NO:7, wherein the analogue hybridizes to a nucleic acid defined by nucleotides 1-2526 of SEQ ID NO:7 under stringent hybridization conditions and maintains seed-coat, or seed-coat associated promoter activity.

This invention also includes a seed-coat promoter comprising at least 10 contiguous nucleotides of nucleotides 1-2450 of SEQ ID NO:8, or an analogue of the nucleic acid sequence defined by nucleotides 1-2450 of SEQ ID
NO:8õ wherein the analogue hybridizes to a nucleic acid defined by nucleotides 1-2450 of SEQ ID NO:8 under stringent hybridization conditions and maintains seed-coat, or seed-coat associated promoter activity.

The present invention also is directed to a seed-coat promoter comprising at least 10 contiguous nucleotides of nucleotides 1-5514 of SEQ ID
NO:9, or an analogue of the nucleotides sequence defined by nucleotides 1-5514 of SEQ ID NO:9, wherein the analogue hybridizes to a nucleic acid defined by nucleotides 1-5514 of SEQ ID NO:9 under stringent hybridization conditions and maintains seed-coat, or seed-coat associated promoter activity.
Brief Description of the Drawings Figure 1 depicts the fluorogenic analyses of GUS expression in the plant T218. Each bar represents the average one standard deviation of three samples. Nine different tissues were analyzed: leaf (L), stem (S), root (R), anther (A), petal (P), ovary (0), sepal (Se), seeds 10 days post anthesis (Si) and seeds 20 days post-anthesis (S2). For all measurements of GUS activity, the fraction attributed to intrinsic fluorescence, as determined by analysis of untransformed tissues., is shaded black on the graph. Absence of a black area at the bottom of a histogram indicates that the relative contribution of the background fluorescence is too small to be apparent.

Figure 2 shows the cloning of the GUS fusion in plant T218 (pT218) and construction of transformation vectors. Plant DNA is indicated by the solid line and the pror.noterless GUS-nos gene is indicated by the open box.
The transcriptional start site and presumptive TATA box are located by the closed and open arrow heads respectively. DNA probes #1, 2, 3 and RNA
probe #4 are shown. The EcoRI fragment in pT218 was subcloned in the pBIN19 polylinker to create pT218-1. Fragments truncated at the Xbal SnaBI

and Xbal sites were also subcloned to create pT218-2, pT218-3 and pT218-4.

Abbreviations for the endonuclease restriction sites are as follows: EcoRI
(E), HindI1I (H), XbaI (X), SnaBI (N), SmaI (M), SstI (S).

Figure 3 shows the expression pattern of promoter fusions during seed development. GUS activity in developing seeds (4-20 days postanthesis (dpa)) of (Fig. 3a) plant T218 (*-*) and (Fig. 3b) plants transformed with vectors pT218-1 (0-0), pT21.8-2 (0-0), pT218-3 (0-0) and pT218-4 (0-A) which are illustrated in Figure 2.. The 2 day delay in the peak of GUS activity during seed development, seen with the pT218-2 transformant, likely reflects greenhouse variation conditions.

Figure 4 shows GUS activity in 12 dpa seeds of independent transformants produced with vectors pT218-1 (0), pT218-2 (0), pT218-3 (0) and pT218-4 (A). The solid markers indicate the plants shown in Figure 3 (b) and the arrows indicate the average values for plants transformed with pT218-1 or pT218-2.

Figure 5 shows the mapping of the T218 GUS fusion termini and expression of the region surrounding the insertion site in untransformed plants.
Figure 5(a) shows the mapping of the GUS mRNA termini in plant T218. The antisense RNA probe from subclone #4 (Figure 2) was used for hybridization with total RNA of tissues from untransformed plants (10 g) and from plant T218 (30 g). Arrowheads indicate the anticipated position of protected fragments if transcripts were initiated at the same sites as the T218 GUS
fusion.
Figure 5 (b) shows the RNase protection assay using the antisense (relative to the orientation of the GUS coding region) RNA probe from subclone e (Figure 7) against 30 g total RNA of tissues from untransformed plants. P, untreated RNA probe; -, control assay using the probe and tRNA only; L, leaves from untransformed plants; 8, 10, 12, seeds from untransformed plants at 8, 10, and 12 dpa, respectively; T10, seeds of plant T218 at 10 dpa; +, control hybridization against unlabelled in vitro-synthesized sense RNA from subclone c (panel a) or subclorie e (panel b). The two hybridizing bands near the top of the gel are end-labelled DNA fragment of 3313 and 1049 bp, included in all assays to monitor losses during processing. Molecular weight markers are in number of bases.

Figure 6 proviides the nucleotide sequence of pT218 (top line) (SEQ ID
NO: 1) and pIS-1 (bottom line). Sequence identity is indicated by dashed lines.
The T-DNA insertion site is indicated by a vertical line after bp 993. This site on pT218 is immediately followed by a 12 bp filler DNA, which is followed by the T-DNA. The first nine amino acids of the GUS gene and the GUS

initiation codon (*) are shown. The major and minor transcriptional start site is indicated by a large aind small arrow, respectively. The presumptive TATA
box is identified and is in boldface. Additional putative TATA and CAAT
boxes are marked with boxes. The location of direct (1-5) and indirect (6-8) repeats are indicated by arrows.

Figure 7 shows the base composition of region surrounding the T218 insertion site cloned fi-om untransformed plants. The site of T-DNA insertion in plant T218 is indicated by the vertical arrow. The position of the 2 genomic clones pIS-1 and pIS-2, and of the various RNA probes (a-e) used in RNase protection assays are indicated beneath the graph.

Figure 8 shows the Southern blot analyses of the insertion site in Nicotiana species. DNA from N. tomentosiformis (N tom), N. sylvestris (N
syl), and N. tabacum (N tab) were digested with HindIII (H), Xbal (X) and EcoRI (E) and hybridized using probe #2 (Figure 2). Lambda HindII1 markers (kb) are indicated.

Figure 9 shows the AT content of 5' non-coding regions of plant genes.
A program was writtein in PASCAL to scan GenBank release 75.0 and to calculate the AT contents of the 5' non-coding (solid bars) and the coding regions (hatched bars) of all plant genes identified as "Magnoliophyta"
(flowering plants). The region -200 to -1 and + 1 to +200 were compared.
Shorter sequences we:re also accepted if they were at least 190 bp long. The horizontal axis shows the ratio of the AT content (%). The vertical axis shows the number of the sequences having the specified AT content ratios Figure 10 shoNvs a Northern analysis of the expression of several of the genes of the present invention within developing seed coats, embryo, pod, flower, root, stem ancl leaf tissues. Figure 10 (a) shows the expression of SC4;

Figure 10 (b) shows the expression of SC20; Figure 10 (c) shows the expression of SC21, Figure 10 (d) shows the expression of Ep locus peroxidase within these tissues. Figure 10 (e) shows the expression of HP (hydrophobic protein) in leaf, flower, pod, seed coat, embryo, stem or root tissues.
Figures 10 (f) and (g), total RNA was isolated from leaf, flower, pod shells, seed coat, embryo, stem or root tissue. Equal amounts of RNA (10 g) were vacuum blotted to nylon and probed with HPS cDNA. Ribosomal RNA (rRNA), visualized by stainirig with ethidium bromide, is shown as control. Figure 10 (f), RNA from tissues at early (E) mid (M) or late (L) stages of development were compared for HP gene expression. All samples shown are from dull seeded phenotype (cv Harosoy 63). Figure 10 (g), RNA from pod tissues of dull (cv Harosoy 63) and shiny (cv. Williams 82) seeded soybeans were compared for HP gene expression.

Figure 11 shovvs the restriction maps obtained from Figure 11 (a) SC20;
Figure 11 (b) SC21; Figure 11 (c) HP (hydrophobic protein) genomic region, and Figure 11 (d) SC4. Included in Figure 11 (c) are restriction enzyme sites for BamHI, BgIII, HindIII,, and XbaI; the HP ORF; TATA box consensus signals; and the position of direct repeats of 12 bp or longer.

Figure 12 shows the morphology of the seed coat of Glycine max.
Figure 12 (a) shows the structures present at six days after anthesis (DAF);
Figure 12 (b), at 12 DAF; and Figure 12 (c) at 18 DAF.

Figure 13 shows in situ hybridization results obtained with seed coats of Glycine max at different developmental stages, and probed as follows: Figure 13 (a) seed coat at 3 days after anthesis (DAF), probed with SC4; Figure 13 (b) seed coat at 9 DAF, probed with SC20; Figure 13 (c) seed coat at 15 DAF, probed with SC21; Figure 13 (d) seed coat at 18 days after anthesis, probed with a soybean peroxidase, corresponding to the Ep locus. Figures 13 (e), (f) and (g) were obtained from cross sections of developing soybean seeds (cultivar Maple Presto, EpEp). Hybridization of 31S-probe to complementary mRNA
appears as bright white signal in these dark field microscopy images. Figure 13 (e) 6 DAF (DPA, days post anthesis), Figure 13 (f) 9 DAF, and Figure 13 (g) 12 DAF. Scale bars are 100 :m. Emb, embryo; F, funiculus; HG, hourglass cells; PC, pericarp; SC, seed coat.

Figure 14 shows light micrographs of a seed-coat obtained from soybean. Figure 14 (a) shows a plastic embedded section of the seed-coat near the hilum at 21 daf and. stained with Toluidine Blue O. Note the association of the membranous endocarp with the seed-coat pallisade. Figure 14 (b) shows a wax-embedded section of a soybean seed-coat as 12 daf probed with 35S-labelled Hydrophobic Protein (HP) antisense RNA, and counter stained with Toluidine Blue O. Note strong specific localization of the prbbe within the membranous endocarp. Pallisade (p), hourglass cells (h), counterpallisade (c), arial cells (a), stellate parenchyma (s), thin walled parenchyma (n), thickwalled parenchyma (k), pod parenchyma (d), and membranous endocarp >. Figures 14 (c) and (d). show localization of HP mRNA transcript by in situ hybridization. Cross sections of soybean pods containing immature seeds (dull phenotype, HPS (+), cv Maple Presto). Hybridization of'SS labelled HP probe to complementary mRNA appears as bright white signal in these dark field microscopy images. E, embryo; Ep, inner epidermal layer of endocarp; Ex, exocarp; F, funiculiis; M, mesocarp; Sc, seed coat; Sm, sclerenchyma layer of endocarp. Bar = 100 m. Figure 14 (c), Expression at 6 DPA (days post anthesis). Figure 14 (d), Expression 12 DPA.

Figure 15 shows the Soybean hydrophobic protein (HP) cDNA and deduced amino acid sequences. Figure 15 (a), the cDNA and amino acid sequence of HP. The pre-protein signal sequence is underlined. Figure 15 (b) shows the deduced amino acid sequence of HP pre-protein. Alternate N-terminal residues are boxed, as determined by peptide microsequence analysis.
Figure 15 (c) shows a Kyle-Doolittle hydrophilicity plot of HP (Lasergene). In this plot, positive values indicate greater hydrophilic character. Also represented are the ttiree domains of the HP pre-protein and the length of the mature peptide. Figu;re 15 (d) shows a schematic comparison of HP domain structure to three other plant proteins. Bold numbers indicate the length in amino acid residues for the domain segments. The pattern of spacing between the eight cysteine residues within the hydrophobic domains is also shown below each protein. Sequenc:es for tobacco N16 polypeptide (D86629), maize proline rich hydrophobic protein (PRHP) (X60432), and Arabidopsis lipid transfer protein 1(LTP1) (M80567) were retrieved from GenBank.

Figure 16 shows scanning electron micrographs of representative 'Dull' and 'Shiny' seeded soybean cultivars. Scale bars are included in the figures.
The lowest magnification (x18), Figure 16 (a) is a view of the entire seed. The large oval shaped scar on the seed surface is the hilum, corresponding to the point of detachment of the mati.ue seed from the funiculus. Figure 16 (b), x100, and Figure 16 (c) x500, are focused outside of hilum region.

Figure 17 shows a silver stained SDS-PAGE analysis of protein extracts from seed tissues and surface. Lanes marked 'M' indicate protein standards, and their corresponding mass in kilodaltons is also provided. Figure 17 (a), Soluble protein extracts from the embryo, seed coat, and seed surface of a dull phenotype (cv Harosoy 63). Each sample at approximately 1 g of total protein. Figure 17 (b), Seed surface protein extracts of a dull phenotype (cv Harosoy 63) with different concentrations of dithiothreitol (DTT) present in the sample loading buffer=, as indicated at the top of each lane. Figure 17 (c), Seed surface protein extracts of dull (D), shiny (S), and bloom (B).

Figure 18 shows restriction fragment length polymorphisms between dull and shiny phenotypes. Genomic DNA from dull (cv Harosoy 63) and shiny (cv Williams 82) soybeans with abundant (+) or trace (-) amounts of HPS on the seed surface, was digested with restriction enzymes, electrophoretically separated, blotted, and hybridized to HP cDNA probe. The size of hybridizing fragments was estimated by comparison with standards and is shown on the left.
Figure 19 shows the nucleotide sequence and deduced amino acid sequence of SC4 cDNA, and the sequence comparisons between SC4 protein and BURP proteins. :Figure 19 (a), 5' and 3' untranslated sequences are in lowercase lettering. The stop codon is shown with an asterisk and two polyadenylation signals are underlined. Two copies of a ten amino acid repeat is also underlined. Concensus sequnences for N-glycosylation (NNT; NSSN;
and NGTV) are also underlined. Figure 19 (b), amino acid alignment of the carboxyl terminus of the SC4 protein with the BURP domain (A) and the amino terminus of the SC4 protein with the conserved segments of the second domain (B) of several BURP domain proteins. Pg1p is not included in panel B as the second domain of this protein does not contain a conserved segment. Gaps were introduced to optimize the alignment. Conserved amino acids are shown in bold face. Amino acids of each protease are numbered from the precursor sequence.
Figure 19 (c) shows the structual similarity between SC4 protein and the BURP
domain proteins.

Figure 20 shows Northern blot analysis of SC4 and SC20 mRNA

accumulation in seed coat embryo and pod organs of soybean. 10 /.zg total RNA
from seed coat, embryo and pod organs between 6-24 days past anthesis were hybridized with radiolabled probes. For day 6, total RNA was prepared from whole seeds. Each blot was hybridized with a SC4 cDNA probe, Figure 20 (a), a SC20 cDNA probe Figure 20 (b), and an 18S rRNA probe Figure (c).

Figure 21, shows the localization of SC4 mRNA in Seed coat organs of soybean by in situ hybridization. Transections of seed coats at 3 days past anthesis (dpa) and 6 cipa. Hybridization to Antisense, Figure 21 (a), and sense, Figure 21 (b) SC4 labelled RNA probes. Abbreviations, II - inner integument, 01 outer integument, P pod. Bar equals 100Acm.

Figure 22, shows Southern blot analysis of SC4. Figure 22 (a) shows Southern analysis of the gene family composition of sc4 in soybean. Figure 22 (b) shows Southern analysis of sc4 in diverse plant species. Hybridized filter was washed under conditions of low stringency, twice at 52 C for 15 min in 2x SSC, 0.1 %SSC, 0.1 % SDS and once at 52 C for 30 min in 0. lx SSC, 0.1 %
SDS.
Figure 23 reveals the characterization of sc2O and the SC20 protein.
Figure 23 (a) is a restriction map of sc2O. Figure 23 (b) shows the nucleotide sequence and deduceci amino acid sequence of sc20 cDNA. The stop codon is shown with an asterisk and the polyadenylation signal is underlined. The concensus sequences for N-glycosylation are also underlined. Figure 23 (c) shows the hydrophobic plot of SC20 protein, where hydrophobic regions possess a positive sign, and hydrophilic regions possess a negative sign. In Figure 23 (d), alignment of SC20 protein with other subtilases is shown. D, H
and S regions represent amino acid sequences around the catalytic aspartate, histidine and serine residues of the subtilases. The catalytic residues are labelled with an asterisk. N region represents amino acid sequence around the conserved asparagine residue, of subtilases. # indicates the conserved asparagine. AF70, cucumisin, P69B, Ag12, subtilisin BPN', kex2, furin are from Picea abies, Cucumis melo L., Lycopersicon esculentum, Alnus glutinosa, Bacillus subtilis, Saccharomyces cerevisiae, and Homo sapiens respectively.

Conserved amino acid,s are shown in boldface. Amino acids of each protease are numbered from the precursor sequence.

Figure 24 shows localization of SC20 mRNA in seed coats of soybean by in situ hybridization. Transection of seed coats at 12 days past anthesis hybridized to Antisense, Fivure 24 (a), and Sense, figure 24 (b), SC20 radiolabelled RNA probes. Abbreviations: H Hilumi, II inner integument, 01 outer integument, * thick walled parenchyma, ** thin walled parenchyma. Bar equals 100,um.

Figure 25 shows Southern blot analysis of sc2O. Figure 25 (a) and (b), Southern analysis of the gene family composition of sc2O in soybean under conditions of medium stringency (twice at 52 C for 15 min in 2x SSC, 0.1 %SDS, and once at 52 C for 30 min in 0. lx SSC, 0.1 % SDS), Figure 25 (a), and high stringency (once at 62 C for 30 min in 0. lx SSC, 0.1 % SDS) Figure 25 (b). Figure 25 (c) shows Southern analysis of sc4 in diverse plant species. genomic DNA was digested with EcoRI. Hybridization used a radiolabelled SC20 cDNA probe. The filter was washed under conditions of medium stringency, tvvice at 52 C for 15 min in 2x SSC, 0.1 % SDS and once at 52 C for 30 min in 0.lx SSC, 0.1 % SDS.

Detailed Description of the Preferred Embodiments T-DNA tagging with a promoterless (3-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity only in developing seed coats. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proxirr.ial to the GUS gene. Deletion analyses localized the cryptic promoter to an. approximately 0.5 kb region between a Xbal and a SnaBI restriction endonuclease site of the 5' flanking tobacco DNA. This region spans from nucleotide 1 to nucleotide 467 of SEQ ID NO: 1.

Other work, based on the differential screening of seed coat libraries has led to the identificatioii of several other genes that are differentially expressed within, or tissues that are directly associated with, the seed coat of plants.
These genes include SC4 (SEQ ID NO's: 3 and 9, cDNA and genomic sequences, respectively), SC20 (SEQ ID NO's: 4 and 8, cDNA and genomic sequences respectively), SC21 (SEQ ID NO: 5, cDNA sequence), and their associated promoters (see SEQ ID NO 9 and 8 for promoters of SC 4 and SC20, respectively; also Figure 12). Furthermore, the isolation of genes encoding highly expressed seed coat proteins led to the identification of a seed-coat specific peroxidase from the Ep locus and associated promoter (Ep genomic sequence, SEQ ID NO:2), as well as a gene encoding a seed-coat localized hydrophobic protein (HP, cDNA sequence SEQ ID NO:6) and associated promoter (within genomic sequence, SEQ ID NO:7, also see Figure 11 (c)). Thus, the present invention includes promoters, genes and proteins isolated from several plant species, that are preferentially expressed, or specific to seed-coat tissues, as well as promoters, genes and associated proteins obtained from tissues associated with the seed-coat.

The term cryptic promoter means a promoter that is not associated with a gene and thus does not control expression in its native location. These inactive regulatory sequences are buried in the genome but are capable of being functional when positioned adjacent to a gene.

The DNA sequence of an aspect of the present invention includes the DNA sequence of SEQ! ID NO: 1, the promoter region within SEQ ID NO: 1 (for example from nucleotide 1 to 476), and analogues thereof. Similarly, another aspect of this invention includes a DNA sequence of SEQ ID NO:2, the promoter region of this sequence (nucleotides 1-1532), and analogues thereof.
Another aspect of this invention includes a DNA sequence of SEQ ID NO:7, the promoter region (nucleotides 1-2526), and analogues thereof, a DNA
sequence of SEQ ID NO 8, the promoter region (nucleotides 1-2450) and analogues thereof, and a DNA sequence of SEQ ID NO:9, the promoter region (nucleotides 1-5514) and analogues thereof.

Analogues include those DNA sequences which hybridize under stringent hybridization conditions (see Maniatis et al., in Molecular Cloning (A
Laboratory Manual), Cold Spring Harbor Laboratory, 1982, p. 387-389) to the DNA sequence of SEQ ID NO: 1, 2, 7, 8 or 9 provided that said sequences maintain the seed coat, or seed-coat associated promoter activity. An example of one such stringent :hybridization conditions may be hybridization at 4XSSC
at 65 C, followed by washing in 0.1XSSC at 65 C for an hour, or at 62 C for 30 min in 0. lx SSC, 0.1 % SDS. Alternatively an exemplary stringent hybridization condition could be in 50% formamide, 4XSSC at 42 C. With the use of Digoxigenin labelled probes, stringent hybridization may include 65 C

in 0.25 M Na2HPO4 (pH 7.2), 20% SDS, 1 mM EDTA and 0.5% blocking reagent (Boehringer Mannheim) followed by washing at 22 C in 20 mM
Na2HPOa (pH 7.2), 1% SDS and 1 mM EDTA and washes in the same solution at 68 C. Analogues also include those DNA sequences which hybridize to the sequence of SEQ ID NO: 1, 2, 7, 8 or 9 under relaxed hybridization conditions provided that said sequences maintain the seed-coat promoter activity.
Examples of such non-hybridization conditions includes hybridization at 4XSSC
at 50 C or with 30-40% formamide at 42 C. Alternate conditions of medium stringency include washing the filter twice at 52 C for 15 min in 2x SSC, 0.1 %
SDS and once at 52 C: for 30 min in 0. lx SSC, 0.1 % SDS.

Furthermore, another aspect of this invention is directed to the identification and characterization of seed-coat promoters (see Figure 11) and their corresponding genes of cDNA's (SEQ ID NO's: 3-6), as characterized by Southern or in situ hybridization analysis of the expression patterns of genes expressed under the control of seed-coat promoters within developing seed coats (Figures 13 and 14). Furthermore, restriction maps of the promoter and downstream regions of several seed-coat genomic clones is presented (Figure 11).

Proteins of interest may be expressed in seed coat tissues by placing a gene capable of expressing the protein of interest under the control of the DNA
promoters of this invention. Genes of interest include but are not restricted to herbicide resistant genes, genes encoding viral coat proteins, or genes encoding proteins conferring biological control of pest or pathogens such as an insecticidal protein for example B. thuringiensis toxin. Other genes include those capable of modifying the production of proteins that alter the taste of the seed and/or that affecit the nutritive value of the seed.

By "seed-coat'"' it is meant tissues typically found within, and associated with, the seed-coat of developing or mature angiosperm seeds. With out wishing to limit the types of tissues found within a seed-coat, this region of the seed typically comprises a range of cell types including, and bounded by, an inner endothelium, and an outer epidermis or palisade cell layer. Within these inner and out cell layers, there may be found parenchyma-like cells, for example thin, or thick walled parenchyma, or stellate parenchyma, vascular tissue, hypodermis, hour-glass cells (osteosclereids), and one or more integuments, including the inner and outer integuments. However, it is to be understood that other cell types found within the region between the inner endothelium and outer epidermis may also be considered to comprise the seed-coat, for example, but not limited to the hilum, and funiculus comprising arial cells. Furthermore, it is to be understood that "seed-coat" also refers to tissues associated with, or adhering to the seed coat, for example the membranous endocarp (of the inner ovary wall), as this cell type adheres to the seed-coat and remains in associationi with the seed coat (see for example Figure 15 (a), (b), and Figure 16). Therefore, as used herein, tissues that are associated with, or that adhere to, the seed-coat are referred to as "seed-coat associated tissues", or "tissues associated with the seed-coat" It is contemplated that other cell types may also associate with seed-coat tissues in addition to those disclosed above and that the tissues identified above should not be considered limiting in any manner.
By "seed-coat gene" it is meant a gene that is differentially expressed within the seed-coat as detected under stringent conditions (as defined above).
Examples of such a gene include, but are not limited to SC4, SC 20, SC 21, or Ep locus peroxidase. However, the product of the gene may be exported from the cell to an exterior location of a seed-coat cell, including the surface of the seed-coat itself. An e:xample, which is not to be considered limiting in any manner, of a gene product that is synthesized in within a seed-coat associated cell, and that is localized onto the surface of the seed coat, is the hydrophobic protein (HP; see Figures 14 (a) (b) and Figure 16).
A "seed-coat promoter" is a promoter that is differentially active within cells of the seed-coat. When operably linked with a gene under its control, a seed-coat promoter confers expression to a gene within the seed-coat, which can be detected under stringent conditions (as defined above). Seed coat associated promoter refers to a promoter that is active in tissue associated with the seed coat as defined above.

By "differentially expressed" it is meant the expression of a gene under the control of a promeRer, as detected by standard means, within a specified tissue or organ. Such standard means for detecting expression include, but are not limited to, Northeirns, or in situ hybridizations and the like performed under stringent conditions, or reporter gene expression. For example, a gene that is differentially expresseci in seed-coat tissues is detectable within seed-coat tissues, and displays little or no expression in other tissues such as root, stem.

By "preferentially expressed" it is meant the expression of a gene under the control of a promoter, as detected by standard means, wherein the majority of expression is detected within a specified tissue or organ. Such standard means for detecting expression include, but are not limited to, Northerns, or in situ hybridizations and the like performed under stringent conditions, or the expression of reporter genes. For example, a gene that is preferentially expressed in seed-coat tissues is detectable within seed-coat tissues, but may exhibit some expression within other tissues such as root, stem.

By "seed-coat localized" or "localized onto the seed-coat" it is meant a gene product that, as a result of some property of the amino acid sequence of the gene product, is targeted within, or onto seed-coat tissues, respectively.
Properties of an amino acid sequence that may direct the targeting of a protein within or onto seed-coat tissues include, but are not limited to, signal sequences that direct intracellular, and extracellular localization of a protein, and also hydrophobic regions within a protein, that results in localization of the protein onto the seed coat. An example, which is not to be considered limiting, of a protein that is localized onto the seed-coat, is the hydrophobic protein (HP).
HP is localized on the outside of the seed-coat following its synthesis within the membranous endocarp, and appears to be involved with the adherence of the, endocarp to the seed-coat (see Figure 16).
Development of the Soybean coat The seed coat of Glycine max undergoes dramatic changes in the first two and a half weeks after anthesis (flowering). At six days after anthesis (DAF; see Figure 12 (a)), the seed coat has a distinct epidermis (10), consisting of thin-walled cuboidal cells; an outer integument (20), consisting of up to a dozen layers of thin-walled parenchyma, and containing vascular tissue (recurrent vascular bundle) in the subhilar region; an inner integument (30), consisting of up to 6 layers of deeply-staining thick-walled parenchyma; and an endothelium (40), consisting of thin-walled cuboidal cells.

At 12 days after anthesis (Figure 12 (b)), there is a distinct hypodermis (15) of thin-walled cuboidal cells directly beneath the epidermis (10); the outer integument (20) has differentiated into an upper layer of thin-walled parenchyma (25), and a lower layer of thick-walled parenchyma (27); the inner integument (30), while still having very thick, deeply staining cell walls, has become stretched, and is compressed to about 3 cells thick; the endothelium (40) is also retained. Also evident in Figure 12 (b) is the endosperm (50), and the developing cotyledons (60).

By 18 days after anthesis (Figure 12 (c)), the epidermal cells have divided and elongated to form thick-walled macrosciereids, forming a palisade layer (13). The hypodermis has differentiated into osteosclereids: thick walled cells with a characteristic I-shape (hourglass cells; 17). A prominent vascular region (70) has developed in the thin-walled parenchyma (25) of the outer integument which stops before reaching the region of the seed opposite the hilum; the thick-walled parenchyma (27) is retained. The inner integument (30) has become completely stretched and crushed, leaving a single, deeply staining wall layer directly above the endothelium (40). The hilum region contains a well-developed counter-palisade (80), and a tracheid bar (90). The seed coat remains attached to the funiculus (100). The sub-hilar region contains well-develope:d vascular tissue (recurrent vascular bundles; 70) and stellate parenchyma (110).

At maturity, the seed coat consists of the palisade layer (13), hourglass cells (17), a partially crushed layer of parenchyma (what remains of the outer integument), and an eridothelium (40). The remnant of the inner integument (30) is often not distinguishable. The tissues of the hilum although compressed, are retained.

The stages of seed-coat development are also identified in Tables 1 and 2.

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=o vC ... v o tn SUBSTITUTE SHEET (RULE 26) Early development of the tobacco seed coat .At 6 days after anthesis, the tobacco seed coat consists of an epidermis of very large, thin walled cells; a layer of parenchyma cells up to 6 cells thick; and an endothelium of thin-=walled, cuboidal cells. By 10 days after anthesis, the inner walls of the epidermis have thickened significantly, with 2-3 layers discernible; the thin-walled parenchyma has become reduced to 3-4 cells thick due to stretching of the layer as the seed expands; and the endothelial cells have become thinner and elongated. At 22 days after anthesis, the epidermal cells have stretched and elonigated to accommodate the expanding seed, and the parenchyma and endottielium have elongated and fused into a crushed layer with few individual cells distinguishable.

Seed-coat crvntic nromote There are several lines of evidence that suggest that the seed-coat expression of GUS activity in the plant T218 is regulated by a cryptic promoter.
The region surrounding the promoter and transcriptional start site for the GUS
gene are not transcribed in untransformed plants. Transcription was only observed in plant T218 when T-DNA was inserted in cis. DNA sequence analysis did not uncover a long open reading frame within the 3.3 kb region cloned. Moreover, the region is very AT rich and predicted to be noncoding (data not shown) by the: Fickett algorithm (Fickett, 1982, Nucleic Acids Res.
10, 5303-5318) as implemented in DNASIS 7.0 (Hitachi). Southern blots revealed that the insertion site is within the N. tomentosiformis genome and is not conserved among related species as would be expected for a region with an important gene.

As this is the first report of a cryptic promoter specific to seed-coat tissues in plants, it is irnpossible to estimate the degree to which cryptic promoters may contribute to the high frequencies of promoterless marker gene activation in plants. It is interesting to note that transcriptional GUS
fusions in Arabidopsis occur at niuch greater frequencies (54%) than translational fusions (1.6%, Kertbundit et al., 1991, Proc. Nati. Acad. Sci. USA 88, 5212-5216).
The possibility that cryptic promoters may account for some fusions was recognized by Lindsey et al. (1993, Transgenic Res. 2, 33-47).

The results disclosed herewith confirms others (Gheysen et al., 1987, Proc. Natl. Acad. Sci. USA 84, 6169-6173 and 1991, Genes Dev. 5, 287-297) that T-DNA may insert into A-T rich regions as do plant transposable elements (Capel et al., 1993, Nucleic Acids Res. 21, 2369-2373). We illustrate that promoters of plant genes are also A-T rich raising speculation that gene insertions into these regions could facilitate the rapid acquisition of new regulatory elements during gene evolution.

The insertion of functional genes into the nuclear genome and acquisition of new regulatory sequences has already played a major role in the diversification of certain genes and the endosymbiosis of organelles. In plants, most organellar proteins are nuclear encoded due to the ongoing transfer of their genes into the nucleus (Palmer, 1991õ In Bogorad L and Vasil IK (eds) The Molecular Biology of Plastids, Academic Press, San Diego, pp 5-53). Recently, it has been shown that the cox 2 gene of cowpea (Nugent and Palmer, 1991, Cell 66, 473-481) and soybean (Covello and Gray, 1992, EMBO J. 11, 3815-3820) were transferred from mitochondria to r.iucleus without promoters by RNA intermediates. The ' results disclosed herewith, with T-DNA-mediated gene fusions reveal the facility with which promoters can be acquired by incoming genes. The presence of cryptic promoters and diverse regulatory elements in the intergenic regions may insure that genes rapid:ly achieve the features needed to meet the demands of complex multicellular organisms.

Other seed-coat. and seed-coat-associated promoters Transcripts encoding seed coat specific genes were isolated from seed-coat cDNA libraries. These cDNA transcripts were then used to identify the corresponding structural genes and associated promoters from genomic DNA
libraries. These promoters, genes and gene products have been isolated and characterized Examples of such genes include, but are not limited to, SC4, SC20, SC21, a peroxidase cloned from the Ep locus, and HP (hydrophobic protein). It is to be understood that this seed-coat library comprises tissues typically found within the seed-coat and tissues adhering to the seed-coat such as the membranous endocarp and cells found in the funicular region such as arial cells (see above for full defuzition of seed-coat).

Ep locus Peroxidase The amount of peroxidase activity present in seed coats may vary substantially among different cultivars. The presence of a single dominant gene Ep causes a high seed coat peroxidase phenotype. Homozygous recessive epep plants are - 100-fold lower in seed coat peroxidase activity which results from a reduction in the amount of peroxidase enzyme present, primarily in the hourglass cells of the subepidermis (Gijzen et al., 1993). In plants carrying the Ep gene, peroxidase is heavily concentrated in the hourglass cells (osteosclereids;
which forni a highly differentiated cell layer with thick, elongated secondary walls and large intercellular spaces).
A seed-coat peroxidase gene, corresponding to the Ep locus, was obtained from a soybean seed-coat library. The genomic DNA sequence comprises four exons spanning bp 1533-1752 (exon I), 2383 -2574 (exon 2), 3605-3769 (exon 3) and 4033-4516 (exon 4) and three introns comprising 1752-2382 (intron 1), 2575-3604 (intron 2) and 3770-4516 (intron 3), of SEQ ID NO:2. Features of the upstream regulatory region of the genomic DNA include a TATA box centred on bp 1487; a cap signa132 bp down stream centred on bp 1520. Also noted within the genomic sequence are three polyadenylation signals centred on bp 4520, 4598, 4663 and a polyadenylation site at bp 4700. The promoter region of the genomic sequence comprises nucleotides 1-1532 of SEQ ID NO:2.

Expression of Ep is first detected at 6 DPA in the thin-walled parenchyma of the outer integument, adjacent to the thick-walled parenchyma, and flanking the hilum region. By 9 DPA a thin band of expression extends around the entire seed coat, at the junction of the thin-and thick-walled parenchyma. Expression shifts to the hourglass cells as they begin to develop, at 12 DPA (see Figures (e), (f) and (g)).

Expression of a gene under the control of the Ep (peroxidase) promoter (nucleotides 1-1532 of SEQ ID NO:2, is observed within the seed-coat from 6 to 18 days after anthesis is shown in Figures 13 (d) to (g).
Hydrophobic Protein (HP) Soybean HP is an 8.3 kD protein consisting of 80 amino acids rich in hydrophobic residues and entirely lacking methionine, phenylalanine, tryptophan, lysine and histidine residues (see Figure 15). The amino acid sequence shows no significant homology to any known proteins (Odani et al., 1987, Eur J Biochem 162, 485-491).

To determine the composition of proteins deposited on the soybean seed surface, seeds were washed with a detergent-buffer solution and the extracted peptides were separated by SDS-PAGE. Protein extracts from the seed coat and embryo were also prepared for comparison. These results are shown in Figure 17 (a). The embryo and seed coat extracts contained many proteins covering a wide molecular mass range. In contrast, extracts from the seed surface were dominated by a few low molecular mass proteins. Figure 17 (b) demonstrates that HP
extraction and separation by SDS-PAGE is dependant on dithiothreitol (DTT).
Even though HP is an abundant seed constituent and a potent allergen, there have been no studies on the expression or localization of the protein or any description of the corresponding gene. This is the first report on the isolation and characterization of HP cDNA (SEQ ID NO:6) and the corresponding genomic clone (SEQ ID NO:7), the pattern of gene expression (Figure 10 (e)), and the localization of the protein (Figure 14 (b)) and its effect on seed luster (Figure 16). Figure 14 (c) shows that the presence of surface protein is related to the luster, or light reflective properties of the seed surface. Surface extracts from shiny seeded phenotypes usually contained far less protein than dull seeded extracts. Moreover, there were large differences in the amount of protein present on the seed surfaces of the two bloom phenotypes examined.

These results also show that the outermost components of the seed coat are in fact derived from the inner layer of the pod wall (see Figure 14 (a)).

The cDNA and genomic copies of the seed-coat associated HP gene were obtained from lambda libraries prepared from cultivar Harosoy 63. The genomic DNA sequence comprises a promoter region from 1-2525 of SEQ ID NO:7.
Within this promoter region are located clustered direct repeats (between 1-586;
see also Figure 11 (c). and a TATA box located at position 2442-2447. The ORF for HP is between 2526-2882, with the translational start site at 2526, followed by a signal sequence from 2526-2642, and the mature protein from 2643-2882. Also noted within the genomic sequence are six polyadenylation signals and a polyadenylation site at bp 3193.

Developmental and tissue specific expression patterns for the HP gene were determined by RNA blot analysis and in situ hybridization. Representative RNA blots, probed with HP cDNA, are shown in Figures 10 (e) and (f). These results show that HP is highly expressed in the pod during the mid to late stages of seed development. Hybridization signals were also observed in seed coat RNA
samples. No expression was evident in the flower, leaf, embryo, stem, or root.
We also compared HP transcript levels of two different seed luster phenotypes that differed in the amount of HP present on their seed surfaces. Figure 10 (g) shows that HP mRNA levels are several fold greater in dull seeded plants that accumulate large amounts of HP on the seed surface when compared to shiny seeded plants that have only trace amount of HP on the seed surface. Faint signals, corresponding to low HP transcript levels, were detectable in shiny seeded phenotypes after prolonged exposure times (not shown).

Localization of HP gene expression by in situ hybridization is shown in Figures 14 (b), (c) and (d). At six days post anthesis (DPA) expression of HP
is limited to the membrarious inner layer of the pericarp. By 12 DPA expression is very strong and the inrier epidermis is showing signs of becoming detached from the rest of the pericarp and, in places, is adhering to the seed surface.
Tissue sections from this stage of development also showed strong hybridization signals in the sclerenchyma, itidicating that HP expression occurs throughout the endocarp. Portions of membranous endocarp adhere to the seed during the course of development (see Figures 14 (a) and 16) and thus constitute a newly identified component of the seed coat of mature, fully developed soybeans. The deposition of this material alters the physical properties and the composition of the seed surface, as shown by SDS-PAGE analysis (Figures 17 (a), (b) and (c)) and by scanning electron microscopy (Figure 16). A comparison of dull- and shiny-seeded cultivars reveals that the HP gene controls this phenotypic trait in soybeans.

In Figure 14 (b) can be seen the expression of a gene under the control of the HP promoter, The promoter (nucleotides 1-2526 of SEQ ID NO:7) is active within the membranous endocarp associated with the outer seed-coat.

SC 4. 20 and 21 Genes expressin,g specifically in seed coat tissue were isolated from a seed coat cDNA library obtained from seed coats in later stages of development.

The deduced protein sequence from the SC4 cDNA (Figure 19 (a); SEQ
ID NO:3) consists of 289 amino acids and has a molecular mass of 31.9 kDa and a predicted pI of 7.95. 'Three puatative glycosylation sites are present at positions 92, 128 and 269. The putative polypeptide encoded by SC4 exhibits similarity with proteins that comprises a BURP domain (see Figure 19 (b)). The BURP
domain is a long carboxyl terminal domain containing a number of highly conserved amino acids (Hattori J. et al., 1998. Mol. Gen. Genet. 259: 424-428).
The genomic sequence of sc4 is provided in SEQ ID NO:9 (also see Restriction map Figure 11 (d)) and comprises a promoter from nucleotides 1-5514 of SEQ
ID NO:9.

The expression of a gene under control of the SC4 promoter (nucleotides 1-5514 of SEQ ID NO::9) within soybean seed coat at 3 days after anthesis is shown in Figure 13 (a). The activity of the promoter is localized within the inner integument (arrow). Other areas of brightness in this figure include the recurrent vascular bundles in the funiculus, and the trichomes of the pod (the bright areas are due to the birefringence of crystalline areas in the cell walls, and are also present in the negative control; data not shown).

RNA samples from seed coat, embryo, stem, root, leaf, pod and flower were hybridized with a radiolabelled SC4 cDNA probe (Figure 10 (a)) to determine organ specificity of the expression of SC4. The sc4 transcript was only expressed in the seed coat organ. It was estimated that the size sc4 mRNA
was 1.2 kb (data not shown).

Northern blot arialysis was carried out to determine the temporal expression pattern of sc4. RNA from seed coat, embryo and pod organs between 6-24 dpa were hybridized with a radiolabelled SC4 cDNA probe. No gene expression was observed in any of the embryo development stages examined (Figure 20 (a)). sc4 expression was apparent in the seed by 6 dpa. After 6 dpa the expression of sc4 in the seed coat increased - 4-fold to its maximum detected level between 9-12 dpa. By 15 dpa sc4 expression had decreased by - 2.5-fold dpa and continued to decline to just detectable levels by 18 dpa (Fig. 3.7).

Expression of sc4 could. only be detected in the seed coat at 21-24 dpa when the filter was over-exposed. Gene expression of sc4 in the pod was detected from 21 dpa only after over-exposure of the filter (data not shown).

To analyse the distribution of sc4 expression with respect to cell differentiation during seed coat development in situ hybridization was performed on seed sections from 3-24 dpa seeds. sc4 was expressed throughout the inner integument of the seed coat at 3 dpa (Figures 13 (a) and 21). By 6 dpa the expression pattern of se4 had changed, and was localized to the outer integument parenchyma but not to the vascular tissue embedded within this layer. sc4 expression in the outer integument was maintained until 18 dpa after which time no further expression was detected (see Table 4 in Examples). In concurrence with northern blot analysis, the in situ hybridization results revealed that sc4 expression increased to a maximum between 9-12 dpa and decreased thereafter (Table 4, in Examples). In addition, expression of sc4 was not observed in the embryo of seed at 3-6 dpa.

Expression of a gene under the control of the SC4 promoter (1-5514 of SEQ ID NO:9) is seen in Figures 13 (a) and 21.

Southern blot analysis was carried out to examine the gene family composition of sc4. Soybean genomic DNA was cleaved with Eco RI, Hind III
and Xba I. which do not: have recognition sites in the SC4c cDNA sequence.
Under conditions of low to high stringency (i.e., from 40-10 C below Tm of the probe ) the SC4 cDNA probe hybridized to a single band (Figure 22) and therefore sc4 appears to be a single gene.
Southern blot analysis was also performed to determine the occurrence of sc4 within the following plant species: pea (Pisum sativum), canola (Brassica napus), oat (Avena sativa), onion (Allium cepa), pepper (Capsicum annuum), Mimosa sp. (Mimosa pudica), black spruce (Picea mariana (Mill) B.S.P.), birch (Betula pendula Roth). The genomic DNA was digested with Eco RI. Under all stringency conditions it was observed that the radiolabelled SC4 cDNA probe hybridized to only soybean genomic DNA (Figure 22 (b)). Further analysis of more related species to soybean need to be carried out.

The open reading frame of SC20 encodes a putative protein of 770 amino acid residues with a calculated molecular mass of 82.688 kDa and a predicted pI
of 6.93. The predicted protein has ten potential N-glycosylation sites (Figure (b)). The hydropathy profile (Figure 23 (c)) of SC20 protein revealed that the first 23 amino acids constitute a hydrophobic region typical of an eukaryotic signal peptide. From noirthern blot analysis, the SC20 cDNA clone hybridizes to a-- 2.5 kb transcript.

The genomic sc20 clone is 7235 bp in length (see Figure 23 (a) for restriction map, and SEQ ID NO:8). Alignment of sc20 genomic and SC20 cDNA sequences revealed that sc2O contained eight introns of 94 bp, 101 bp, 185 bp, 80 bp, 154 bp, 112 bp, 110 bp and 93 bp respectively (Figure 23 (a)).
A
search of the 5' upstreaun region of sc20 revealed three potential transcription start sites at positions 1085, 1156 and 2272. The promoter region of sc2O
spans nucleotides 1-2450 of SEQ ID NO:8.

Sequence comparisons (Figure 23 (d)) revealed that the putative polypeptide encoded by SC20 was similar to plant proteins belonging to the Pyrolysin family in the clan of serine proteases known as the subtilases (Barrett A.J. and Rawlings N.D., 1995. Arch. Biochem. Biophys. 318:247-250; Siezen, R. J. and Leunissen, J. A. M. 1997. Protein Sci. 6: 501-523.). The SC20 protein comprises 3 domains: a signal peptide of 23 residues followed by a prosequence of 93 resiclues and a mature domain of 654 residues. The predicted mature domain of SC20 has a calculated molecular weight of 69.918 kDa and an isoelectric point of 6.34.

Northern blot analysis was carried out to determine specificity of sc20 expression in various soybean organs i.e., seed coat, embryo, stem, root, leaf, pod and flower (Figure 10 (b)). sc20 has seed coat-specific expression as its mRNA was detected ortly in the seed coat organ. The sc20 transcript was determined to be approximately 2.5 kb (data not shown). Even after prolonged exposure of the filter, no sc20 transcripts was detected in any of the other plant organs.

Northern blot analysis was performed to determine the temporal gene expression pattern of sc:20 in seed coat, embryo and pod organs of soybean.
Total RNA prepared from organs between 6- 24 dpa were probed with a radiolabelled SC20 cDNA probe. sc20 expression was detected at 9 dpa and rose 1.5 fold to its maximuin observed level at 12 dpa (Figure 24). By 18 dpa accumulation of sc20 n:zRNA had decreased 4-fold. Prolonged exposure of the filter enabled detection of sc20 expression at 6 dpa and 21-24 dpa. No gene expression was observed at any stage of embryo or pod development examined even after prolonged exposure of the filters. This confirmed that sc20 expression was seed coat-specific.

In situ hybridization was carried out to analyse the spatial gene expression pattern of sc20 within the seed coat between 3-24 dpa. Seed sections were hybridized with radiolabelled sense and anti-sense SC20 RNA probes. No birefringent cell structui-es were evident in the seed sections used (Figure 24).

Gene expression of sc20 was localized to the thick-walled parenchyma of the outer integument (see Figures 13 (b) and 24). The temporal expression pattern of 9-21 dpa expression with an observed peak at 12 dpa was almost identical to that determiined by northern blot analysis (Table 4, in Examples).
sc20 transcripts were not detected in the embryo between 3-6 dpa. The in situ hybridization results of the seed sections concur with the northern blot analysis that within the seed org~m sc20 is expressed only in the seed coat organs.
Expression of gene under control of the SC20 promoter (1-245 of SEQ

ID NO:8) is seen in Figures 13 (b) and 24.
SouthÃrn blot analysis was performed to ascertain whether sc2O is a single gene or a member of a gene family. Soybean genomic DNA was cleaved with Eco RI, Hind III, Xba I and Eco RV which have three, four, two and one recognition site(s) respectively in the sc20 clone (see Figure 23 (a)). Under conditions of high stringency to detect genes with at least 90% similarity to sc20 the probe hybridized to a single band (Figure. 25 (b)). Under medium stringency conditions to observe genes with 80% similarity to sc20 it was observed that the SC20 probe annealed to 2-3 bands for each digest (Figure 25 (a)). Under conditions of low stringency i.e., 40 C below Tm the SC20 probe hybridized to several more bands froni each digest (data not shown). This suggested that sc20 is a member of a small gene family composed of 2-3 members and that the soybean genome contains several genes which are more distantly related to sc20.

Southern blot arialysis was also performed to determine the distribution of sc20 among a number of diverse plant species i.e., pea (Pisum sativum), canola (Brassica napus), oat (Avena sativa), onion (Allium cepa), pepper (Capsicum annuum), Mimosa sp. (Mimosa pudica), black spruce (Picea mariana (Mill) B.S.P.), birch (Betula pendula Roth). The genomic DNA was restricted with Eco RI. The SC20 cDNA probe hybridized to only the genomic DNA of soybean (Figure 25 (c)) irrespective of stringency conditions utilized. It is possible that the gene may exist in rnore species more closely related to soybean.

The expression of a gene under the control of SC21 promoter (see Figure 11 (b)) within seed coat tissues at 15 days after anthesis is shown in Figure (c). Note specific localization of the probe in the thin-walled parenchyma of the outer integument, including the area immediately surrounding the tracheid bar (arrow).
The nucleotide sequences of SC21 (SEQ ID NO:5) and SC17 were identical apart from the position of the poly (A) tail and were just less than 65 %
similar to a Cicer arientinum (chickpea) mRNA for an unknown protein.

The expression of genes under the control of seed-coat promoters of this invention are shown in Figures 10, 13, 14, 21 and 24.

The results of these and other experiments indicating the expression patterns of these genes is summarized in Table 4 within the Examples section.

The promoters of the present invention can be used to control the expression of any given gene spatially and developmentally within developing seed coats, or seed-coat associated tissues. Some examples of such uses, which are not to be considered limiting, include:

1. Modification of storage reserve yields in seed coats, such as starch by the expression of yeast invertase to mobilize the starch, or increasing starch levels by increasing the sink strength by enhancing carbon unloading into seeds, by expressing invertase in specific seed coat tissues, or reduce starch levels by inhibit starch biosynthesis through the expression of the antisense transcript of ADP-glucose pyrophosphorylase.

2. Modification of seed colour contributed by anthocyanin pigments or condensed tannins in the seed coats by expression of antisense transcripts of the phenylalanine ammonia lyase or chalcone synthase genes.

3. Modification of fibre content in seed-derived meal by expression of antisense transcripts of the caffeic acid-o-methyl transferase or cinnamoyl alcohol dehydrogenase genes.

4. Inhibition of seed coat maturation by expression of ribonuclease genes to allow for increased seed size, and to reduce the relative biomass of seed coats, and to aid in dehulling of seeds.

5. Expression of genes in seed coats coding for insecticidal proteins such as a: amylase inhibitor or protease inhibitor.

6. Partitioning of seed metabolites such as glucosinolates into seed coats for fungal or insect resistance.

7. Production of high value proteins in seed coats for use as pharmaceuticals or for use in industrial processes.

8. Control af seed borne diseases by expressing antifungal antiviral, or anti-bacterial proteins within the seed coat Furthermore, modifications of the nucleotide, or amino acid, sequence of HP, or the preparation of chimeric gene constructs comprising the regulatory region of HP associated with a gene of interest will result in:
- alterations in the textural, visual, chemical or other properties of the seed coat, including the seed surface;

- the production of plants that are less susceptible to seed borne and pod diseases by expressing heterologous proteins in tissues of the ovary wall;
- lessening the health hazard of seed dust exposure by genetic selection or transformation, to produce plants with reduced allergenic protein expression on the seed surface Thus this invention is directed to such promoter and gene combinations.
Further this invention is directed to such promoter and gene combinations in a cloning vector, wherein the gene is under the control of a seed coat specific -promoter and is capable of being expressed in a plant cell transformed with the vector. This invention further relates to transformed plant cells and transgenic plants regenerated from such plant cells. The promoter and promoter gene combination of the present invention can be used to transform any plant cell for the production of any transgenic plant. The present invention is not limited to any plant species.

The following list sumrnarises the nucleotide sequence data in the SEQUENCE
LISTING of the present application:
pT218 genomic DNA sequence is found in SEQ ID NO: 1;
Ep genomic DNA sequence is listed in SEQ ID NO:2;
SC4 cDNA sequence is presented in SEQ ID NO:3;
SC20 cDNA sequence is in SEQ ID NO:4;
SC21 cDNA sequence is presented in SEQ ID NO:5;
HP cDNA is listed in SEQ ID NO:6;
HP genomic DNA sequence is found in SEQ ID NO:7;
SC20 genomic I)NA sequence is listed in SEQ ID NO:8; and SC4 genomic DNA sequence is presented in SEQ ID NO:9.

While this invenition is described in detail with particular reference to preferred embodiments thereof, said embodiments are offered to illustrate but not limit the invention.

EXAMPLES
Characterization of a Seed Coat-Specific GUS Fusion Transfer of binary constructs to Agrobacterium and leaf disc transformation of Nicotiana tabacum SRi were performed as described by Fobert et al. (1991, Plant Molõ Biol. 17, 837-85 1). Plant tissue was maintained on g/ml kanamycin sulfate (Sigma) throughout in vitro culture.

Nine-hundred aiid forty transgenic plants were produced. Several hundred independent transformants were screened for GUS activity in developing seeds using the fluorogenic assay. One of these, T218, was chosen for detailed study because of its unique pattern of GUS expression.

Fluorogenic and histological GUS assays were performed according to Jefferson (Plant Mol. Biol. Rep., 1987, 5, 387-405), as modified by Fobert et al.
(Plant Mol. Biol., 1991, 17, 837-851). For initial screening, leaves were harvested from in vitro grown plantlets. Later flowers corresponding to developmental stages 4 and 5 of Koltunow et al. (Plant Cell, 1990, 2, 1201-1224) and beige seeds, approximately 12-16 dpa (Chen et al., 1988, EMBO
J. 7, 297-302), were collected from plants grown in the greenhouse. For detailed, quantitative analysis of GUS activity, leaf, stem and root tissues were collected from kanamycin resistant Fl progeny of the different transgenic lines grown in vitro. Floral tissues were harvested at developmental stages 8-10 (Koltunow et al., 1990, Plant Cell 2, 1201-1224) from the original transgenic plants. Flowers of these plants were also tagged and developing seeds were collected from capsules at 10 and 20 dpa. In all cases, tissue was weighed, immediately frozen in liquid nitrogen, and stored at -80 C.
Tissues analyzed by histological assay were at the same developmental stages as those listed above. Different hand-cut sections were analyzed for each organ. :For each plant, histological assays were performed on at least two different occasions to ensure reproducibility. Except for floral organs, all tissues were assayed in phosphate buffer according to Jefferson (1987, Plant Mol.
Biol.
Rep. 5, 387-405), with 1 mM X-Gluc (Sigma) as substrate. Flowers were assayed in the same buffer containing 20% (v/v) methanol (Kosugi et al., 1990, Plant Sci. 70, 133-140)õ

Tissue-specific patterns of GUS expression were only found in seeds.
For instance, GUS activity in plant T218 (Figure 1) was localized in seeds from 9 to 17 days postanthesis (dpa). GUS activity was not detected in seeds at other stages of development or in any other tissue analyzed which included leaf, stem, root, anther, ovary, petal and sepal (Figure 1). Histological staining with X-Gluc revealed that GUS expression in seeds at 14 dpa was localized in seed coats but was absent from the embryo, endosperm, vegetative organs and floral organs (results not shown).

The seed coat-specificity of GUS expression was confirmed with the more sensitive fluorogenic assay of seeds derived from reciprocal crosses with untransformed plants. T:he seed coat differentiates from maternal tissues called the integuments which do not participate in double fertilization (Esau, 1977, Anatomy of Seed Plants. New York: John Wiley and Sons). If GUS activity is strictly regulated, it must originate from GUS fusions transmitted to seeds maternally and not by pollen. As shown in Table 3, this is indeed the case. As a control, GUS fusions expressed in embryo and endosperm, which are the products of double fertilization, should be transmitted through both gametes.
This is illustrated in Table 3 for GUS expression driven by the napin promoter (BngNAPI, Baszczynki and Fallis, 1990, Plant Mol. Biol. 14, 633-635) which is active in both embryo and endosperm (data not shown).

Table 3. GUS activity in seeds at 14 days post anthesis.

Cross GUS Activity nmole MU/min/mg Protein T218 T218 1.09 + 0.39 T218 WTO 3.02 + 0.19 WT T218 0.04 0.005 WT WT 0.04 t 0.005 NAP-5b NAP-5 14.6 t 7.9 NAP-5 WT 3.42 t 1.60 WT NAP-5 2.91 ~ 1.97 a WT, untransformed plants b Transgenic tobacco plants with the GUS gene fused to the napin, BngNAP1, promoter (Baszczynski and Fallis, 1990, Plant Mol.
Biol. 14, 633-635).

Cloning and Analysis of the Seed Coat-Specific GUS Fusion Genomic DNA was isolated from freeze-dried leaves using the protocol of Sanders et al. (1987, Nucleic Acid Res. 15, 1543-1558). Ten micrograms of T218 DNA was digestect for several hours with EcoRi using the appropriate manufacturer-supplied buffer supplemented with 2.5 mM spermidine. After electrophoresis through a 0.8% TAE agarose gel, the DNA size fraction around 4-6 kb was isolated, purified using the GeneClean kit (BIO 101 Inc., LaJolla, CA), ligated to phosphatase-treated EcoRI-digested Lambda GEM-2 arms (Promega) and packaged in vitro as suggested by the supplier. Approximately 125,000 plaques were transferred to nylon filters (Nytran, Schleicher and Schuell) and screened by plaque hybridization (Rutledge et al., 1991, Mol.
Gen.

Genet. 229, 31-40), using the 3' (termination signal) of the nos gene as probe (probe #1, Figure 2). This sequence, contained in a 260 bp SstI/EcoRI
restriction fragment from pPRF-101 (Fobert et al., 1991, Plant Mol. Biol. 17, 837-851), was labelled with [a 32P]-dCTP (NEN) using random priming (Stratagene). After plaque purification, phage DNA was isolated (Sambrook et al., 1989, A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press), mapped and subcloned into pGEM-4Z (Promega). The EcoRI fragment and deletions shown in Figure 2 were inserted into pBIN19 (Bevan, 1984, Nucl.
Acid Res. 12, 8711-8721). Restriction mapping was used to determine the orientation of the fusion in pBIN19 and to confirm plasmid integrity. Plants were transformed with a derivative which contained the 5' end of the GUS gene distal to the left border repeat. This orientation is the same as that of the GUS
gene in the binary vector pB1101 (Jefferson, 1987, Plant Mol. Biol. Rep. 5, 387-405).

The GUS fusion in plant T218 was isolated as a 4.7 kb EcoRI fragment containing the 2.2kb promoterless GUS-nos gene at the T-DNA border of pPRF120 and 2.5 kb of 5' flanking tobacco DNA (pT218, Figure 2), using the rnos 3' fragment as probe (probe #1, Figure 2). To confirm the ability of the flanking DNA to activate the GUS coding region, the entire 4.7 kb fragment was inserted into the binary transformation vector pBIN19 (Bevan, 1984, Nucl. Acid Res. 12, 8711-8721), as shown in Figure 2. Several transgenic plants were produced by Agrobacterium-mediated transformation of leaf discs. Southern blots indicated that each plant contained 1-4 T-DNA insertions at unique sites.
The spatial patterns of GUS activity were identical to that of plant T218.

Histologically, GUS staining was restricted to the seed coats of 14 dpa seeds and was absent in embryos and 20 dpa seeds (results not shown). Fluorogenic assays * Trademark of GUS activity in developing seeds showed that expression was restricted to seeds between 10 and 17 dpa, reaching a maximum at 12 dpa (Figure 3 (a) and 3 (b)). The 4.7 kb fragment therefore contained all of the elements required for the tissue-specific and developmental regulation of GUS expression.
7'o locate regions within the flanking plant DNA responsible for seed coat-specificity, truncated derivatives of the GUS fusion were generated (Figure 2) and introduced into tobacco plants. Deletion of the region approximately between 2.5 and 1.0 kbõ 5' of the insertion site (pT218-2, Figure 2) did not alter expression compared with the entire 4.7 kb GUS fusion (Figures 3b and 4).
Further deletion of the DNA, to the SnaBI restriction site approximately 0.5 kb, 5' of the insertion site (pT218-3, Figure 2), resulted in the complete loss of GUS
activity in developing seeds (Figures 3b and 4). This suggests that the region approximately between 1.0 and 0.5 kb, 5' of the insertion site contains elements essential to gene activation. GUS activity in seeds remained absent with more extensive deletion of plant DNA (pT218-4, Figures 2, 3b and 4) and was not found in other organs including leaf, stem, root, anther, petal, ovary or sepal from plants transformed with any of the vectors (data not shown).

The transcriptiorial start site for the GUS gene in plant T218 was determined by RNase protection assays with RNA probe #4 (Figure 2) which spans the T-DNA/plant DNA junction. For RNase protection assays, various restriction fragments from pIS-1, pIS-2 and pT218 were subcloned into the transcription vector pGEM-4Z as shown in Figures 7 and 2, respectively. A
440bp HindIII fragment of the tobacco acetohydroxyacid synthase SURA gene was used to detect SUR4 and SURI3 mRNA. DNA templates were linearized and transcribed in vitro withi either T7 or SP6 polymerases to generate strand-specific RNA probes using the I'romega transcription kit and [a 32P]CTP as labelled nucleotide. RNA probes were further processed as described in Ouellet et al.
(1992, Plant J. 2, 321 -330). RNase protection assays were performed as described in Ouellet et al., (1992, Plant J. 2, 321-330), using 10-30 g of total RNA per assay. Probe digestion was done at 30 C for 15 min using 30 g ml-' RNase A. (Boehringer Mannheim) and 100 units ml-' RNase Tl (Boehringer Mannheim). Figure 5 shows that two termini were mapped in the plant DNA.
The major 5' terminus is situated at an adenine residue, 122 bp upstream of the T-DNA insertion site (Fiigure 6). The sequence at this transcriptional start site is similar to the consensus sequence for plant genes (C/TTC 1 ATCA; Joshi, 1987 Nucleic Acids Res. 15, 6643-6653). A TATA box consensus sequence is present 37 bp upstream of this start site (Figure 6). The second, minor terminus mapped 254 bp from the insertion site in an area where no obvious consensus motifs could be identified (Figure 6).

The tobacco DNA upstream of the insertion site is very AT-rich (> 75 %, see Figure 7). A search for promoter-like motifs and scaffold attachment regions (SAR), which are often associated with promoters (Breyne et al., 1992, Plant Cell 4, 463-471; Gasser and Laemmli, 1986, Cell 46, 521-530), identified several putative regulatory elements in the first 1.0 kb of tobacco DNA
flanking the promoterless GUS gene (data not shown). However, the functional significance of these sequences remains to be determined.

Cloning and Analysis of the Insertion Site from Untransformed Plants A lambda DASH genomic library was prepared from DNA of untransformed N. tabacum SRl plants by Stratagene for cloning of the insertion site corresponding to the gene fusion in plant T218. The screening of 500,000 plaques with probe #2 (Figure 2) yielded a single lambda clone. The EcoRI and XbaI fragments were subcloned in pGEM-4Z to generate pIS-1 and pIS-2.
Figure 7 shows these two overlapping subclones, pIS-1 (3.0 kb) and pIS-2 (1.1 kb), which contain tobacco DNA spanning the insertion site (marked with a vertical arrow). DNA sequence analysis (using dideoxy nucleotides in both directions) revealed that the clones, pT218 and pIS-1, were identical over a length of more than 2.5 kb, from the insertion site to their 5' ends, except for a 12 bp filler DNA insert of unknown origin at the T-DNA border (Figure 6 and data not shown). The presence of filler DNA is a common feature of T-DNA/plant DNA junctions (Gheysen et al., 1991, Gene 94, 155-163). Gross rearrangements that sornetimes accompany T-DNA insertions (Gheysen et al., 1990, Gene 94, 155-163; and 1991, Genes Dev. 5, 287-297) were not found (Figure 6) and therefore could not account for the promoter activity associated with this region. The region of pIS-1 and pIS-2, 3' of the insertion site is also very AT-rich (Figure 7).

To determine whether there was a gene associated with the pT218 promoter, more than 3.3 kb of sequence contained with pIS-1 and pIS-2 was analyzed for the presence of long open reading frames (ORFs). However, none were detected in this region (data not shown). To determine whether the region surrounding the inserticin site was transcribed in untransformed plants, Northern blots were performed with RNA from leaf, stem, root, flower and seeds at 4, 8, 12, 14, 16, 20 and 24 dpa. Total RNA from leaves was isolated as described in Ouellet et al., (1992, Plant J. 2, 321-330). To isolate total RNA from developing seeds, 0.5 g of frozen tissue was pulverized by grinding with dry ice using a mortar and pestle. The powder was homogenized in a 50 ml conical tube containing 5 ml of buffer (1 M Tris HCI, pH 9.0, 1 % SDS) using a Polytron homogenizer. After two extractions with equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1), nucleic acids were collected by ethanol precipitation and resuspended in water. The RNA was precipitated overnight in 2M LiCI alt 0 C, collected by centrifugation, washed in 70%
ethanol and resuspended in water. Northern blot hybridization was performed as described in Gottlob-McHugh et al. (1992, Plant Physiol. 100, 820-825). Probe #3 (Figure 2) which spans the entire region of pT218 5' of the insertion did not detect hybridizing RNA. bands (data not shown). To extend the sensitivity of RNA detection and to include the region 3' of the insertion site within the analysis, RNase protection assays were performed with 10 different RNA probes that spanned both strands of pIS-1 and pIS-2 (Figure 7). Even after lengthy exposures, protected fragments could not be detected with RNA from 8, 10, 12 dpa seeds or leaves of untransformed plants (see Figure 5 for examples with two of the probes tested). The specific conditions used allowed the resolution of protected RNA fragments as small as 10 bases (data not shown). Failure to detect protected fragments was not due to problems of RNA quality, as control experiments using the same samples detected acetohydroxyacid synthase (AHAS) SURA and SURB mRNA which are expressed at relatively low abundance (data not shown). Conditions used in the present work were estimated to be sensitive enough to detect low-abundance messages representing 0.001-0.01 % of total mRNA levels (Ouellet et al., 1992, Plant J. 2, 321-330). Therefore, the region flanking the site of T-DNA insertion does not appear to be transcribed in untransformed plants.

Genomic Origins of the Insertion Site Southern blots were performed to determine if the insertion site is conserved among Nicaticzna species. Genomic DNA (5 jig) was isolated, digested and separated b;y agarose gel electrophoresis as described above.
After capillary transfer on to nylon filters, DNA was hybridized, and probes were labelled, essentially as described in Rutledge et al. (1991, Mol. Gen. Genet.
229, 31-40). High-stringency washes were in 0.2 x SSC at 65 C while low-stringency washes were in 2 x SSC at room temperature. In Figure 8, DNA of the allotetraploid species N. tabacum and the presumptive progenitor diploid species N. tomentosiformis and N. sylvestris (Okamuro and Goldberg, 1985, Mol.
Gen. Genet., 198, 290-298) were hybridized with probe #2 (Figure 2). Single hybridizing fragments of identical size were detected in N. tabacum and N.
tomentosiformis DNA digested with HindIII, XbaI and EcoRI, but not in N.
sylvestris. Hybridizations with pIS-2 (Figure 8) which spans the same region but includes DNA 3' of the insertion site yielded the same results. They did not reveal hybridizing bands, even under conditions of reduced stringency, in additional Nicotiana species including N. rustica, N. glutinosa, N.
megalosiphon and N. debneyi (data not shown). Probe #3 (Figure 2) revealed the presence of moderately repetitive DNA specific to the N. tomentosiformis genome (data not shown). These results suggest that the region flanking the insertion site is unique to the N. tomentosiformis genome and is not conserved among related species as might be expected for regions that encode essential genes.

Cloning of seed-coat genes from Soybean:
a) Isolation of seed-coat cDNA clones A seed coat cDNA library was constructed in Lambda GEM-4 from poly(A)+ mRNA isolated from soybean [Glycine max (L.) Merrill] seed coats. A
sample of the total ampl:ified library was used to sub-clone inserts from the original lambda vector iinto pBK-CMV (Stratagene). Random clones were selected from this mass excision for plasmid purification and single-run DNA
sequencing to construct an expressed sequence tag (EST) database.

For differential screening, an additional cDNA library was constructed from cultivar Maple Presto (EpEp) seed coats. The seed coats were harvested from seeds of four fresh weight groups: <50 mg, 50-100 mg, 150-250 mg and > 250 mg, to represent all developmental stages. Total RNA was isolated from the seed coats using Trizole reagent (BRL) from which poly (A) + RNA was isolated using Oligotex resin (Qiagen). First and second strand cDNAs were synthesized using the Ri.boclone cDNA synthesis kit and then cloned into a lambda GEM-4 vector (Promega). This seed coat library was differentially screened with positive and negative cDNA probes to identify genes preferentially expressed in the seed coat. The positive probe was derived from poly (A)+
mRNA isolated from seed coat tissues while the negative probe was made from poly (A)+ mRNA from, seedling, flower bud, leaf, pod and root tissue. The cDNA library was screened with cDNA synthesized from RNA using oligo(dT)15 primer, and hybridizations were carried out in Denhardt's solution (Sambrook et al. (1989) Molecular Cloning, Second Edition) at 65 C; wash 4 x 30 minutes 0.1X SSC 0.1% SDS at 65 C.

T'wenty-one positive clones were identified after plaque purification. The Lambda vector GEM-4 contains a complete pGEM1 plasmid. During the cloning procedure the cDNA is inserted into the Lambda vector at the multicioning site of this plasmid. The ent:ire pGEM1 plasmid, containing the cDNA insert, can be removed from the Lambda vector by digestion with Spel and then can be relegated to form a functional plasmid. Except for SC 11 and SC 19, the insert was removed from pGE:M1 by digestion with Xbal and EcoRl and ligated into an alternative plasmid vector pGEM4-Z. Following this protoco121 seed coat clones were used to tran.sform E. coli DH5a. No transformants were obtained with seed coat clones SC:7 and SC10 and so these clones were not processed further.

Seed surface pro'teins were obtained from soybean. A single seed was placed in a 2 mL plastic capped test tube and surface proteins were extracted by adding 0.5 mL of a buffer-detergent solution containing 10 mM Tris-Cl (pH 7.5) 0.5 % SDS, and 20 mM DTT, and placing the tube in a boiling water bath for 2 min. The contents of the tube were mixed and an aliquot was withdrawn and centrifuged for 5 min at 14,000 g. Freshly prepared loading buffer containing mM DTT was added to the sample and proteins were electrophoretically separated on 15 % acrylarnide gels in the presence of SDS (see Figure 17) using a modified Laemmli system, as described by Fling and Gregerson (1986, Anal Biochem 155:83-88). Fixation and visualization of the proteins by silver staining followed the method of Blum et al., (1987 Electrophoresis 8: 93-99). The amino terminal of the major peptides (indicated as HPS in Figure 17 (b)) were micro-sequenced from the blotted proteins according to Moos et al., (1988 J. Biol.
Chem 263: 6005-6008). The resulting amino acid sequences were identical and matched existing sequer.ices in the GenBank protein database for HP (Odani et al., 1987 Eur J. Biocheni 162, 485-491). Both peptides had alternative N-terminal residues of Ala or Ile, as has been previously noted for HP. The different electrophoretic mobilities of the two peptides could not be accounted for from the microsequencing analysis, but may be due to differences in glycosylation.

Several different soybean varieties were also compared by SDS-PAGE
analysis (see Figure 17 (c)).

To obtain the cD]!VA transcript of HP, sequences in the seed coat expressed sequence tag database were searched for reading frames corresponding to the HP amino acid sequence. Using this strategy, several identical cDNA
transcripts were isolated from the cDNA library obtained from Harosoy 63 seeds described above that included in their reading frames peptide sequences exactly matching HP. The encoded products of these DNA sequences were identified using the BLASTX program at the NCBI site.

b) Characterization of cDNA clones Sequence analysis Agarose gel electrophoresis of Xbal /EcoRl digests of the 19 remaining plasmid clones indicated that the inserts ranged in size from approximately 350bp to 1600bp, including the poly A tail. Inserts of the seed-coat clones were characterized by double stranded dideoxy sequencing of the 5' and 3' ends of the clones. A preliminary classification of the seed coat cDNA clones was made on the basis of sequence homology in the 3' and 5' ends of the clones. Based on sequence similarity with each other these 19 clones were grouped into 7 groups of clones. Sequence siniilarity was found between four of these groups and GenBank (with proline rich protein and three peroxidase groups). The three remaining groups had no sequence similarity with GenBank. SC4, SEQ ID

NO:3 (found to be the same as SCl), SC20, SEQ ID NO:4 (found to be the same as SC15), and SC21, SEQ ID NO:5, each represent one clone of each of the three groups which did riot exhibit similarity with anything in the GenBank database.

The 1119 bp nuclleotide sequence of SC4 (SEQ ID 3, Figure 19 (a); also see Restriction Map Figure 11 (d)) does not represent the full-length cDNA
clone as it does not contain an ATG codon for translation initiation. Two typical polyadenylation signals (AATAAA) are located at positions 1096 and 1102. The deduced protein sequence from the SC4 cDNA (Figure 19 (a)) consists of 289 amino acids and has a molecular mass of 31.9 kDa and a predicted pI of 7.95.
Three puatative glycosylation sites are present at positions 92, 128 and 269.
The putative polypeptide encoded by SC4 exhibits similarity with proteins that comprises a BURP domain (e.g. RD22, an Arabidopsis thaliana dehydration-responsive protein (Yamaguchi-Shinozaki K. and Shinozaki, K.
1993. Mol. Gen. Genet., 238: 17-25); PG1(3, a Lycopersicon esculentum polygalacturonase isoenzyme 1(3 subunit (Zheng L. et al., 1992. Plant Cell. 4:
1147-1156); Sali3-2, a Glycine max L aluminium-induced protein (Ragland M.
and Soliman, K.M. 1997. Plant Physiol. 114: 395); USP, a Viciafaba unknown seed protein (Baumlein H. et al., 1991. Mol. Gen. Genet. 225: 459-467) and ADR6, a Glycine max L auxin-induced protein (Datta N. et al., 1993. Plant Mol.
Biol. 21: 859-869); see Figure 19 (b)). The BURP domain is a long carboxyl terminal domain containing a number of highly conserved amino acids (Hattori J.
et al., 1998. Mol. Gen. Genet. 259: 424-428). The carboxyl terminal of the conceptual SC4 protein sequence contains the following conserved amino acids which are typical of the BURP domain proteins: two phenylalanine residues, two cysteine residues and four cysteine-histidine motifs which are also in the conserved alignment of CHX10CHX25-27CHX25-26 CH, where X is any amino acid (Figure 19 (b)). This BURP domain proteins also share a similar structural make-up of 3-4 domains (Figure 19 (c)) i.e., an amino-terminal domain containing a hydrophobic sequence, a second domain which may or may not be conserved, a third domain consisting of tandem repeats of a short amino acid sequence (not all BURP domain proteins have this domain) and a long carboxyl-terminal BURP domain (Hattori J. et al.,1998. Mol. Gen. Genet. 259: 424-428).
The tandem repeats which make up the third domain do not appear to have a common concensus sequence between the different BURP domain proteins. In addition to the BURP domain, the putative SC4 protein shares sequence similarity between its am.ino terminus and the conserved segment of the second domain possessed by several of the BURP domain proteins (Figure 19 (b)). It was also determined that the SC4 protein has a region containing two copies of the repeated sequence ESRSIXXYAG where X is any amino acid (Figure 19 (a)) which is similar to the staructural organization of the third domain of several BURP domain proteins. :Due to the extent of structural and sequence similarity between the SC4 protein and the BURP domain proteins it is likely that SC4 also contains a hydrophobic amino terminal if it was full-length.

The SC20 cDNA clone was sequenced (Figure. 23 (b)) and found to consist of 2447 bp with one 2310 bp open reading frame starting at nucleotide position 13 and ending at 2322. The TAG stop codon may be leaky as plants have tRNAs capable of niisreading it. However, any readthrough will be terminated by a second stop codon TGA which is immediately adjacent to UAG.
The 3' untranslated region contains one putative polyadenylation signal (AATAAA) located 21 nt after the stop codon.

The open reading frame of SC20 encodes a putative protein of 770 amino acid residues with a calculated molecular mass of 82.688 kDa and a predicted pI
of 6.93. The predicted protein has ten potential N-glycosylation sites (Figure (b)). The hydropathy profile (Figure 23 (c)) of SC20 protein revealed that the first 23 amino acids constitute a hydrophobic region typical of an eukaryotic signal peptide. From northern blot analysis, the SC20 cDNA clone hybridizes to a-- 2.5 kb transcript (data not shown). SC20 was used to obtain the genomic clone which was from a soybean cv. Harovinton genomic library.
Sequence comparisons (Figure 23 (d)) revealed that the putative polypeptide encoded by SC20 was similar to a Picea abies (black spruce) AF70 protein (Sabala et al., 1997. Physiol. Plant. 99: 316-322); cucumisin, (Yamagata Y. et al. 1994 J. Biol. Chem. 269: 32725-32731) from Cucumis melo L. (musk melon); a pathogen-induced protein, P69B, (Tornero P. et al., 1997 J. Biol.
Chem. 272: 14412-12219) from Lycopersicon esculentum (tomato) and a nodule-specific protein, Ag12, (Ribeiro A. et al., 1995 Plant Cell. 7: 785-794) from Alnus glutinosa (black alder). These plant proteins belong to the Pyrolysin family in the clan of serine proteases known as the subtilases (Barrett A.J. and Rawlings N.D., 1995. Arch. Biochem. Biophys. 318:247-250; Siezen, R. J. and Leunissen, J. A. M. 1997. Protein Sci. 6: 501-523.). The SC20 protein contains the conserved catalytic residues aspartate, histidine and serine as well as the highly conserved asparagine residue which is involved in stabilizing substrate binding. Moreover, the order of these four conserved residues in the SC20 protein is also a characteristic feature of subtilases. The SC20 protein also has a large sequence insertion between the conserved asparagine and serine residues found in plant subtilases but not in other subtilase members such as subtilisin BPN'(Power S.D. et al., 1986 PNAS. 83:3096-3100), kex2 (Mizuno K. et al.', 1988 Biochem. Biophys. Res. Comm. 156: 246-254) or furin (van de Ven W.J.M. et al.,1990 Mol. Biol. Rep. 14: 265-275). Based on sequence similarity between the N-termini of mature plant subtilases and the SC20 protein (data not shown) it was predicted that the SC20 protein had a mature domain starting from residue 1.17. Therefore the SC20 protein appears to be composed of 3 domains:
a signal peptide of 23 residues followed by a prosequence of 93 residues and a mature domain of 654 residues. The predicted mature domain of SC20 has a calculated molecular weight of 69.918 kDa and an isoelectric point of 6.34.
HE
The cDNA sequence for HP (pHPScDNAl) is (SEQ ID NO:6) shown in Figure 15. The 700 bp transcript includes 30 bp of 5' untranslated region (UTR), an open reading frame (ORF) of 119 amino acids, and 313 bp of 3' UTR.
Several polyadenylation signals were identified in the 3' UTR. The final 80 residues of deduced amino acid sequence exactly match the peptide sequence reported for the hydrophobic protein (Odani et al., 1987, Eur J Biochem 162, 485-491). Thus, the HP cDNA transcript indicates that hydrophobic protein is translated with a leader sequence of 39 amino acids.

Northern blot analysis Northern analysis, using the cDNA inserts of each clone as a probe, was performed to investigate: the expression pattern of the 19 seed coat clones.

RNA isolation from leaf, stem, pod and flower tissue was optimized based on a protocol adapted from Tripure Isolation reagent kit (Boehringer Mannheim). Plant tissue was frozen in liquid nitrogen and homogenized with the Tripure reagent (a monophasic solution of phenol and guanidine thiocyanate).
After the addition of chloroform the sample is centrifuged so that it separates into three phases. RNA is recovered from the upper aqueous phase by isopropanol precipitation. Due to the problem of polysaccharide contamination which increases with seed maturity, the isopropanol precipitation step was carried out in the presence of high salt which effectively maintains the polysaccharides in a soluble form whilst the RNA is precipitated.

Total RNA from seed-coat, embryo and root tissue was isolated as described by Fobert et al. (Plant J. 1994 6:567-577). Plant tissue was frozen in liquid nitrogen and homogenized in 1M Tris-HCI, pH9, 1 % SDS buffer. The sample was extracted twiice with equal volume phenol:chloroform:isoamyl alcohol (25:24:1), nucleic acids were collected by ethanol precipitation, collected by ethanol precipitation and resuspended in water. The RNA was precipitated overnight in 2M LiCl at 0 C, collected by centrifugation and resuspended in water.

RNA was denatured and size fractionated by formaldehyde gel electrophoresis and transferred onto nylon filters. Northern hybridization was carried out using radioactively labeled cDNA probes with hybridization in modified Church's buffer (Church and Gilbert (1994) PNAS USA 81:
1991-1995) at 65 C; wash 2 x 30 minutes 0.1X SSC 0.1 % SDS at 65 C. From this analysis, it was observed that SC4, and SC20 have seed coat specific expression. Ep locus peroxidase has preferential expression within seed-coat tissues, and SC21 was only expressed in seed coat, stem, root and flower tissues.
The results are shown in Figure 10 (a) - (d).

HE

For the analysis of HP expression, total RNA was isolated from roots, stems, leaves, flowers, pods, seed coats, and embryos dissected from soybean plants at various stages of development, according to published methods (Warig and Vodkin (1994) Plant Mol Biol Rep 12, 132-145). The RNA samples were quantitated by measuring absorbence at 260 nm, and by electrophoretic separation in formaldehyde gels and comparison to known standards. Samples of total RNA (10 g each) were blotted to nylon membrane using a vacuum manifold apparatus and fixed by UV cross-linking. Digoxigenin-labelled cDNA
was prepared according to instructions of the manufacturer (Boehringer) and used to probe the RNA blots,. Results, Figures 10 (e) and (f) show that the HP gene is highly expressed in the pod tissues during the later stages of development.
Hybridization signals were also noted in RNA samples derived from seed coat tissue, but not in RNA samples from the leaf, flower, embryo, stem or root.
These results, together with the data from the in situ hybridizations (see below) and the scanning electron microscopy analysis, indicate the HP gene is specifically expressed iri the endocarp of the ovary wall. Pieces of this tissue detach from the pod wa;ll and adhere to the seed surface during development, thus becoming a component of the mature seed coat (see Figure 14 (a).

RNA samples from seed coat, embryo, stem, root, leaf, pod and flower were hybridized with a radiolabelled SC4 cDNA probe (Figure 10 (a)) to determine organ specificity of the expression of SC4. The sc4 transcript was only expressed in the seed coat organ. It was estimated that the size sc4 mRNA
was 1.2 kb (data not shown).

Northern blot analysis was carried out to determine the temporal expression pattern of sc4. RNA from seed coat, embryo and pod organs between 6-24 dpa were hybridized with a radiolabelled SC4 cDNA probe. At 6 dpa the seed is too small to separate the seed coat and embryo organs and so total RNA
was isolated from an enitire seed. sc4 expression was already apparent in the seed by 6 dpa. No gene: expression was observed in any of the embryo development stages examined (Figure 20 (a)). sc4 mRNA transcripts were not observed in the embryo of 3-6 dpa seed sections examined by in situ hybridization using a radiolabelled SC4 antisense RNA probe (data not shown).
Therefore the sc4 expre:ssion observed at 6 dpa in the seed tissue is likely to be seed coat derived. After 6 dpa the expression of sc4 in the seed coat increased - 4-fold to its maximuin detected level between 9-12 dpa. By 15 dpa sc4 expression had decreased by - 2.5-fold dpa and continued to decline to just detectable levels by 18 dpa (Fig. 3.7). Expression of sc4 could only be detected in the seed coat at 21-24 dpa when the filter was over-exposed. Gene expression of sc4 in the pod was detected from 12-21 dpa only after over-exposure of the filter (data not shown).

Northern blot analysis was carried out to determine specificity of sc20 expression in various soybean organs i.e., seed coat, embryo, stem, root, leaf, pod and flower (Figure 10 (b)). sc20 has seed coat-specific expression as its mRNA was detected only in the seed coat organ. The sc20 transcript was determined to be approximately 2.5 kb (data not shown). Even after prolonged exposure of the filter, no sc20 transcripts was detected in any of the other plant organs.

Northern blot analysis was performed to determine the temporal gene expression pattern of sc20 in seed coat, embryo and pod organs of soybean.
Total RNA prepared from organs between 6- 24 dpa were probed with a radiolabelled SC20 cDNA probe. sc20 expression was detected at 9 dpa and rose 1.5 fold to its maximum observed level at 12 dpa (Figure 24). By 18 dpa accumulation of sc20 mRNA had decreased 4-fold. Prolonged exposure of the filter enabled detection of sc20 expression at 6 dpa and 21-24 dpa. No gene expression was observed at any stage of embryo or pod development examined even after prolonged exposure of the filters. This confirmed that sc20 expression was seed coat-specific.
b) In situ hybridization To analyze the distribution of the clones mRNA expression with respect to cell differentiation during development, in situ hybridization, on sections from 3, 6, 9, 12, 15, 18, 21 and 24 DAF seeds was used. Seeds or parts of seeds were fixed in. FAA fixative (50% ethanol, 5% acetic acid and 3.7 % formaldehyde), dehydrated in an ethanol./ tertiary butyl alcohol series and infiltrated and embedded in paraplast plus. Sections (5-l0 m) were cut on a microtome, transferred onto Superfrost slides which are positively charged to allow better adherence of the sections to the slide surface. Prior to in situ hybridization the samples were dewaxed in a xylene/ethanol series. In situ hybridization was carried out with 35S-labelled cDNA sense and anti-sense probes following the method of Cox and Goldberg (1998).

For the in situ analysis of Ep expression, flowers were tagged on days of full anthesis when the banner petal was fully extended and harvested at three day intervals from 1-30 days post anthesis (DPA), and at 45 DPA [19]. Tissue samples were fixed in a solution of 3.7% formaldehyde, 50% ethanol, and 5%
acetic acid for 3 h at room temperature, dehydrated in an ethanol series (50, 60, 70, 80, 90, 95, 100%) then infiltrated with t-butyl alcohol (TBA) in ethanol in a stepwise series (25, 50, 75, and 100%), followed by infiltration with Paraplast and several changes of pure melted Paraplast at 57 C. After infiltration, samples were placed in blocks and allowed to harden. Sections of 8-10 :m were cut on a rotary microtome and affixed to glass slides. Prior to hybridization, sections were de-waxed in xylene, and re-hydrated in an ethanol series (100, 95, 85, 70, 50, 30, 15, 0% ethanol in distilled RNAse free water).

Localization of RNA was performed with 35S-labelled RNA probes generated from Ep cDNA clones. The prehybridization, hybridization, and wash conditions followed published methods (Cox K.H., and Goldberg R.B. 1988, Analysis of plant gene expression. In Shaw CH (ed), Plant Molecular Biology:
A Practical Approach, pp. 1-35. IRL Press, Oxford). Briefly, sections were treated with Proteinase K and acetylated with acetic anhydride in triethanolamine.

The sections were then hybridized with 35S-RNA probes overnight at 42 C, washed, and dehydrated in an ethanol series before application of Kodak NTB-2 track emulsion. After 1 week at 4 C, slides were developed in Kodak D-19 developer, fixed in Kodak Fix, and stained in Toluidine Blue O(0.05 % in 50 mM Acetate buffer, pH 4.5). Slides were then dehydrated in an ethanol and xylene series, and mounted in Permount TM. Slides were photographed on Kodak EPL 400 slide film, using dark field optics .

The expression of a gene under the control of the Ep (peroxidase) promoter (nucleotides 1-1532 of SEQ ID NO:2, is localized within the hourglass cells (arrow; Figure 13(d)) within the seed-coat at 18 days after anthesis. Expression of Ep is first detected at 6 DPA in the thin-walled parenchyma of the outer integument, adjacent to the thick-walled parenchyma, and flanking the hilum region (Figure 13 (e)). By 9 DPA a thin band of expression extends around the entire seed coat, at the junction of the thin-and thick-walled parenchyma (Figure 13 (f)).
Expression shifts to the hourglass cells as they begin to develop, at 12 DPA
(Figure 13 (g)).

HP
For the analysis of HP (Figures 14 (c) and (d)), tissue samples were fixed in a solution of 50 % ethanol, 5 % acetic acid, 3.7 % formaldehyde for 3 h at room temperature, dehydrated in an ethanol series (50, 60, 70, 80, 90, 95, 100 %) then infiltrated with t-butyl alcohol (TBA) in a stepwise series (25, 50, 75;
and 100 % TBA in ethanol), followed by infiltration with Paraplast by gradual addition of increasing amounts of Paraplast to 100 % TBA, followed by several changes of pure melted Paraplast at 57 C. After infiltration, samples were placed in blocks and allowed to harden. Sections of 8 to 10 m were cut on a rotary microtome and affixed to glass slides. Prior to hybridization, sections were de-waxed in xyiene, and re-hydrated in an ethanol series (100, 95, 85, 70, 50, 30, 15, 0 % ethanol in distilled RNAse free water). Sections were then treated with Proteinase K and acetylated with acetic anhydride in triethanolamine. Sections were hybridized with -5S-RNA probes overnight at 42 C, then washed and dehydrated in an ethanol series before application of Kodak NTB-2 track emulsion. After 1 week at 4 C, slides were developed in Kodak D-19 developer, fixed in Kodak Fix, and briefly stained in Toluidine Blue 0 before dehydrating in an ethanol and xylene series, then mounting in Permount. Slides were photographed on Kodak EPL 400 slide film, using dark field optics.

The expression of a gene under the control of the HP promoter (nucleotides 1-2526 of SEQ ID NO:7) is localized within the membranous endocarp (arrow, Figure 14 (b)) at 12 days after anthesis. At six days post anthesis (DPA) expression of HPS is limited to the membranous inner layer of the pericarp. By 12 DPA expression is very strong and the inner epidermis is showing signs of becomrng detached from the rest of the pericarp and, in places, is adhering to the seed surface. Tissue sections from this stage of development also showed strong hybridization signals in the scierenchyma, indicating that HP
expression occurs throughout the endocarp.

To analyse the distribution of sc4 expression with respect to cell differentiation during seed coat development in situ hybridization was performed on seed sections from 3-24 dpa seeds. The seed sections were hybridized with radiolabelled sense and antisense SC4 RNA probes which were detected by exposure of the sections, to photographic emulsion. Within the seed sections the antisense or sense RNA probes can be localized by observing the accumulation of silver grains (produced in the emulsion by the emitted (3-particles) under dark-field illumination with a light microscope. Cell walls of some plant structures can be birefringent (i.e., reflect light) under dark-field illumination. Two birefringent areas can be observed in both the hilum and the funiculus of the seed sections in Figure 21 therefore any expression or lack there-of by sc4 will be masked in these locations.

sc4 was expressed throughout the inner integument of the seed coat at 3 dpa (Figure 21). By 6 dpa the expression pattern of sc4 had changed, and was localized to the outer integument parenchyma but not to the vascular tissue embedded within this layer. sc4 expression in the outer integument was maintained until 18 dpa after which time no further expression was detected (see Table 4 below). In concurrence with northern blot analysis, the in situ hybridization results revealed that sc4 expression increased to a maximum between 9-12 dpa and decreased thereafter (Table 4). In addition, expression of sc4 was not observed in the embryo of seed at 3-6 dpa.

The expression of a gene under control of the SC4 promoter (nucleotides 1-5514 SEQ ID NO:9) within soybean seed coat at 3 days after anthesis is also shown in Figure 13 (a). Expression is localized within the inner integument (arrow; Figure 13 (a)). Other areas of brightness in this figure include the recurrent vascular bundles in the funiculus, and the trichomes of the pod (the bright areas are due to the birefringence of crystalline areas in the cell walls, and are also present in the negative control; data not shown).

In situ hybridization was carried out to analyse the spatial gene expression pattern of sc20 within the seed coat between 3-24 dpa. Seed sections were hybridized with radiolabelled sense and anti-sense SC20 RNA probes. No birefringent cell structures were evident in the seed sections used (Figure 24).

Gene expression of sc20 was localized to the thick-walled parenchyma of the outer integument (see Figures 13 (b) and 24). The temporal expression pattern of 9-21 dpa expression with an observed peak at 12 dpa was almost identical to that determined by northern blot analysis (Table 4, in Examples).
sc20 transcripts were not. detected in the embryo between 3-6 dpa. The in situ hybridization results of the seed sections concur with the northern blot analysis that within the seed orga:n sc20 is expressed only in the seed coat organs.

Expression of gene under control of the SC20 promoter (1-2450 of SEQ
ID NO:8) is seen in Figures 13 (b) and 24.

The expression of a gene under the control of SC21 (see Figure 11 (b)) within seed coat tissues at 15 days after anthesis is localized in the thin-walled parenchyma of the outer integument, including the area immediately surrounding the tracheid bar (arrow; Figure 13 (c)).

c) Construction of genomic libraries In order to characterise the gene corresponding to seed coat cDNA
clone(s), several genomic libraries were constructed in A. vectors from total DNA
isolated from etiolated seedlings of various soybean cultivars. Two soybean genomic libraries were constructed in 1Lambda FixII (Stratagene, La Jolla, CA) from the total DNA isolated from etiolated seedlings of soybean [Glycine max (L.) Merrill ] cvs. Harosoy 63 and Harovinton. The DNA was partially digested with Bgl JI prior to ligation into the cloning vector.
Genomic clones corresponding to the cDNA clone SC4 and SC20 were obtained. Lambda DNA was isolated from each plaque. An -- 8 kb Xba I
fragment from the SC201ambda clone and an - 8 kb Sac I fragment from the SC4 lambda clone, identified by southern blotting, were ligated into pBlueScript-SK (Stratagene, La Jolla, CA) and transformed into E. coli TOP 10 cells.

Southern blot analysis of genomic soybean DNA, was carried out with 7 seed coat cDNA probes to determine similarities between clones and whether the clones represent a single gene or a gene family. Southerns were also performed to determine the occurrence of the seed-coat specific genes within other dicotyledonous and monocotyledonous plant species. Soybean genomic DNA
was cleaved with several restriction enzymes and the resulting DNA fragments were size fractionated using agarose gel electrophoresis, denatured and transferred to nylon filters. Hybridization was carried out with radiolabelled cDNA probes.
Isolation of genomic clones Initially, soybear.i genomic libraries were screened for the presence of the seed coat clone using the polymerase chain reaction with primers specifically designed from each cDNA sequence. This helped to target potential libraries for the isolation of genomic clones. The chosen genomic library was then screened using nucleic acid hybridization with cDNA probes. For genomic library screening hybridization conditions involved using modified Church's buffer (Church and Gilbert (1994) PNAS USA 81: 1991-1995) at 65 C; wash 0.1X

SSC 0.1 % SDS at 52-55 C. Probes were random primed in presence of 32PdCTP using standard protocols.

A seed-coat peroxidase gene, corresponding to the Ep locus, was obtained from a soybean seed-coat library. The genomic DNA sequence comprises four exons spanning bp 1533-1752 (exon I), 2383 -2574 (exon 2), 3605-3769 (exon 3) and 4033-4516 (exon 4) and three introns comprising 1752-2382 (intron 1), 2575-3604 (intron 2) and 3770-4516 (intron 3), of SEQ ID NO:2. Features of the upstream regulatory region of the genomic DNA include a TATA box centred on bp 1487; a cap signal 32 bp down stream centred on bp 1520. Also noted within the genomic sequence are three polyadenylation signals centred on bp 4520, 4598, 4663 and a polyadenylation site at bp 4700. The promoter region of the genomic sequence comprises nucleotides 1-1532 of SEQ ID NO:2 (see co-pending US patent application serial No. 08/723,414 and 08/939,905, both of which are incorporated by reference).
HE

For the isolation of the genomic HP gene, a genomic library was constructed from DNA isolated from the soybean cultivar Harosoy 63. The DNA
was purified and partially digested with Bgl H prior to ligation into the cloning vector lambda FixII (Stratagene). The resulting library was amplified and screened with the hydrophobic protein cDNA probe (pHPScDNAl). A positive clone was identified, purified, and found to contain a 14 kb insert. The entire insert was sub-cloned into pBluescript KS(+) and named pHPS1. The HP gene was determined by PCR analysis to lie near one end of the 14 kb Bgl II
fragment (for restriction map see Figure 11 (c)). This region of the pHPS1 insert was sequenced by primer wa:lking, and 3368 bp of this sequence data is disclosed here (SEQ ID NO:7). Aside from the polyadenylation site, the cDNA sequence (pHPScDNAl) exactly matches a stretch of sequence encoded on the genomic clone (pHPS1), indicating that this gene contains no introns. Additionally, a TATA box consensus signal was identified 81 bp upstream from the ATG
translation start site.

A genomic clone corresponding to SC4 cDNA clone was isolated from the soybean genomic library Harosoy 63 (Bgl II digest). The genomic sc4 clone is 8310 bp in length (SEQ ID NO:9). The promoter region is found between nucleotides 1-5514 of SEQ ID NO:9. The restriction map is provided in Figure 11 (d).

A genomic clone corresponding to SC20 cDNA clone was isolated from soybean genomic library prepared from cv Harovinton (GigapackGold packaging). The genomic sc20 clone is 7235 bp in length (see Figure 23 (a), SEQ ID NO:8). Alignment of sc20 genomic and SC20:2 cDNA sequences revealed that sc20 contained eight introns of 94 bp, 101 bp, 185 bp, 80 bp, bp, 112 bp, 110 bp and 93 bp respectively (Figure 23 (a)). A search (www.hgc.lbl.gov/cgi-bin/promoter.pl) of the 5' upstream region of sc20 revealed three potential transcription start sites at positions 1085, 1156 and 2272.
The promoter region is found between nucleotides 1-2450 of SEQ ID NO:8. The restriction map of SC20 is presented in Figure 11 (a) and 23(a).

A genomic clone corresponding to SC21 cDNA clone was isolated from the soybean genomic libr-ary prepared from Harosoy 63 (EcoRl digest). The DNA of the SC21 genomic clone was digested with several restriction enzymes, fractionated by agarose gel electrophoresis and transferred onto nylon membrane.
Hybridizations were carried out using radiolabelled cDNA. A restriction map of this clone is presented in Figure 11 (b).

Southern analysis Southern blot analysis was carried out to examine the gene family composition of sc4. Soybean genomic DNA was cleaved with Eco RI, Hind III
and Xba I. which do not have recognition sites in the SC4c cDNA sequence.
Under conditions of low to high stringency (i.e., from 40-10 C below Tm of the probe ) the SC4 cDNA probe hybridized to a single band (Figure 22) and therefore sc4 appears to be a single gene.

Southern blot analysis was performed to ascertain whether sc20 is a single gene or a member of a gene family. Soybean genomic DNA was cleaved with Eco RI, Hind III, Xba I and Eco RV which have three, four, two and one recognition site(s) respectively in the sc20 clone (see Figure 23 (a)).

Hybridization was carried out with radiolabelled SC20 cDNA probe which could anneal from the middle of exon 6 to the Eco RI site on exon 9. For each digest the probe was expected bind to only one of the resulting sc20 restriction fragments. Under conditions of high stringency to detect genes with at least 90%
similarity to sc20 the probe hybridized to a single band (Figure. 25 (b)).
Under medium stringency conditions to observe genes with 80% similarity to sc20 it was observed that the SC20 probe annealed to 2-3 bands for each digest (Figure (a)). Under conditions of low stringency i.e., 40 C below Tm the SC20 probe hybridized to several more bands from each digest (data not shown). This suggested that sc20 is a:member of a small gene family composed of 2-3 20 members and that the soybean genome contains several genes which are more distantly related to sc20.

Southern blot analysis was performed to determine the occurrence of the seed-coat genes within the following plant species: pea (Pisum sativum), canola 25 (Brassica napus), oat (Avena sativa), onion (Allium cepa), pepper (Capsicum annuum), mimosa (Mimosa pudica), black spruce (Picea mariana (Mill B.S.P.), birch (Betula pendula Roth). Genomic DNA was cleaved with EcoRI and the resulting DNA fragments were fractionated using agarose gel electrophoresis, denatured and transferred to nylon filters. Hybridization was carried out with radiolabelled SC4 (Figure.22 (b)), SC20 (Figure 25 (c)), SC21, Ep locus peroxidase, and HP cDNA probes, using modified Church's buffer at 65 C. The filters were washed with 2XSSC, 0.1 %SDS at 42 C for 30 minutes, followed by 0.1XSSC, 0.1%SDS at 65 C for 30 minutes. SC4, SC20 and Ep locus peroxidase cDNA hybridized to the genomic DNA of soybean only. SC21 cDNA hybridized to the genomic DNA of both soybean and oat. HP cDNA
hybridized to the genomic DNA of soybean.
Analysis of promoter activity The developmental expression of genes under the control of SC4, SC20 SC21 and the peroxidase: promoter were further characterized during development of the seed coat by in situ hybridization as described above. The results are summarized i:n Table 4.

Developmental analysis of SC20 indicates that the promoter is highly active at 12 DAF within the outer integument and thick walled parenchyma, however, activity of the SC20 promoter is detectable from about 9 DAF (as per Figure 13 (b)) to about 1.8 DAF, and is partially detected at 21 DAF.

The SC4 promoter is active from about 3 daf (also see Figure 13 (a)) to about 6 DAF within the inner integument, and then is highly active at 9 DAF
within the outer integument and stellate parenchyma, and strongly active at 12 DAF in these same tissues. The SC4 promoter is still active within the outer integument up to 18 DAF.

The SC21 promoter is active throughout seed coat development during all stages examined, from 3 about DAF to about 24 DAF, with strongest activity noted from about 9 DAF to about 15 DAF (also see Figure 14 (c)). The gene under the control of the SC21 promoter is expressed primarily within the outer integument and derived tissues.

. i ~

The EP (peroxidase) promoter is active from about 6 DAF to about 24 DAF.
Expression of the peroxidase gene, from about 12 DAF to about 24 DAF, is predominantly within cells of the outer integument, and the hourglass cells (see also Figure 13 (d)).

The HP promoter is active from about 9 daf through to about 24 daf. The promoter is active within the membranous endocarp throughout this period of time (see also Figure 14 (b)).

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SUBSTITUTE SHEET (RULE 26) Seed Surface Analysis of Dull and Shiny Soybean Varieties Seed surface proteins of several different soybean varieties were compared by SDS-PAGE analysis. A single seed was placed in a 2 mL plastic capped test tube and surface proteins were extracted by adding 0.5 mL of a buffer-detergent solution (10 mM Tris-Cl (pH 7.5) 0.5% SDS, 20 mM DTT) and placing the tube in a boiling water bath for 2 min. The contents of the tube were mixed and a sample was withdrawn and centrifuged for 5 min at 14,000 g.
The proteins in the supernatant were electrophoretically separated on 15 %
acrylamide gels in the presence of SDS (Fling and Gregerson (1986) Anal Biochem 155, 83-88) and detected by silver staining. This analysis revealed that the 8.3 kD hydrophobic protein is by far the most abundant protein molecule occurring on the seed surface of 'Dull' seeded varieties. Only trace amounts of hydrophobic protein was detected on the surface of 'Shiny' seeded soybean varieties (results not shown).

Analysis of seed coat tissues using light microscopy indicated that the membranous endocarp of the pod wall remains in association with the seed-coat (Figure 14 (a). Scanning electron microscopy (SEM) of the seed surface of soybeans also showed obvious differences between 'Dull' (e.g.cultivar Clark) and 'Shiny' (e.g. cultivar Williams 82) varieties (see Figure 16). Whole seeds were sputter coated with gold and examined by SEM at several magnifications.
When viewed with the naked eye, 'Dull' varieties present a surface with a powder-like coating whereas 'Shiny' types appear to have a smoother and more light-reflective surface. Examination by SEM at low magnification (18 X) reveals that the surface of 'Dull' types is uniformly covered with small, dimple-like indentations and bits; of adhering material. These indentations are also visible on 'Shiny' types, but the surface is virtually free of adhering material. At higher SEM magnifications, the surface of 'Dull' types appears rough and ragged whereas the 'Shiny' seeded soybeans have a relatively smooth and undulating surface.

Without wishing to be bound by theory, it appears that the adhering material on the 'Dull' seeded types are remnants of the membranous endocarp tissue and is rich in hydrophobic protein. The expression of the hydrophobic protein in the endocarp causes bits of this tissue to stick to the seed surface, resulting in the 'Dull' phenotype. Lack of expression similarly may result in the 'Shiny' phenotype. The hydrophobic protein may be involved in the adherence of the endocarp to the seed surface.

Analysis of 'Dull' and 'Shiny' Seeded Varieties Total genomic DNA was extracted from 'Dull' or 'Shiny' seeded varieties and amplified by PCR using primers targeted to the HP gene. The resulting amplification products were clearly polymorphic between the two genotypes. Good amplification of target segments of DNA were regularly observed when template DNA was from 'Dull' types whereas DNA from 'Shiny' types produced multiple products or products that were shorter or longer than expected, depending on the primer combination. These results indicate that different alleles the HP gene occurs in 'Dull' and 'Shiny' types of soybean.
This allelic variation causes profound differences in seed surface morphology and composition.

To compare HP gene structure in two different seed luster phenotypes that were also different i:n the amount of HP present on the seed surfaces, we hybridized genomic DNA blots with probes derived from the HP cDNA
sequence under high stringency conditions.

Soybean genomic DNA was isolated from frozen, lyophilized tissue according to the method of Dellaporta et al., (1983). Restriction enzyme digestion of 30 tcg DNAõ separation on 0.5 % agarose gels and blotting to nylon membranes followed staiidard protocols (Sambrook et al., 1989). Digoxigenin labelled cDNA was prepared and used to probe DNA blots according to the instructions provided by the manufacturer (Boehringer Mannheim).

Hybridization was carried out at 65 C for 16 h in 0.25 M Na2HPO4 (pH 7.2), 20% SDS, 1 mM EDTA and 0.5% blocking reagent (Boehringer Mannheim).
Filters were then washed 4 x 15 min at 22 C in high stringency wash solution (20 mM Na2HPO4 (pH 7.2), 1% SDS and 1 mM EDTA), followed by 3 x 15 min washes in the same solution at 68 C.

A typical result from such a Southern analysis is shown in Figure 18.
Genomic DNA blots from cultivars that accumulated large amounts of HP on the seed surface produced strong hybridization signals. These intensely hybridizing fragments are not present in genomic DNA from plants that have only trace amounts of HP on the seed surface. However, several fainter signals are also present in DNA blots from both types of plants. These results indicate that sequences related to the HP cDNA are prevalent in the soybean genome, and that the HP gene structure is polymorphic among soybean cultivars. Soybean types that accumulate large amounts of HP on the seed surface possess additional copies of this gene.

The present invention has been described with regard to preferred embodiments.
However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing form the scope of the invention as described in the following claims.

SEQUENCE LISTING
<110> Miki Dr., Brian L
Gizen Dr., Matk Miller Dr., Shea Boutilier Dr., Kim Hu., Ming Bowman, LuAnn Batchelor, Anthea <120> Seed-Coat Promoters, Genes and Gene Poducts <130> 08-869947W0 <140> 2,325,819 <141> 1999-04-13 <150> 09/059,090 <151> 1998-04-13 <160> 12 <170> PatentIn Ver. 2.0 <210> 1 <211> 1070 <212> DNA
<213> Nicotiana tabacum <400> 1 tctagacttg tcttttcttt acataatcct cttcttcttt tttttgttag tttcttctgt 60 tttatccaaa aaacgaatta ttgattaaga aatacaccag acaagttttt tacttctttt 120 tctttttttt tttgtggtaa aaaattacac ctggacaagt ttatcacgaa aatgaaaatt 180 gctatttaag ggatgtagtt ccggactatt tggaagataa gtgttaacaa aataaataaa 240 taaaaagttt atacagttag atctctctat aacagtcatc cttatttata acaatacttt 300 actataaccg tcaaatttat tttgaaacaa aattttcatg ttatgttact ataacagtat 360 tttattatag caaccaaaaa atatcgaaac agatacgatt gttatagagc gatttgattg 420 tatcattatc cacatatttt cgtaagccca attactcctc ctacgtacga tgaaagtaaa 480 ccaatttaaa gttgcaaaaa tccaatagat ttcaatactt cttcaactgg cgttatgtta 540 ggtaatgact cctttttaac ttttcatctt taatttgaag tttctttcat taaaagaaag 600 tttctagaag agaagtgttt taacacttct agctctacta ttatctgtgt ttctagaaga 660 aaaatagaaa atgtgtccac ctcaaaaaca actaaaggtg ggcaaatctc cacctattta 720 ttttattttg gattaattaa gatatagtaa agatcagtta taaacggagt tttgagttga 780 tacagtgaat tttaagatgt gtaccgattt aactttattt acatttatgt ttcgcacata 840 taagaagtcc gatttggaaa tactagattt tgtcaatcag gcaattcatg tggttgaaga 900 atttaagtta tatacaatga tgatataaag aatttttata ctattagtgc aaattaatcg 960 attactaaaa attattattc tattaattta tgctatcgtg cctccccaac ccgtcgaccg 1020 cggtacccgg tggtcagtcc cttatgttac gtcctgtaga aaccccaacc 1070 <210> 2 <211> 4700 <212> DNA
<213> Glycine max <400> 2 tagataaaaa aatgggatat aatttttctc agatgttgtt tatactgttt ttttaatcag 60 aattaaaatt cctctttaat tatcgacata attttttttg gtgaatatta tcgacataat 120 tatttaatac aaatttttat tgtacataga agtgatactt caattttaat attggagaac 180 agtacgaaaa cataaaaaaa ctgttattag aagaaaaaaa tatatggaaa aggttagcta 240 catatattag ctaaattagt tgttctaatt ggctatataa accctattgt actctttgta 300 atctcacctt tttcatttaa atacatttct actttttaag ttctatattt tctctcaatt 360 ttcttcgata aaccatgaaa tttaacatgg tatatcagcg ataccaccca ctttgaaagc 420 catgtatggc tagtatgggc agccaaaatt tgccctggtt caagcaaagc aagtgtttat 480 atagatgtga cttttgttga ggaactcatg ccaatggtac tgattgtgaa actgagaaaa 540 ctaatttgga gaatttgaat tatgatcatt aaatactcct ctcctgacta ccttcgtccc 600 tcaaatttgt accatcatta tttcccaaaa atttgattac aatgcactaa ttaatgaatg 660 tttcttacat tatcatatta tcatatctga cattttgttt ttacttttta taataattat 720 tttaaaaagt catacatgca aataattttt taatagttta cagttaaatt tttacagtaa 780 aaatgcatga aaattaaact ttatttttcc aagtcatcat ttagtcaaat cccaaaacaa 840 tgattatttt ttgcaaatga atgtttattg aacatttaaa tgtagcctaa ttaattctgg 900 ttatggtgtc aatgttccaa aacctaatgc aagatcttag caagtacata catagatcta 960 attttaaact tatctttacg caagagatat aaagattata catctagttt taaacattaa 1020 cttttgtttt tgtgttaaaa aacagtaaca ttttcttaat tttgtagagt gacgtgctcc 1080 aaccatatta acgaagattt taattggtat tcaagttcat gaacttagta aataagtttt 1140 ggtcttcagt tttcaatttt cattacaaca tttatgtaaa atatcaacgt tttctgaaat 1200 ttgttgcttg tgtgctccaa ccacatttaa gagattatag aaattaattt tcaagaagat 1260 aatgattcct actcttgctg gccctaccat agtacaataa atccactcat aaatcaacaa 1320 gtcgtcgtca taggcaattg ggcatcatat cataaacaat acgtacgtga tattatctag 1380 tgtctctcag tttactttat gagaaattat ttttctttaa aaaaagttaa ttaataaaaa 1440 catttgcgat accgtgagtt acaagaaatc cgccgaattc atctctataa ataaaaggat 1500 ctatatgaga ggtaaaatca tattaactca aaatgggttc catgcgtcta ttagtagtgg 1560 cattgttgtg tgcatttgct atgcatgcag gtttttcagt ctcttatgct cagcttactc 1620 ctacgttcta cagagaaaca tgtccaaatc tgttccctat tgtgtttgga gtaatcttcg 1680 atgcttcttt caccgatccc cgaatcgggg ccagtctcat gaggcttcat tttcatgatt 1740 gctttgttca agtacgtact tttttttttc cttccaaaat gccctgcata tttaacaaga 1800 ttgctttgtt cacctagaaa aatgtgtttt tttcaacgat cttacgtacg tttgtttggt 1860 ttgaaaaata aatcagaaag agatcaagaa aatagctaga aagaaagcaa cgttttttta 1920 aaaggtattt agtgtgagaa aaatattaaa actgaagaga aagaaattaa ataagctttt 1980 cttgaatgat atttacatgt cttattaact taaagtcacc ttttttcttt aagttgtgct 2040 tgaagaaaaa agatgtcttt cagtttagtt ttgattaatg ctaattatat ttttaattaa 2100 ttaattaata ctatatatct atttaccata ttaattatta ctatatttca tgatgacaac 2160 agacaagtat tctaaagagg tatcggtaga tgattaattt ttttataaaa aaatcttttg 2220 cgtgtataga tattctttta taattggtgc agaaacttgt aatgctaatt gcaattaatc 2280 ttacattgat taactaatag ctataatcaa tatttaggtt aggtatagga gacaaatcaa 2340 gtgatctgaa caaattaagt tgttatattt gcattgtgac agggttgtga tggatcagtt 2400 ttgctgaaca acactgatac aatagaaagc gagcaagatg cacttccaaa tatcaactca 2460 ataagaggat tggacgttgt caatgacatc aagacagcgg tggaaaatag ttgtccagac 2520 acagtttctt gtgctgatat tcttgctatt gcagctgaaa tagcttctgt tctggtaatt 2580 aataactcct aattaattcc caaccattaa aaagttgcat gattggattc aaaattctat 2640 ggtattgggg ttctgatata aatttgtaat taaattgcac taaaaaaaat tatcatatac 2700 ttttaataaa aaaaatttat ctaatttaat ttattattaa aactattttt aaaattcaat 2760 cctaactctt ttttaatcgg agcatgtaag ctggcaccca ccgtatatcg ttggaagatg 2820 ctataaaacc atttaattaa tggatggaat cagtcaaaac atttaattca aaatactctt 2880 aattgtgatt agtaatcatg ttcgggcaag ttacgttgtg tataattaat ttgacttaat 2940 cagataaaaa aacaaatgga cgcaagccgg ttggtataga tatcactggc ctgtagaata 3000 tgtggttttt cacgtttaaa taaaagctag ctactatatt atatttagtc tttttttttc 3060 ttaaacccat ttaacgtgat ttattgactg tgaaacatgt ttccacacac aggcttagaa 3120 actcctcgca actaacatct ccaaaatttg actatttatt tatgaagata attcatctat 3180 gatgttcaac tctattatat atatgtatca tcgcagtatt aagaattata atagtcaaat 3240 atagaagtat atcgggtaaa tgtagttgca tgtgcgacct gtttcgtgta aaatgcttat 3300 tctatatagc tttttttatt ggaaaataac gatgaactaa aaacgaaagg gtatcatata 3360 gtttgacttt tatgttagag agagacatct taatttggtc atatgttaaa taattaatta 3420 caatgcatac acaaatattt atgccatatc taaaaaatga taaaatatca taggtatact 3480 caactatatg atatccccat aacagaaatt gtacttttct tcaggcaatg aacttaacat 3540 ttctgtttgc taaaaacaaa catccactta aagtggttca acatatttat gtaataattt 3600 acagggagga ggtccaggat ggccagttcc attaggaaga agggacagct taacagcaaa 3660 ccgaaccctt gcaaatcaaa accttccagc acctttcttc aacctcactc aacttaaagc 3720 ttcctttgct gttcaaggtc tcaacaccct tgatttagtt acactctcag gtatacataa 3780 tcaatttttt atttgctatt agctagcaat aaaaagtctc tgatacagac atatttagat 3840 aaattaattt ctccataaac atttataata aaattatcaa tttatgtact taaaaattat 3900 ggattgaagc tcttttcatc caacttttac taaagttaag gtgcatataa tataaaataa 3960 actatctctt gtttcttata aaaagattga agataagtta aagtctactt ataaatcatt 4020 aatatatgta taggtggtca tacgtttgga agagctcggt gcagtacatt cataaaccga 4080 ttatacaact tcagcaacac tggaaaccct gatccaactc tgaacacaac atacttagaa 4140 gtattgcgtg caagatgccc ccagaatgca actggggata acctcaccaa tttggacctg 4200 agcacacctg atcaatttga caacagatac tactccaatc ttctgcagct caatggctta 4260 cttcagagtg accaagaact tttctccact cctggtgctg ataccattcc cattgtcaat 4320 agcttcagca gtaaccagaa tactttcttt tccaacttta gagtttcaat gataaaaatg 4380 ggtaatattg gagtgctgac tggggatgaa ggagaaattc gcttgcaatg taattttgtg 4440 aatggagact cgtttggatt agctagtgtg gcgtccaaag atgctaaaca aaagcttgtt 4500 gctcaatcta aataaaccaa taattaatgg ggatgtgcat gctagctagc atgtaaaggc 4560 aaattaggtt gtaaacctct ttgctagcta tattgaaata aaccaaagga gtagtgtgca 4620 tgtcaattcg attttgccat gtacctcttg gaatattatg taataattat ttgaatctct 4680 ttaaggtact taattaatca 4700 <210> 3 <211> 1119 <212> DNA
<213> Glycine max <400> 3 caatgctgcg ttaactccta gacattactg ggaaacgatg cttccaagaa ctcccttgcc 60 gaaagcaatc acagagctac taagccttga aagtaggtcc atatttgaat atgccgggaa 120 tgatgaccag tcagaaagta ggtccatatt aggatacgct ggctataatc aagacgagga 180 tgatgtgagc aaacacaata tacaaatctt caacaggttg tttttcttgg aagaggacct 240 gcgtgctggc aaaatattca acatgaagtt cgtcaacaac acaaaagcca cagtcccgtt 300 gctaccgcgc caaatttcga aacaaatacc gttctcagaa gataaaaaga agcaagtgtt 360 ggcgatgctt ggcgtggaag cgaactcaag caacgccaag atcatagcgg agaccattgg 420 tctttgccaa gagcctgcaa cggagggaga aaggaaacac tgcgcgactt cgttggagtc 480 catggttgat ttcgtcgttt ccgcgctcgg gaagaacgtt ggtgctttct caacagagaa 540 agaaagggaa actgagtctg gaaagtttgt agtggtgaaa aatggggtga ggaagttggg 600 agatgataag gttattgcct gtcatccaat gagttaccct tatgttgtgt ttgggtgtca 660 tctagtgcca aggagtagcg ggtatttggt gcgcttgaag ggagaagatg gggttcgagt 720 gaaagcagtt gttgcgtgcc acagagacac gtcaaagtgg gaccataatc atggggcatt 780 caaagtgctc aatcttaagc ctgggaatgg tacagtatgc catgtcttca ctgaggggaa 840 tcttctttgg cttccaaatt agattaatta ccatatacat atttgtcctt gttctatcct 900 taaataagtg gaatcacctg aagaattgtg cgtaatgagt tgtttgtctt tgtggaaatt 960 gttatctgtc ttgcatcacc aaataggtat atataaaata acaggagcgt ggtatttgtt 1020 gcacaaaaat ggatttcaac cgatcaaaaa aatatagcct ttaccaatta gaagggtttg 1080 gctttgttag caaataataa aaataaaata tcttgatgg 1119 <210> 4 <211> 2447 <212> DNA
<213> Glycine max <400> 4 caaagtttta acatgaaagg caataataca cttttgttgc atttattcta cactactctc 60 ttcctgtttc ttgtagtgtc aagttcatct tcaacaggga atgaaagtaa cgatgacact 120 aacagtaaag aagtttatat cgtgtacatg ggagctgcag attcaacaaa agcttctctt 180 aaaaatgagc acgctcagat tctgaattca gtgctaagaa ggaatgagaa tgccctagta 240 cggaactaca agcatggttt ctctgggttc gcagctcgtc tatcaaaaga ggaggcaaac 300 tcaattgctc agaaacctgg tgtggtgtct gttttccctg accccattct gaagctccac 360 actacacgtt catgggattt cctcaaaagc caaactcgtg tcaatatcga caccaaacca 420 aatacgctgt ccggttcttc tttttcttca tcagacgtca ttcttggcgt cttagacaca 480 ggcatatggc cagaggcggc gagttttagc gacaagggtt tcggtcctgt tccatcccga 540 tggaaaggca cctgcatgac atcaaaagac ttcaattcct cttgttgtaa caggaagata 600 attggcgcga ggttttaccc taacccagag gagaaaacgg caagggattt caacggacat 660 gggactcacg tttcgtcgac ggcagtgggc gtgccggtga gtggcgcatc gttctatggt 720 ctggcggcgg ggacggcaag gggtgggtcc cctgagtcaa ggttggcggt ttacaaagtg 780 tgtggggctt ttgggtcatg tcctgggtcg gccattcttg cggggtttga cgatgccatt 840 cacgacggag tggatatctt gtcgctgtcg ctcggtggat tcggtggaac caaaaccgat 900 ttgaccaccg acccgattgc gattggagca ttccactccg tccagcgcgg catcctggtg 960 gtctgcgccg ccgggaacga cggagaacca ttcaccgttc tcaacgacgc accttggatt 1020 ttaaccgttg cagcttccac catcgaccgt gatcttcaat ccgacgtggt cttgggtaat 1080 aaccaagtcg tcaagggaag agccataaat ttctcccctc ttttaaattc tcccgattat 1140 ccaatgatat atgctgagtc tgctgccagg gcaaatatct ccaacataac tgatgcaaga 1200 caatgccacc cagattcatt agatccaaaa aaagtcatag ggaagattgt ggtttgtgat 1260 ggaaaaaatg acatttatta ttcaactgat gagaaaattg tcatagtgaa ggcgttggga 1320 ggaataggtc tggttcatat tactgatcaa tctggatcag tagcatttta ttatgtggac 1380 ttcccagtaa cagaggtaaa atcaaaacat ggcgacgcaa tcctccagta catcaactca 1440 actagccatc cagtgggaac aatactagca acagttacaa ttcctgatta taagcctgct 1500 ccccgggtgg gttatttttc atcaagaggg ccttcattga ttacaagcaa tgttctcaag 1560 cctgatattg cagccccggg agttaacatt ctcgctgcat ggtttggaaa tgacacatca 1620 gaggttccaa aaggaagaaa gccctcacta tatcgcatac tctcaggaac ttccatggct 1680 actccacatg tttcagggct tgcatgcagt gtcaaaagaa aaaaccccac ttggagtgcc 1740 tccgcaatca aatctgccat catgacttca gcaattcaaa atgacaattt gaagggtccc 1800 ataacaacgg attcagggtt gatagccaca ccttatgact atggagcagg ggcaattaca 1860 acatctgaac cattgcaacc ggggctagtt tatgaaacca acaacgttga ctacttgaac 1920 tatttgtgtt acaatggact taacataacc atgataaagg tcatctccgg aactgtcccc 1980 gagaatttca attgtcccaa ggattcgagc tctgatctca tctccagcat caactaccct 2040 tccatagcag taaacttcac tggcaaagca gacgcggtcg tgagtagaac tgtcacaaac 2100 gttgacgaag aagatgaaac agtgtacttc cccgttgttg aagctcctag tgaagtaatt 2160 gtcacactct ttccatataa tcttgagttt acgacaagta ttaaaaaaca aagctacaat 2220 attactttca gaccgaagac ctccttgaag aaagatttgt ttggatctat cacttggagt 2280 aacgacaaat atatggttcg aattcctttt gtattaacta aatagtgaaa ttaaaaagta 2340 gcgatgaata aatgcaagct aagttcttcg tggtgcctac actcgagtcc tgattattta 2400 ttattcatat gccttctgtt ttaatttatt tattatactt tcagcct 2447 <210> 5 <211> 829 <212> DNA
<213> Glycine max <400> 5 gccttaaggc aacgacagcg agttcttctg ttgttcgttg actccaagga cggggtctta 60 gttggtggct tcgtggtttc cttctttggt ggcttcgtgg ttgttgtctt tgtcatcctt 120 gttgttgatg tcttcttcgg ttcggtctcc tcggatttct tcaacgtcaa ctctggctcc 180 tccaccactg tttcctctgt ctccttctct tcggtcgtcg tcttctcttt agtctcctct 240 tttgacttct tcactttctt cttcgtcttc tcggatttct ttgatgactt tgtctttgtc 300 ttagtcgtgg tcgtcgttgg tgtctccttc tcttgtttgg tcgactctgt caccttcggg 360 gtcatctcca aggacaactc ttttgacttc gaatctgaat acctgtcaca ctcactcttt 420 ttgttcaaat ttttaccagg atcaacaccc ctagtaatcc agatggtaaa acgtacagta 480 caacttttcg aaaaaaaaaa taaataaaga aatcaaatga aataataatt atataataat 540 aatatactac caattcagaa accaacataa tacctcccat atggatgcac actcgtgttg 600 tgaaccgagg tgtagtcgca cgaatggtgc accactgtac tctttaagat cctcaacaca 660 taacttcact cgttcgacga cgaccacgac ctcaaatccc tactcccaaa taaaataacc 720 gtcccaatgt actcaaacac tactacacaa taacacaaag aaatccaaac ttatttacta 780 taaatgaaat tttttttttt ttttttagat ctagctggcg tcgggttga 829 <210> 6 <211> 700 <212> DNA
<213> Glycine max <400> 6 taagctttca agagacaaac tgctttgaaa aatgggatcc aaggttgttg catccgttgc 60 ccttctcctc tccatcaaca ttcttttcat ttccatggtt agctccagca gccactacga 120 tccacagccc caaccttctc acgtcactgc tcttattaca cgacctagtt gtccggatct 180 gagtatttgc ctcaatattt taggcgggtc tctaggaacc gtggatgatt gttgtgccct 240 catcggtggt cttggtgaca ttgaagccat tgtgtgcctt tgcatccaac tcagggccct 300 cggaatatta aaccttaacc gtaatttgca gttaatatta aactcctgtg gacgaagcta 360 cccgtcaaac gccacttgcc cccgaaccta agaacagaat atgtatggca ctaattacca 420 tattacttcg tatcatggtg tttgtttgtt tgtctgtgtt taaagttaag gatgttatac 480 ccttcgtgcc tgctacatat atatagtggg cactataata ttaccaataa attaacgtcc 540 atatataaga ataataataa ataaataaat atttctatac aaataaaggt tacgtaatgt 600 tgttgttctc gtggatgggg atcttatctt cctcctcgct atctttgttt atcgtatttc 660 agtgaaagtt gttcaataaa agtcctttgt tcaacaagta 700 <210> 7 <211> 3368 <212> DNA

<213> Glycine max <400> 7 tctttcgatc aatactaata aagtcttatt tgccttccag agacaattga gtccgttggc 60 acgcagagac aaattatggt aatttgcccc tttttgaaga cttcaatgtc tttcgatcaa 120 gactattaaa gtcttctttg ccttctagag acaaattatg gtcatctgat tctttttgaa 180 tacttcaatg tctttcaatc aagacaatca aagttttttc gaatacttca aagtcttctt 240 tgccttccgg agacaattaa gtctgttgga acgcagagac aaattatgat catctacccc 300 atttcgaaga cttcaatgtc tttcgataaa gactattaaa gtcttctttg ccttccggag 360 acaatcaagt tcattggcac gtagagacaa attatggtca tctgcctctt ttcgaatact 420 tcaatgtctt tcgattaaga ctatcaaagt cttcttttcc ttccggagac aatcaggttc 480 tttggcacgc agagacaaat tatgttcatc tgcctctttt cgaagacttc aatgtctttc 540 gatcaagact atcaaagtct tctttgcctt ccgaagacaa tcaagtctgt tggcacgcaa 600 agtttgagga aaaattggac gaagatcggg acaaatggac cgtatggttt gacggagcgt 660 caaacattct aggccatggc attggggcag tattggtctc tccggacaat caatgtgtac 720 ctttcacaac caggctagga ttcgactgca ccaacaacga tcttcggtgc aattactcaa 780 atcggggaac taaggtggaa tgaagttgtc gtcattttgg gcaaggacaa gggagttgtt 840 ggagctaacc atggcatagc aaaggagatt gagggagagg ataattaggg catcaacacc 900 catggaaccc atttaatttc ccaacataga ttgatagaaa tattattgca gtctctcttg 960 atagcttaaa tattgatgag caagtgctct tgcttgtggt ttctagctga actttacagg 1020 tacgagtata agattactaa acttgtttcg atcctgaacc cgaactcgcc tgtcacttaa 1080 aatttttaaa atttttgcat aatttaatca aaaggcataa aatttttatt actagttaat 1140 ttttttttta gaatttttac ataatttaat attattttct taactatttt ttagatacac 1200 gcgctataat aaatttattt atatatatgt agttaaaaat aaatgtttaa taatcaattt 1260 attttttcta aaatcaaatt tttaatattt ttttacaaaa aaatattttt ctaagttgaa 1320 tcgtgtatgg gacggggtca gggatacccg atatctgacg ggtacgagga tgagacaata 1380 aacttaaatc cgtcgagtat tggatacgag tatgggaata tgttggggag tcggggtaag 1440 gaattgagga aacaatatcc atacccaccc gccctattgt catgtctaga cactacaaag 1500 aagggttaaa gaaacctaag ttaaaatagt agattatatg acatttagtc ctgtaaaaag 1560 aagaagagaa agatgtagaa aattttcaag aaagatatca agttaaataa tatttttcaa 1620 agtttgattt ttaattatat caaacaacgt agtgtgattc atgtaattgg tgacttacct 1680 actagtataa aaatttgttc tctttgttgt tgttgcatgt atggaatgaa ttttaaaaaa 1740 atcataaata taatttgaaa tcattttaaa attatgtaaa atcatttcga attattgatc 1800 tagattaaac aattacttag tgtaacaaga gaatttttgc ttagatttaa actttaatct 1860 ggctagcacc tagagattta tttttgtaat gatccatgac aatatcataa ttatgataat 1920 atatgtcata atttaaattt gtattcatct ttctttaaaa aatatacttg aaagtgttaa 1980 attgtacttc aaagatttag catattagtt tagttctgga taataaatta aaattattat 2040 tctcaaaaat gagataattc tttcatgtac aattcttcat acatagtatc aaatgtcttc 2100 ttcattttat gacacatgcc ttttaatttt tatattaata aaattaattt tttattaaat 2160 taaataatat tttaatctct ttaatgcttg aattaatata ttttttttta aaaaactaag 2220 catgacaagg tatttacaat ttactctaga aataatatac actaattaac acaagaataa 2280 gtatttttca aaatattttt tttttcatac aaaccacaag tatctgcaac aaaacttcct 2340 ttgagtgttt aagagagtta catacccaaa acagaaatgt gggaccgttg atcatcacac 2400 caattcaatt tattcagacg ctcgctttgt ggtaattggc ctataaattg tatcccaaac 2460 ttcagttaga caacaaaagc acttgttcac caattaagct ttcaagagac aaactgcttt 2520 gaaaaatggg atccaaggtt gttgcatccg ttgcccttct cctctccatc aacattcttt 2580 tcatttccat ggttagctcc agcagccact acgatccaca gccccaacct tctcacgtca 2640 ctgctcttat tacacgacct agttgtccgg atctgagtat ttgcctcaat attttaggcg 2700 ggtctctagg aaccgtggat gattgttgtg ccctcatcgg tggtcttggt gacattgaag 2760 ccattgtgtg cctttgcatc caactcaggg ccctcggaat attaaacctt aaccgtaatt 2820 tgcagttaat attaaactcc tgtggacgaa gctacccgtc aaacgccact tgcccccgaa 2880 cctaagaaca gaatatgtat ggcactaatt accatattac ttcgtatcat ggtgtttgtt 2940 tgtttgtctg tgtttaaagt taaggatgtt atacccttcg tgcctgctac atatatatag 3000 tgggcactat aatattacca ataaattaac gtccatatat aagaataata ataaataaat 3060 aaatatttct atacaaataa aggttacgta atgttgttgt tctcgtggat ggggatctta 3120 tcttcctcct cgctatcttt gtttatcgta tttcagtgaa agttgttcaa taaaagtcct 3180 ttgttcaaca agtgattcct tctctctctg tctttctttt cactttcgta ttttctttag 3240 gtataaggtg gcaaaaatag acaggaatat cgatcttgtg ataaaattaa aatcggtttg 3300 ctgatgtttt aattagttag aaaaaagaag acatatattt atcgtaattc ctgttcatga 3360 ttataaga 3368 <210> 8 <211> 7235 <212> DNA
<213> Glycine max <400> 8 gtcgactcga tctcaaattt tatttcattt aaaataaaac ataatttaat tttcgtctct 60 cttccttatt gtatcattat aaaagtagga aaacaaatat aaattagaac aaacataata 120 ttaattaata agaataattt gtttgttgct ttgaattttc tattctaata acattaggta 180 gtaataaaat taagttgagt ttcatttttt tgaaagaatt aacttaataa ttgtatattt 240 ttgtttaagt ataatatttt agataatgta ttatcacatt aaaaatttag agtgatagac 300 aaattatgtt tattaatcaa tattatgttt atttaattgt ttgttttaag ttaggtttgt 360 tttcatattt tttttagtgt ttttattata atgagaaaaa aaatggagga taaaagataa 420 aaattatatt atattttact cttaaataaa acaaaatttg gagtcctaaa aattagttta 480 atgaaaactt gttgacatag gtctaatcta ttcaataatc atgttagctt atttgtgctc 540 ttggactcct tcattaacgg taatagatga atgaatttat tgcatttctt tcacttttga 600 ttactaaaat ttaaattttc atttttcgaa aatgaattcg ttactatttt atacatttaa 660 aaataaaaat gattatttca tcttataatt atcaaataat atgcctgtga tgatgatttt 720 tttcaaaaat tgaaaatgtc ttattgccta gttaggctat agaatctttt tggctcatct 780 caatcgcatg gcgacatggt taccgcgttc attgggtaat tatatttatg tatttaaatt 840 aattctatta aaaccaattc tattaaagct gaaaactaaa atgacactta atttctgtaa 900 gagtcgtgta attagcgagc cagagtaatg caaccaaaga gcttctttcc ccaccttaat 960 tcctttataa tgaatttgat tgataccata catagctaag cttttttttc tttcgattta 1020 catagccaag ctttagctgc tatatataat atgttgttta tataattttg acatgttgca 1080 ttacatgtta ctaactggtt gcaacct.cca gttttggtta tcgaatatat gctggccggc 1140 accaactaca aaaaatttct gtatggctgg ctactaatac tatatcttca aatgtctcac 1200 tttacatgga ctgagtgcat aacgggcaat tgcagtgctc ataaggaata cgcatgcaat 1260 cattttatca tcgcagattg ttgcttttcc ttcactgtaa aaaaaaataa aaataacaat 1320 taattgagca ccgaatgaac tagaccgatt gcatgcatta taatacatga agaaaattat 1380 ttatactaat cttttgtttc aagttttaaa gttgactttt ttgttttaca agcacaataa 1440 caatcaaata accaataatt taatcattaa aaagataaac aaactcatta gataaaataa 1500 taattatgat aaaatatgtt taagtcctct atatttaagt tgaattttgt ttttagtctt 1560 tcaaattgtg cattttagtc tctgaaattt tattttaaaa aaatagtctt tgggcttact 1620 ctccaaaatt tgcacatttt aattttaggt tattgattaa agattaaaaa catttttttt 1680 tatcaaattt agggaccaaa ttacttgata taaatactca agaactaaaa acaaatttca 1740 ataaaaatat aagggattaa aaatatattt tattctgata actaacaatc atcaaatttt 1800 atgacaatca tatgtaacct tttataatat aattgtaaga ataattaatt ttaataaata 1860 aataaataaa tgagacttat aatagtaaat acatatgttt gcctaggtaa taacaatgat 1920 aacaagtaat agaattatca ttcttattgt atctattaat taatatatta ttagatgtat 1980 taattagtat atatattttt tactataaat agtatatatt attaattatt atacatttat 2040 taaagtatat tcattaaaga ttttgaggga ggggagctga agccactaac ccccgtaaat 2100 ccgtccttgc acaaaacacg acatgagaat ggttttgtat actccacaat ttaatatcca 2160 ataaaataat ttctttttta ttttaattag gaaactccaa gattgcttta acttatacaa 2220 aatctgaata acacaaaaaa ataaaaaatc ggaattaaat gttgcccgat tgttgactaa 2280 cttatacaaa atcttattta aatgcttaaa atcgtgtcat aatataatga atatatattt 2340 gcaaatatat atttattata taattgcaaa tatatattct aattttgagt ataaataaca 2400 gcatgtgagg gtgcagcaaa acacacactg agtgcaacaa agttttaaca tgaaaggcaa 2460 taatacactt ttgttgcatt tattctacac tactctcttc ctgtttcttg tagtgtcaag 2520 ttcatcttca acagggaatg aaagtaacga tgacactaac agtaaagaag tttatatcgt 2580 gtacatggga gctgcagatt caacaaaagc ttctcttaaa aatgagcacg ctcagattct 2640 gaattcagtg ctaagaaggt acgtataatt acataatatt attattatat gggcaccaat 2700 taattaattt gatgattgat gtgtttacat attttgtgtg aatgaattga aggaatgaga 2760 atgccctagt acggaactac aagcatggtt tctctgggtt cgcagctcgt ctatcaaaag 2820 aggaggcaaa ctcaattgct cagaaacctg gtgtggtgtc tgttttccct gaccccattc 2880 tgaagctcca cactacacgt tcatgggatt tcctcaaaag ccaaactcgt gtcaatatcg 2940 acaccaaacc aaatacgctg tccggttctt ctttttcttc atcagacgtc attcttggcg 3000 tcttagacac aggttgtcca taatcaaaaa aaaaaaaaaa acatgatata tatgtgtgtg 3060 tttcattttt taaaaatgtt aataataata tatacaaaaa tggaatattt caggcatatg 3120 gccagaggcg gcgagtttta gcgacaaggg tttcggtcct gttccatccc gatggaaagg 3180 cacctgcatg acatcaaaag acttcaattc ctcttgttgt aacaggtaaa ctaaaatgtg 3240 aaaccataat aataataata ataataataa taaatatata aaggcgaacg ttattaatta 3300 ttaattatta ttagaaaaaa ggtgatttca gcttgctgtt taagaaggtt tggaatgaat 3360 cctatttaat taggtagtgg atggaataac ggttaggttt gtatttatag gaagataatt 3420 ggcgcgaggt tttaccctaa cccagaggag aaaacggcaa gggatttcaa cggacatggg 3480 actcacgttt cgtcgacggc agtgggcgtg ccggtgagtg gcgcatcgtt ctatggtctg 3540 gcggcgggga cggcaagggg tgggtcccct gagtcaaggt tggcggttta caaagtgtgt 3600 ggggcttttg ggtcatgtcc tgggtcggcc attcttgcgg ggtttgacga tgccattcac 3660 gacggagtgg atatcttgtc gctgtcgctc ggtggattcg gtggaaccaa aaccgatttg 3720 accaccgacc cgattgcgat tggagcattc cactccgtcc agcgcggcat cctggtggtc 3780 tgcgccgccg ggaacgacgg agaaccattc accgttctca acgacgcacc ttggatttta 3840 accgttgcag cttccaccat cgaccgtgat cttcaatccg acgtggtctt gggtaataac 3900 caagtcgtca aggtacctac atattctact ttaaatcggt gcagtgcaac taatgtcatc 3960 ttttctcatc gttgataatt attaaacttc agggaagagc cataaatttc tcccctcttt 4020 taaattctcc cgattatcca atgatatatg ctgagtctgc tgccagggca aatatctcca 4080 acataactga tgcaaggtac gtactctaaa aaccatttgt cgtttcgtat tggacaaact 4140 tcaaatcaag caatcaacta agcaataaca aacaagtgtt tcatcaccaa ttatatgtaa 4200 tactcatata taacctctta gcaaatgatt aaatcatttg tcacatgcag acaatgccac 4260 ccagattcat tagatccaaa aaaagtcata gggaagattg tggtttgtga tggaaaaaat 4320 gacatttatt attcaactga tgagaaaatt gtcatagtga aggcgttggg aggaataggt 4380 ctggttcata ttactgatca atctggatca gtagcatttt attatgtgga cttcccagta 4440 acagaggtaa aatcaaaaca tggcgacgca atcctccagt acatcaactc aactaggtaa 4500 ggatattata tagcacttga aagaagcaac attcttgatt aattttagaa tttgctttga 4560 tcacgagtta ttttctttta attctttgtg catatatgta atataaagcc atccagtggg 4620 aacaatacta gcaacagtta caattcctga ttataagcct gctccccggg tgggttattt 4680 ttcatcaaga gggccttcat tgattacaag caatgttctc aaggtatgat atgacgatcg 4740 atagaattat acatatcaat catcatcctc aatatgctca ttgctcaaac actaaacaga 4800 acattcattc tttctttctt tctttctttc tagcctgata ttgcagcccc gggagttaac 4860 attctcgctg catggtttgg aaatgacaca tcagaggttc caaaaggaag aaagccctca 4920 ctatatcgca tactctcagg aacttccatg gctactccac atgtttcagg gcttgcatgc 4980 agtgtcaaaa gaaaaaaccc cacttggagt gcctccgcaa tcaaatctgc catcatgact 5040 tcaggtcacc catttgataa tgtgatctaa gtaagtaatg tgatccagca aaatgtacca 5100 taccaactca tatcattcta taaattaata tgtatgcagc aattcaaaat gacaatttga 5160 agggtcccat aacaacggat tcagggttga tagccacacc ttatgactat ggagcagggg 5220 caattacaac atctgaacca ttgcaaccgg ggctagttta tgaaaccaac aacgttgact 5280 acttgaacta tttgtgttac aatggactta acataaccat gataaaggtc atctccggaa 5340 ctgtccccga gaatttcaat tgtcccaagg attcgagctc tgatctcatc tccagcatca 5400 actacccttc catagcagta aacttcactg gcaaagcaga cgcggtcgtg agtagaactg 5460 tcacaaacgt tgacgaagaa gatgaaacag tgtacttccc cgttgttgaa gctcctagtg 5520 aagtaattgt cacactcttt ccatataatc ttgagtttac gacaagtatt aaaaaacaaa 5580 gctacaatat tactttcaga ccgaagacct ccttgaagaa agatttgttt ggatctatca 5640 cttggagtaa cgacaaatat atggttcgaa ttccttttgt attaactaaa tagtgaaatt 5700 aaaaagtagc gatgaataaa tgcaagctaa gttcttcgtg gtgcctacac tcgagtcctg 5760 attatttatt attcatatgc cttctgtttt aatttaattt attatacttt cagcctctct 5820 aatatgtttt tttcttttgc aaatatataa gctgacttac tatttacact caaaattagt 5880 tccaacttat tcactagccg tttgccctca gcttaattaa aaaaaagaaa tgtgatttaa 5940 ttacattaat tatagctgga tcgtagtaac ctcggatttt tacacgggtt ggtaattcaa 6000 catcaatttc atgcttcaaa tgcaaactcc tcaaaagtag ttgcagacta aaatgatgaa 6060 tttttaacaa aacttgtaca aaggtaaggg ggaactaggg aagtgagctc ataaaataag 6120 gaaactgttt cgactgagtt ttgagtaagg tgtggctgaa ttttggcttg agttgtggtg 6180 agctgtagca gagtttcgac tttgttgtgg taagctatga cttagttttg acgaattgtg 6240 gtgagttgtg gtcgaatgga atcttggatc tcctaatccg gtgtaggaga agtacctaca 6300 aaaaggactc caacaatcaa ctcaattgga tccgagatac ttatgtatcg atgtatgaaa 6360 tgattaaaac ataccttgtt gtgttttatt tatgtcaata tatacatatt cgacattaag 6420 gaggttacac taactagata gtcctctaag cgttgcgtga tcgtgaaaag ttagtgatca 6480 tcactatcga gttcataggt tgtacggtac ataaatccca ctaatcgagt aactatcaag 6540 ttcatggggg ttgtacggta cataagtctg tcagattccc atgatgggta ttgctaagtt 6600 gaataaatcg ggcatataca ttacacgagt ttaagatgat ttaattttcc ttatatatca 6660 ttattttata ctgggtgagt gttttctttg aaaaactgag gtgtggatcc aacctcttga 6720 gaagtgcttt taaaaaaatg aggtttggat cctatctctt gagaggtgct tgaaaaagct 6780 tatcaaaata gttattggca ttcaatgttg ttttcaaccg agggcaatta aacacacctt 6840 tgattagtgg gcaattgaag ttagaaagaa gcttataaag gatagtatta ttatactatt 6900 acaaccaagc aaacaatgtt tacttcaaag ttgatacctt attgatataa tgtattattt 6960 actatgagtt aaaatgtaat ttgaaaaaaa aaatgtaact gaaaatgttc ggatgatttt 7020 catattttgt aaaaaaaaaa aaaaaagaaa aaaaagttat taggctaatt tttgcaaagt 7080 acattttggt atgaaaaacc aaaaacagaa gtaatgcatt ttgtgccatg gcagaatgca 7140 gagtatctac ccaggattca ttatgaacaa cttaatgcta agcatcatca gttaaccccc 7200 caaattaatc ttgaaagcta caattctatt ctaga 7235 <210> 9 <211> 8310 <212> DNA
<213> Glycine max <400> 9 gagctccgcg aaatttgtta tggccatact cttccttgcg agccctcttg gtctcttgtt 60 caagggctct tgcggtagtt gcattctctt cccgtaattt ggcacactcc ttccggatgt 120 gtgtagcggc taacttgaac ttctccttgg caagtttcgc ctttcctaac tcgtttttga 180 gagcttggac ttcttcgtct tcttccggtg cttcgaaact gtctttgctg acgactttta 240 acttggcgag ccaatctaaa cctcgtattt gaactttcag ccattcatga taaccaccaa 300 tgatgccatt acgaatgccc ctaagttctt gatctttcct taacggggtt tcccatgcct 360 tatggattct ttgtatagcc ttgaaatttt gcatgccgaa atctctcaca aggaaaggag 420 aaatcctttc ttccatcggt gttcccctca tggggtaccc tagttgtctt atagcgagcg 480 cgggattgta gttgatacaa cccctcgttc ttatcagtgg aatgtttggg taccctccac 540 atgagaaaag gactccctcc tttccttcct tccatcgggg gaaccaacta attgttctac 600 ctcctatctc ggccaagagt tggtcccaat ctattcctct cttttcagca cacgagtgat 660 ggctttggag cggacatgga tgcctcgtgt tttgctggaa caggtgtgaa accaaccaaa 720 cacagagagc gggcaagcaa cagatgatcc atgcgctact cttctcgcac cttcggtcaa 780 atgtgtcaaa taaatctgcc aagacagcta ccaccgggct ttcctgtttg gaaagaggac 840 gaccccaaag attagcaatg ctaacacatc cataaacggg acccaatctt cttgatttgc 900 catatccctc gccttgtctt ctaggtactt ccgtggtagg cccgctatgc cgtttcgagt 960 ttgttttacg cggtccaaac ctcttgctga atctttgacc acggtcgcaa ttctgctcaa 1020 agaggggaga cacccggaga aaagatatga ttttcttccc ccgagaggac atcctagaat 1080 ctcctcaaat tcttcaatgg tcggtaccaa ttggaagtct ccgaacgtga agcatctcaa 1140 aggctggtcg tagtattggg tgagtgacgc aatggcctct atggatacct ctggtatggt 1200 caaatctaag atcgatcttg gattgttgtt ggttttgttg aagtattttc taaccttttg 1260 aatatgttac taatgtcaaa tttattattt gttataaaat ctttttgtct gggtttattt 1320 tctagggttt tctttaattt ttccagaaaa actttctttt cctggggatc aggtatagaa 1380 ttgattgcct caaacaagag atcgatctct ccgaaattgc acacagtggt caatgtctca 1440 gcatatctgt tttatagttg tggattctat aagtcaattt agattattgt taattaccta 1500 agttattgtt ttaaattcat acataattaa ctttgtctta acaacaacag acatctgact 1560 gagacaagta tgtgagcggg gaattctgat gtgtatggat tcctcgagcc acagtctatt 1620 cagagatctg ggcaatcgca gtttgaatca gaaagttaca tcaagagttg gatgcagagt 1680 tcaaaacgcg atgtctacct tggagcctac ctgaatgggt aagtcaaata aaacaactaa 1740 atttaaataa tatataatac tacaataacc catattcatc tccactgcag cggacacagg 1800 aaaatggtct ttattctgcc taaggaaaac cttgttgtct ggttttgttc cttgcataac 1860 aggtcagaca actaccttaa gggaataatt aacaggtcag tgttgttttt aatacatttg 1920 cattagcata actcaacaac atcaatattt taatgttcct cgtattcaac agtgctttga 1980 aaggacttga tgatactcca caatctaaat ccaaggctgg tgctaggtgg atcgttgtta 2040 aggtacgtga tttaaataaa acttctactt atatatactg cttatgtgtg tgtacactaa 2100 ttgtagttaa cgtttaatta atatccaaat ttcattatgt atttagtgta atagacaaaa 2160 aggaagcact gagtgcggct attacatcat acactagatg tccatgataa tcttaggaac 2220 ttttaggaat aattgggaaa ccgtaattgt ttatttcaaa caaagtttat tttcttgtca 2280 ttggtattac attattaact tatgttttat ttgatcatgc agtattttaa cgatgttaaa 2340 ccattggaag caaagagatt gaaggtgctt cgcatccagt gggcacaatt ttatctcaaa 2400 gttacaaatc aaagttagga tgttagggaa ctatttcatt ttaggttact taattagttt 2460 actaccttgt tttacatttt tgccaattgc tatgccattt gaacattgaa tattcttaat 2520 tgatagttat taataaatca tttattcata gttatcaatt gatagtttac tatagcttta 2580 tactaaaaac cgtttaaaag cagaatgcaa atgatatatg ctgtgatcta tggtctgatt 2640 tcaattttac aggttcaatt ttgagttttt ttgtaaaaac aaaaagtgta ttaaaaaaaa 2700 ctaaaaacaa aatcggtttt ttacaaaaat cgatgttaac atacaactta acatcggttt 2760 ctcaaaaaac cgatgtttgt gtatattaac attgattttt gtaaaaaacc aatattaacc 2820 tatgaaaatt taacattggt ttttatacaa aaccgatgtt aacttataga ttaacatcag 2880 ttttgtataa aaacttatgt taacgtttct aacttaacat caatttttat agaaaaccga 2940 tgttaatata tgagttaaca tcgattttga tacaaaactg atgttaatct ataagctaac 3000 atcggttttg tagataaccg atattttaaa ttttacgtta aactgatgtt aacgataata 3060 ctttcaacat cgattaaaaa tcgatgtaga aagtcgtaaa taaccgatgt agaaaatcta 3120 ttttctaata gtgtccatct caatatatgt ttggatagtg tcggcattac cctggtttga 3180 ctaatgcttt tgcactcaaa ttgtcacacc ataaaatcgg tgttctgaga atgggcaagc 3240 taatcttctt gatccattga gaacctaatt ttcattattt ttttacctaa acataaaaac 3300 acaaaaatat aagaaaataa aataaaatcc ttatctaaat tcagaacata aaataaataa 3360 catctttcat gttaatattt taaaattcat tattttgaag ccaaaatggg ttaaaattta 3420 taacaagtga cttaaaatta aaatttccaa aaataagtag aaaattagat aatttaaaaa 3480 ttaccaaact cattttatcc ttcatgactg ttatgtactg atgcagtgta attacccaca 3540 ttataaatgg taattacaaa ctagtactac cttataaatt aattaccccc attctcctat 3600 cttacaaata aaattaaaaa tacggacaaa catggcagtg gtgtggtttc gctttaaaaa 3660 taaaaatata caaacatgaa agtgatgtgt tttcgttgtt atatataata atgctcacga 3720 tcgagactca actaaatgat cacgcaaatg tttttcttag gatgaagcct gttgctaatt 3780 tcttcatctc aaaatccggt cattacgcaa tttatagatt tgtcgagatt ttggcttctt 3840 ctttaaattt gcgcataaaa acttatcaat gtatacctta tggctgcatg aaattaccta 3900 ttcttatggt tgcatattaa atgttcatac tttttatatt attttattta gattaaattg 3960 taattttgat ccctattttt aaattaaggc attccattaa tggaccattt atgtgacaaa 4020 agattaaata acttaaaaat acaaaaaggt cttagtataa attaaattta aaatatttta 4080 tataaagata aaataataaa accataaaat acttacttta tttagttaat tttataaata 4140 ctattttaca tgtatcatta attagtccag aattattaat ttttttaatt ataaaaattt 4200 ataaattgaa ataaaatatt taatatttaa atatatttaa catttaattt tacatgtact 4260 tatttttctt ataaaatttt attttaaaat aaatcaacta aaatttgttg ttattttagt 4320 ataattttaa aaaaatacat aaactaatat atagattatt ttaaaattat tttatctaat 4380 ttatgaaaat taagtagatt taaaataaca tttaactaat ctaaaattta aaatttatta 4440 cttgttataa ttaaaataaa ctttttcatt atttatatat aaaaataaat gtagataatt 4500 ttatcaatta cataaaataa tttatatata actaatttac atattaattt atttaatttt 4560 cctcatttat ataattgata ttgaaaaact tatttactaa atatttttta tagttaaaat 4620 aaaaatatta acatgttttt ttcctaaaaa cattaaatat tcaaatttta ttcatttatt 4680 taattaaaat aaaggtaatt tacatattaa aataaattac ataatttgta taatttatat 4740 attaataaaa aaatatataa attaatttaa attagtcaaa taaaaataaa aatatataaa 4800 ctattcaata tatatatata tatatatata tatatatata tatatatata taatataatg 4860 acatacaaga aaattaaaag gatttataaa aaggatttat atattaaata ttttttaatt 4920 cataaatttt tataatttaa aaaattaact catacataaa taatatttaa aaaattagtt 4980 aaaaaaataa atatcttaat agtttgttat tttaatttta aataagagat tataagttta 5040 attcttatta ttttttttca taattttaac tcttaccaag tatttttttc actttgttta 5100 atctctattc tctctctctt tatatatata tatatatata tatataataa atttctaatc 5160 aactaaatag tgtatatttt acatgcagag tacataaaaa ttaaaattgg gtataatagt 5220 tattgtacgt catctcactc actcttcctc ttagagtcta tatatatgca atatctctgg 5280 tctccctatc accttctctt ctctaagcac tttacttttt ttttcagcca tggagtttca 5340 ttgccttcca atatttcttt atctcaatgt gagcataaac cttccttttt catctcgttt 5400 tttaacctgt attgcgtgaa gttggatttc ttcattaatt atcattttaa ttatagcaat 5460 aagtatcaag tgatgggttt ttgcatgaat tatgtagttg atgttgatga cagccaatgc 5520 tgcgttaact cctagacatt actgggaaac gatgcttcca agaactccct tgccgaaagc 5580 aatcacagag ctactaagcc ttggtgagta agaaatcaaa ttgaaataaa ataagcaaca 5640 cttttgtaat taaatctgaa acgataaatg tacgtaaaca aaaaatagaa ttagaaacca 5700 atgcaaacag ctataagcct ttttttgtaa tagctaatct gccttaatta aaccattgaa 5760 tgactcaata tgttcatctt tttttccttt tagtagaata ctgcccttct tcatattcga 5820 tatactattg gcttccttag tgaattctta ttctaatggt tgcatagctg tgttctcact 5880 aagcctcctt tcccatgtct ttgattctat gaaacgaaac tttcaaaact tggaatcacc 5940 tattaagtac aataaagaaa taattaattt tatttttatc ctttaattat tttatgaatt 6000 tggttccttt attttatttg atatattttt tacaaatttg gttttctcat tattttaatt 6060 gtttattttt tgttcatctc ttaacttaat atccaacatt tcataaatat gctgacataa 6120 tactagatta ttatatcacc tgtggggata ttcatgttgc acaattagtg gaggctcaaa 6180 ttcgtgaaaa aataaataaa atttggagag accatattca ccaaatcaga atagtataca 6240 gagacaaaaa aaaaaaaaaa aaacaaatta tgcaacgaat gaaaagttaa ccattatatt 6300 tagtattgag taatttgaag tcagtgtgag acatggcaac gtagttaact tactttaatt 6360 aaaaatatga ataattaaaa taggaatata taacggattc ggattctctc cagcaatgat 6420 gaggacgtag gattaatttt atggctgaga gtaaatccaa aaagatacgc aaaccacgta 6480 tcaccgctac tgcttgacca aatggaagcg acttcaaacc aaaattagga caaagaaaat 6540 aaaaatacaa atgtcaataa actcaatata caaagacaga gtgtaagttt tactaaatgg 6600 aaagctggaa ttgaagtaaa agctttacat atatattgct tgctaaacaa ttttgaattt 6660 tttttatctt ggattcttgc taaacaatta attatatcta ttaatatttc tctaataata 6720 tgggatacac ttattattat tattattatt attattatta ttattattat tatttgattt 6780 aaatataatt ttgatatttg actaatatta aatttatttt aaacaatcac aaatttcgaa 6840 taattaatga taatattaag ttattttcat atttgactag tatcaaaaag ttatttacac 6900 atttgactta ctgttatttt attttagttc tctagttttt aaaagtataa taagttagtt 6960 ctttcatttc ttttctattt tccattttaa ttaaaaaaaa ttcaattttt attttatttt 7020 gtaacaatgg gctgaaaata gctactaaaa ttttggctgt gtttttcttt accaatcaga 7080 aagtaggtcc atatttgaat atgccgggaa tgatgaccag tcagaaagta ggtccatatt 7140 aggatacgct ggctataatc aagacgagga tgatgtgagc aaacacaata tacaaatctt 7200 caacaggttg tttttcttgg aagaggacct gcgtgctggc aaaatattca acatgaagtt 7260 cgtcaacaac acaaaagcca cagtcccgtt gctaccgcgc caaatttcga aacaaatacc 7320 gttctcagaa gataaaaaga agcaagtgtt ggcgatgctt ggcgtggaag cgaactcaag 7380 caacgccaag atcatagcgg agaccattgg tctttgccaa gagcctgcaa cggagggaga 7440 aaggaaacac tgcgcgactt cgttggagtc catggttgat ttcgtcgttt ccgcgctcgg 7500 gaagaacgtt ggtgctttct caacagagaa agaaagggaa actgagtctg gaaagtttgt 7560 agtggtgaaa aatggggtga ggaagttggg agatgataag gttattgcct gtcatccaat 7620 gagttaccct tatgttgtgt ttgggtgtca tctagtgcca aggagtagcg ggtatttggt 7680 gcgcttgaag ggagaagatg gggttcgagt gaaagcagtt gttgcgtgcc acagagacac 7740 gtcaaagtgg gaccataatc atggggcatt caaagtgctc aatcttaagc ctgggaatgg 7800 tacagtatgc catgtcttca ctgaggggaa tcttctttgg cttccaaatt agattaatta 7860 ccatatacat atttgtcctt gttctatcct taaataagtg gaatcacctg aagaattgtg 7920 cgtaatgagt tgtttgtctt tgtggaaatt gttatctgtc ttgcatcacc aaataggtat 7980 atataaaata acaggagcgt ggtatttgtt gcacaaaaat ggatttcaac cgatcaaaaa 8040 aatatagcct ttaccaatta gaagggtttg gctttgttag caaataataa aaataaaata 8100 tcttgatgga taaatggttg ctaagttgat taagattgtg gcagaatacc aagtcaatga 8160 atagtccatc acaagcatca aaagaaaaga taatgattcc ttgaaaatag aaagcacttt 8220 gtgttttgaa ttcaaaatgc acactggaga gttgttggag gttacaagcc agatcgagtc 8280 gactcccttt agtgagggtt aattgagctc 8310 <210> 10 <211> 119 <212> PRT
<213> Glycine max <400> 10 Met Gly Ser Lys Val Val Ala Ser Val Ala Leu Leu Leu Ser Ile Asn Ile Leu Phe Ile Ser Met Val Ser Ser Ser Ser His Tyr Asp Pro Gln Pro Gln Pro Ser His Val Thr Ala Leu Ile Thr Arg Pro Ser Cys Pro Asp Leu Ser Ile Cys Leu Asn Ile Leu Gly Gly Ser Leu Gly Thr Val Asp Asp Cys Cys Ala Leu Ile Gly Gly Leu Gly Asp Ile Glu Ala Ile Val Cys Leu Cys Ile Gln Leu Arg Ala Leu Gly Ile Leu Asn Leu Asn Arg Asn Leu Gln Leu Ile Leu Asn Ser Cys Gly Arg Ser Tyr Pro Ser Asn Ala Thr Cys Pro Arg Thr <210> 11 <211> 286 <212> PRT
<213> Glycine max <400> 11 Asn Ala Ala Leu Thr Pro Arg His Tyr Trp Glu Thr Met Leu Pro Arg Thr Pro Leu Pro Lys Ala Ile Thr Glu Leu Leu Ser Leu Glu Ser Arg Ser Ile Phe Glu Tyr Ala Gly Asn Asp Asp Gln Ser Glu Ser Arg Ser Ile Leu Gly Tyr Ala Gly Tyr Asn Gln Asp Glu Asp Asp Val Ser Lys His Asn Ile Gln Ile Phe Asn Arg Leu Phe Phe Leu Glu Glu Asp Leu Arg Ala Gly Lys Ile Phe Asn Met Lys Phe Val Asn Asn Thr Lys Ala Thr Val Pro Leu Leu Pro Arg Gln Ile Ser Lys Gin Ile Pro Phe Ser Glu Asp Lys Lys Lys Gln Val Leu Ala Met Leu Gly Val Glu Ala Asn Ser Ser Asn Ala Lys Ile Ile Ala Glu Thr Ile Gly Leu Cys Gln Glu Pro Ala Thr Glu Gly Glu Arg Lys His Cys Ala Thr Ser Leu Glu Ser Met Val Asp Phe Val Val Ser Ala Leu Gly Lys Asn Val Gly Ala Phe Ser Thr Glu Lys Glu Arg Glu Thr Glu Ser Gly Lys Phe Val Val Val Lys Asn Gly Val Arg Lys Leu Gly Asp Asp Lys Val Ile Ala Cys His Pro Met Ser Tyr Pro Tyr Val Val Phe Gly Cys His Leu Val Pro Arg Ser Ser Gly Tyr Leu Val Arg Leu Lys Gly Giu Asp Gly Val Arg Val Lys Ala Val Val Ala Cys His Arg Asp Thr Ser Lys Trp Asp His Asn His Gly Ala Phe Lys Val Leu Asn Leu Lys Pro Gly Asn Gly Thr Val Cys His Val Phe Thr Glu Gly Asn Leu Leu Trp Leu Pro Asn <210> 12 <211> 770 <212> PRT
<213> Glycine max <400> 12 Met Lys Gly Asn Asn Thr Leu Leu Leu His Leu Phe Tyr Thr Thr Leu Phe Leu Phe Leu Val Val Ser Ser Ser Ser Ser Thr Gly Asn Glu Ser Asn Asp Asp Thr Asn Ser Lys Glu Val Tyr Ile Val Tyr Met Gly Ala Ala Asp Ser Thr Lys Ala Ser Leu Lys Asn Glu His Ala Gln Ile Leu Asn Ser Val Leu Arg Arg Asn Glu Asn Ala Leu Val Arg Asn Tyr Lys His Gly Phe Ser Gly Phe Ala Ala Arg Leu Ser Lys Glu Glu Ala Asn Ser Ile Ala Gln Lys Pro Gly Val Val Ser Val Phe Pro Asp Pro Ile Leu Lys Leu His Thr Thr Arg Ser Trp Asp Phe Leu Lys Ser Gln Thr Arg Val Asn Ile Asp Thr Lys Pro Asn Thr Leu Ser Gly Ser Ser Phe Ser Ser Ser Asp Val Ile Leu Gly Val Leu Asp Thr Gly Ile Trp Pro Glu Ala Ala Ser Phe Ser Asp Lys Gly Phe Gly Pro Val Pro Ser Arg Trp Lys Gly Thr Cys Met Thr Ser Lys Asp Phe Asn Ser Ser Cys Cys Asn Arg Lys Ile Ile Gly Ala Arg Phe Tyr Pro Asn Pro Glu Glu Lys Thr Ala Arg Asp Phe Asn Gly His Gly Thr His Val Ser Ser Thr Ala Val Gly Val Pro Val Ser Gly Ala Ser Phe Tyr Gly Leu Ala Ala Gly Thr Ala Arg Gly Gly Ser Pro Glu Ser Arg Leu Ala Val Tyr Lys Val Cys Gly Ala Phe Gly Ser Cys Pro Gly Ser Ala Ile Leu Ala Gly Phe Asp Asp Ala Ile His Asp Gly Val Asp Ile Leu Ser Leu Ser Leu Gly Gly Phe Gly Gly Thr Lys Thr Asp Leu Thr Thr Asp Pro Ile Ala Ile Gly Ala Phe His Ser Val Gln Arg Gly Ile Leu Val Val Cys Ala Ala Gly Asn Asp Gly Glu Pro Phe Thr Val Leu Asn Asp Ala Pro Trp Ile Leu Thr Val Ala Ala Ser Thr Ile Asp Arg Asp Leu Gln Ser Asp Val Val Leu Gly Asn Asn Gln Val Val Lys Gly Arg Ala Ile Asn Phe Ser Pro Leu Leu Asn Ser Pro Asp Tyr Pro Met Ile Tyr Ala Glu Ser Ala Ala Arg Ala Asn Ile Ser Asn Ile Thr Asp Ala Arg Gln Cys His Pro Asp Ser Leu Asp Pro Lys Lys Val Ile Gly Lys Ile Val Val Cys Asp Gly Lys Asn Asp Ile Tyr Tyr Ser Thr Asp Glu Lys Ile Val Ile Val Lys Ala Leu Gly Gly Ile Gly Leu Val His Ile Thr Asp Gln Ser Gly Ser Val Ala Phe Tyr Tyr Val Asp Phe Pro Val Thr Glu Val Lys Ser Lys His Gly Asp Ala Ile Leu Gln Tyr Ile Asn Ser Thr Ser His Pro Val Gly Thr Ile Leu Ala Thr Val Thr Ile Pro Asp Tyr Lys Pro Ala Pro Arg Val Gly Tyr Phe Ser Ser Arg Gly Pro Ser Leu Ile Thr Ser Asn Val Leu Lys Pro Asp Ile Ala Ala Pro Gly Val Asn Ile Leu Ala Ala Trp Phe Gly Asn Asp Thr Ser Glu Val Pro Lys Gly Arg Lys Pro Ser Leu Tyr Arg Ile Leu Ser Gly Thr Ser Met Ala Thr Pro His Val Ser Gly Leu Ala Cys Ser Val Lys Arg Lys Asn Pro Thr Trp Ser Ala Ser Ala Ile Lys Ser Ala Ile Met Thr Ser Ala Ile Gln Asn Asp Asn Leu Lys Gly Pro Ile Thr Thr Asp Ser Gly Leu Ile Ala Thr Pro Tyr Asp Tyr Gly Ala Gly Ala Ile Thr Thr Ser Glu Pro Leu Gln Pro Gly Leu Val Tyr Glu Thr Asn Asn Val Asp Tyr Leu Asn Tyr Leu Cys Tyr Asn Gly Leu Asn Ile Thr Met Ile Lys Val Ile Ser Gly Thr Val Pro Glu Asn Phe Asn Cys Pro Lys Asp Ser Ser Ser Asp Leu Ile Ser Ser Ile Asn Tyr Pro Ser Ile Ala Val Asn Phe Thr Gly Lys Ala Asp Ala Val Val Ser Arg Thr Val Thr Asn Val Asp Glu Glu Asp Glu Thr Val Tyr Phe Pro Val Val Glu Ala Pro Ser Glu Val Ile Val Thr Leu Phe Pro Tyr Asn Leu Glu Phe Thr Thr Ser Ile Lys Lys Gln Ser Tyr Asn Ile Thr Phe Arg Pro Lys Thr Ser Leu Lys Lys Asp Leu Phe Gly Ser Ile Thr Trp Ser Asn Asp Lys Tyr Met Val Arg Ile Pro Phe Val Leu Thr Lys

Claims (22)

WE CLAIM:

1. An isolated nucleotide sequence comprising the nucleic acid sequence defined by SEQ ID
NO:7, a nucleotide sequence that hybridizes to the nucleic acid sequence of SEQ ID NO:7, or a nucleotide sequence that hybridizes to a complement of the nucleotide sequence of SEQ ID
NO:7, wherein hybridization condition is selected from the group consisting of:
hybridizing overnight in a solution comprising 4 × SSC at 65°C
and washing one hour in 0.1 × SSC at 65°C;
hybridizing overnight in a solution comprising 4 × SSC at 65°C
and washing 30 minutes in 0.1 × SSC, 0.1%SDS at 62°C;
hybridizing overnight in a solution comprising 50% formamide, 4 × SSC at 42°C and washing one hour in 0.1 × SSC at 65°C;
hybridizing overnight in a solution comprising 50% formamide, 4 × SSC at 42°C and washing 30 minutes in 0.1 × SSC, 0.1 %SDS at 62°C; and hybridizing overnight in a solution comprising 0.25M Na2PO4 buffer at pH 7.2, 20%
SDS, and 1 mM EDTA, and 0.5% blocking reagent, at 65°C and washing at 22°C in a solution comprising 20mM Na2HPO4, 1% SDS and 1 mM EDTA, followed by washing at 68°C in a solution comprising 20mM Na2HPO4, 1% SDS and 1 mM EDTA;
wherein the nucleotide sequence exhibits either promoter activity, imparts a dull luster to a seed coat, or both.

2. The isolated nucleotide sequence of claim 1 comprising nucleotides 1-2525 of SEQ ID
NO:7, a nucleotide sequence that hybridizes to nucleotides 1-2525 of SEQ ID
NO:7, or a nucleotide sequence that hybridizes to a complement of nucleotides 1-2525 of SEQ ID NO:7, wherein the nucleotide sequence exhibits promoter activity.

3. A vector comprising a heterologous gene encoding a protein, and the nucleotide sequence of claim 2, wherein the heterologous gene is under the control of the nucleotide sequence.

4. A plant cell which has been transformed with the vector as claimed in claim 3.

5. A transgenic plant cell transformed with the nucleotide sequence as claimed in claim 2, operatively linked to a heterologous gene encoding a protein.

6. A method of expressing a protein of interest on a seed coat, the method comprising:

i) transforming a desired plant with the vector of claim 3 comprising the desired heterologous gene encoding the protein of interest to produce a transformed plant;

ii) selecting the transformed plant; and iii) growing the transformed plant to produce seed expressing the protein of interest.

7. A method of modifying storage reserves yields, seed colour, fibre content, or seed metabolites in a seed coat, the method comprising:

i) transforming a desired plant with the vector of claim 3 comprising the desired heterologous gene encoding a protein of interest having an activity that modulates either storage reserves within the seed coat, seed colour, fibre content or seed metabolites to produce a transformed plant;

ii) selecting the transformed plant; and iii) growing the transformed plant to produce seed expressing the protein of interest.

8. A method of increasing seed size or decreasing the biomass of a seed coat, the method comprising:

i) transforming a desired plant with the vector of claim 3 comprising the desired heterologous gene encoding a protein of interest having an activity that increases seed size or decreases the biomass of a seed coat to produce a transformed plant;

ii) selecting the transformed plant; and iii) growing the transformed plant to produce seed expressing the protein of interest.

9. A method of imparting fungal, insect, viral or bacterial resistance in a seed, the method comprising:

i) transforming a desired plant with the vector of claim 3 comprising the desired heterologous gene encoding a protein of interest having an activity that imparts fungal, insect, viral, or bacterial resistance to produce a transformed plant;

ii) selecting the transformed plant; and iii) growing the transformed plant to produce seed expressing the protein of interest.

10. A method of modifying the textural, visual, or chemical properties of a seed coat, the method comprising:

i) transforming a desired plant with the vector of claim 3 comprising the desired heterologous gene encoding a protein of interest that modifies the textural, visual, or chemical properties to produce a transformed plant;

ii) selecting the transformed plant; and iii) growing the transformed plant to produce seed expressing the protein of interest.

11. A method of modifying storage reserve yields, seed colour, fibre content, or seed metabolites in a seed coat, the method comprising:

i) transforming a desired plant with the vector of claim 3 comprising the desired heterologous gene encoding an antisense transcript that modulates either storage reserves within the seed coat, seed colour, fibre content or seed metabolites to produce a transformed plant;

ii) selecting the transformed plant; and iii) growing the transformed plant to produce seed expressing an antisense transcript.

12. A method for localizing production of a protein of interest in a seed coat associated tissue comprising, providing a plant transformed with the nucleotide sequence of claim 2 operatively linked to a heterologous gene encoding the protein of interest, and producing seed from the plant, such that the protein of interest is expressed in the seed coat associated tissue.

13. The method of claim 12, wherein the protein of interest is expressed within the membranous endocarp.

14. The method of claim 12, wherein the protein of interest is expressed within the inner layer of the pericarp.

15. The method of claim 12, wherein the protein of interest is localized onto the seed coat.

16. The isolated nucleotide sequence of claim 1 comprising nucleotides 2526-3368 of SEQ
ID NO:7, a nucleotide sequence that hybridizes to nucleotides 2527-3368 of SEQ
ID NO:7, or a nucleotide sequence that hybridizes to a complement of nucleotides 2526-3368 of SEQ ID NO:7, wherein the nucleotide sequence imparts a dull luster to a seed coat.

17. A vector comprising the nucleotide sequence as defined in claim 16 under the control of a promoter.

18. A plant cell which has been transformed with the vector as claimed in claim 17.

19. An isolated protein encoded by the nucleotide sequence of claim 16.

20. The isolated protein of claim 19 further characterized as having a molecular mass of about 3.2 kD.

21. A method for modifying luster of a seed coat comprising, transforming a plant with the nucleotide sequence of claim 16 under the control of a seed coat promoter, and producing seed from the plant, wherein the luster of the seed of the plant, when compared to a similar plant that does not comprise the nucleotide sequence, is reduced.

22. A method for imparting a dull luster to a seed comprising, transforming a plant with the nucleotide sequence of claim 16 under the control of a seed coat promoter, and producing seed from the plant, wherein the luster of the seed of the plant, when compared to a similar plant that does not comprise the nucleotide sequence, is dull.