TOMATO FRUIT PROMOTERS

Abstract: Promoters isolated from potato which cause expression of a gene of choice in tomato fruit; tomato plant cells and plants containing them.

Description: TOMATO FRUIT PROMOTERS Recent advances in genetic engineering have provided the requisite tools to transform plants to contain foreign genes. It is now possible to pro- duce plants which have unique characteristics of agronomic and crop processing importance. The ability to chose the tissues in which to express such foreign genes and the time during plant growth to obtain expression is possible through the choice of a regulatory sequence which turns on the gene, called the promoter. A wide range of promoters are known for various plants, plant tissues, and developmental stages. The tomato is a very important plant for genetic engineering. It is readily transformed to express foreign genes and has many characteristics which are known to be improved by certain genes. It is also a valuable crop plant in many countries and was the first transgenic food crop approved for sale in the U.S. Promoters useful in expressing foreign genes in tomato and other fruits are known. For example, U.S. Patent 4,943,674 (Houck et al., July 24, 1990, incorporated herein by reference) discloses the 2A11 promoter as useful in expression of a heterologous gene in tomato fruit. The E4 and E8 promoters (Deikman, et al. (1988) The EMBO Journal 7:3315-3320), as well as the promoter for polygalacturonase are known to be fruit specific. However, there is a need for additional strong promoters which will cause expression of a gene in tomato fruit, particularly those having varying expression patterns through the growth and maturity of the fruit. It is an object of the present invention to provide promoters which will function as fruit-specific in tomatoes. It is a still further object of the present invention to provide tomato plant cells and plants containing these promoters and genes encoding desired proteins or antisense sequences for down-regulating proteins or sense expression for co-suppression of protein production. SUMMARY OF THE INVENTION The present invention provides a method of obtaining expression of proteins in a fruit-enhanced manner in tomatoes by the use of promoters isolated from potatoes. These promoters have surprisingly been found to function in tomato cells to provide a high level of expression of a desired protein or antisense mRNA. The present invention also provides transformed tomato plant cells containing a recombinant, double-stranded DNA molecule comprising in sequence: (a) a promoter selected from the group consisting of potato patatin promoters, potato ADPGPP promoters, and potato granule bound starch synthase promoters; (b) a structural DNA sequence that causes the production of an RNA sequence; and (c) a 3' non-translated region which functions in plant cells to cause transcriptional termination and the addition of poly- adenylated nucleotides to the 3' end of the RNA sequence, wherein said promoter is heterologous with respect to said structural DNA. Whole tomato plants containing this DNA construct are also provided. As used herein, the term "fruit-enhanced" means functioning most strongly or exclusively in fruit tissue or cells. As used herein, the term "potato patatin promoter(s)" means a promoter, isolated from a potato genome, associated with the DNA which encodes a patatin protein. As used herein, the term "potato ADPGPP promoter(s)" means a promoter, isolated from a potato genome, associated with the DNA which encodes either the large or small subunit of the enzyme complex ADPglucose pyrophosphorylase. As used herein, the term "potato granule bound starch synthase promoters)" means a promoter, isolated from a potato genome, associated with the DNA which encodes a granule bound starch synthase. DETAILED DESCRIPTION OF THE INVENTION The expression of a plant gene which exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region which adds polyadenylate nucleotides to the 3" end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the promoter. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complimentary strand of RNA. Surprisingly it has been found that certain promoters from potato will cause transcription of DNA in the fruit of tomatoes. Particularly of interest are promoters which normally cause preferential expression in the tubers. These include patatin promoters, ADPGPP promoters, and granule bound starch synthase promoters. The expression pattern of these promoters in tomato fruit can be confirmed by fusion to the β-glucuronidase (GUS) gene and by following the expression of the GUS enzyme during development in transgenic fruit. Results are given below in Example 1. These promoters have also been fused to the C P-glgC16 construct described in WO 91 19806 (Kishore et al., corresponding to U.S.S.N. 08/120,703, incorporated herein by reference), which increases ADPglucose pyrophosphorylase activity. Results of transformation of tomatoes with three of these constructs are shown in Example 2. Alter¬ natively, in order to increase sucrose content in fruit, one might want to inhibit the action of the native plant ADPGPP gene(s) by incorporating an antisense sequence (or a sense sequence for cosuppression) corresponding to one or both of the subunits of the native ADPGPP behind a promoter of the present invention. Other genes which might be usefully fused to a promoter of the present invention include sucrose phosphate synthase (SPS), which is thought to control the overall rate of sucrose biosynthesis in plant cells, and sucrose synthase. Expression of an SPS or sucrose synthase gene, driven by a promoter of the present invention, may result in a developing fruit with stronger sink activity. .Another possible use is with an invertase gene. Expression of invertase in a sink cell such as in a fruit is another method for increasing the ability of a cell to act as a stronger sink by breaking down sucrose to metabolites that can be used in carbon utilization pathways, e.g., starch biosynthesis. More sucrose is then mobilized into the sink tissue. Alternatively, the promoters may be used to drive genes which alter the ripening characteristics of fruit, such as ACC synthase or ACC deaminase. There may be many other genes which need fruit-specific promoters of varying strengths and growth profiles, for which the promoters of the present invention would be very useful. Other promoters which drive expression of proteins found in tubers would also be expected to work in tomato plants with preferential expression in fruit. A number of such genes with tuber- specific or -enhanced expression, from which the promoters can be obtained, are known. These include the sucrose synthase gene (Salanoubat and Belliard, 1987, 1989) and the major tuber proteins including the 22 kd protein complexes and proteinase inhibitors (Hannapel, 1990). Other promoters which are contemplated to be useful in tomato fruit expression include those that show enhanced or specific expression in potato tubers, that are promoters normally associated with the expression of starch biosynthetic or modification enzyme genes, or that show different patterns of expression within the potato tuber. Examples of these promoters include those for the genes for soluble starch synthase, the branching enzymes, diproportionating enzyme, debranching enzymes, amylases, starch phosphorylases, pectin esterases, the 40 kD glycoprotein, ubiquitin, aspartic proteinase inhibitor, the carboxypeptidase inhibitor, tuber polyphenol oxidases, putative trypsin inhibitor and other tuber cDNAs. Plant Transformation/Regeneration A double-stranded DNA moK ule containing one of the promoters of the present invention can be inserted into the genome of a plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella, et al. (1983) Nature 303:209; Klee, H.J., et al. (1985) Bio/Technology 3:637-42; and EPO publication 120,516 (Schilperoort et al.). In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen. A particularly useful Agrobacterium-b&sed plant transformation vector for use in transformation of dicotyledonous plants is plasmid vector pMON505 (Rogers, S.G. et al. (1987) "Improved Vectors for Plant Trans¬ formation: Expression Cassette Vectors and New Selectable Markers" in Methods in Enzvmologv. ed. Wu and Grossman, pp 253-277, San Diego: Academic Press). Binary vector pMON505 is a derivative of pMON200 in which the Ti plasmid homology region, LIH, has been replaced with a 3.8 kb Hindlll to Smal segment of the mini RK2 plasmid, pTJS75 (Schmid- hauser, T.J. and D.R. Helinski. (1985) J. Bacteriol. 164-155). This segment contains the RK2 origin of replication, oriV, and the origin of transfer, oriT, for conjugation into Agrobacterium using the tri-parental mating procedure (Horsch, R.B. and H. Klee. (1986) PNAS U.S.A. 83:4428-32). Plasmid pMON505 retains all the important features of pMON200 including the synthetic multi-linker for insertion of desired DNA fragments, the chimeric NOS/NPTII'/NOS gene for kanamycin resistance in plant cells, the spec- tinomycin/streptomycin resistance determinant for selection in E. coli and A. tumefaciens, an intact nopaline synthase gene for facile scoring of trans- formants and inheritance in progeny and a pBR322 origin of replication for ease in making large amounts of the vector in E. coli. Plasmid pMON505 contains a single T-DNA border derived from the right end of the pTiT37 nopaline- type T-DNA. Southern analyses have shown that plasmid pMON505 and any DNA that it carries are integrated into the plant genome, that is, the entire plasmid is the T-DNA that is nserted into the plant genome. One end of the integrated DNA is located between the right border sequence and the nopaline synthase gene and the other end is between the border sequence and the pBR322 sequences. Another particularly useful Ti plasmid cassette vector is pMON- 17227. This vector is described by Barry et al. in WO 92/04449 (corres¬ ponding to U.S.S.N. 07/749,611, incorporated herein by reference) and contains a gene encoding an enzyme conferring glyphosate resistance which is an excellent selection marker gene for many plants, including tomato. The gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (CTP2) and expressed from the FMV promoter as described therein. When adequate numbers of cells (or protoplasts) containing the gene of choice driven by a promoter of the present invention are obtained, the cells (or protoplasts) are regenerated into whole plants. Choice of methodology for the regeneration step is not critical, with suitable protocols being available for many tomato varieties. The following examples are provided to better elucidate the practice of the present invention and should not be interpreted in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications, truncations, etc. can be made to the methods and promoters described herein while not departing from the spirit and scope of the present invention. EXAMPLE 1 Two of these promoters have been fused to the GUS gene and trans¬ formed into tomatoes. Plasmids pMON17279 (described in Example 2), containing the potato small subunit .ADPGPP promoter, and pMON17327, containing the potato patatin 1.0 (or patl.O) promoter were used in the assay. The plant transformation vector pMON17327, containing the patatin 1.0 promoter-GUS expression cassette, was constructed as follows: the patatin 1.0 promoter (as in pMON20113, described below) was cloned as a Hindlll-Bglll fragment into pMON10098, disclosed in U.S. Patent 5,254,801, (replacing the enhanced CAMV35S promoter) to form pMON16952. The E. coli uidA (GUS) gene was then cloned as Bglll- EcoRI fragment downstream of the patatin 1.0 promoter in pMON16952 to form pMON17266. The CMoVb-CTP2 CP4 EPSPS - E9 3' glyphosate selection cassette as described for pMON17227 was then cloned into the Notl-Xhol sites (replacing the Nptll selectable marker cassette) to form pMON17327. Tomato plant cells were transformed utilizing Agrobacterium strains by the method as described in McCormick et al. (1986) Plant Cell Reports 5:80-84. In particular, cotyledons are obtained from 7-8 day old seedlings. The seeds are surface sterilized by the following procedure: 1) soak seeds in water for 15 min; 2) soak in 70% EtOH for 1 minute, then rinse with sterile water; 3) soak in 1 N NaOH for 20 minutes; 4) rinse 2 times in sterile water; 5) soak in 25% Chlorox with Tween 20 for 25 min; 6) rinse in sterile, deionized water 3 times. The seeds are germinated in phyta trays (Sigma) on Davis germination media, as described above, with the addition of 25 mg L ascorbic acid. The seeds are incubated for 2-3 days at 28 °C in the dark, and then grown in the growth chamber at 25 °C, 40% humidity under cool white lights with an intensity of 80 einsteins m-2 s -ι. The photoperiod is 16 hrs of light and 8 hrs of dark. Seven to eight days after initiating germination, the cotyledons are explanted as described above. The cotyledons are pre-cultured on "feeder plates" composed of Calgene media, plus acetosyringone and 1 mM galac- turonic acid, containing no antibiotics, using the conditions described above. Cotyledons are then inoculated with a log phase solution of Agro¬ bacterium containing the plasmids described above. The concentration of the Agrobacterium is approximately 5x108 cells/ml. The cotyledons are allowed to soak in the bacterial solution for eight minutes and are then blotted to remove excess solution on sterile Whatman filter disks and are subsequently replaced to the original feeder plate where they are allowed to co-culture for 2-3 days. Cotyledons are transferred to selection plates containing Davis regeneration media with 2 mg 1 zeatin riboside, 500μg/ml carbenicillin, and lOOμg/ml kanamycin. After 2-3 weeks, cotyledons with callus and/or shoot formation are transferred to fresh Davis regeneration plates containing carbenicillin and kanamycin at the same levels. The experiment is scored for transformants at this time. The callus tissue is subcultured at regular 3 week intervals and any abnormal structures are trimmed so that the developing shoot buds will continue to regenerate. Shoots develop within 3- 4 months. Once shoots develop, they are excised cleanly from callus tissue and are planted on rooting selection plates. These plates contain 0.5X MSO containing 50μg/ml kanamycin and 500μg ml carbenicillin. These shoots form roots on the selection media within two weeks. If no shoots appear after 2 weeks, shoots are trimmed and replanted on the selection media. Shoot cultures are incubated in percivals at a temperature of 22°C. Shoots with roots are then potted when roots are about 2 cm in length. The plants are hardened off in a growth chamber at 21°C with a photoperiod of 18 hours light and 6 hrs dark for 2-3 weeks prior to transfer to a greenhouse. In the greenhouse, the plants are grown at a temperature of 26°C during the day and 21°C during the night. The photoperiod is 13 hours light and 11 hours dark and allowed to mature. When a glyphosate resistance gene is used instead of NPTII (conferring kanamycin resistance) the method differs as follows: Cotyledons are transferred to selection plates containing Davis regeneration media with 2 mg/1 zeatin riboside, 500μg/ml carbenicillin, and lOOμg/ml cefotaxime. After 8 days, tissue is moved to fresh regeneration medium containing 0.03 mM glyphosate, 500 μg/ml carbenicillin, and 100 μg/ml cefotaxime. .After 2-3 weeks, cotyledons with callus and/or shoot formation are transferred to fresh Davis regeneration plates containing glyphosate (0.05 mM), carbenicillin and cefotaxime. The experiment is scored for transformants at this time. The callus tissue is subcultured at regular 3 week intervals and any abnormal structures are trimmed so that the developing shoot buds will continue to regenerate. Shoots develop within 3-4 months. Once shoots develop, they are excised cleanly from callus tissue and are planted on rooting selection plates containing the same level of glypho¬ sate, carbenicillin, and cefotaxime, but with a reduced level of zeatin (1 mg/L). Benlate is also incorporated into the medium. These shoots form roots on the selection media within three weeks. Whole plants are then obtained as described above. Regen, rated tomato plants were observed for evidence of expres¬ sion of GUS throughout their life cycle. The results indicated that both promoters caused expression in tomato fruit throughout development. Expression was detected from the onset of fruit development to maturity. Expression was relatively uniform throughout the fruit with strong expres¬ sion detectable in all cell types including the seed. Expression appeared slightly stronger in green fruit. Both promoters also caused some expres¬ sion in the leaves and the stems of the transgenic tomato plants. Patl.O promoter expression was restricted to the cells near the vascular region of the leaf and was less intense than the expression in fruit. The small subunit ADPGPP promoter caused some expression in leaves and stems in a area surrounding the vasculature in cortical cells. Expression levels in these cells were equivalent to those detected in fruit. EXAMPLE 2 The promoters of the present invention were fused to the chimeric CTP-GZg C16 gene (and suitable 3' sequences) disclosed in WO 91/19806, and the expression cassettes were moved into a plant transformation vector, as described below. The patatin 3.5 promoter was obtained from the plasmid pBI240.7 (Bevan et al., Nucleic Acids Res. 14(ll):4625-4638, 1986). The majority of the 3.5 promoter was excised from pBI240.7, from the HindlH site (-3500) to the Xbal site at -337, and combined with the remainder of the promoter, from the Xbal site to a Bglll site at +22 (formerly a Dral site), in a triple ligation into a vector which provided a Bglll site to form pMON17280. This latter plasmid then served as the vector for the triple ligation of the complete 3.5 promoter and the plastid target peptide-^Z^Clβ fusion from pMON20102, described in WO 91/19806, to form the tuber expression cassette (in pMON17282). This cassette, consisting of the patatin 3.5 promoter, the plastid target peptide-G/^C16 fusion, and the NOS 3 * sequences, was introduced into the plant transformation vector pMON- 17227, a Ti plasmid vector disclosed and described by Barry et al. in WO 92/04449, on a NotI fragment to form pMON17316. The patatin 1.0 promoter was excised from the pBI241.3 plasmid as a Hindlll-BamHI fragment (The pBI241.3 plasmid contains the patatin- 1 promoter segment comp -.sing from the AccI site at 1323 to the Dral site at 2289 [positions refer to the sequence in Bevan et al., 1986] with a HindlH linker added at the former and a BamHI linker added at the latter position; Bevan et al., 1986) and ligated together with the CTP1- glgC16 fusion (the Bglll-SacI fragment from pMON20102) and a pUC- type plasmid vector cut with HindlH and Sad (these cloning sites in the vector are flanked by NotI recognition sites). The cassette was then introduced, as a NotI site in pMON886, as disclosed in U.S. Patent 5,254,801, incorporated by reference herein, such that the expression of the glgClβ gene is in the same orientation as that of the NPTII (kanamycin) gene. This derivative is pMON20113. Another vector containing the patatin 1.0 promoter was prepared from pMON10098, also disclosed in U.S. Patent 5,254,801. pMON10098 was digested with Hindlll and Sad. To this was added the HindlH-BamHI promoter segment described above and the CTPl-glgC16 fusion (the Bglll- SacI fragment from pMON20102). This resulted in the vector pMON- 16951, which contains the patatin 1.0 promoter-CTP-^ C16 cassette in a double border binary vector also containing the NPTII gene. To make this a glyphosate selection vector, the NPTII gene was removed by digesting with Not I and Xho I, and the CP4 expression cassette as described for pMON17227 above was inserted. This resulted in the vector pMON17320, which contains the FMV-CP4 cassette as a selectable marker and the patatin cassette, all in a binary, double border plasmid. The promoter for the potato tuber ADPGPP small subunit gene, SEQ ID NO: 1, (Nakata et al. (1992) J. Cell. Biochem. Suppl. 16F, Abstract Y311, p. 266). was obtained as a Xbal-Bglll fragment of the genomic clone 1-2 and inserted into the Xbal and BamHI site of Bluescript II KS-. The promoter fragment used consists of the portion from the Clal site about 2.0 kb extending from the putative initiating methionine to the Hindlll site located 12 bp before this ATG. A Bglll site was placed adjacent to this HindlH site by subcloning through another pUC vector, and was linked through this latter site to the fusion of the CTP targeting and the glgClβ coding sequences. This cassette, with a NOS 3' sequence, was introduced into two vectors to provide differing transformation selec¬ tion markers. pMON17279 contains both GUS and the NPTII (kanamycin) selection cassettes and pMON17354 conk-ins the glyphosate selection cassette as in pMON17227, described above. The promoter for the potato granule bound starch synthase (GBSS) gene (SEQ ID NO:2) was isolated from Russet Burbank DNA by PCR based on the sequence published by Rohde et al. (1990) J. Genet. & Breed. 44:311-315. PCR primers were designed to introduce a Hindlll cloning site at the 5' end of the promoter, and a Bglll site downstream of the transcription start site. The resultant 1.2 Kb promoter fragment was ligated into pMON10098, described in U.S. Patent 5,254,801, incorporated herein by reference, in place of the E35S promoter, and fused with a Bglll-SacI fragment containing the CTPl-glgC16 chimeric gene of pMON- 20102, described above. The E35S-NPT II-Nos cassette was removed from this plasmid and replaced with the FMV-CP4 expression cassette, as described above, resulting in the plasmid pMON16996. Promoters for the large subunit of potato tuber ADPGPP were isolated from two varieties of potato, Russet Burbank (SEQ ID NO:3) and Desiree (SEQ ID NO:4). The clones were identified using plaque hybridi¬ zation with a probe from the 5' end of a cDNA from the large subunit of .ADPglucose pyrophosphorylase. The translational start sites (ATG) of these clones were identified by plant consensus (Lutcke et al. (1987), EMBO J. 6(l):43-48). PCR primers were used to introduce an BamHI site at the 3' end downstream of the ATG and a HindlH site at the 5' end of both promoters. The resulting 600 bp Russet Burbank promoter and 1600 bp Desiree promoters were each ligated into pMON10098 plasmids in place of the E35S promoter, and fused with a Bglll-SacI fragment from pMON20102 containing the CTP-g IgClβ chimeric gene. The E35S-NPTH- Nos cassette was removed from these plasmids and replaced with a Notl- Sall fragment of pMON17227, discussed above, resulting in pMON21522 (Russet Burbank) and pMON21523 (Desiree). Tomato tissue was prepared and transformed with plasmids pMON- 17279, pMON17320, and pMON17316, and tomato plants were regen¬ erated as described in Example 1. One or more lines transformed with each vector were grown in plots of twenty plants under commercial condi- tions in a California field. Five pounds of fruit were gathered from each plot and processed. The concentrated juice was analyzed for soluble solids (Brix), paste viscosity (Bostwick), total solids, and serum viscosity. The data are reported in Table 2. The control lines (UC204C) were non- transgenic controls. The results indicate that the small subunit ADPGPP promoter (from pMON17279) causes high expression ofglgClβ in green fruit with a 2- to 10-fold decrease in red fruit. This results in significant starch in the ripe fruit (0.3-0.4% fresh weight in one line) and in viscous juice extracted from the fruit with an concomitant increase in measured solids. The patatin 1.0 promoter and the patatin 3.5 promoter are weaker, but definitely active in tomato fruit. Table 2 ι___ Brix Bostwick %Tot,solids Ser.visc. 17320-8674 4.0 14.55 4.78 6.421 17316-9302 4.3 16.85 4.98 5.893 17279-10120 4.6 15.40 5.36 7.951 17279-10245 4.5 14.00 5.30 6.455 17279-10246 4.7 13.00 5.53 6.796 17279-10272 4.6 15.15 5.32 7.998 17279-10309 4.3 14.70 5.14 6.079 17279-9858 4.3 10.90 5.33 6.521 17279-10076 4.6 12.40 5.49 6.435 17279-10205 4.2 13.05 5.04 4.558 17279-10279 4.2 13.55 4.98 4.096 17279-10307 4.0 11.30 4.98 4.061 17279-10356 4.4 12.75 5.22 4.848 UC204C control 4.1 15.80 4.85 6.518 UC204C control 4.1 15.80 4.67 5.561 UC204C control 4.0 14.65 4.76 5.967 UC204C control 4.0 14.65 4.78 5.652 UC204C control 4.1 14.60 4.78 5.564 UC204C control 4.2 14.45 4.87 5.463 All publications and patents mentioned in this specification are herein incorporated by reference as if each individual publication or patent was specifically and individually stated to be incorporated by reference. From the foregoing, it will be seen that this invention is one well adapted to attain all the ends and objects hereinabove set forth together with advantages which are obvious and which are inherent to the invention. It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and sub¬ combinations. This is contemplated by and is within the scope of the claims. SEQUENCE LISTING ( 1 ) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Monsanto Company (B) STREET: 800 North Lindbergh Boulevard (C) CITY: St. Louie (D) STATE: Missouri (E) COUNTRY: United States of America (F) POSTAL CODE (ZIP): 63167 (G) TELEPHONE: (3134)694-3131 (H) TELEFAX: (314)694-5435 (ii) TITLE OF INVENTION: Tomato Fruit Promoters (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/334,639 (B) FILING DATE: 04-NOV-1994 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2196 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATCGATTATT GGTTTATCGG GTTTTGATCG TTATCGGTTC GGTTTAACCG TTAAAATTTG 60 ACACAAAAAT AAAAATTGAA AAGCACTTAG AAACAAGGTG ACAAACCTAA TAAACCATGC 120 ACATGAGTTC ACAAGTTACA TCTTGCTAAA AAACAAACAC TTTTACATTG TAGAATAACC 180 AAGTGTCTGG GACAACCAAA AATGAAAGTA GGAAACCAAA CTCTAAGTCA AGGACTTTAT 240 ATACAAAATG GTATAACTAT AATTATTTAA TTTACTATTG GGTTATCGGT TAACCCGTTA 300 AGAACCGATA ACCCGATAAC AAAAACAATC AAAATCGTTA TCAAAACCGC TAAACTAATA 360 ACCCAATACT GATAAACCAA TAACTTTTTT TTTATTCGGG TTATCGGTTT CAGTTCGGTT 420 TTGAACAATC CTAGTGTCCT AATTATTGTT TTGAGAACCA AGAAAACAAA AACTGACGTC 480 GCAAATATTT CAGTAAATAC TTGTATATCT CAGTGATAAT TGATTTCCAA GATGTATAAT 540 TATCATTTAC GTAATAATAG ATGGTTTCCG AAACTTACGC TTCCCTTTTT TCTTTTGCAG 600 TCGTATGGAA TAAAGTTGGA TATGGAGGCA TTCCCGGGCC TTCAGGTGGA AGAGACGGAG 660 CTGCTTCACA AGGAGGGGGT TGTTGTACTT GAAAATAGGC ATTTATTCCG TTCGCAAACC 720 TATCATGTTC CTATGGTTGT TTATTTGTAG TTTGGTGTTC TTAATATCGA GTGTTCTTTA 780 GTTTGTTCCT TTTAATGAAA GGATAATATC TCGTGCCAAA AATAAGCAAA TTCGGTACAT 840 AAAGACATTT TTTTTCTTTC GTGGATTTTC TGTTTATGGA GTTGTCAAAT GTGGAATTTA 900 TTTCATAGCA TGTGGAGTTT CCTCCTCTCC TTTTTCATGT GCCCTTGGGC CTTGCCTGTT 960 TCTTGCACCG CAGTGTGCCA GGGCAGTCGG CAGATGGACA TAAATGGCAC ACCGCTCGGC 1020 TCGTGGAAAG AGTATGGTCA GTTTCATTGA TAAGTATTTA CTCGTATTCG GCGTATACAT 1080 CAAGTTAATA GAAAGTAAAC ACATATGATA TCATACATCC ATTAGTTAAG TATAAATGCC 1140 AACTTTTTAC TTGAATCGCT GAATAAATTT ACTTACGATT AATATTTAGT TGTGTGTTCA 1200 AACATATCAT GCATTATTTG ATTAAGAATA AATAAACGAT GTGTAATTTG AAAACCAATT 1260 AGAAAAGAAG TATGACGGGA TTGATGTTCT GTGAAATCAC TGGCAAATTG AACGGACGAT 1320 GAAATTTGAT CGTCATTTAA ACATATCAAC ATGGCTTTAG TCATCATCAT TATGTTATAA 1380 TTATTTTCTT GAAACTTGAT ACACCAACTC TCATTGGGAA AGTGACAGCA TAATATAAAC 1440 TATAATATCA ATCTGGCAAT TTCGAATTAT TCCAAATCTC TTTTGTCATT TCATTTCATC 1500 CCCTATGTCT GCCTGCAAGT ACCAATTATT TAAATACAAA AATCTTGATT AAACAATTCA 1560 TTTTCTCACT AATAATCACA TTTAATAATA AACGGTTCAT ACACGTGCGT CACCTTTTTT 1620 TCGATTTTCT CTCAAGCGCA TGTGATCATA TCTAACTCTT GTGCAAACAA GTGAAATGAC 1680 GTCCATTAAT AAATAATCTT TTGAATACCT GTTCA'J.TTTA ATTTATTTGG ATTTGCTAAG 1740 GATTTTTTTT AGTTTTTGAG ATTTTTTATA ATTTTAAATT AAAAAAAATA AGTTAAATAT 1800 ATCGAAAATG TCTTTTAATC TTATTTTTGA AAAAGATAAT TAGCTCAAAC AAATTAAAAT 1860 TGGTAACTAT TTTTCGGAAA AATAATGATT CTTATTGTAC ATTCTTTTTC ATCGATTAGA 1920 TATTTTTTTT AAGCTCAAGT ACAAAAGTCA TATTTCAATC CCCAAAATAG CCTCAATCAC 1980 AAGAAATGCT TAAATCCCCA AAATACCCTC AATCACAAAA AGTGTACCAA TCATAACTAT 2040 GGTCCTCTGT AAATTCCAAC AAAATCAAGT CTATAAAGTT ACCCTTGATA TCAGTACTAT 2100 AAAACCAAAA ATCTCAGCTG TAATTCAAGT GCAATCACAC TCTACCACAC ACTCTCTAGT 2160 AGAGAAATCA GTTGATAACA AGCTTTGTTA ACAATG 2196 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1226 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AAGCTTAAGC GGTAACGAGA TAGAAAATTA TATTACTCCG TTTTGTTCAT TACTTAACAA 60 ATGCAACAGT ATCTTGTACC AAATCCTTTC TCTCTTTTCA AACTTTTCTA TTTGGCTGTT 120 GACAGAGTAA TCAGGATACA AACCACAAGT ATTTAATTGA CTCCTCCGCC AGATATTATG 180 ATTTATGAAT CCTCGAAAAG CCTATCCATT AAGTCCTCAT CTATGGATAT ACTTGACAGT 240 TTCTTCCTAT TTGGGTGTTT TTTTCCTGTT AAGTGGAACG GAGACATGTT ATGATGTATA 300 CGGGAAGCTC GTTAAAAAAA AAAAAACAAT AGGAAGAAAT GTAACAAACA TTGAATGTTG 360 TTTTTAACCA TCCTTCCTTT TAGCAGTGTA TCAATTTTGT AATAGAACCA TGCATCTCAA 420 TCTTAATACT AAAAAATGCA ACTTAAGATA GGCTAAACCA AGTAAAGTAA TGTATTCAAC 480 CTTTAGAATT GTGCATTCAT AATTTGATCT TGTTTGTCGT AAAACATTAG AAAATATATT 540 TACAGTAATT TGGAATACAA AGCTAAGGGG GAAGTAACTA CTAATATTCT AGTGGAGGGA 600 GGGACCAGTA CCAGTACCTA GATATTATTT TTAATTACTA TAATAATAAT TTAATTAACA 660 CGAGACATAG GAACGTCAAG TGGTAGCGGT AGGAGGGAGT TGGTTTAGTT TTTTAGATAC 720 TAGGAGACAG AACCGGACGG GCCCATTGCA AGGCCCAAGT TGAAGTCCAG CCGTGAATCA 780 ACAAAGAGAG GGCCCATAAT ACTGTCGATG AGCATTTCCC TATAATACAG TTGCCTTCCA 840 CTAAGGGATA GTTACCCGCA ATTCTCTTGA CACGTGTCAC TGAAACCTGC TACAAATAAG 900 GCAGGCACCT CCTCATTGAC ACTCACTCAC TCACTCACTC ACACAGCTCA ACAAGTGGTA 960 ACTTTTACTC ATCTCCTCCA ATTATTTATG ATTTCATGCA TGTTTCCCTA CATTCTATTA 1020 TGAATCGTGT TATGGTGTAT AAACGTTGTT TCATATCTCA TCTCATCTAT TCTGATTTTG 1080 ATTCTCTTGC CTACTGTAAT CGGTAATAAA TGTGAATGCT TCCTCTTCTT CTTCTTCTCA 1140 GAAATCAATT TCTGTTTTGT TTTTGTTCAT CTGTAGCTTA TTCTCTGGTA GATTCCCCTT 1200 TTTGTAGACC ACACATCACA AGATCT 1226 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 591 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AAGCTTGATA TCGAATTCCT GCAGCCCGGG GGATCTCCTT AAAACTTTTT CTGAATTACT 60 TTTCAAGATT CTTGATTCTG CACCACTAGC AATTTCCATT TTTCTTTCAG TGATTTTGGT 120 TACTTATTTG ACATTCTTGT TTTCAAGATC CAACATCATC ACTTTCCAGG TTCAAAATCT 180 TGTTTTTTTT CTTTTTTCTT TTAATGCTCT ATATTGTGGA AGTCCACAGG TGAATTTTTA 240 CGATATGGGT TTACCACTTA GCTTTCTTGT AATATTTTAT CAATTTTAGA AAATATATGT 300 GTGAAATACC TAATTTTACG TAGAGATCAT GGGTTCATAT GCGTAAAGAT TCATGTTTTT 360 GTGGTAATGC TATGAGGTAT TAGTACTGAG CATATAGCTA GCTTGGGTTT TGGGTTTACC 420 GACCAAAAAA AAAAATTAGT GATATTTTCT TTATGTAAAT TATACTTTTC TTGGTTGCTA 480 AAAGATAACA TATACTTTAT TGAGATTTGA ATAAATCTAT TTGATTTAGA TCCATTGATA 540 AATCTTAATC TTATGGGATT ACTGATTTGT TGATTGGCTG CAGAAGGATC C 591 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1705 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AAGCTTGGGT ACCGGGCCCC CCCTGGAGGT CGAGTGCCAT CACATCCAGG GTGTAGGCTC 60 GGGGCGTGAC AAACTTGGTA TCTGAGCTCA GAGTTCAAGA GTCTAAGGTG TCTATAAAGT 120 CTGTGCCTTT AGAGTCCTAG TTATCGGTGT GAAGCGCGCC ACATCTATAA CCAGGAGGCT 180 GCGACATTTA AGAATTATCA TACTTCTTTC ATACTCTTTT CGTGCAATAG AGTTCAACTC 240 CATAAAGTCT CTTTATAATT CATGTTTACG CATATCTTTG AGATCATGCC TCCATGTAGA 300 GTTGTCTGAG GTCGTCCTGC TAGAAGAAAT ATTGATCCTC AGGATCAAGG GGTACCCAAT 360 GCACCAGAAG TGCGACCCCA AGGAAAGGTC ACTAATGTTG AGTTCCAGGA TGTTATACGG 420 ATATTGAGTG AAGTTGTGAC CAACCAAGCT GGACAACAAA GAGGGAATCA ACAAGATGTG 480 GTTGATACAT CCAGAATCCG TGAGTTCTTA AGGATGAATC CTTCAGACTT CACCAATTCA 540 AGAGTCACTG AGGATCTGGA AAACTTTGTG GAAGAGTTGT AGAAGGTTTT TGAGGTTATG 600 CATGTTGTTG ATGCTGAGCG AGTGGAACTA ACTGCATACC AACTGAATGG TGTTGCTAGA 660 GTATGGTACG ACCAATAGAA AAAGAGTAGA GTTGAGGGTG CACAAATTGT GAGTTGGGCA 720 GTGTTTGAAG AGGCCTTCAT GGGGCATTTC TTTTCCCATG AACTATATGG CAAAGGTAAG 780 AGAATTTCCT CACTCTTAAG CAGGAATCCA TGAGTGTGCA TAAGTATAGC CTCAAGTTCA 840 CTCAACTGTC GCCTATGCTC CAGAGATGGC TGTTGATATG AGGAGCAGGA TGGGCTTGTT 900 TGTGTTTGGG TTGTCTCATC TGTCAATCAA AGAAGGTAAG GTTGTGATGT GGATAAAGGA 960 CATGGACATC GAAAGGGTAA TGATCCTTGT GCAACAGGTT GAGGAAGATA AGTTGAGGGA 1020 TAGAGAAGAG TTCTGAAACA AGAGGGCTAA GAACACATGA AATGAGTACG TAAGCAGAAG 1080 AGTAATGCAA ATCGGTTATC TTTTCAATGA AAGCCAAATA AACCTGCTTG ATTGTTTGCA 1140 AGTGCAACCT GTACCAACGA ACAAAGGTGA GTTCAAGAAT CAGAATTCTT AGAAATTCAG 1200 AGCTAGACCT GCACAATCTC AAGGTAGTGT GGCACAAGGA TGTAATGGGA CTCCTGCATG 1260 TGTTAAGTAC GGTAGGAACC ACCCAGGAGC GTGTCATGAT GGCTCTGCTG GTTGCTTCAA 1320 GTGTGGTCAG AATGGTCACT TCATGAGAGA GTGCCTAAAG AANAGGCAAG GTAATAGCAA 1380 TGGGGGCAAT ATATCACAAT CTTCTTCAGT GGCTCCACNA GATAGAGCTG CACCTTGAGG 1440 ATCATGGGTT CATATGCGTA AAGATTCATG TTTTGTGGTA ATGCTATGAG GTATTAGTAC 1500 TGAGCATATA GCTAGCTTGG GTTTTGGGTT TACCGACCAT TTTTTTTAAT TAGTGATATT 1560 TTCTTTATGT ATTTTATACT TTTCTTGGTT GCTTAAAGAT TACATATACT TTATTGAGAT 1620 TTGAATAAAT CTATTTGATT TAGATCCATT GATAAATCTT AATCTTATGG GATTACTGAT 1680 TTGTTGATTG GCTGCAGAAG GATCC 1705

Claims: Claims: 1. A method of causing expression of a protein or an antisense mRNA in the fruit of tomato plants comprising: (1) transforming tomato plant cells with a DNA construct comprising in operative order: (a) a promoter selected from the group consisting of patatin promoters, potato ADPGPP promoters, and potato granule bound starch synthase promoters; (b) a structural DNA sequence that causes the production of an RNA sequence; and (c) a 3' non-translated region which functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3' end of the RNA sequence; and (2) regenerating tomato plants containing said DNA construct. 2. The method of Claim 1 wherein said structural DNA sequence is in antisense orientation. 3. The method of Claim 1 wherein said structural DNA encodes ADPglucose pyrophosphorylase. 4. The method of Claim 3 wherein said ADPglucose pyrophosphorylase is glgClβ. 5. The method of Claim 4 wherein said promoter is the small subunit ADPGPP promoter. 6. A tomato plant cell comprising a recombinant, double- stranded DNA molecule comprising in sequence: (a) a promoter selected from the group consisting of patatin promoters, potato ADPGPP promoters, and potato granule bound starch synthase promoters; (b) a structural DNA sequence that causes the production of an RNA sequence; and (c) a 3' non- translated region which functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3' end of the RNA sequence. 7. The tomato plant cell of Claim 6 wherein said structural DNA sequence is in antisense orientation. 8. The tomato plant cell of Claim 6 wherein said structural DNA sequence encodes ADPglucose pyrophosphorylase. 9. The tomato plant cell of Claim 8 wherein said ADPglucose pyrophosphorylase is glgClβ. 10. The tomato plant cell of Claim 9 wherein said promoter is the small subunit .ADPGPP promoter. 11. A tomato consisting of cells of Claim 6. 12. A tomato consisting of cells of Claim 10.

Inventors: BARRY GERARD FRANCIS, EDWARDS JANICE WETTSTEIN, KISHORE GANESH MURTHY, STARK DAVID MARTIN, BARRY, GERARD, FRANCIS, EDWARDS, JANICE, WETTSTEIN, KISHORE, GANESH, MURTHY, STARK, DAVID, MARTIN
Applicants: MONSANTO CO, MONSANTO COMPANY
Application year: 1995
Main classification: C12N15
IPC codes: A01H5, C12N5, C12N9, C12N15