Fruit specific promoters

TFM7 and TFM9, promoters for expression of a gene of choice in fruits such as tomato; DNA molecules, plant cells and plants containing them.

BACKGROUND OF THE INVENTION 
Recent advances in genetic engineering have provided the requisite tools to 
transform plants to contain foreign genes. It is now possible to produce 
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, the solids content of tomato fruit can be increased by 
expressing an ADPglucose pyrophosphorylase gene behind a fruit specific 
promoter. (Kishore, PCT Appl. WO 91/19806). The promoter from the 2A11 
genomic clone (Pear, et al. (1989) Plant Mol. Biol. 13:639-651) will 
control expression of ADPglucose pyrophosphorylase 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, the last three are limited to expression during a late stage in 
the development of the tomato fruit and so are known as red fruit 
promoters. The 2A11 promoter will cause expression during early stages, 
but is weaker than desired for some genes. Therefore, there is a need for 
stronger promoters which will cause expression of a gene during the 
development of the green fruit. 
It is an object of the present invention to provide such promoters. It is a 
further object of the present invention to provide promoters which will 
function as fruit-specific in tomatoes and other fruit-bearing crops. It 
is a still further object of the present invention to provide DNA 
constructs containing these promoters and a gene encoding a desired 
protein or the antisense sequence for a less desirable protein. 
SUMMARY OF THE INVENTION 
The present invention provides two fruit-specific promoters which provide 
for expression at greater levels during early development of the fruit 
body of a plant. The two promoters are (1) TFM7 which is a DNA fragment of 
about 2.3 kb, of which 1.4 kb of the 3' end is shown in SEQ ID NO:1; and 
(2) TFM9 which is a DNA fragment of about 900 bp, of which bp of the 3' 
end is shown in SEQ ID NO:2. 
The present invention also provides a recombinant, double-stranded DNA 
molecule comprising in sequence: 
(a) a promoter selected from the group consisting of TFM7 and TFM9; 
(b) a structural DNA sequence that causes the production of an RNA sequence 
which encodes a desired protein; 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, 
wherein said promoter is heterologous with respect to said structural DNA. 
Plant cells and whole plants containing this DNA construct are also 
provided. 
Plants in which this DNA construct may be used include, but are not limited 
to, tomato, strawberry, and raspberry.

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. 
Novel fruit specific promoters exhibiting high and specific expression 
during the development of the tomato fruit have been isolated. A 
differential screening approach utilizing a tomato fruit cDNA library was 
used to identify suitable cDNA clones that expressed specifically in green 
fruit. cDNA probes, prepared from mRNA extracted from fruit at early and 
late developing stages and from combined leaf+stem tissue of tomato, were 
used. Clones that expressed abundantly in green fruit and that showed no 
detectable expression in leaves were identified. Genomic Southern analysis 
indicated a small (1-2) gene copy number. The promoters for these cDNA 
clones were then isolated by screening a tomato genomic clone bank. The 
expression pattern of these promoters is confirmed by fusion to the 
.beta.-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 been fused to the CTP-glgC16 construct described in WO 
91/19806. Results of transformation of tomatoes with the TFM7 construct 
are shown in Example 2. Alternatively, in order to increase sucrose 
content in fruit, one might want to inhibit the action of the plant ADPGPP 
gene by incorporating an antisense sequence corresponding to one or both 
of the subunits of ADPGPP. Use of the promoters of the present invention 
could be a convenient means to do this at the early fruit stage. 
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. 
Expression of an SPS gene, driven by TFM7 or TFM9, 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. 
Expression of invertase in the proper tissue and cellular compartments 
when the fruit is a strong sink, i.e., in a green fruit, is highly 
desirable. 
Lastly, the use of the promoters of the present invention with a gene for 
sucrose synthase would be desirable for the reasons given for SPS. 
Plant Transformation/Regeneration 
A double-stranded DNA molecule 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-based plant transformation vector for 
use in transformation of dicotyledonous plants is plasmid vector pMON505 
(Rogers, S. G. et al. (1987) "Improved Vectors for Plant Transformation: 
Expression Cassette Vectors and New Selectable Markers" in Methods in 
Enzymoloay, ed. Wu and Grossman, pp 253-277, San Diego: Academic Press). 
Binary vector pMON505 is a derivative of pMON200 in which the Ti plasmid 
hornology region, LIH, has been replaced with a 3.8 kb HindIII to SmaI 
segment of the mini RK2 plasmid, pTJS75 (Schmidhauser, 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 Agrobacteriurn 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 
spectinomycin/streptomycin resistance determinant for selection in E. coli 
and A. tumefaciens, an intact nopaline synthase gene for facile scoring of 
transformants 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 inserted 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 and contains a gene 
encoding an enzyme conferring glyphosate resistance which is an excellent 
selection marker gene for many plants. 
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 hosts from tomato and peppers. 
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 genes described herein while not departing from the spirit and scope 
of the present invention. 
EXAMPLE 1 
Two of the green fruit promoters, described above and designated TFM7 and 
TFM9, were isolated and characterized from a Lycopersicon esculentum cv. 
VF36 genomic library. For each of these a partial sequence of the 5' 
terminus, untranslated and promoter regions from which the promoter was 
derived is herein provided. SEQ ID NO:1 is for TFM7. The 2.3 kb promoter 
fragment has as a 5' end the internal XbaI site and extends to the 
putative translation initiation point (modified by placing a BglII 
recognition site at this latter point). SEQ ID NO:2 is for TFM9. The 
.about.900 bp TFM9 promoter fragment extends from the internal SalI site 
to the putative translation initiation point (modified by placing a BglII 
recognition site at this latter point). 
Each of these promoters has been fused to the GUS gene and transformed into 
tomatoes. Regenerated tomato plants were observed for evidence of 
expression of GUS throughout their life cycle. The results are shown in 
Tables 1 and 2. All values given therein represent the mean with standard 
error from at least 3 fruit harvested from 3 or 4 R1 GUS positive plants 
from a transgenie line. GUS activity readings from several floridade 
wild-type control plants are also indicated. Developmental and tissue 
stages are Immature Green 1 (2-3 cm fruit), Immature Green 2 (4-5 cm 
fruit), Mature Green fruit (at least 2 locular cavities are filled), 
Turning (10-20% of the fruit is pink or red) and young leaf. "ND" means no 
detectable units recorded. In Table 1, transgenic lines 11541, 11420,and 
11305 are selected lines containing the pTFM7/GUS/nos construct. In Table 
2, transgenic lines 11256, 11269, and 11290 are selected lines containing 
the pTFM9/GUS/nos construct. 
TABLE 1 
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Flor- 
11541 11420 11305 idade 
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Imm Gm 1 
58620 .+-. 3371 
21670 .+-. 7555 
6338 .+-. 773 
12 
Imm Gm 2 
71887 .+-. 5657 
19933 .+-. 6401 
5805 .+-. 900 
14 
Mature Gm 
45243 .+-. 8666 
14723 .+-. 12636 
6334 .+-. 1358 
26 
Turning 34937 .+-. 6273 
4780 .+-. 1470 
5293 .+-. 901 
39 
Leaf 49 .+-. 70 
19 .+-. 32 103 .+-. 61 
ND 
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TABLE 2 
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Flor- 
11256 11269 11290 idade 
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Imm Gm 36283 .+-. 4822 
109682 .+-. 23956 
20737 .+-. 535 
12 
Imm Gm 31793 .+-. 7382 
104445 .+-. 22885 
13530 .+-. 1091 
14 
2 
Mature 14663 .+-. 1650 
89115 .+-. 34585 
5377 .+-. 1491 
26 
Gm 
Turning 
8468 .+-. 2171 
33003 .+-. 5159 
2309 .+-. 883 
39 
Leaf 210 .+-. 101 
498 .+-. 166 
83 .+-. 22 
ND 
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EXAMPLE 2 
TFM7 and TFM9 were fused to the chimeric CTP-GlgC16 gene (and suitable 3' 
sequences) disclosed in WO 91/19806, and the expression cassettes were 
moved into a plant transformation vector, as discussed below. 
The TFM7 promoter was ligated into the vector pMON999 for ease of 
manipulation, resulting in the plasmid pMON16987. Fusion with the 
CTP-glgC16 chirneric gene was achieved through a triple ligation of the 
Hind III-Bgl II TFM7 promoter fragment from pMON16987, with a Bgl II-Sac I 
fragment from pMON20102 (contains the chimeric gene, disclosed in WO 
91/19806), and placing this into the binary plant transformation vector 
pMON10098 (See FIG. 11 in WO 91/19806) digested with Hind III and Sac I. 
This plasmid, pMON16989, was subsequently used to transform tomato variety 
UC204C. 
The TFM9 promoter was fused to CTPi-glgC16 essentially as described above. 
The SalI-BamHI TFM9 promoter fragment plus GUS was ligated into pEMBL18+ 
cut with the same enzymes to give pMON22701. The TFM9 promoter could then 
be removed as a Hind III-Bgl II fragment (from pMON22701) and ligated with 
the CTPl-glgC16 Bgl II-Sac I fragment from pMON20102 into Hind III-Sac I 
digested pMON10098, resulting in pMON22709. This plasmid was used to 
transform tomato variety UC204C. 
Tomato plant cells were transformed utilizing Agrobacterium strains by the 
method as described in McCormick et al. (1986). 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 minutes; 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 minutes; 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.degree. C. in 
the dark, and then grown in the growth chamber at 25.degree. C., 40% 
humidity under cool white lights with an intensity of 80 einsteins 
m-.sup.2 s-.sup.1. 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 
galacturonic acid, containing no antibiotics, using the conditions 
described above. 
Cotyledons are then inoculated with a log phase solution of Agrobacterium 
containing the plasmids described above. The concentration of the 
Agrobacterium is approximately 5.times.10.sup.8 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/l zeatin riboside, 500 .mu.g/ml 
carbenicillin, and 100 .mu.g/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 0.5.times. MSO 
containing 50 .mu.g/ml kanamycin and 500 .mu.g/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.degree. 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.degree. 
C. with a photoperiod of 18 hours light and 6 hours dark for 2-3 weeks 
prior to transfer to a greenhouse. In the greenhouse, the plants are grown 
at a temperature of 26.degree. C. during the day and 21.degree. C. during 
the night. The photoperiod is 13 hours light and 11 hours dark and allowed 
to mature. 
Fruit from plants transformed with pMON16989 (TFM7 promoter) have been 
obtained and tested. TFM7 causes high expression of glgC16 enzyme in the 
green fruit (&gt;0.1% extracted protein), but glgC16 expression is very weak 
or undetectable in the ripe fruit. TFM7 results in starch in the ripe 
fruit in some lines, while controls always have an iodine score of `0`, 
indicating no starch. Juice from these fruits is very viscous, and soluble 
solids are increased in many of the lines. Comparison data is shown in 
Table 3. Soluble solids and starch rating were measured in serum from hot 
break tomato juice. Starch was measured by adding one drop of an iodine 
solution to filtered serum, and measuring color intensity on a 0-4 scale 
where yellow=0 (no starch) to dark blue=4 (high starch). 
TABLE 3 
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LINE % BRIX IODINE STAIN 
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16989-10712 5.7 0 
16989-10714 6 2 
16989-10223 5.6 4 
16989-10381 6.7 4 
UC204C 5.8 0 
UC204C 6.3 0 
UC204C 5.4 0 
UC204C 5.4 0 
UC204C 5.4 0 
UC204C 6.7 0 
UC204C 5.2 0 
UC204C 6 0 
UC204C 6 0 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1478 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
AGCCTTGTGTTAGGGGGTATTCAAACCTTCTTTGACTGAAAATTTTATTATTTATACATG60 
TTTAAAATTACTTTTTAATCTATATATAATAGATATCAATCCTTCATTTAATTGTATTTT120 
TGTATTAATTCTATAAATATTAAATTACTTTATTAAAAATTCTAATTCTGTCACTCGTCA180 
TTTCATAATATTCTTGACGGTGATGGTAGTGATAATTACGTTGATTGGAGCCACATGGGC240 
CGCTACTTTTTAAAAGGATGAACCTTGGAATGTAGTGAATGTTGAGTCTCATAGCTCACT300 
CACGGACTCAACAGCAAAATCTGTCCTCTTTTTCCCTTCTCCAATTCACATACTGTCACT360 
TGGACAAATAATATTTGAAAATTTTGGCCTAAAGTTAGGTTTGGAGCCGTATGGTAATTT420 
GATACACAAATTATTATATAATTGATATATCAGGTATATATATCAAGTTGTCGCTTCTTC480 
GTTCATTGTTTCTCTCACTAAAATTTTCAATTCACTTTTTAAAAAATCGATAAATTTTTA540 
ATATAACTTTACATAACATATTCAAAATTACAAAAATAAAGGATATTTTTATATGTTTAT600 
TTTTAATGTAAGATTAAATATTTAGAATTCTTTTTAAGAACGGTACAAGCAAATTAAAAG660 
AGAGAAGGTATATTAGTGGGCCTATGTATCTTTGATATCATATGCCTCTCAAAGAGCATC720 
CTGATGAGTCTATATATCTTTGTTGATAGTGATTTAACCATTTATGTATGTACGTAGTAC780 
TAAGACATGTTAAATAAGATCCTAGAGAAAGATTTTTGGAAAAGTGAAAACAGCAATAAA840 
GAAAAGTCATTTAAACACTTTCCAACAAACATTTGGTAATCGATTTTAATTACCCACTTA900 
AACAAAACTATTTGTACGTAAAATGTTTAAGTAGAAAAGAGATTTTTTTAAAAAAAAAAA960 
GAAGGCAAGAGGTCATATATCTGACCCTTCCTTAAATCCCCGCGTATAACACTTTCTTTT1020 
TTTTGTGTGTGTATGTTCAGGAACATTTGTATTTTCTATTTGAAATTTCTCATTAAGTCA1080 
AATTCGAAATCTTTTAAATAATGTAGAGAAATCTCATTATATTTAACAATCCCACTTGAT1140 
GAATTCCTAAACATTTTCTATAAAATAACACTAAATCTTTAATTATACATATTACATACC1200 
TAACTCAAGCAATCTTGTCGGAAAAATCATTAGAAAAGAATTGGAAATAGGGAAATAAAT1260 
AGACATATTTTGGTTAGTATCTTTGTCTATAAGAATGGGTGTGTTAAAGAGCTAGTGCCA1320 
TAGTGTACCATTCTATTGGTAGCATTTGGCAAGAGTTATTCCCTCTCTCCATACCAATGG1380 
AGAAGTTTAATCTTGCTAGAGTCTTATTGTTGCTTCTTCAACTTGGAACTTTGTTCATTG1440 
CCCATGCATGTCCTTATTGTCCATATCCTCCTTCCACC1478 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 450 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
AAATAAATATTTCAAAGTAAATTGTTACTCCCTCTATCCCATACTCTTTTCTTTTTTTAA60 
TCGATTTCTTACTCTAATTGAACTATTGGAGACAACTTAAATGTAAATTTTTTTTTTCTT120 
TATCAAAATGATTGGCTGCTATATAAATATCTAATGGTTATTATACATAAATTTTAATAT180 
TTTTTATAAAAAAATATCGAGCTAAATCATATCGTTTAAATATAGAGATGTGTTATTTAT240 
TTAAAAATTAATTTTAAAAAAGTGAATATTGTAAATTAGGATGAAAGAGTATTATATTGG300 
TTGTCGCAGTATAAATACCCTGCATGCCATTACATTTGTTCAATCATCTTTGCAACGATT360 
TGTGTGCTTTAGCTTCCTTACATAACATGGCTTCTATAACTAAAGCCTCATTACTTATCC420 
TTTTCCTCTCCTTGAATCTCCTTTTCTTCG450 
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