Strawberry fruit promoters for gene expression

Promoters isolated from genomic DNA of strawberry plants are disclosed. The promoters are capable of tissue-specific expression in transgenic plants. A plant promoter that is a nucleic acid region located upstream of the 5' end of a plant DNA structural coding sequence that is transcribed at high levels in ripening fruit. This promoter region is capable of conferring high levels of transcription in ripening fruit tissue and in developing seed tissues when used as a promoter for a heterologous coding sequence in a chimeric gene. The promoter and any chimeric gene in which it may be used can be used to obtain transformed plants or plant cells. Chimeric genes including the isolated promoter region, transformed plants containing the isolated promoter regions, transformed plant cells and seeds are also disclosed.

TECHNICAL FIELD 
The present invention relates to fruit tissue gene expression for the 
modification of fruit phenotype. The invention is exemplified by the use 
promoters from Fragaria sp. that express selectively in receptacle tissue. 
BACKGROUND 
One of the goals of plant genetic engineering is to obtain plants having 
improved characteristics or traits. Many different types of 
characteristics or traits in plants are considered advantageous. Those of 
particular importance with regard to fruit bearing plants include control 
of fruit ripening, improvements in the nutritional characteristics of the 
edible portions thereof, resistance to plant diseases, resistance to 
insects, cold tolerance and enhanced stability or shelf-life of the 
ultimate consumer product obtained from the plant. 
At least two key components are required to stably engineer a desired 
trait, or control of such a trait, into a plant. The first key component 
comprises identifying and isolating the gene(s) which either encode(s) or 
regulate(s) a particular trait. The second component comprises identifying 
and isolating the genetic element(s) essential for the actual expression 
and/or selective control of the newly isolated gene(s) so that the plant 
will manifest the desired trait and, ideally, manifest the trait in a 
controlled or controllable manner. This second component, which controls 
or regulates gene expression, typically comprises transcriptional control 
elements known as promoters. Although a generic class of promoters which 
drive the expression of heterologous genes in plants have been identified, 
a broad variety of promoters active in specific target tissues or cells of 
plants remain to be described. The identification of such target or 
tissue-specific promoters is critical to the introduction of the 
above-mentioned tissue-specific improvements in plants such as fruit 
bearing plants. 
Several promoters useful in expressing heterologous genes in selected 
fruits have already been identified. For example, the E4 and E8 promoters 
(Deikman, et al.), the kiwifruit actinidin promoter (Lin, et al.) and 
promoter for polygalacturonase are known to be fruit specific. U.S. Pat. 
No. 4,943,674 (Houck et al., Jul. 24, 1990) discloses a 2All promoter as 
useful in expression of a heterologous gene in tomato fruit. These 
promoters, however, have been isolated from fruit tissue which comprises 
mature or maturing ovaries (hereinafter referred to as "traditional 
fruit"). As such, these traditional fruit promoters would be ineffective 
in controlling desired traits in such accessory fruit bearing plants as 
strawberry, apple, pear, quince and the like wherein the major portion of 
the edible fruit comprises receptacle tissue (see An Introduction to Plant 
Biology. 2nd Edition, Braungart & Arnett, eds., C.V. Mosby Co. 1965). 
Similarly, to date, genes thought to be active in fruit tissue have been 
isolated from traditional fruit tissue instead of receptacle containing 
tissue. Promoters involved in fruit expression have been identified in PCT 
application WO 97/27295. 
There exists a need for receptacle tissue selective promoters which provide 
for increasing or decreasing expression during fruit development, 
maturatioin and ripening in the art. Access to such receptacle tissue 
selective promoters would enable the genetic engineering of fruit tissue 
from commercially important plants such as strawberry, apple, and pear. 
Two cDNAs have been previously identified as receptacle tissue selective 
(Reddy and Poovaiah, 1990, Plant Molecular Biology, 14: 127-136 and 
Wilkinson et al., 1995, Plant Molecular Biology 27:1097-1108). The 
promoters for these two cDNAs were cloned and sequenced. Expression of 
reporter genes in strawberry plants will be used as an assay of the tissue 
specificity of the isolated promoters. Of particular interest are 
promoters which provide for recepticle tissue selective expression of 
genes. Also of interest is the ability to enhance or modify the properties 
of other promoters. 
Relevant Literature 
Reddy and Poovaiah, Plant Mol. Biol., (1990) 14: 127-136, reports on the 
cloning of a cDNA for an auxin repressed mRNA from strawberry. Wilkinson, 
et al., Plant Mol. Biol., (1995) 27:1097-1108 report the identification of 
mRNAs in strawberry with enhanced expression in ripening fruit, for 
example RJ39. 
SUMMARY OF THE INVENTION 
The present invention provides novel promoters, termed "SAR5 and RJ39", 
which cause decreasing or increasing tissue-selective expression of 
heterologous DNA in the receptacle tissue of plants. 
The present invention also provides novel chimeric genes comprising a 
receptacle tissue-selective promoter operably coupled to a heterologous 
DNA sequence. 
The present invention furthermore provides a method for expression of a 
heterologous gene, the improvement which comprises the use of an accessory 
fruit plant promoter which causes decreasing or increasing 
tissue-selective expression in seed, sink and receptacle tissue of plants 
during fruit development, maturation and ripening, said accessory fruit 
plant promoter having a sequence selected from the group consisting of 
those sequences shown in SEQ ID NOS. 1 and 2 and sequences substantially 
homologous thereto. 
Novel transformed plant cells and transgenic plants comprising the 
heterologous genes of the present invention or produced by the methods of 
the present invention are additionally provided.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the subject invention, nucleic acid constructs are 
provided which are active in the receptacle tissue of plants, and in 
particular, accessory fruit bearing plants. The novel promoter sequences 
of the present invention provide for increasing or decreasing expression 
of heterologous genes during fruit development, maturation and ripening. 
The phrase "heterologous gene" means that the DNA coding sequence does not 
exist in nature in the same gene with the promoter to which it is now 
attached. The promoter sequences of the present invention now provide an 
opportunity to engineer agriculturally and commercially important traits 
into a class of fruits, fruit tissue and fruit bearing plants. More 
specifically, this class of fruits includes those plants comprising 
accessory fruit and other plants in which regulation of receptacle 
function or engineered expression in receptacle tissue is desirable. 
Constructs can be included in a transcriptional cassette or an expression 
cassette in which downstream from the regulated transcriptional initiation 
region is a nucleotide sequence of interest which provides for regulated 
modification of plant phenotype, by modulating the production of an 
endogenous product, as to amount, relative distribution, or the like, or 
production of an exogenous expression product to provide for a novel 
function or product. One or more introns also may be present. Depending 
upon the manner of introduction of the nucleic acid construct into a host 
plant, other DNA sequences may be required such as sufficient T-DNA from 
an Agrobacterium plasmid for transfer to a plant host. Plant hosts of 
particular interest are fruit plants, such as strawberry. 
In one embodiment, DNA sequences for promoters are provided which are 
active in strawberry plants. Strawberry plants are an important commercial 
fruit crop in many temperate regions of the world and are especially 
suitable for improvement through genetic engineering techniques, such as 
clonal propagation, versus conventional breeding and selection. The high 
heterozygosity and polyploidy associated with commercial lines of 
strawberry plants hinder the improvement of such plants through 
traditional breeding methods. In contrast, clonal propagation of 
strawberry plants provides for stable transformation of a single dominant 
gene for a desired trait into a commercially important genotype without 
sexual recombination. The novel promoters of the present invention now 
provide an opportunity to engineer into such receptacle fruit bearing 
plants as strawberry such commercially and agriculturally desirable traits 
including delayed fruit ripening, increased sugar content, modified color 
and fungal resistance as more specifically described hereinafter. 
In another embodiment of the present invention, receptacle tissue-selective 
promoter sequences are isolated from a genomic library created from 
Fragaria vesca DNA. Specifically, a probe is hybridized to Fragaria vesca 
genomic DNA fragments under medium to high stringency hybridization 
conditions (Maniatis et al., 1982). The identified genomic fragments are 
then isolated and purified. Confirmation of receptacle tissue-selective 
activity can then be achieved by transforming plants, or plant tissue with 
chimeric genes containing said substantially homologous sequences in 
accordance with the examples hereinafter. 
In one important embodiment of the present invention, two distinct, novel 
promoters, each individually able to direct high level transcription of a 
second DNA sequence expressively coupled thereto in receptacle tissue of 
accessory fruit bearing plants, are provided. These promoters are 
designated SAR5 and RJ39 (also referred to as RJ39C). Nucleotide sequences 
of these promoters are provided in SEQ ID NOS. 1 and 2 respectively. It is 
understood by those of ordinary skill in the art that the DNA sequences 
shown in any of SEQ ID NOS. 1, and 2 include any promoter active in 
developing, maturing and ripening fruit receptacle tissue having a DNA 
sequence substantially homologous to any one of said promoter sequences. 
Strawberry fruit develops from receptacle tissue at the base of flowers. 
From the base of the flower, the receptacle tissue develops into ripened 
fruit receptacle tissue through the stages of: small green, large green, 
green-white, turning and full red stages. 
Novel fruit selective promoters exhibiting decreasing or increasing and 
tissue selective expression during the development of the strawberry fruit 
have been isolated. An mRNA, RJ39, has been identified as having increased 
expression during fruit ripening, selectively in strawberry fruit 
receptacle tissue (Wilkinson et al., Plant Mol. Biol. (1995) 
27:1097-1108). Expression of the RJ39 mRNA was observed at low levels in 
fruit in the green-white stage of fruit development. Expression of RJ39 
increased during fruit ripening through the full-red stage of development. 
The SAR5 mRNA is expressed during fruit development and maturation. SAR5 
was identified as being repressed by auxin, and exhibited decreasing 
expression during strawberry fruit ripening (Reddy and Poovaiah, Plant 
Mol. Biol. (1990) 14:127-136). Expression initiated during flower bud 
development and increased during flower development and early fruit 
development, then decreased linearly during fruit maturation, and was 
expressed at very low levels or absent in turning and full red fruits. 
These mRNAs expressed abundantly in the ripening receptacle tissue of 
accessory fruit plants (RJ39) or early in fruit development and maturation 
(SAR5) and showed little or no detectable expression in leaf tissues. The 
low number of hybridizing fragments, and the lack of sequence variability 
indicated a low gene copy number. The cDNA clones of RJ39 and SAR5 were 
used to isolate genomic clones which contained a genomic copy of the cDNA, 
and additional nucleic acid sequences corresponding to the transcriptional 
initiation region. Expression controlled by these promoters may be 
confirmed by fusion to the .beta.-glucuronidase (GUS) gene and following 
the expression of the GUS enzyme during various stages of fruit 
development, maturation and ripening in transgenic fruit. 
The promoters of the present invention may be used to increase the sugar 
content in fruit. In particular, one may inhibit the action of the plant 
glucose-6-phosphatase gene by controlling transcription of an antisense 
sequence corresponding to one or both of the subunits of 
glucose-6-phosphatase. 
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 SAR5 or RJ39 may result in a 
developing fruit with higher carbohydrate composition. 
The use of promoters from the present invention with other genes such as 
ADP Glucose pyrophosphorylase, glgC16, encoding a starch synthesizing 
enzyme, may also be of interest. Expression of glgC16 driven by SAR5 or 
RJ39 may result in a developing fruit with higher carbohydrate 
composition. 
Another possible use is with the invertase gene. Expression of invertase in 
a sink cell such as in a fruit is a 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. 
The use of the promoters of the present invention with a gene for sucrose 
synthase would be desirable for the reasons given for SPS. 
Other genes may be used in constructs with the SAR5 and RJ39 promoter 
sequences to achieve resistance to pathogens. For example, genes encoding 
for the production of phytoalexins (e.g. hydroxystilbenes), the expression 
of disease resistance genes, such as R genes, defense induction genes, 
such as avr genes, and genes for resistance to insects. 
The promoters of the present invention may also be used in constructs 
containing two genes of complementary functions. For example, plants may 
be transformed with a construct containing two genes which may be 
complementary to each other to increase sugars in a fruit. One gene may be 
required in early fruit development, and the second gene product may act 
on the product of the early gene later in fruit ripening. 
For example, the promoter SAR5 may be used to drive glgC16 early in fruit 
development to increase the amount of starch in developing fruit, and the 
RJ39 promoter may be used to drive SPS late in fruit development to 
further increase soluble solids in the fruit receptacle tissue. 
Alternatively, the promoters may be used together to drive the same gene. 
By using the SAR5 and RJ39 promoters to drive the same gene, a sustained 
level of gene expression may be achieved during fruit development. For 
example, invertase may be driven by both SAR5 and RJ39 to provide for a 
constant, increased sink strength during fruit development and ripening. 
Plants containing two or more promoter-gene fusions may be produced by 
several methods to one skilled in the art. A single construct containing 
two promoter-gene fusions may be used for transformation, or 
alternatively, two promoter-gene constructs may be used (cotransform). In 
addition, transgenic plants containing one of the promoter-gene fusions 
may be used as explant material to retransform with the second 
promoter-gene fusion. Transgenic plants containing two promoter-gene 
fusions may also be obtained by crossing transgenic plants containing one 
promoter-gene fusion integrated into it's genome. 
By using the promoter sequences provided herein, one of ordinary skill in 
the art is now able to isolate, or chemically or enzymatically synthesize, 
by conventional methodologies, promoters having sequences essentially 
identical to those sequences described herein and promoters substantially 
homologous thereto. For example, the isolation of such promoter sequences 
can be achieved by using conventional techniques to synthesize a 
hybridization probe comprising all or a portion of a promoter sequence set 
forth in any of SEQ ID NOS. 1 and 2. The hybridization probe is preferably 
about 20 to 600 nucleotides in length. 
A double-stranded DNA molecule containing one or more 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, L., et al., Klee, H. J., et al., 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 nucleotide sequence of interest is inserted downstream from and under the 
regulation of the transcriptional initiation region. The nucleotide 
sequence of interest provides for modification of plant phenotype for 
example by altering the production of an endogenous product, as to amount, 
relative distribution, or the like, or by encoding a structurally or 
functionally novel gene product. The nucleotide sequence may have any open 
reading frame encoding a peptide of interest, for example, an enzyme, or a 
sequence complementary to a genomic sequence, where the genomic sequence 
may be an open reading frame, an intron, a non-coding leader sequence, or 
any other sequence where the complementary sequence inhibits 
transcription, messenger RNA processing, e.g., splicing, or translation. 
The nucleotide sequence of interest may be synthetic, of natural origin, 
or combinations thereof. Depending upon the nature of the nucleotide 
sequence of interest, it may be desirable to synthesize the sequence with 
plant preferred codons. The plant preferred codons may be determined from 
the codons of highest frequency in the proteins expressed in the largest 
amount in the particular plant species of interest. 
The termination region is one which is functional in a plant host cell. In 
addition to containing at least one terminating sequence, the termination 
region can include a poly A signal. In view of the relative 
interchangeability of the termination regions, the selection of a 
termination region for use in the expression construct is primarily based 
on convenience. The termination region and the transcriptional initiation 
region, or the termination region and nucleotide sequence of interest can 
originate from the same or different sources. Convenient termination 
regions are available from the Ti-plasmid of A. tumefaciens, such as the 
octopine synthase gene and nopaline synthase gene termination regions. 
Additional DNA sequences can be included in the transcription cassette, for 
example, adapters or linkers for joining the DNA fragments in the proper 
orientation and, as appropriate, in the proper reading frame. Other DNA 
sequences may be needed to transfer transcription constructs into 
organisms used for transforming plant cells, e.g., A. tumefaciens. In this 
regard, the use of T-DNA of the Ti- or Ri- plasmids as a flanking region 
in a transcription construct is described in EPO Application No. 116,718 
and PCT Application Nos. WO84/02913, 02919 and 02920. See also 
Herrera-Estrella, Nature (1983) 303:209-213; Fraley et al., Proc. Natl. 
Acad. Sci, USA (1983) 80:4803-4807; Horsch et al., Science (1984) 
223:496-498; and DeBlock et al., EMBO J. (1984) 3:1681-1689. 
The expression constructs of the present invention, which contain the 
regulated 5'-untranslated regions of two receptacle selective genes from 
strawberry, are transformed into plant cells to evaluate their ability to 
function with a structural gene other than the open reading frame that is 
natively associated with the 5'-untranslated region and to ascertain their 
expression characteristics. 
A variety of techniques are available for the introduction of DNA into a 
plant cell host. These techniques include transformation employing 
Ti-plasmid DNA and A. tumefaciens or A. rhizogenes as the transforming 
agent, protoplast fusion, injection, electroporation, and the like. The 
transcription construct normally is joined to a marker that allows for 
selection of transformed cells in the treated population, for example, 
resistance to antibiotics such as kanamycin, G418, bleomycin, 
chloramphenicol and others. 
Any plant variety may be employed as a host cell in accordance with this 
invention. Of particular interest are agricultural fruit crops, such as 
strawberries and tomatoes, although the use of the RJ39 and SAR5 
transcriptional initiation regions in other plants, including other 
fruit-bearing plants is also considered. Examples of plants in which the 
promoters of the present invention may find use include, but are not 
limited to, any sink tissue of plants including strawberry, raspberry, 
tomato, potato tuber, tobacco, soybean, cotton boll, and cotton seed. 
The transformed plant host cells are used to regenerate plants. See, e.g., 
McCormick et al., Plant Cell Reports (1986) 5: 81-84. These plants are 
then grown and pollinated with either the same transformed strain or with 
different strains, and the resulting hybrid having the desired phenotypic 
characteristic identified. Two or more generations may be grown to ensure 
that the desired phenotypic characteristic is stable maintained and 
inherited and then seeds harvested to ensure the desired phenotype or 
other property has been achieved. 
The embodiments described above and the following examples are provided to 
better elucidate the practice of the present invention. It should be 
understood that these embodiments and examples are provided for 
illustrative purposes only, and are not by way of limitation of the scope 
of the invention. 
The following experimental protocol describes the identification and 
isolation of the promoter of a gene differentially expressed in the 
receptacle tissue of plants. One skilled in the art will recognize that 
substitutions and alterations may be made in the components, conditions, 
and procedures presented herein without departing from the scope or 
intention of the protocol. The recombinant DNA techniques employed are 
familiar to those skilled in the art of manipulating and cloning DNA 
fragments and employed persuant to the teachings of Sambrook et al. 
EXAMPLES 
Example 1 
Isolation of Promoters 
A. Isolation of Strawberry Genomic DNA 
Strawberry genomic DNA was isolated from Fragaria vesca (2n=14) and 
Fragaria x ananassa (variety Redcoat) as described here. Fresh or frozen 
strawberry leaf tissue was ground in a chilled mortar and pestle, and 2 
volumes (v/w of fresh tissue) of DNA Extraction Buffer (500 mM Sorbitol, 
100 mM Tris, 5 mM EDTA, 2% .beta.-mercaptoethanol, pH 7.5) was added, and 
ground again briefly. Homogenate was transfered to a centrifuge tube and 
2.5 volumes of Nuclei Lysis Buffer (200 mM Tris, 50 mM EDTA, 2M NaCl, and 
2% CTAB (hexadecyltrimethylammonium bromide), pH 7.5) was added with 0.5 
volumes 5% Sarkosyl solution. Homogenates were mixed briefly by inversion, 
then incubated at 65.degree. C. for 15 minutes. DNA solutions were mixed 
again at room temperature for 5 minutes by inversion. Samples were 
chloroform extracted one time with an equal volume of chloroform/ isoamyl 
alcohol (24:1). The samples were centrifuged at 12,000 rpm for 15 minutes, 
and the top, aqueous phase was transfered to a new tube. The genomic DNA 
was precipitated by adding one volume of isopropanol, incubated for 30 
minutes on ice and centrifuged for 10 minutes at 12,000.times.g. DNA 
pellets were washed with 70% ethanol, and air dried. Two additional steps 
were used to remove contaminating polysaccharides. Dried pellets were 
resuspended in TE, and 1/4 volume 5M NaCl was added. Samples were then 
incubated on ice for 30 minutes, and centrifuged for 10 minutes at 
12,000.times.g. The supernatant was transfered to a new tube and the DNA 
precipitated with 2.5 volumes of ethanol. The dried pellets were 
resuspended in TE. Potassium acetate (2M final concentration) was added, 
and samples were incubated again on ice for 30 minutes and centrifuged for 
10 minutes at 12,000.times.g. DNA in the supernatant was precipitated, 
dried, and resuspended in TE. 
B. Isolation of SAR5 Promoter 
A genomic clone of the SAR5 gene was obtained by PCR amplification of 
strawberry genomic DNA using the primers designed according to the 
sequence of SAR5 (Reddy and Poovaiah, 1990, Plant Molecular Biology, 
14:127-136). The forward PCR primer SAR5-5C1 
(5'-TCGAATTCAGAGCAAAGATGGTTCTGC-3', SEQ ID NO:3) contains SAR5 encoding 
sequence from the 5' end of the cDNA, including the ATG start codon 
(underlined above) and restriction cloning sites. SAR5-3N2 
(5'-ACCTCGAGGGATCCTCATCACTTGTCG-3', SEQ ID NO:4) is the reverse primer 
containing complementary sequences to bases 369 to 386 in the 
3'-untranslated region (numbering according to Reddy and Poovaiah) and 
restriction cloning sites. The genomic DNA was prepared from strawberry 
(variety Redcoat) leaf tissue using the method described in 1A above. PCR 
amplification was carried out according to the manufacturer's 
recommendations (Perkin-Elmer) at 40 cycles of 94 degrees C. 1 min., 49 
degrees C. 1 min., and 72 degrees C. 1 min. The PCR product was purified 
from an agarose gel slice using a Prep-A-Gene kit (BioRad), digested with 
EcoRI and BamHI, and cloned into the EcoRI and BamHI sites of pBluescript 
SK- (Stratagene) creating the clone pCGN8023. Analysis of sequence 
obtained using an ABI automated sequencer demonstrated that a complete 
genomic DNA copy of SAR5 was obtained and contained two introns. 
In order to reduce some of the complexities involved in cloning from an 
octoploid species, such as Fragaria x ananassa (variety Redcoat, 8n=56), 
DNA from a diploid species, Fragaria vesca was used to clone the SAR5 
promoter. Strawberry genomic DNA was prepared from Fragaria vesca as 
described in example 1A above. DNA was digested with the restriction 
enzyme BglII and the digestion products were separated on a 0.7% agarose 
gel. The digested, fractionated DNA fragments were transferred to a 
positively charged nylon membrane (Nytran) by capillary blotting overnight 
in 10.times. SSC buffer (Southern transfer). A radioactive probe was 
prepared from the first 450 bp of the SAR5 genomic clone (up to the BglII 
site) using a Prime-It kit (Stratagene) and the filter was incubated 
overnight in hybridization solution at 60 degrees C. The filter was 
subsequently washed to eliminate non-specific probe binding, with the 
final wash being 0.25.times. SSC, 0.1% SDS at 60 degrees C. The results 
after X-ray film exposure indicated that SAR5 is a single-copy gene in F. 
vesca. 
F. vesca genomic DNA was digested with BglII in combination with EcoRI, 
PstI, XhoI, or SpeI, followed by Southern blotting and hybridization as 
described above. From the size of the hybridizing bands in each lane, a 
restriction map of the SAR5 promoter region (extending down to the 
internal BglII site) was generated. The restriction map indicated that a 
promoter fragment of approximately 6 kb (PstI), 3.6 kb (SpeI), 2 kb 
(XhoI), or 0.5 kb (EcoRI) could be obtained by cutting with the 
appropriate enzyme in combination with BglII. 
F. vesca genomic DNA was cut with XhoI and BglII, fractionated on a 0.7% 
agarose gel, and the gel region around 2.5 kb (2kb promoter+450 bp coding 
region) excised with a razor blade. DNA from the gel piece was purified 
using a Prep-A-Gene kit and ligated to XhoI- and BamHI-digested 
pBluescript SK- DNA. The ligation mix was transformed into competent 
E.coli cells which were then plated out onto selection media. 
Ampicillin-resistant colonies were patched in a grid format to new plates 
and then lysed onto nitrocellulose filters for in situ hybridization 
(Sanmbrook et al. Molecular Cloning). 
A probe corresponding to the first 450 bp of the SAR5 genomic clone was 
prepared using an ECL Labeling and Detection kit and used to screen the 
filters according to the manufacturer's instructions (Amersham). After 
X-ray film exposure, one positive colony was identified. This colony was 
inoculated into liquid media for overnight growth and plasmid DNA was 
prepared using a Qiaprep Spin Miniprep kit (Qiagen). The plasmid DNA was 
analyzed by restriction enzyme analysis and then sequenced using an ABI 
automated sequencer. The clone was verified as containing 5' flanking 
sequence (the SAR5 promoter) and a portion of the SAR5 coding region (up 
to the internal BglII site). The clone was designated pGSAR5-16 
A BamHI restriction site was introduced upstream of the SAR5 initiation 
codon (ATG) by PCR amplification of the above promoter clone with the 
primers pSAR5-BH1 (5'-TAGGATCCGTCTTTGCTCTGAACTC-3', SEQ ID NO:5) and 
pSAR5-R4 (5'-ACCTTGTACCTAAGAAAGCC-3', SEQ ID NO:6). The PCR product was 
purified from an agarose gel slice using a Prep-A-Gene kit, digested with 
XbaI and BamHI, and cloned into the XbaI and BamHI sites of pBluescript 
SK-, yielding the plasmid pSAR5PCR/pSK- (also referred to as the truncated 
or TSAR5 promoter,). 
To facilitate other cloning experiments, the TSAR5 promoter was subcloned 
as an XbaI to EcoRI fragment into the XbaI to EcoRI sites of pUC119, 
creating the plasmid pCGN8046. The full-length SAR5 promoter, referred to 
as FSR or pCGN8047 (SEQ ID NO:1), was recreated by cloning the upstream 
region from the original promoter clone as an XhoI to XbaI fragment into 
the SalI to XbaI sites pCGN8046. Plasmids containing the reconstructed 
full-length promoter with a BamHI site introduced just upstream of the 
SAR5 initation codon were identified by restriction enzyme analysis. 
B. Isolation of RJ39 Promoter 
The strawberry ripening-induced cDNA clone RJ39 was originally isolated by 
Wilkinson, Lanahan, Conner, and Klee (Plant Molecular Biology 
27:1097-1108, 1995) using a polymerase chain reaction differential display 
technique to compare mRNA populations from white vs. full-red berries. 
Northern analyses revealed that the RJ39 mRNA is strongly fruit-enhanced 
or fruit-specific, with expression levels increasing as the fruit develop 
from the small green to full-red stage. Little or no expression was 
detected in leaf, petiole, or root tissues. The cDNA clone that was 
isolated was not full-length (571 bp vs. roughly 850 bp transcript on 
Northern blots), and the translation product of this cDNA showed no 
significant homology to any known proteins. 
A genomic clone containing the RJ39 coding region and 5' flanking sequences 
(promoter) was obtained in a manner similar to that described for the SAR5 
promoter. Strawberry genomic DNA was prepared from Fragaria vesca (2n=14) 
as described in Example 1A. An aliquot of DNA was digested with the 
restriction enzyme XbaI and then the DNA fragments were separated on a 
0.7% agarose gel. The digested, fractionated DNA fragments were 
transferred to a positively charged nylon membrane (Nytran) by capillary 
blotting overnight in 10.times. SSC buffer (Southern transfer). A 
radioactive probe was prepared from the first 400 bp of the RJ39 cDNA 
clone (up to the XbaI site) using a Prime-It kit (Stratagene) and the 
filter was incubated overnight in hybridization solution at 60 degrees C. 
The filter was subsequently washed to eliminate non-specific probe 
binding, with the final wash being 0.25.times. SSC, 0.1% SDS at 60 degrees 
C. The results after X-ray film exposure indicated that RJ39 is a 
single-copy gene in F. vesca. 
Aliquots of F. vesca genomic DNA were digested with XbaI in combination 
with EcoRI, PstI, XhoI, SpeI, BglII, HindIII, EcoRV, SphI, or SalI, 
followed by Southern blotting and hybridization as described above. From 
the size of the hybridizing bands in each lane, it was determined that the 
only restriction site inside of the 5' flanking XbaI site was EcoRI (band 
of approximately 1.1 kb). All other restriction enzymes tested gave the 
same size band (approximately 1.9 kb) as F. vesca genomic DNA cut with 
XbaI alone. In order to obtain a functional promoter fragment, a genomic 
clone of approximately 1.8-2 kb, which contains the entire RJ39 coding 
region and roughly 900 bp of promoter sequence, should be obtained. 
F. vesca genomic DNA was cut with XbaI, fractionated on a 0.7% agarose gel, 
and the gel region between 1.6-2 kb excised with a razor blade. DNA from 
the gel piece was purified using a Prep-A-Gene kit and ligated to 
SpeI-digested, alkaline phosphatase-treated pBluescript SK- DNA. The 
ligation mix was transformed into competent E.coli cells which were then 
plated out onto selection media containing IPTG and X-GAL. White, 
ampicillin-resistant colonies were patched in a grid format to new plates 
and then lysed onto nitrocellulose filters for in situ hybridization 
(Sambrook et al., Molecular Cloning) 
A probe corresponding to the first 400 bp of the RJ39 cDNA clone was 
prepared using a Prime-It kit (Stratagene) and used to screen the filters 
in a manner similar to that used for the Southern blots. The hybridization 
temperature was 65 degrees C. and the final wash was 0.15.times. SSC, 0.1% 
SDS at 65 degrees C. After X-ray film exposure, several positive colonies 
were identified. These colonies were inoculated into liquid media for 
overnight growth and plasmid DNA was prepared using a Qiaprep Spin 
Miniprep kit (Qiagen). The plasmid DNA was analyzed by restriction enzyme 
analysis and 2 clones were selected. Sequence analysis of the two clones 
demonstrated that both clones contained 5' flanking sequence (the RJ39 
promoter) and the complete RJ39 coding region (down to the XbaI site in 
the 3' flanking sequence). One clone, designated pRJ39C#N-91, was selected 
for further cloning. 
A BglII restriction site was introduced upstream of the RJ39 initiation 
codon (ATG) by PCR amplification (with Pfu polymerase) of pRJ39C#N-91 with 
the primers RJ39C-3N1 (5'-AACTGCAGATCTAGTGTGGCAGTAGGTCTG-3', SEQ ID NO:7) 
and T7 promoter primer (5'-TAATACGACTCACTATAGGG-3', SEQ ID NO:8). The PCR 
product was purified from an agarose gel slice using a Prep-A-Gene kit, 
digested with BamHI, and ligated to SmaI- and BamHI-digested pUC119 
creating the plasmid pCGN8051. 
Example 2 
Preparation of Plant Expression Constructs 
The expression construct pCGN8014 was used as a cloning vector for plant 
transformation. The vector is a derivative of the vector pMON18354 which 
is described in provisional U.S. patent application Ser. No. 60/036,131, 
and regular application claiming priority, therein application number 
09/008,979 filed Jan. 20, 1998, which disclosure is incorporated herein by 
reference. The pMON18354 was modified by transposing the fragment 
containing the nopaline synthase (nos 5') promoter, neomycin 
phosphotransferase (nptII) kanamycin resistance gene, and nos termination 
(nos 3') sequences from between the right border and SRE49 promoter to the 
3' postion between the .beta.-glucoronidase (GUS) reporter gene, nos 3' 
elements and the left border sequence. The modification yielded the vector 
pCGN8014. 
Another binary vector for plant transformation, pCGN5928, was constructed 
using the neomycin phosphotransferase (nptII) kanamycin resistance gene 
driven by the nopaline synthase transcriptional initiation region (nos 5') 
and transcription termination (nos 3') sequences (Fraley et al., Proc. 
Natl. Acad. Sci (1983) 80:4803-4807 and Depicker et al., J. Molec. Appl. 
Genet. (1982) 1: 562-573). Both the nos 5' and nos 3' were PCR amplified 
from the Agrobacterium tumafaciens strain C58 and linked together with the 
nptII gene from pCGN783 (Houck, et al., Frontiers Appl Microbiol (1988)4) 
as an EcoR I fragment to form pCGN5908. The nos 5'-nptII-nos3' fragment 
was then cloned into pCGN1541, containing ori322, Right border (0.5 Kb), 
lacZ, Left Border (0.58 Kb), as an Xho I fragment between the Right 
border-lacz and Left border sequences to create the intermediate pCGN5910. 
The ColEI and pRi origins of replication as well as the Gentamycin 
resistance gene were aquired from a Not I deleted derivative of pCGN1532 
(McBride and Summerfelt, Plant Molecular Biology, (1990), 14:269-276) as a 
BamH I fragment to create pCGN5924. Finally, a linker containing unique 
restriction sites was synthesized and cloned into the Asp 718/Hind III 
(within the lacZ sequence) sites of pCGN5924 to create the binary vector 
pCGN5928. 
Plant transformation vectors were constructed to test the strength and 
tissue specificity of TSAR5 and FSR promoters in transgenic plants using 
the GUS reporter gene as a marker. Vector pCGN8045 was created by cloning 
the FSR promoter from pCGN8047 as a HindIII to BamHI fragment into the 
HindIII to BglII sites of pCGN8014. The order of genetic elements in the 
T-DNA are: RB-pFSR-GUS-nos3'-nos5'-nptII-nos3'-LB. Vector pCGN8054 was 
created by cloning the TSAR5 promoter piece from pCGN8046 as an XbaI to 
BamHI fragment into the XbaI to BglII sites of pCGN8014. The order of 
genetic elements in the T-DNA are: 
RB-pTSAR5-GUS-nos3'-nos5'-nptII-nos3'-LB. These vectors were transformed 
into an appropriate Agrobacterium strain and can be used to generate 
transgenic strawberry plants for evaluation of the promoters. 
A binary vector was constructed to test the strength and tissue specificity 
of the RJ39 promoter in transgenic plants using the beta-glucuronidase 
(GUS) reporter gene as a marker. Vector pCGN8052 was created by cloning 
the RJ39 promoter from pCGN8051 as an XbaI to BglII fragment into the XbaI 
to BglII sites of pCGN8014. The order of genetic elements in the T-DNA 
are: RB-pRJ39C-GUS-nos3'-nos5'-nptII-nos3'-LB. (The RJ39 promoter has also 
been referred to as RJ39C). This vector was transformed into an 
appropriate Agrobacterium strain and can be used to generate transgenic 
strawberry plants for evaluation of the promoter. 
Two additional DNA fusion constructs were prepared, one containing the full 
length SAR5 promoter controlling glgC16, and one containing the truncated 
SAR5 promoter controlling glgC16. The constructs were prepared as follows. 
The plant transformation vector pCGN8040 contains glgC16 under the control 
of the truncated SAR5 promoter. The truncated SAR5 promoter (TSAR5) was 
cloned as a PstI-BamHI fragment into the PstI and BglII sites of pCGN8222 
creating the plasmid pCGN8039. The TSAR5 promoter replaces the TFM7 
promoter in pCGN8222. PCGN8222 was created by cloning the glgC16 encoding 
sequence from pMON18345 (described in U.S. Pat. No. 5,608,150) as a 
BglII-SacI fragment into pMON18337 (described in provisional U.S. patent 
application Ser. No. 60/036,131 and regular application claiming priority, 
therein application number 09/008,979 filed Jan. 20, 1998). The 
TSAR5-glgC16-nos 3' fragment was cloned from pCGN8039 as a PstI-NotI 
fragment into the binary vector pCGN5928, SseI-NotI sites, to create the 
plant expression vector pCGN8040. 
The plant transformation vector pCGN8042 also contains glgC16, but is under 
the control of the full length SAR5 promoter. The full length SAR5 
promoter (FSR) was cloned from pCGN8047 as a PstI-BamHI fragment into 
pCGN8222, PstI-BglII sites, to form the plasmid pCGN8041. The 
fSAR5-glgC16-nos 3' fragment was cloned as a PstI-NotI fragment into the 
SseI-NotI restriction sites of pCGN5928 yielding the plant expression 
vector pCGN8042. 
Plant transformation vectors were transformed into Agrobacterium 
tumefaciens strain LBA4404 by the method of Holsters et al., Molecular and 
General Genetics (1979) 163:181-187. 
Example 3 
Plant Transformation 
Transgenic strawberry plants may be obtained using the methods of Nehra, N. 
S. et al. (Plant Cell Rep. (1990) 9:10-13 and Plant Cell Rep. (1990), 
9:293-298), Matthews, H. V. et al., (In Vitro Cell. Dev. Biol. (1995) 
31:36-43 and WO 95/35388, Dec. 28, 1995) or as described in U.S. patent 
application Ser. No. 60/071,773, filed Jan. 19, 1998 
Plant transformation vector pCGN8045 was transformed into strawberry plants 
using the following method: 
Strawberry Micropropagation Transformation Protocol A 
Plant Material 
In vitro strains BHN FL90031-30 or BHN 92664-501 (CA-adapted) strawberry 
cultures are grown in presterilized Magenta GA7 boxes (Magenta Co., 
Chicago, Ill.) containing micropropagation medium (Table 3). Each unit of 
tissue contains two to three apical meristems, and two units are placed in 
each jar. The cultures are then incubated at about 22.degree. C., with 
cool white light with 16/8 photoperiod at about 34-40 mEinsteins m-2 
sec-1. About every four weeks, each unit of tissue is subdivided into two 
to four clumps and placed on fresh media of the same composition. Ideal 
stock material for explanting is available at two to three weeks after the 
last subculture. 
TABLE 1 
______________________________________ 
Selection medium A 
Component Concentration 
______________________________________ 
MS salts/MS vitamins (Sigma M0404) 4.4 g/L Glucose 
20 g/L 
Washed Agar 8 g/L 
Thiodiazuron 2.3 mg/L 
Indoleacetic acid 1.75 mg/L 
Timentin 500 mg/L 
Cefotaxime 100 mg/L 
Kanamycin 50 mg/L 
pH adjusted to 5.7 
______________________________________ 
TABLE 2 
______________________________________ 
Elongation medium A 
Component Concentration 
______________________________________ 
MS salts/MS vitamins (Sigma M0404) 4.4 g/L Glucose 
20 g/L 
Washed agar 8 g/L 
Timentin 500 mg/L 
Cefotaxime 100 mg/L 
Indoleacetic acid 0.45 mg/L 
Galacturonic acid 2.5 mg/L 
Kanamycin 50 mg/L 
pH adjusted to 5.7 
______________________________________ 
TABLE 3 
______________________________________ 
Micropropagation Medium 
Component Concentration 
______________________________________ 
MS salts (Sigma 0153) 2.2 g/L 
MS vitamins (Sigma M3900) 1 mL/L 
MgSO4.7H2O 0.2797 g/L 
CaCl2.2H2O 0.2739 g/L 
KH2PO4 0.5950 g/L 
H3BO3 18.6 mg/L 
NaMoO4.2H2O 0.7 mg/L 
Iron stock 5 mL/L 
Myo-inositiol 100 mg/L 
Ascorbic acid 100 mg/L 
N6-benzylaminopurine 1 mg/L 
Indolebutyric acid 0.37 mg/L 
Sucrose 30 g/L 
Washed agar 8 g/L 
pH adjusted to 5.8 
______________________________________ 
Agrobacterium Preparation 
Four days prior to co-cultivation, Agrobacterium tumefaciens strain LBA4404 
containing plant expression vectors were streaked from a frozen glycerol 
stock AB plate (AB media supplemented with 15 g Difco Bacto Agar, Table 4) 
containing 150 g/L streptomycin, 100 mg/L gentamycin and 100 mg/L 
kanamycin. Twenty-four hours prior to co-cultivation single colonies were 
placed into 5 mL of MG/L media (Table 5). Cultures were grown overnight at 
30.degree. C., 200 rpm agitation. 
TABLE 4 
______________________________________ 
AB media 
Component Amount 
______________________________________ 
20X AB Stocks [120 g/L K2HPO4, 46 g/L NaH2PO4.H2O, 
40 g/L 
NH4Cl, 6 g/L KCl] 50 mL 
1 M MgSO4 1 mL 
0.1 M CaCl2 1 mL 
20% Glucose (w/v) 25 mL 
FeSO4.7H2O (0.25 mg/mL) 10 mL 
______________________________________ 
TABLE 5 
______________________________________ 
MG/L media 
Component Concentration 
______________________________________ 
Mannitol 5 g/L 
L-glutamic acid 1 g/L 
KH2PO4 0.25 g/L 
NaCl 0.10 g/L 
MgSO4.7H2O 0.10 g/L 
Biotin 1 mg/L 
Tryptone 5 g/L 
Yeast extract 2.5 g/L 
pH adjusted to 7.0 
______________________________________ 
Explant Inoculation 
Explanting and Pre-Culture Steps 
Small folded leaves about 2-4 mm in length possessing a vibrant green, 
glassy appearance are excised at the petiole. They are placed into a petri 
dish containing about 1-1.5 mL of sterile water and a sterilized WHATMAN 
filter paper. The basal portion of the leaves is removed with a single cut 
such that 3 leaflets are produced from each leaf. The leaflets (explants) 
are placed onto the preculture plates (Table 6). The preculture plates are 
prepared using solid medium and pipetting 1 mL of TXD liquid medium which 
has been supplemented with 200 mM acetosyringone and 100 mM galacturonic 
acid onto the solid plate. Two sterilized WHATMAN filter papers are placed 
onto the plate. Approximately 50 explants are placed onto each preculture 
plate. The plates are placed under low light conditions for about three 
days by placing in an aluminum foil covered box. 
TABLE 6 
______________________________________ 
Preculture/co-culture medium with overlay 
Component Concentration 
______________________________________ 
MS salts/MS vitamins (Sigma M0404) 0.44 g/L Glucose 
30 g/L 
Washed agar 8 g/L 
Thiadazuron 2.2 mg/L 
Indoleacetic acid 1.75 mg/L 
Acetosyringone 39.28 mg/L 
Galacturonic acid (100 mM) 4 mL 
pH adjusted to 5.7 
______________________________________ 
The overlay is 1 mL/plate of TXD liquid medium containing 200 mM 
acetosyringone, 100 mM galacturonic acid, and 2 sterile WHATMAN 8.5 cm 
filter papers. 
Inoculation and Co-Culture Steps 
The Agrobacterium suspension is diluted to 5.times.10.sup.8 bacteria/mL 
with MG/L media just immediately prior to use. The explants are removed 
from the preculture plate and allowed to sit in 5 mL of bacterial 
suspension for 5 minutes. The explants are then removed from the bacterial 
suspension and blotted dry on sterile paper towels and placed back on the 
preculture plate. The explants are spread out uniformly adaxial side down 
so that all are in good contact with the filter paper and are not 
overlapping. These plates are then co-cultured under low light conditions 
for an additional 3 days. 
Tissue Selection and Regeneration 
The explants are moved to delay medium (Table 7) for 3 days, adaxial side 
down. The explants are stored under low light conditions during the delay 
period. 
TABLE 7 
______________________________________ 
Delay medium A 
Component Concentration 
______________________________________ 
MS salts/MS vitamins (Sigma M0404) 4.4 g/L Glucose 
20 g/L 
Washed agar 8 g/L 
Thidiazuron 2.3 mg/L 
Indoleacetic acid 1.75 mg/L 
Timentin 500 mg/L 
Cefotaxime 100 mg/L 
pH adjusted to 5.7 
______________________________________ 
TABLE 8 
______________________________________ 
Rooting Medium A 
Component Concentration 
______________________________________ 
MS salts (Sigma 0153) 2.2 g/L 
MS vitamins (Sigma M3900) 1 mL/L MgSO4.7H2O 0.2797 g/L 
Cacl2.2H2O 0.2739 g/L 
KH2PO4 0.5950 g/L 
H3BO3 18.6 mg/L 
NaMoO4.2H2O 0.7 mg/L 
Iron stock 5 mL/L 
Myo-inositiol 100 mg/L 
Ascorbic acid 100 mg/L 
Indolebutyric acid 0.37 mg/L 
Timentin 500 mg/L 
Cefotaxime 100 mg/L 
Glucose 20 g/L 
Washed agar 8 g/L 
pH adjusted to 5.7 
______________________________________ 
After the three day delay, the explants (about 50 per plate) are 
transferred adaxial side down onto selection medium A (Table 1) and are 
cultured for about 3 weeks in the light (20-40 mEinsteins m-2 sec-1). 
After about 3 weeks, the explants are placed on selection medium B (Table 
9). Subcultures are performed every 3 weeks. By 6 weeks, transformed 
explants will produce green shoots and green callus. Only explants which 
contain this shooting material and green callus should be moved. If the 
explants associated with the shoots and green callus are still green and 
healthy, then the entire explant should be moved together with the 
regenerating material. By 9 to 12 weeks, green actively growing shoot 
units can be picked from the explants and placed by themselves on 
selection medium B (Table 9). Each actively dividing unit represents an 
independent event. Once the unit has tripled in size, individual shoots 
can be placed on elongation medium (Table 6). This step may take three to 
six weeks. Shoots are rooted on rooting medium (Table 8). This step 
requires approximately two to three weeks. 
TABLE 9 
______________________________________ 
Selection medium B 
Component Concentration 
______________________________________ 
MS salts/MS vitamins (Sigma M0404) 4.4 g/L Glucose 
30 g/L 
Washed agar 8 g/L 
Thiadazuron 3.4 mg/L 
Indoleacetic acid 0.45 mg/L 
Timentin 500 mg/L 
Cefotaxime 100 mg/L 
Kanamycin 50 mg/L 
pH adjusted to 5.7 
______________________________________ 
Shoots are potted into 6-pack containers of Sunshine mix #1 (80% peat). The 
containers are placed into a misting tent on trays with dome lids for 3 
days. Subsequently, the dome lids are tilted halfway to allow for airflow 
for 3 more days. The dome lid is removed after 6 days and plants stay 
under the misting tent for an additional 10 to 15 days. Plants are misted 
until they are taken out of the misting tent. 
Plants are then taken out and set on a bench for 7 days and transplanted 
into 6 inch pots of 25% of each: peat, sand, pumice, and redwood mulch. 
Greenhouse day temperatures range from about 20-24.5.degree. C. and the 
night temperatures are about 10-14.5.degree. C. There is no artificial 
light, and light intensity is decreased from the end of May to the end of 
September by use of a shade cloth. 
Example 4 
Determination of Gene Expression 
Plants transformed with promoter-GUS fusions may be examined for gene 
expression using methods well known in the art. Expression in response to 
exogenously applied auxin can also be examined using methods known in the 
art. The expression of the GUS enzyme would be expected to be similar to 
the expression of the SAR5 and RJ39 mRNAs. 
One method involves GUS staining of plant tissue and examining the staining 
pattern. Plant tissue and fruit of plants transformed with promoter-GUS 
constructs may be harvested at different developmental stages and stained 
for .beta.-glucoronidase activity. Fruit tissue may be sectioned and 
stained by infiltrating the tissue with GUS staining buffer (50 mM 
Potassium Phosphate (pH 7) 1 mg/ml X-Gluc 
(5-bromo-4-Chloro-3-indole-.beta.-D-glucorinide) and 0.1% Trition X-100). 
Tissue is allowed to stain in the staining buffer overnight at 37.degree.. 
The tissue is then destained using washes of 70% ethanol for 1 hour, then 
100% ethanol. The tissue may then be examined visually for staining. 
In order to quantify the expression, assays to determine the GUS enzyme 
activity may also be used (Jefferson, R. A., Plant Mol. Biol. Reporter 
(1987) 5:387-405). Total protein is extracted from plant tissue using GUS 
extraction buffer (50 mM Sodium Phosphate, pH 7.0, 10 mM 
.beta.-mercaptoethanol, 10 mM EDTA, 0.1% Sodium Lauryl Sarcosine and 0.1% 
Triton X-100). Samples are centrifuged, and supernatant containing the 
protein is transferred to a new tube. Assays are carried out in Assay 
buffer (1 mM MUG (4-methyl umbelliferyl .beta.-D-glucorinide in GUS 
extraction buffer). Fluorescence is measured using a fluorometer, and 
relative expression may be determined. 
In addition, gene expression may be examined at the transcription level 
using Northern hybridizations. Total or poly(A)+ RNA may be isolated from 
fruit tissues, as well as other tissues, at different developmental 
stages. The RNA is then separated on a denaturing agarose gel and 
transfered to nylon membrane (Sambrook et. al., 1989). Hybridizations may 
then be performed using a labelled probe, and the hybridized membrane is 
then exposed to radiographic film. Expression of the mRNA transcript may 
be determined by observing the hybridization pattern of the membrane. 
The above examples provide for the SAR5 and RJ39 promoters which may be 
used to provide for increasing or decreasing fruit receptacle tissue 
selective expression of heterologous genes during fruit development, 
maturation and ripening. Such a pattern of expression is particularly 
desirable for expression of genes for traits such as increased sugar 
content and disease resistance. 
All publications and patent applications mentioned in this specification 
are indicative of the level of skill of those skilled in the art to which 
this invention pertains. All publications and patent applications are 
herein incorporated by reference to the same extent as if each individual 
publication or patent application was specifically and individually 
indicated to be incorporated by reference. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 8 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 2061 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Fragaria vesca 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: promoter 
&lt;222&gt; LOCATION: (1)..(2061) 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: unsure 
&lt;222&gt; LOCATION: (897) 
&lt;223&gt; OTHER INFORMATION: position 897 can be re - #presented by any 
nucleotide 
- - &lt;400&gt; SEQUENCE: 1 
- - ctcgagcact aaacaagtta agtaatctcc ctatctctgt tacaagcttg ta - 
#ttctttgg 60 
- - ttgtgtacta accaatgtca ctgtcatatt gatccggctt aagatgttac tg - 
#atgcttat 120 
- - gggcggtgaa ggtggatatt ccaaaggtaa ggttgttata caataccgaa ga - 
#gtatacat 180 
- - tctatgccta aactcccaat tttttttttt aattttttgg tgacgagaaa aa - 
#ccattgtg 240 
- - gttctttcct cccatcatgt atggtgtcct aatattggtg ttattcacct gg - 
#aaaaattc 300 
- - gaaccaagaa acgtttgaac tagctgtgtt cttgactgga gagaatgaat ca - 
#actacgga 360 
- - ttttgagaaa caaatactat ccaacaaaga aaatgaggga gaaatgtcca tc - 
#tgctaaaa 420 
- - ttgttggcgt gaccgcaaca gagagtccac ggcccactga gaaccttgac gg - 
#gcgaaggc 480 
- - gcactgcaat atcaggggtg gggcccatct caatgcatgc aatgcctaag gc - 
#ccatattt 540 
- - ctttttgatc ccacggccca gttcacttac cacgactact atatgttagt tt - 
#tgttttta 600 
- - tttcttatag gtttagctca cttagctgga agtcagtctc acgtttatta ta - 
#ggaataat 660 
- - aaagttttgt atcgggaaaa actcaatata catgaagccc gagctacata ac - 
#taggttct 720 
- - aaaaatcggt ctgggcggcc gcctagacgc tcgacgatgg ctgcccgtct cg - 
#atttcaag 780 
- - caaatcgtgt ggtctgggcg gctgtccgat tttcgattga ttactcgggc tg - 
#ggcggcga 840 
- - ttactcaggc taggcggcga ttactcgggt tgggcgggtg ggcgggttgg cg - 
#ggacnaga 900 
- - ttctctcaac tggaaatcgt cttcgatatt gtccacgtcc atgagagata gg - 
#aacctttg 960 
- - gagcctgagt tgaagacgat agagaagaag atgaagggaa cagaataagg ag - 
#acgtgggt 1020 
- - ttttagttct ttatgataat tgagatgagt tatctattac attaggctta at - 
#ctaattgg 1080 
- - gtctggaaag atattgactt tactgatggg tattctttta atagctttta ca - 
#caaattaa 1140 
- - tccaataatt gttatccgtt tttctaagga caaggtttag gatatagttt at - 
#taggtgat 1200 
- - taaaaattat ttaatattat attaaatgat tatttaagta aataattgct tg - 
#ttataata 1260 
- - attatatgca ttatttatat aattatctat tattaaaaat aaaatattta ta - 
#caaaaata 1320 
- - taaatccgat taatccccga ttaatctttt gggcgctatc cgcccgacta ac - 
#gcctaaca 1380 
- - ttttttaaaa ccttgtacct aagaaagcct ataacatcat gaatcaatat ca - 
#tgcaaaat 1440 
- - cgtttaaaga aaacgtctga ttccaactct gtccatagga gttgaattca ga - 
#atccggag 1500 
- - aatctgaatt taattctttc ttttattatt ttcgttcatt ctaaacgatg tt - 
#aaaaaaat 1560 
- - ttaggacatg agacttaata tctagagcag tgtcacactt ataccaaagt ag - 
#ggaaccaa 1620 
- - cgtgtcacta ttaatcacat gtctcccatc attctgggcc cttcttgctc ta - 
#cggaatag 1680 
- - gacaagtatt catatagtaa gggtacactt gtgaaccaag cctcgtctca ct - 
#aaatttct 1740 
- - ctatgaatta tctttacata gcgggacccc agtttaccaa gtcttcatcc tt - 
#atccatgt 1800 
- - tttcctcctt tttccactct ccaaattatc ctacaaccag tcgtagaatt ag - 
#gaattacc 1860 
- - accctgaggt tgaaactaaa gacacttgga agtagcagaa cggtgaagaa cc - 
#ccattatc 1920 
- - aaatcagtaa ctttctaata gtaaacccca agatattttt agctaccaag ct - 
#ctcttata 1980 
- - tatacaacca tccaggaaag acccagaaca cactaaaaag gaagagatcg ag - 
#aaagaaag 2040 
- - aaagagttca gagcaaagat g - # - # 
2061 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 874 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Fragaria vesca 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: promoter 
&lt;222&gt; LOCATION: (1)..(874) 
&lt;223&gt; OTHER INFORMATION: RJ39C promoter 
- - &lt;400&gt; SEQUENCE: 2 
- - tctagacttg tatgcattac agcacgagca gttcatttca tgcatgacat ga - 
#aaacaacg 60 
- - cgtggtgcaa tctactaaac cacgtgtacg gctaatctca ggggtttttc ta - 
#gggtttag 120 
- - aatgtttcaa acatctatat taggttttgg agtttgcata gatacgttca aa - 
#agttaata 180 
- - gcaaatttga aggtgtgcaa tgttaacatt gacctgttaa cttgtgattt ct - 
#agctgtaa 240 
- - aacagataac gctttagcta atgctaagtt ttcacatttt cattttcggg ct - 
#aaatatat 300 
- - ggttgtgagt aatgtttcag agatactttt aactttttga aagatatagt ct - 
#cttaatgg 360 
- - atatatatga gatgtaattt tatgagttat ctgtttcgat ttgaattcac gt - 
#ttaggttt 420 
- - aggattagct agcactttaa cattatcctg aaatatcttg gaaacaaatt tg - 
#gtgttgtc 480 
- - ctaacaataa tattattgtg ttgaaatttg cacattgcat ttttatgtgt tg - 
#aaatttta 540 
- - tctaaaatgc ttggtggagc ataataattt gaaaagagaa agagtcaata tg - 
#aatccgat 600 
- - gcattcttgt cgtccagatt aactatttaa caaatatcca aatctatcta ta - 
#tggttata 660 
- - aattatattt attttctaaa tttacttcca tgtttttaat ttgccaggtc at - 
#cgatctaa 720 
- - ttacacggta gagaatactc atgaaactgg aattctgaat attcacgtca gc - 
#acagatat 780 
- - tagctggctc tgcttatctt tctctctatc caacactgtg attcaaaccc cc - 
#attaaatc 840 
- - cagacctact gccacactgt ccctttcttc catg - # - 
# 874 
- - - - &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: PCR 
primer 
SAR5-5C1 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: primer.sub.-- bind 
&lt;222&gt; LOCATION: (1)..(27) 
- - &lt;400&gt; SEQUENCE: 3 
- - tcgaattcag agcaaagatg gttctgc - # - # 
27 
- - - - &lt;210&gt; SEQ ID NO 4 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: PCR 
primer 
SAR5-3N2 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: primer.sub.-- bind 
&lt;222&gt; LOCATION: Complement((1)..(27)) 
&lt;223&gt; OTHER INFORMATION: complementary to SAR5-5C1 - #sequence I.D. #3 
- - &lt;400&gt; SEQUENCE: 4 
- - acctcgaggg atcctcatca cttgtcg - # - # 
27 
- - - - &lt;210&gt; SEQ ID NO 5 
&lt;211&gt; LENGTH: 25 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: Primer 
pSAR5-BH1 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: primer.sub.-- bind 
&lt;222&gt; LOCATION: (1)..(25) 
- - &lt;400&gt; SEQUENCE: 5 
- - taggatccgt ctttgctctg aactc - # - # 
25 
- - - - &lt;210&gt; SEQ ID NO 6 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: Primer 
PSAR5-R4 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: primer.sub.-- bind 
&lt;222&gt; LOCATION: (1)..(20) 
- - &lt;400&gt; SEQUENCE: 6 
- - accttgtacc taagaaagcc - # - # 
- # 20 
- - - - &lt;210&gt; SEQ ID NO 7 
&lt;211&gt; LENGTH: 30 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: Primer 
RJ39C-3N1 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: primer.sub.-- bind 
&lt;222&gt; LOCATION: (1)..(30) 
- - &lt;400&gt; SEQUENCE: 7 
- - aactgcagat ctagtgtggc agtaggtctg - # - # 
30 
- - - - &lt;210&gt; SEQ ID NO 8 
&lt;211&gt; LENGTH: 20 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence: T7 
Promoter 
Primer 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: primer.sub.-- bind 
&lt;222&gt; LOCATION: (1)..(20) 
- - &lt;400&gt; SEQUENCE: 8 
- - taatacgact cactataggg - # - # 
- # 20 
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