WO2001077310A1

WO2001077310A1 - A promoter cv20oxp which regulates the integument-specific expression of a gibberellin 20-oxidase gene in developing seeds of watermelon and a method for generating seedless fruits using the promoter

Info

Publication number: WO2001077310A1
Application number: PCT/KR2000/001127
Authority: WO
Inventors: Gyn-Heung An; Hong-Gyu Kang; Sung-Hoon Jun; Junyul Kim; Ki-Hwan Jung; Yong-Joo Park; Sang-Yeb Lee
Original assignee: Dongbu Hannong Chemical Co., Ltd.
Priority date: 2000-04-08
Filing date: 2000-10-09
Publication date: 2001-10-18
Also published as: KR20010090954A; AU2000279655A1; KR20020093028A

Abstract

The present invention relates to a testa specific gene, which is isolated from the developing seed of ∫i⊃Citrullus lanatus∫/i⊃, a promoter and a method for generating seedless fruits by using the promoter of the said testa specific gene. More specifically, the present invention provides a Cv20ox gene, gibberellin 20-oxidase(GA 20-oxidase) gene showing the tissue-specific expression pattern, which is strongly expressed in the integumental tissue at seed development of watermelon and is weakly expressed in the inner tissue of seed, a promoter regulating the integument-specific expression of the said gene and a method for generating seedless fruits by introducing plant growth regulation gene ligated to the said promoter and regulating the overexpression of plant growth regulation composition in the testa.

Description

A PROMOTER Cv20oxP WHICH REGULATES THE INTEGUMENT-SPECIFIC

EXPRESSION OF A GIBBERELLIN 20-OXIDASE GENE IN DEVELOPING

SEEDS OF WATERMELON AND A METHOD FOR GENERATING SEEDLESS

FRUITS USING THE PROMOTER

Technical Field

The present invention relates to the Cv20ox gene, a promoter of the integument-

specific Cv20ox gene isolated from the developing seeds of the Citrullus lanatus, and a

method for producing seedless plants using the promoter.

More particularly, the present invention relates to the Cv20ox gene encoding a GA

20-oxidase — which shows the tissue-specific expression pattern. For example, the GA

20-oxidase is strongly expressed in the integument tissues and weakly in inner parts of the

seeds during the seed development of the Citrullus lanatus, a Cv20ox promoter that

regulates the integument-specific expression of the Cv20ox gene, and a method for

producing seedless plants using the Cv20ox promoter.

Background Art

Up to the present, many researchers have developed transgenic plants that are genetically modified for the improvement of the resistance to disease and harmful insects,

and the increase of harvest. The public is already being provided with the genetically

modified plants that have proven to be safe. Also, it is focused on the development of

seedless plants as a part of theses studies. Although a seed is essential for the

reproduction, when being consumed, it becomes an inconvenient factor for the consumers.

For this reason, the consumers tend to prefer the seedless fruits. Moreover, the seedless

fruits have the advantage of being able to be obtained under environmental conditions that

are difficult for pollination and fertilization.

In order to have the utility as the seedless plants, it requires: 1) the

parthenocarpy — the development of fruit formation and growth without fertilization, 2) the

production of seedless parthenocaφic fruits, and 3) the production of fruits with viable

seeds when flowers are pollinated.

Some of the methods of developing these parthenocarpic plants are known. For

example, the method of using a synthetic plant growth factor (Liparl et al, Ada.

Hort.229:307-312_? 1988), the method of using mutants capable of the parthenocaφic

development (Lin S. et al, J. Horod. 75 : 62-66, 1988), and the method of using plants

altered in their polyploid (Kihara H., Plant Growth Substance-Inducing Application in

Agriculture, Tata McGray Publishing Company Ltd., New Delhi, India, 1981) are known.

Among the methods mentioned above, the most common method is to treat the

synthetic phytohormones on flower buds. This method uses the principle that the increase of plant hormones in the ovary by pollination and fertilization stimulates a cell

division and leads to facilitate fruit formation and growth. More particularly, an artificial

treatment with the synthetic hormone increases the plant hormone concentration in the

ovary at the early stage of flower and fruit development and, thus, promotes a normal fruit

growth — even if pollination does not occur. However, the method — which uses the

synthetic plant growth factors — requires high culturing skills because of the following

reasons: first, if the synthetic plant growth hormones are not treated on right time, a fruit

drop may happen; second, the growth hormone should be treated only to the female

flowers once or twice; and third, the pericaφ may crack after fruit setting. Also, said

methods require intensive labor and high cost. More significantly, the synthetic

chemicals may become part of the human diet.

Another method for producing seedless fruits is to use the mutants altered in the

chromosome number, such as triploids. Dr. Jang-Chun Woo of Korea developed this

method. The seedless watermelon is a typical seedless fruit that was produced by this

method.

In this method by Dr. Jang-Chun Woo, the watermelon with tetraploid is produced

from a normal watermelon with diploid, and cross-fertilized with a watermelon with

diploid to produce a watermelon with triploid. The watermelon with triploid produced

by this method cannot produce normal pollens and ovaries during reproductive division

and, thus, forms a seedless watermelon. However, since the growth of the watermelon was unstable, its pericaφ grew to be thick or cracked — which resulted in an increase of

the deformed fruit formations and a reduction of sugar content. For these reasons, this

method is no longer used.

Beside the said methods above, a method for producing seedless fruits using some

gene manipulation has been reported, especially, in the tobaccos and the eggplants. In

this method, the plant growth regulatory gene (iaaM) — hich is linked to the downstream

of the ovule-specific gene (DejΗ9 of Antirrhinum majus) promoter — was introduced into

plants and over-expressed in the ovule. Then a state, similar to the pollinated state, was

made without the increase of the endogenous phytohormone by pollination etc. to promote

the fruit growth and to produce the seedless fruits (Giuseppe et al., Nature Biotechnology

15:1398-1402,1997). Thus, the method for producing the seedless fruits using the ovule-

specific gene promoter is known to be a useful method because problems do not exist.

However, in order to apply this method in other plants, ovule-specific expression genes

and the position of their promoters have to be located. Until now, this method has been

applied to the tobaccos and the eggplants, but has not been applied to the watermelons and

the other fruits — which have higher values as goods from other seedless fruits. Therefore,

the development of a new promoter — which can be applied to other plants — has become

necessary in order to produce other seedless fruits such as seedless watermelon etc.

Gibberellins are hormones which play significant roles in the plant growth and development, including seed germination, stem growth, flower formation, flower growth,

fruit setting, seed development and biosynthesis of anthocyanin (Hooley R., Plant Mol.

Biol. 26 :1529-1555, 1994).

Gibberellins are biosynthesized from tetracyclic diteφene ent-kaurene by two

types of enzymes — the monooxygenase and the dioxygenase (Hedden P. et al, Annu. Rev.

Plant. Physiol. Plant Mol. Biol. 48:431-460, 1997). In the early stage of the biosynthesis,

GAι and GA₅₃ are synthesized by the monooxygenase. These gibberellins are converted

to the physiologically-active Cχ₉-GAs by two main dioxygenases, gibberellin (GA) 20-

oxidase and 3β -hydroxylase. The GA 20-oxidase forms a Cχ skeleton by the sequential

oxidation of a C₂o of the GAι₂ and the GA₅₃. The 3β -hygroxylase catalyzes the 3β -

hydoxylation of the Cι skeleton — resulting in the formation of the GAs that are

biologically active.

The genomic DNA and cDNA clones encoding the GA 20-oxidase and the 3β -

hydroxylase — which are involved in biosynthesis of the GA — have been isolated from

several of the plant species. Also, their enzymatic activities have been measured with the

recombinant enzymes produced in the transformed Escherichia coli (Hedden & Kamiya,

Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:431-460,1997).

It has been reported that the expression of the two dioxygenases was repressed in

feedback regulation by the GA treatment(Chiang et al, Plant Cell. 7:195-201, 1995). It has also been demonstrated that the expression of the GA 20-oxidase in the Arabidopsis

(Xu et al, Proc. Natl Acad. Sci. USA. 42: 6640-6644,1995) and the spinach (Wu et al,

Plant Physiol, 110:547-554, 1996) was enhanced under a long-day conditions. In

addition, through the studies about the GA 20-oxidases in Arabidopsis, bean, pea, and

tomato plants, it has been demonstrated that these plants contained several types of the GA

20-oxidase genes — of which the expression was regulated spatially and temporally during

development and, also, which involved in different regulation processes of the GA such as

stem elongation, flower formation, and fruit setting (Phillips et al, Plant Physiol,

108:1049-1057, 1995).

It has been reported that some of the GA 20-oxidases are expressed specifically in

the seeds and the fruits. For example, the YAP169 of the arabidopsis is specifically

expressed in the siliques (Phillips et al, Plant Physiol, 108:1049-1057, 1995), the Pv85-

26 of bean in the developing seeds(Garcia-Martinez et al., Plant Mol. Biol, 33: 1073-1084,

1997), and the M3-8 of the Marah macrocarpus in the embryos and the endosperm of the

developmg seeds (MacMillan et al., Plant Physiol, 113: 1369-1377, 1997).

Especially, since developing seeds contain more abundant GAs than any of the

other plant organs, they have been used frequently in the genetic and the biochemical

studies for elucidating the the GA biosynthetic pathways in the plants (Graebe et al., Annu.

Rev. Plant Physiol, 38: 419-465, 1987). However, little was known about the roles of

the GAs and the exact location of the synthesis in the developing seeds. Recently, it just has been reported that the GAs are involved in the early- and the late-stages of the seed

development in some of the plant species (Euwens & Schwabe et al., J. Exp. Bot, 26: 1-14,

1975), and the pattern of the seed development are changed in several of the GA-deficient

mutants of the pea plants. For example, it has been observed that the expression of the

GA was dramatically reduced in the developing seeds, and the development of the seeds

was halted in the lh-2 mutation (Swain et al., Plant J., 12: 1329-1338, 1993; Swain et al.,

Planta, 195: 426-433, 1995). The physiological, the biochemical and the genetic

analyses in the gibberellin-deficient mutants, the Is-1 and the Ih-2, have revealed that the

gibberellin biosynthesized in the embryo and/or the endosperm is necessary for the seed

development in the early several days after the pollination (Ait-Ali et al., Plant J., 11: 443-

454, 1997). Furthermore, the seed development is very important in the pericarp

development associated with the plant hormones such as the gibberellins and the auxins

etc. (Nan Huizen et al., Plant Physiol. 115: 123-128, 1997). In the case of the tomatoes,

the gibberellin was necessary for the fruits and the seed development during a transient

period after the fertilization (Groot et al., Physiol. Plant. 67: 315-319, 1987).

When the present inventors noticed that the utility of the gene expressed

specifically in the seed and its promoter, they attempted to develop a method for producing

the seedless fruits by searching for the seed-specific gene and its promoter. As a result,

the inventors completed the present invention by searching a promoter of the integument- specific Cv20ox gene in developing seeds in the Citrullus lanatus and identifying that the

promoter regulates the integument-specific expression of the gene. Therefore, the

Cv20ox promoter provided by the present invention can be used in a method for producing

the seedless fruits — more specifically, the seedless plant of the gourd family, such as the

watermelon or the melon that have a high preference, by linking it to the plant growth

regulatory gene and introducing it into the plants and over-expressing the gene in those

plants.

Disclosure of Invention

An objective of the present invention is to provide a Cv20ox gene encoding the

GA 20-oxidase expressed specifically in the integument during the seed development in

the Citrullus lanatus.

Another objective of the present invention is to provide a Cv20ox promoter, which

regulates the integument-specific expression.

An objective of the present invention is also to provide a method for producing

the seedless fruits using the above promoter. More particularly, it is an objective to

provide a method for producing the seedless fruits by linking a plant growth regulatory

factor to the above promoter and inducing an over-expression of the plant growth

regulatory factor in the integuments. Hereinafter, the present invention will be described in more detail.

The present invention provides the integument-specific Cv20ox gene isolated from

the developing seeds of the Citrullus lanatus and its promoter regulating the integument-

specific expression of the gene.

The cDNA of the Cv20ox gene consists of 1,420 nucleotides (See SEQ. ID NO: 3)

and encodes a GA 20-oxidase protein consisting of 379 amino acid residues (See SEQ. ID

NO: 4. It has typical conserved regions of 2-oxoglutarate-dependent dioxygenases,

including a Fe²⁺-binding motif consisting of His-239, Asp-241, and His-295, and the

putative 2-oxoglutarate-binding motif. Additionally, the amino acid sequence of the GA-

20 oxidase — which is encoded by said gene — has high homology with the GA 20-oxidases

that were previously found in other plants. Particularly, it has a homology of 76% and

63% with the GA 20-oxidases in M. macrocarpus and the pumpkin, respectively. The

protein also shares >50% identity with the GA 20-oxidases of pea, bean, arabidopsis,

spinach, and lettuce (See Fig. la and lb).

The DNA and the RNA blot analyses of the Citrullus lanatus seeds have shown

that there are one or two copies of the Cv20ox gene within the Citrullus lanatus genome

(See Fig. 2). This gene was never expressed in the other reproductive and vegetative

organs except in the developing seeds. The gene exhibits the spatial expression pattern.

More particularly, among the various tissues of the seeds, it was strongly expressed in the integument tissues — while it was expressed weakly in the inner part of the seeds (See Fig.

3a and 3b).

According to the result of the in situ hybridization analysis, most of the Cv20ox

transcript localized predominantly on the inner layer of the integument during the seed

development in the Citrullus lanatus (See B, E, and G in Fig. 4). The Cv20ox gene

expression was initiated at three days after the pollination, which was maintained until 15

days after the pollination (See Fig. 5).

To investigate the effects of the GA₃ treatment on the Cv20ox gene expression, the

expression-level of the gene under the GA₃ treated conditions and the untreated conditions

was compared with the expression level of the Sue synthase gene as a control. As a

result, the expression of the Cv20ox was strongly repressed by the GA₃ treatment, while

the expression of the Sue synthase was not affected (See Fig. 6a). The result illustrates

that the expression of the GA 20-oxidase gene is feedback-regulated by the excessive GAs,

similar to the result reported previously in several plants.

In another embodiment of the present invention, the expression pattern of the

Cv20ox after treating with the CPPU — an inducer of the parthenocaφy in plants — was

compared with the seeds of normal pollination by the northern blot analysis. As a result,

it was observed that the CPPU treatment resulted in an earlier expression of the Cv20ox

gene compared with the pollinated controls. The earlier induction of the gene was

probably due to the promoted seed and the fruit development by the CPPU treatment. It was observed that this gene transcript was expressed strongly in the integument and

weakly in the inner parts of the seeds (See Fig. 6b). Since the CPPU-treated seeds did

not develop embryos or an endosperm, it suggested that the GA 20-oxidase transcript

observed in the inner parts to be probably expressed in the blast cells such as micella or

transfer cells.

Furthermore, the present invention provides the promoter that regulates the

integument-specific expression of said Cv20ox gene that was isolated from the developing

seeds in the Citrullus lanatus.

The region of the Cv20ox promoter consists of the nucleotide sequences

represented by SEQ ID NO: 5, in which the putative TATA box (TATAAATC box) is

present in 132 nucleotides upstream-region from the translation start codon, and it contains

three stretches of more than 19 bps A/T nucleotides as an A/T-rich region. These

distributions of the nucleotide sequences is similar to that of the 5' upstream-region of the

ConA gene, one of the major seed-storage protein genes of the Canavalia gladiata known

to include highly A/T-rich sequences. Moreover, since the A/T-rich sequences of the 5'

upstream-region was known to play a significant role in the transcriptional activation

(Yamamoto et al., Plant Mol Biol 27: 729-741, 1995) — the region of the Cv20ox promoter

is useful in identifying the mediating-signal that regulates the Cv20ox gene expression.

This promoter can be used in a method for producing the seedless fruits by linking it to a plant growth regulatory factor, introducing it into the plant, and then regulating the over-

expression of the plant growth regulatory factor in the integument.

In order to investigate the integument-specific activity of the Cv20ox promoter, the

inventors have set up a transient expression system by linking this promoter to a reporter

gene — which can be easily expressed and confirmed in its expression pattern without

passing through the complex steps of the plants. Thus, the expression vector with the

GUS (β-glucuronidase) — a reporter gene — fused to the downstream of the Cv20ox

promoter was made. Then it was inserted to the various organs of the Citrullus lanatus and

was examined by the expression pattern by the GUS staining. As a result, this promoter

activity was detectable only in the integument tissues of the developing seeds. It was,

especially, very strong in the layer close to the nucellus. On the other hand, the promoter

activity was not detectable in the non-reproductive organs — such as leaves, roots,

hypocotyls, and cotyledons.

The promoter having the same nucleotide sequences with more than the continuous

23 base pairs in the nucleotide sequences of the Cv20ox promoter represented by SEQ ID

NO: 5 belongs to the scope of the present invention. The promoter, of which the

sequence of more than the continuous 23 base pairs is identical to the nucleotide sequence

of the Cv20ox promoter and a partial nucleotide sequence not affecting the function of the

promoter regulation is substituted, modified, deleted, or added, can regulate the

integument-specific expression and be used for the production of the seedless fruits etc. Theoretically, the probability that more than the continuous 23 base pairs will be identical

to each other is very unlikely — one out of 4²³ (7.03 x 10¹³). In addition, as a result of the

Genbank search (http://www.ncbi.nlm.nih.gov/BLAST/), it was found that the sequence of

more than a continuous 23 base pairs showing the identity to the sequence of more than

another continuous 23 base pairs of the Cv20ox promoter — in specific genes — did not

exist. Therefore, it can be confirmed that along with the base sequence of SEQ ID NO: 5,

the sequence having the same nucleotide sequences with more than a continuous 23 base

pairs in the nucleotide sequences of SEQ ID NO: 5 belongs to the scope of the Cv20ox

promoter of the present invention.

Moreover, the present invention provides a method for producing the seedless

fruits using this Cv20ox promoter.

More particularly, the method for producing the seedless fruits of the present

invention using this Cv20ox promoter comprises of the following steps: (1) constructing

an expression vector by making a gene encoding plant growth regulatory material linked

to the downstream of the promoter regulating integument-specific expression of the

Cv20ox gene in the plant; (2) transforming the plant with the expression vector; (3)

selecting a transgenic plant and inducing ^differentiation; (4) adjusting to the soil; and (5)

inducing the fruit formation and the growth by keeping the natural pollination of the

flower of said transgenic plant repressed.

The known method, a method for getting the seedless fruits from the transgenic plants transformed with the plant growth regulatory gene linked functionally to the ovary-

specific promoter (Giuseppe et al, Nature Biotechnology 15: 1398-1402, 1997), can be

referred to the above method.

In the method mentioned above, genes such as iaaM, iaaH, or ipt etc. can be used

as a plant growth regulatory gene. It is preferable to use the iaaM gene encoding

tryptophan mono-oxigenase among them. The iaaM gene encodes tryptophan mono-

oxigenase that oxidizes tryptophan to indolactamide. The produced indolactamide alone

cannot activate auxin — a plant growth hormone — but it is slowly converted — either

chemically or enzymatically — to IAA, the major form of the auxin in plants by the plant

hydrolases. Subsequently, said iaaM gene comes to be over-expressed by the Cv20ox

promoter that regulates the integument-specific expression in the ovary. As a result, the

concentration of the plant growth hormone comes to increase to set and grow the seedless

fruits — similar to the pollinated or the fertilized state even if the normal pollination does

not occur. In case of using the iaaM gene for production of the seedless fruits, there is an

advantage that the seedless fruits can be produced without the exogenous treatment of the

plant hormone — even under the environmental conditions that are adverse for the

pollination and the fertilization. When compared with the conventional method,

profitable results can be obtained in the respects of the labor that is required for producing

the seedless fruits and the costs that is required for regulating the temperature of the

greenhouses on cultivation of the seedless fruits. In the method for producing the seedless fruits mentioned above, along with the

gene promoter — which is represented by SEQ ID NO: 5 and expressed integument-

specifically in the watermelon — also the nucleotide having the identity with the promoter

of SEQ ID NO: 5 in more than a continuous 23 base pairs — which is isolated from the

watermelon and the other plants — can be used as the Cv20ox promoter. In the present

invention, the "promoter" can be used as a general meaning, that is, a meaning of the

nucleotide sequences located in the upstream from the transcription start site to which the

DNA-dependent RNA polymerase recognizes or binds directly or indirectly for the

transcription initiation. Preferably, it can be also used as a meaning of the untranslated

signal-regions, including both the intron- and the promoter-region. Thus, the

untranslated signal-region in addition to the promoter-region of SEQ ID NO: 5 can be

used as the Cv20ox promoter for producing the seedless fruits.

In the process concerning the construction of the transgenic plants, re-

differentiation, the root induction, and the soil adjustment in said method for producing the

seedless fruits, a known method in the art for transformation of plants can be used.

In the process producing the seedless fruits from the transgenic plants produced

through the steps mentioned above, the fruit setting and the growth are achieved by the

over-expression of the iaaM under the conditions that the pollination of the transgenic

plants is inhibited. It is preferable to inhibit the pollination and the fertilization by

wrapping the flowers with paper bags etc. for approximately one month before dehiscence of anthers when the height of the flowers reaches around 2-3 cm.

The method for producing the seedless fruits using the Cv20ox promoter of the

present invention can be applied to other various plants — preferably, to the plants of the

gourd family such as the watermelon and the melon. Also, the method can be applied to

any plants in which the said promoter can be functionally expressed. It is not restricted

for the plants mention above.

Another method for producing the seedless fruit using the Cv20ox promoter of the

present invention is to repress the normal expression of the Cv20ox gene by linking the

gene to the promoter in a reverse directio — resulting in the repression of the seed

development. The said protein involves in the early- and the late-stages of the seed

development as an enzyme that synthesizes the C₁₉-GAs having the physiological activity

by oxidizing the GA]₂ or the GA₅₃ that are synthesized by the ono-oxyganase. As

confirmed in the Gibberellin(GA)-deficient mutants, the expression of the GA is essential

for the seed development. Therefore, linking the said gene to the promoter in the reverse

direction and repressing the expression of the gene to repress the seed development can

produce the seedless fruits.

In the preferred embodiment of the present invention, the pGA2257 expression

vector was constructed by linking the Cv20ox gene to the Cv20ox promoter in the reverse

direction, which, in turn, links the T7'-5' termination sequence to said gene — which is

linked to the promoter. Then, it is inserted into the region between the left- and the right- border of the T-DNA in the pGA810 vector (An et al., Plant Molecular Biology Manual,

Kluwer Academic, Dordrecht, Belgium, 1988). The expression vector includes the

tetracycline and the kanamycin resistance gene as a selective marker. The E.coli JM83

transformed with said pGA2257 was deposited to the Korean Culture Center of

Microorganisms on September 29, 2000 (Accession No: KCCM-10216).

Brief Description of The Drawings

For a thorough understanding of the nature and the puφose of the present invention,

references should be made to the following detailed descriptions taken in connection with

the accompanying drawings in which:

Fig. la and lb illustrates the comparison of the amino acid sequence of the GA 20-

oxidase encoded by the Cv20ox gene with that of the GA 20-oxidase derived from

several plants.

1: C. vulgaris Cv20ox 2: M. macrocarpus M3-8

3: C. maxima cm20oxi 4: P. sativum ps27-12

5: P. vulgaris pv73-l 6: P. vulgaris pvl5-ll

7: L. sativa Ls20oxl 8: L .sativa Ls20ox2

9: P. vulgaris pv85-26 10: A. thaliana at2353

11: A. thaliana atyapl69 12: A. thaliana at2301

13: S. oleracea soU33330 Fig. 2 shows the result of the southern blot analysis of the genomic DNA isolated

from the developing seed of the Citrullus lanatus using the Cv20ox cDNA as a

probe.

E : treated with EcoR I

H : treated with HindΗL

Fig. 3a is a gel photograph showing the result of the northern blot analysis of the

total RNA isolated from the various mature tissues of the Citrullus lanatus to

investigate the tissue-specific expression patterns of the Cv20ox gene.

I : integuments at 8 days after the pollination

S : whole seeds at 8 days after the pollination

IS : inner seed tissues at 8 days after the pollination

FI : fruits at 1 day after the pollination

F2 : fruits at 2 or 3 days after the pollination

F8 : fruit at 8 days after the pollination

T : tendrils L : leaves

M : male flowers O : ovaries before pollination

Fig. 3b is a gel photograph showing that the Cv20ox gene was not expressed in any

other organs at the stage of development, as a result of the northern blot analysis

with the mRNA isolated from the various organs of young plants, except for the

seed, grown for 15 days after sowing. C : cotyledons Hu : upper hypocotyls

Sm : meristem tissue including most of the upper region of hypocotyl

HI : lower hypocotyls R : roots

Fig. 4 shows the tissue-specific expression pattern of the Cv20ox gene in seeds of

the Citrullus lanatus by the in situ hybridization experiment.

A, B and C : seeds at 5 days after pollination

D and E : seeds at 6 days after pollination

F and G : seeds at 4 days after pollination

Nu : nucellus ii : inner layer of integument

oi : outer layer of integument mp : micropylar end

E : globular embryo

Fig. 5 shows the result of the northern blot analysis representing the temporal

expression pattern of the Cv20ox gene of the present invention during the seed

development.

DAP : days after pollination

Fig. 6a is a gel photograph showing the results obtained by treating the GA₃ to the

fruits at 3 days after pollination for 24hrs, harvesting the seeds, extracting the RNA

from it, and, then, performing the northern blot analysis with the RNA — in order to

investigate the effects of the GA₃ on the regulation of the Cv20ox gene expression.

+ : fruits at 3 days after pollination with GA₃ treatment - : fruits at 3 days after pollination without GA₃ treatment

Fig. 6b shows the result of the northern blot analysis showing the comparison of

the Cv20ox gene expression pattern of the seeds harvested after a normal

pollination with that of the seeds harvested after the N-(2-chloro-4-pyridyl)-N'~

phenylurea(hereinafter referred to 'CPPU') treatment.

lane 1: at 2 days after pollination lane 2: at 3 days after pollination

lane 3: at 4 days after pollination lane 4: at 2 days after CPPU treatment

lane 5: at 3 days after CPPU treatment lane 6: at 4 days after CPPU treatment

Fig. 6c shows the result of the northern blot analysis showing the comparison of

the Cv20ox gene expression pattern in the integuments with that in the inner seed

tissues of seeds at 10 days after the CPPU treatment.

Fig. 7 illustrates the cleavage map of the recombinant expression vector,

pGA2118 — in which the GUS (β -glucuronidase) gene is linked to the downstream

of the Cv20ox promoter as a reporter gene — in order to measure the activity of the

Cv20ox promoter of the present invention.

Fig. 8 shows the result of the histochemical detection of the GUS gene expression

after introducing the pGA2118 recombinant vector into the various organs of the

Citrullus lanatus by a particle bombardment.

A: developing seeds at 8 days after pollination

B: inside of the seeds cut in parallel with the plane of the seeds at 8 days after pollination

C: leaves D: roots

E: hypocotyls F: cotyledons

Fig. 9 shows the cleavage map of the binary vector, pGA2257 (accession number:

KCCM-10216), in which the Cv20ox gene is linked to the downstream of the

Cv20ox promoter in the reverse direction and the tetracycline- and the kanamycin-

resistance gene exists as selective markers.

Best Mode for Carrying Out the Invention

The present invention will be described in more detail with the following examples.

It will be appreciated that those skilled in the art, on consideration of this disclosure,

may make modifications and improvements within the spirit and the scope of the present

invention.

Example 1

Isolation of the CV20O cDNA, the GA 20-Oxidase Gene

In the present invention, a cDNA library was constructed from the developing

seeds of the Citrullus lanatus to isolate the GA 20-oxidase gene involved in the biosynthesis of the Gibberellin(GA). Particularly, the FI hybrid of the Citrullus lanatus

(Thunb, van Country Home) was cultivated in a greenhouse and maintained at 30-35 ^°C

during the day and 15-20 ^°C at night. From the seeds of the Citrullus lanatus at

approximately 6-10days after the pollination, the poly (A)⁺ mRNA was extracted by an

oligo(dT)-cellulose chromatography. The cDNA library was constructed with the ZAPII

cDNA cloning kit and the Gigapack HI Gold (Stratagene) according to the

manufacturer's instructions. This cDNA library was amplified in E.coli XL-1 blue

(Stratagene) and the initial number of the plaque-forming units in the library was 4 x 10^6~

which is sufficient to contain low-frequency clones.

To isolate only the cDNA of the GA 20-oxidase gene from the said cDNA, the

PCR was performed using the degenerate primers against the conserved sequences in the

GA 20-oxidases. More particularly, the PCR was performed using the DNA isolated

from the cDNA library of the seeds at 6-10 days after pollination as a template — with each

oligonucleotide represented by SEQ ID NO: 1 and SEQ ID NO: 2 as the forward and the

reverse primer. The PCR reaction was initiated by heating to 94 ^°C for 5 minutes, then

subjected to 40 cycles under the condition as the following: 94 ^°C for 1 minute, 40 ^°C or

50 °C for 1 minute, and 72 ^°C for 1 minute, and completed by amplifying for 10 minute at

72 °C . The resulting 300bp of the PCR reaction was separated by the agarose gel

electrophoresis. Then it was cloned into the pGEM-T Easy vector (Promega). The

cloned DNA sequence was analyzed with the DNA sequence analyzing kit (Big DyeTM Terminator Cycle Sequencing Ready Reaction, PE Applied Biosystems) and the DNA

sequencer (model ABI 373 DNA Sequencer, Applied Biosystems).

The full length Cv20ox cDNA was isolated by screening the cDNA library with the

resulting partial cDNA as a probe, according to the Sambrook method (Molecular Cloning,

Cold Spring Harbor Laboratory Press).

Example 2

Sequence Analysis of the Cv20ox Gene

The sequence obtained in the Example 1 was compared with that of the known the

GA 20-oxidase using the BLAST program of the DNA Data Bank of Japan.

As a result, the amino acid sequence predicted as the GA 20-oxidase gene had a

high homology with the amino acid sequence of the enzyme in other various species.

However, because this clone included only a part of the GA 20-oxidease gene, the cDNA

clone — including full-length ORF predicted as the GA 20-oxidase — was obtained by

screening the cDNA library of the Citrullus lanatus seeds with the 300bp clone as a probe.

The clone consisted of 1,420 nucleotides represented by SEQ ID NO: 3 and encoded a

protein with 379 amino acid residues (Genbank accession no : AF074709). Furthermore,

the said peptide has a Fe²⁺-binding motif consisting of His-239, Asp-241 and His-295, and

a typical consensus sequence in the 2-oxoglutarate-dependent dioxygenase containing 2- oxoglutarate-binding residues.

The sequence analysis of the said peptide has revealed that the Cv20ox peptide

sequences represented by SEQ ID NO:4 was very similar to the amino acid sequences of

the known GA 20-oxidase (See Fig. la and lb). More particularly, the Cv20ox protein

isolated from the seeds of the Citrullus lanatus showed the amino acid homology of 76%

and 63% with the GA 20-oxidase in M. macrocarpus and the pumpkin, respectively. The

said protein also shares >50% identity with the GA 20-oxidases of pea, bean, arabidopsis,

spinach, and lettuce.

Example 3

The Distribution Pattern of the Cv20ox Gene in the Genome

The southern blot analysis of the genomic DNA was performed to identify the

copy-number of the Cv20ox gene in the Citrullus lanatus genome. More particularly, the

total genomic DNA was isolated from the leaves of the 2 weeks-old Citrullus lanatus

plants according to the CTAB method (cetyltrimethylammonium bromide, Rogers SO &

Bendich AJ, Extraction of DNA from plant tissue A6:l-10, 1988). The isolated genomic

DNA was restricted with the HzndHI or EcoRI, separated on a 0.8% agarose gel by

electrophoresis, and transferred to a nylon membrane (Ηybond-N, Amersham). The

transferred DNA was fixed to the nylon membrane by baking the membrane at 80 ^°C for 1 hour. The 300bp cDNA obtained in the Example 1 was labeled with ³²P by the random

priming method (Sambrook et al, Molecular Cloning, 1989) to use as a probe. Then it

was hybridized with the genomic DNA transferred to the membrane.

The pre-hybridizing solution (7% SDS, 0.5M NaPO₄, ImM EDTA, 1% BSA) was

added to the nylon membrane to which the DNA fixed perfectly and incubated in the oven

at 65 ^°C for 2 hour. The labeled DNA probe was denatured by incubating in boiling

water for 5 minutes and, then, added to the hybridizing solution. Then, it was incubated

for 18 hours. Thus, the membrane was washed twice with the solution containing the

0.1X SSC and the 0.1% SDS at 60 ^°C, and exposed to light using X-ray film.

As a result, the ³²P-labeled cDNA probe was hybridized to one or two major bands

(See Fig. 2), indicating that one or two copies of the Cv20ox gene exist in the genomic

DNA of the Citrullus lanatus. The presence of weakly hybridizing bands suggests that

there are additional genes that are related to the Cv20ox.

Example 4

The Tissue-Specific Expression Pattern of the Cv20ox Gene

<4-l> Northern Blot Analysis of the Total RNA and mRNA

To investigate the tissue-specific expression pattern of the Cv20ox gene, the northern blotting was performed with the total RNAs, which was isolated from the various

reproductive and vegetative organs including the whole seeds, the integuments, the inner

seed tissues, the fruits at 1, 2, and 3 days after the pollination, the tendrils, the young

leaves, the male flowers and the ovaries before the pollination (Sambrook et al.,

Molecular Cloning, 1989).

More particularly, the total RNA was extracted from the various organs mentioned

above by using the TRI reagent (Molecular Research Center). The leaf and the root

samples were harvested from the 2 weeks-old plants and the reproductive organ samples

were obtained by dissecting the mature flowers under a dissecting microscope. 10~25 g

of the extracted total RNA was treated with the 10 x MOPS (0.2 M 3-(N-morpholino)

propanesulfonic acid [pH 7.0], 50mM sodium acetate, lOmM EDTA [pH 8.0]), formamide

and formaldehyde in the ratio of 1:1.8:5, making the final volume of 20μJt. In order to

eliminate the secondary structures, after heating this reaction mixture at 65 ^°C for 15

minutes, it was mixed with 2βi of the formaldehyde loading buffer (50% glycerol, ImM

EDTA [pH 8.0], 0.25% bromophenol blue, 0.25% xylene cyanol FF). Then, it was

electrophoresed slowly on 1% agarose gel containing 2.2M formaldehyde at 4N/cm. The

electrophoresed RΝA was soaked in the DEPC-treated water for 1 hour to remove the

formaldehyde and transferred to a nylon membrane by the capillary method.

Subsequently, the pre-hybridizing and the hybridizing reactions were performed as a

southern blot analysis stated above. As a result, the Cv20ox transcript was detected in the position of approximately

1.4kb band (See Fig. 3a). The band was detected strongly in the seeds, especially in the

integument tissues of the seeds, and weakly in the inner part of the seeds. But, the band

was not detectable in any other reproductive organs. The northern blot analysis with the

mRNA — hich was isolated from the cotyledons, the hypocotyls, the shoot meristems,

and the roots grown 15 days after seeding — showed that the Cv20ox transcript was not

detected in any organ at the development stage (See Fig. 3b).

<4-2> In situ Hybridization

To analyze the expression pattern of the Cv20ox gene in the developing seeds, in

situ hybridization in tissues was performed. The Citrullus lanatus seeds were fixed by

treating the FAA fixative solution (50% ethanol[v/v], 0.9 M glacial acetic acid, and

3.7%[v/v] formaldehyde) at 4^°C for 15 hours, dehydrating with ethanol, infiltering with

xylene, and embedding in paraffin (Paraplast X-tra, OXFORD Labware, St. Louis). The

parafilm-fixed seeds were cut into sections of 8-10jMiι and, then, transferred on the

Nectabond-coated slides (Vector Laboratories) and dried at 45 ^°C for 12 hours. The

riboprobes labeled with digoxigenin were prepared using the DIG nucleic acid labeling

kit (Roche Molecular Biochemicals). The sense and the anti-sense RΝA probes of the

Cv20ox cDΝA were synthesized using T7 and T3 RΝA polymerase. The said sections were treated with a solution containing 20/-g/m£ proteinase K (Roche Molecular

Biochemicals) at 37 ^"C for 30 minutes. The samples were acetylated and hybridized in a

solution containing 50% (v/v) formamide, 0.3M NaCl, 20mM Tris-HCl(ρH 7.5), 5mM

EDTA, 5mM Na₂HPO , 10% (w/v) dextrin sulfate, 1 x Denhardf s solution, 0.5mg/m£

yeast tRNA, 80βg/m& salmon-sperm DNA, and 300ng the digoxigenin-labeled riboprobe

at 50 ^°C for 18 hours. After hybridization, to remove the remaining probes, the said

slides were treated with 20//g/m£ RNase (Roche Molecular Biochemicals) at 37 ^°C at 30

minutes, and then washed in 1 x SSC (sodium citrate, sodium chloride) at 55 ^°C for 1

hour and 0.1 x SSC at 55 ^°C for 1 hour. The hybridized probes on the slides were

reacted with an anti-digoxigenin-alkaline phosphatase conjugate (Roche Molecular

Biochemicals). Then, hybridized region was measured by using a nitro-blue tetrazolium

(Roche Molecular Biochemicals) and a 5-bromo-4-chloro-3-indolyl phosphate (Roche

Molecular Biochemicals) as color substrates.

This investigation illustrated that the Cv20ox transcript was localized

predominantly in the inner layer of the integument (See B, E, and G in Fig. 4). This

result is consistent with the northern blot analysis of the example <4-l>. However, the

tissue type that expresses the Cv20ox gene in the inner parts of the seeds of the Citrullus

lanatus was not detectable by the method — probably due to a low-level expression. Example 5

Temporal Expression Pattern of the Cv20ox Gene

To study the temporal expression pattern of the Cv20ox gene in developing seeds

of the Citrullus lanatus, the RNAs isolated from 7 types of seeds at: 3, 4, 6, 8, 10, 12,

and 15 days after the pollination, were analyzed with northern blotting according to the

same method as Example 4.

As a result, the Cv20ox transcript began to appear in the seed at 3 days after the

pollination and increased prominently at 4 days after the pollination. This high-level was

maintained until 15 days after the pollination (See Fig. 5).

j Example 6

Regulation Pattern of the Cv20ox Gene Expression

<6-l> The Effects of GA on the Cv20ox Gene Expression

To examine the factors that influence the expression of the Cv20ox gene isolated

from the seeds of the Citrullus lanatus, the present inventors studied the expression pattern

by treating it with GA₃ and CPPU.

First, to examine whether the Cv20ox transcript level is altered by the GA₃ treatment in developing seeds of the Citrullus lanatus, 300βl of GA in a 0.1% aqueous

Tween 20 solution was treated on a fruit at 3 days after the pollination. After 24 hours,

the seeds were harvested. The RNA was extracted from the GA₃-treated seeds and the

untreated seeds, and northern blot analysis with the RNA was performed. As a control,

the level of the Sue synthase transcript was measured. As a result, the expression of the

Cv20ox was repressed in the GA -treated seeds compared with the untreated seeds, while

there was no change in the level of Sue synthase transcript (See Fig. 6a). This

observation suggests that the expression of the GA 20-oxidase in the seeds of the Citrullus

lanatus be feedback-regulated by the excessively active GA₃ as reported already in several

plants.

<6-2> The Effects of the CPPU on the Cv20ox Gene Expression

To examine the effects of the CPPU on the Cv20ox gene expression, the present

inventors, also, investigated the expression pattern of the Cv20ox in the parthenocaφic

fruits induced by the CPPU treatment. The parthenocaφic fruit development was

induced with lOOmg/1 of CPPU in a 0.1 aqueous Tween 20 solution by applying to the

ovaries at the flowering stage. The northern blot analysis was done according the same

method as in example <6-l> stated above.

As a result, the Cv20ox gene was expressed early in the CPPU-treated seeds compared to the pollinated controls (See Fig. 6b). The earlier expression of the gene was

probably due to the promoted seed and the fruit development by the CPPU-treatment. In

addition, the expression pattern of the Cv20ox at 10 days after the CPPU treatment showed

that the transcript had accumulated primarily in the integument and, also, weakly in the

inner parts of the seeds (See Fig. 6c). Since the CPPU-treated seeds did not develop to

embryos or an endosperm, the GA 20-oxidase transcript — which was observed in the inner

parts as above — as probably present in the nucellar tissue or the transfer cells.

Example 7

Cloning of the Promoter of the Cv20ox Gene

To clone the Cv20ox promoter that regulates the seed-specific expression, about

700 bp of the 5' upstream-region of the Cv20ox promoter was isolated from the total

genomic DNA of the Citrullus lanatus using the PCR Cloning Kit (Takara). 10 ig of the

said isolated genomic DNA was digested with Hind HT, ligated to the HindJR cassette,

and amplified by the PCR. The PCR products were cloned into the Hind III restriction

site of the pGEM-T Easy vector (Promega), thus, to construct the recombinant vector

pGA2044.

The sequence analysis illustrated that the 5' upstream-region of the Cv20ox

promoter consists of the nucleotide sequence represented by SEQ ID NO: 5. The putative TATA box (TATAAATC box) is present in 132 nucelotides upstream-region from

the translation start cordon. The 5' upstream-region was highly A/T-rich, showing 72%

of A/T content, and contained three stretches of more than 19 bp A/T nucleotides. It was

suggested that the A/T-rich sequences of the 5' upstream-region is crucial in

transcriptional activation.

Example 8

Measuring the Activity of the Cv20ox Promoter

To measure the activity of the Cv20ox promoter isolated in Example 7, the present

inventors constructed an expression vector in which the GUS coding-region was fused to

the Cv20ox promoter region.

First, the plasmid pGA2118 was constructed by inserting the 0.7kb promoter

fragment that was obtained from the recombination vector, pGA204 — constructed in

Example 7— into the HindWPst I sites of ρGA1230 (Clontech) containing the GUS

reporter gene, a high-copy-number derivative (See Fig. 7). The said expression vector,

pGA2118, was absorbed to the tungsten particles (M-10, Bio-Rad). The vector-absorbed

particles were inserted into the various organs of the Citrullus lanatus plants that were

previously given a particle bombardment — using the particle bombardment (Biorad

Biolostic PDS-1000/He, Bio-rad Laboratories). A part of the various organs of the plants — containing the expression vector — as cut, then soaked in X-Gluc solution (5-

Bromo-4-Chloro-3-Indolyl-β-D-Glucuronic Acid, lmg/ml) and then incubated at 27 ^°C

for 30 hours in the dark. The transient expression of the fusion gene was measured by

the histochemical detection method that is to observe a blue-region showing the activity of

the GUS reporter gene using a dissecting microscope.

The result shows that the Cv20ox promoter activity was detectable in the

integument of the developing seeds and very strongly in the layer close to the nucellus

(See B in Fig. 8). However, the promoter activity was not detectable in the vegetative

organs — such as leaves (See C in Fig. 8), roots (See D in Fig. 8), hypocotyls (See E in Fig.

8), and cotyledons (See F in Fig. 8). The expression pattern coincided with that of the in

situ localization experiment shown in Example 4.

Example 9

Construction of the Expression Vector for Producing Seedless Plant

The pGA2257 expression vector was constructed by linking the Cv20ox gene to

the Cv20ox promoter in the reverse direction, which, in turn, linked the T7'-5' termination

sequence to the said gene linked to the promoter. Then, it was inserted into the region

between the left- and the right-border of the T-DNA in the pGA810 vector (An et al.,

Plant Molecular Biology Manual, Kluwer Academic, Dordrecht, Belgium, 1988) (See Fig. 9). The expression vector includes the tetracycline- and kanamycin-resistance gene as a

selective marker. The E.coli JM83 transformed with the pGA2257 was deposited to the

Korean Culture Center of Microorganisms on September 29, 2000 (Accession No:

KCCM-10216).

Industrial Applicability

The Cv20ox promoter provided by the present invention can be used in a method

for producing the seedless fruits — ore particularly, the seedless plants of the gourd

family, such as the watermelon and the melon that have a high preference — by linking it to

the plant growth regulatory gene, introducing it into the plants, and over-expressing the

gene in the plants.

INDICATIONS RELATING TO DEPOSITED MICROOGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 136ώ)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 17, 21, 34 ,37, 38 ,line 5, 5, 4, 5, 12 .

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheetD

Name of depositary institution

Korean Culture Center of Microorganisms

Address of depositary institution (including postal code and country)

Korean Culture Center of Microorganisms(KCCM)

361-221, Yurim B/D, Hongje-1-dong, Seodaemun-gu, Seoul 120-091, Republic of Korea

Date of deposit Accession Number

2000. 09. 29. KCCM-10216

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheetD

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

For receiving Office use only For International Bureau use only

0 This sheet was received with the international application p This sheet was received by the International Bureau on:

[Authorized officer [Authorized officer

Form PCT/RO/134(July 1998)

Claims

What is Claimed is:

1. A Cv20ox gene, a gibberellin 20-oxidase gene, which is integument-specifically

expressed in seeds of the Citrullus lanatus.

2. The Cv20ox gene, according to claim 1, has the base sequence represented by SEQ ID

NO: 3.

3. A gibberellin 20-oxidase protein encoded by the gene of claim 1.

4. The Gibberellin 20-xidase protein, according to claim 3, has the amino acid sequence

represented by SEQ ID NO: 4.

5. A Cv20ox promoter regulates the integument-specific expression of the Cv20ox gene

of claim 1.

6. The Cv20ox promoter, according to claim 5, has the base sequence represented by

SEQ ID NO: 5.

7. A promoter regulating the integument-specific expression of the Cv20ox gene has the same sequence with more than the continuous 23 base pairs in the base sequences of the

promoter of claim 6.

8. A method of producing the seedless fruits, comprising the steps of:

1) constructing an expression vector by making a gene encoding the plant

growth regulatory material linked to the downstream of the promoter

regulating the integument-specific expression of the Cv20ox gene in plant;

2) transforming the plant with the expression vector;

3) selecting a transgenic plant and induce redifferentiation;

4) adjusting to the soil; and

5) inducing the fruit formation and the growth by keeping the natural

pollination of the flower of the said transgenic plant repressed.

9. A method of producing seedless fruits, comprising the steps of:

1) constructing a vector for the plant transformation by making a GA 20-

oxidase gene linked in reverse to the downstream of the promoter-

regulating seed-specific gene expression in the plant ;

2) transforming the plant with said vector;

3) selecting a transgenic plant and induce redifferentiation;

4) adjusting to the soil; and 5) inducing the fruit formation and the growth by keeping the natural

pollination of the flower of the said transgenic plant repressed.

10. The method according to claim 9, wherein the vector for the plant transformation is

pGA2257 (accession No.: KCCM-10216), in which the Cv20ox gene is linked in reverse to

the downstream of the Cv20ox promoter.

11. The method according to claims 8 or 9, wherein the promoter in step 1) is the

promoter of the Cv20ox gene, the gibberellin 20-oxidase gene — which is expressed

integument-specifically in the seeds of the Citrullus lanatus.

12. The method according to claim 11, wherein the promoter in step 1) is the Cv20ox

promoter represented by SEQ ID NO: 5.

13. The method according to claims 8 or 9, wherein the promoter in step 1) is the

promoter regulating the integument-specific expression of the Cv20ox gene, which has the

same sequence with more than the consecutive 23 base pairs in the base sequences of SEQ

ID NO: 5.

14. The method according to claim 8, wherein the gene encoding plant growth regulatory material in step 1) is the iaaM, the iaaH or the ipt gene.

15. The method according to claims 8 or 9, wherein the fruit formation and the growth in

step 5) are induced by inhibiting the pollination and the fertilization by wrapping the

flowers with paper bags etc. about 1 month — before dehiscence of anthers when the height

of the flowers reaches approximately 2-3 cm.

16. The method according to claims 8 or 9, wherein the observed plant is the plant of the

gourd family — including Citrullus lanatus and the melon.

17. An expression vector, pGA2257 — in which the Cv20ox gene is linked in reverse to

the downstream of the Cv20ox promoter (accession NO. : KCCM-10216).