KR20020093028A - 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 - Google Patents

Abstract

PURPOSE: Provided are a testa specific gene, which is isolated from the developing seed of Citrullus lanatus, a promoter and a method for generating seedless fruits by using the promoter of the said testa specific gene. CONSTITUTION: The Cv20ox gene, gibberellin 20-oxidase(GA 20-oxidase) gene showing the tissue-specific expression pattern, is strongly expressed in the integumental tissue at seed development of watermelon and is weakly expressed in the inner tissue of seed. The promoter regulates the integument-specific expression of the gene and the method for generating seedless fruits is characterized by introducing plant growth regulation gene ligated to the promoter and regulating the overexpression of plant growth regulation composition in the testa.

Description

A CMO20 gene promoter that regulates the expression of genes specifically for seeding of watermelon seeds and a method for producing a seedless plant using the same A METHOD FOR GENERATING SEEDLESS FRUITS USING THE PROMOTER}

To date, many researchers have developed transgenic plants that have genetically modified a variety of plants to increase their resistance to pests or increase yields. Many genetically engineered plants have been cultivated and supplied to the public. have. As part of this research, attention is also focused on the development of seedless plants. Seeds are necessary for plants to leave offspring, but they are only an inconvenient factor for consumers when consumed as commodities, and in recent years, consumers' preference for seedless fruits is increasing. In addition, seedless fruits generally have the advantage that they can be obtained even under conditions that are difficult to pollination or fertilization.

To be useful as a seedless plant, 1) fruit must be deleted and grown in the absence of unit deletion, that is, without fertilization, 2) fruit must be produced that has no seeds and unit deletion, and 3) pollinated the flowers. In time, fruit with viable seeds should be produced.

As a method of developing such parthenocarpic plants, either synthetic plant growth factors are used (Liparl et al., Acta. Hort . 229: 307-312, 1988), or mutations capable of seedless development. (Lin S. et al., J. Horod . 75: 62-66, 1988), and how to use a mutated plant in chromosomes (Kihara H., Plant growth substance-inducing application in agriculture, Tata McGray Publishing Company Ltd., New Delhi, India, 1981).

The most commonly used method for producing a unit deletion plant as described above is to treat synthetic plant hormones (phytohormones) in the flower buds. In other words, if the concentration of plant hormones in the room is increased by moisture or fertilization, it stimulates cell division and promotes fruit deletion and growth. By increasing the concentration of plant hormones in the room, the fruit can grow normally even if it is not pollinated. However, the method using synthetic plant growth factors may cause fruit drop when the growth hormone is treated incorrectly, and it is difficult to process growth hormone only in female flowers 1 and 2, and fruit fruit is cracked after fruiting. There is a problem, such as a large amount of cultivation techniques are required. In addition, it has been pointed out that the application of such a method requires a lot of labor, is expensive, and above all, a human can consume some of the synthetic chemicals.

Another method for producing seedless fruit is a method using a mutation generated in the number of chromosomes such as a triploid. As a representative seedless fruit produced by the above method, seedless watermelon is developed and grown.

This method was developed by Dr. Jang Chun Woo of Korea. It is a method of forming watermelons with triplex chromosomes by making watermelon chromosomes that are diploids into tetraploids, and then crossing watermelons of tetraploid chromosomes and watermelons of diploid chromosomes to be. The watermelon of the triplex chromosome thus formed does not make normal pollen and eggs during reproductive division, thus forming a seedless watermelon. However, the triploid watermelon is unstable in growth, which causes thickening or cracking of the rind during cultivation, high incidence of malformations, and low sugar content.

In addition to the method of controlling the chromosomes and the method of treating synthetic plant growth hormones, a method of producing seedless fruit using other genetic manipulations has been published. For example, there have been reports on the production of seedless fruits in tobacco or eggplants, which are commonly used to regulate plant growth following the promoter of a gene that is ovule-specific ( defH9 of Antirrhinum majus ). By connecting the gene ( iaaM ) involved in the plant and then overexpressing the plant growth regulator in the room, it makes the state similar to the pollinated state without increasing the endogenous phytohormone due to pollination. It is a method of promoting fruit growth to make seedless fruit (Giuseppe et al., Nature Biotechnology 15: 1398-1402, 1997). As such, the seedless fruit production method using the specific hormone-specific promoter is known as a useful method for the production of seedless plants because no significant problems have been found. The promoter site should be identified. Until now, the method has been applied only to tobacco or eggplant plants, and since no attempt has been made on fruits such as watermelon, which has the highest commercial value as a seedless fruit, development of a new promoter that can be applied to the seedless watermelon has been required. .

Gibberellin, on the other hand, plays an important role in plant growth and development, including seed germination, stem growth, flower formation and growth, fruit-setting, seed development and anthocyanin biosynthesis. Hormone (Hooley R., Plant. Mol. Biol. 26: 1529-1555, 1994).

Gibberellins are biosynthesized from tetracycline diterpene ent- kaurene by two types of enzymes, monooxygenases and dioxygenases (Hedden P. et al., Annu. Rev. Plant.Physiol.Plant Mol. Biol. 48: 431-460, 1997), in the early stages of biosynthesis, GA ₁₂ and GA ₅₃ are synthesized by monooxygenase. It is converted into C ₁₉ -GAs with physiological activity by the deoxygenase GA 20-oxidase and 3β-hydroxylase. GA 20-oxidase involved in the biosynthesis of gibberellin stepwise oxidizes the carbon number 20 of GA ₁₂ and GA ₅₃ to form C ₁₉ skeleton in gibberellin, and 3β-hydroxylase is GA 20-oxidase Catalyzes the 3β-hydroxylation in the C ₁₉ backbone of gibberellins formed by to form gibberellins with biological activity.

Genomic DNA and cDNA clones encoding these two major deoxygenases, GA 20-oxidase and 3β-hydroxylase, which are involved in the biosynthesis of gibberellin, have already been isolated from several species of plants and transformed into Escherichia coli transformants. Enzyme activity was measured using recombinant enzyme produced from (Hedden & Kamiya, Annu. Rev. Plant Physiol. Plant Mol. Biol . 48: 431-460, 1997).

It has also been reported that the expression of these deoxygenases is reduced by feedback regulation by gibberellin (Chiang et al.,Plant Cell.7: 195-201, 1995), Arabidopsis (Arabidopsis; Xu, etc.Proc. Natl. Acad. Sci. USA.42: 6640-6644, 1995) and spinach (Wu et al.,Plant Physiol., 110: 547-554, 1996) The expression of GA 20-oxidase was confirmed to increase its expression level under long-term conditions. In addition, through the study of GA 20-oxidase in Arabidopsis, soybeans, peas and tomatoes, the plants contain several types of GA 20-oxidase genes, and these GA 20-oxidase genes of the plant It has been demonstrated that expression is regulated tissue-specific or temporally upon development and is involved in other regulatory processes of gibberellin, such as stem elongation, flower formation and fruit formation (Phillips et al.Plant physiol, 108: 1049-1057, 1995).

As described above, it has been reported that the GA 20-oxidase found in some plants among the GA 20-oxidases involved in the gibberellin biosynthesis process is specifically expressed in seeds or fruits. For example, Arabidopsis YAP169 is specifically expressed in silique (Phillips et al., Plant Physiol ., 108: 1049-1057, 1995), Pv85-26 of legumes (Garcia-Martinez et al., Plant Mol , Biol ., 33: 1073-1084, 1997) in developing seeds and M3-8 of Marah macrocarpus (MacMillan et al . , Plant Physiol. , 113: 1369-1377, 1997). It was found to be specifically expressed in seed embryos (embryos) and endospores (endosperm).

In particular, developing seeds have been used frequently in genetic and physiological studies to elucidate the process of biosynthesis of gibberellin in plants, as they contain greater amounts of gibberellin than any other plant organ (Graebe et al . , Annu. Rev.). Plant Physiol , 38: 419-465, 1987). However, little is known about the role of gibberellins and the exact site of synthesis in seed development, only recently it has been reported that GAs are involved in the early and late stages of seed development in some plant species (Euwens & Schwabe et al . , J. Exp. Bot. , 26: 1-14, 1975), reported changes in seed development in several gibberellin deficiency mutations in legumes. For example, in the lh-2 mutant, the expression of gibberellin was markedly reduced and seed development was observed to stop (see Wain et al., Plant J. , 12: 1329-1338, 1993; Swain et al. Planta , 195: 426-433, 1995). Physiological, biochemical and genetic analysis of the gibberellin deficiency mutations ls-1 and lh-2 revealed that biosynthesized gibberellin in embryos and / or embryos is required for seed development within the first few days after pollination. (Ait-Ali et al., Plant J. , 11: 443-454, 1997). Moreover, seed development is very important for the development of rinds associated with plant hormones such as gibberellin and auxin (Van Huizen et al., Plant Physiol . 115: 123-128, 1997). Although overcame, gibberellins were necessary for fruit and seed development (Groot et al . , Physiol. Plant . 67: 315-319, 1987).

The present inventors focus on the usefulness of the gene and its promoter specifically expressing the seed as described above, the result of the effort to develop a method of using the same for the production of seedless fruit by searching for the seed and the promoter specific to the seed The present invention was completed by finding a seed specific Cv20ox gene promoter from developing seeds of watermelon ( Citrullus lanatus ) and confirming that the promoter regulates the specific expression of the gene. Therefore, by connecting genes involved in plant growth regulation to the seed-specific Cv20ox gene promoter of the present invention and introducing them into plants, over-expressing the plant growth regulators in seedlings, such as seedless fruits, particularly seedless watermelons or melons that have excellent affinity. It can be usefully used for production methods of making gourds and plants.

Summary of the Invention

It is an object of the present invention to provide a Cv20ox gene encoding GA 20-oxidase which is specifically expressed in epithelial tissues when watermelon seeds are generated.

It is also an object of the present invention to provide a Cv20ox gene promoter that modulates endothelial specific expression.

Another object of the present invention is to provide a method for producing a seedless plant using the promoter. Specifically, an object of the present invention is to provide a method for producing seedless fruit by connecting genes encoding plant growth regulators to the promoters to induce overexpression of these plant growth substances in the epidermis.

Brief description of the drawings

1A and 1B show the amino acid sequence of GA 20-oxidase encoded by the Cv20ox gene of the present invention and the amino acid sequence of GA 20-oxidase derived from various plants,

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

3: C. maxima cm20oxi4: P. sativum ps27-12

5: P. vulgaris pv73-16: P. vulgaris pv15-11

7: L. sativa Ls20ox18: L. sativa Ls20ox2

9: P. vulgaris pv85-2610: A. thaliana at2353

11: A. thaliana atyap16912: A. thaliana at2301

13: S. oleracea soU33330

Figure 2 is a gel photograph showing the results of Southern blot analysis using the Cv20ox cDNA gene as a probe for genomic DNA isolated from the developing seeds of watermelon,

E: Treatment with restriction enzyme Eco RI

H: treated with restriction enzyme Hind III

Figure 3a is a gel photograph showing the results of the northern analysis on the total RNA isolated from several mature tissues of watermelon, in order to investigate the tissue-specific expression of the Cv20ox gene,

I: integumental tissue 8 days after pollination

S: Whole seed on day 8 after pollination

IS: internal seed tissue at 8 days after pollination

F1: fruit on day 1 after pollination

F2: fruit 2 to 3 days after pollination

F8: fruit at 8 days after pollination

T: tendril L: leaf

M: Male flower O: Seeds before pollination

FIG. 3b is a gel photograph showing that the Northern blot analysis of mRNA isolated from various tissues except seed of young plants grown for 15 days after sowing did not express Cv20ox gene in any tissue at developmental stage except seeds.

C: Cotyledon Hu: Upper layer hypocotyl

Sm: stem meristem containing most of the upper part of the hypocotyl

H1: lower layer of hypocotyl R: root

Figure 4 shows the tissue specific expression of the Cv20ox gene in watermelon seeds by in situ hybridization,

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

D and E: seeds at 6 days after pollination

F and G: seed on day 4 after pollination

nu: nucleus ii: inner layer of the outer shell

oi: outer layer of envelope mp: (micropylar end)

e: rectangular boat

5 is a northern blot analysis showing the temporal expression of the Cv20ox gene of the present invention in seed generation ,

DAP: days after pollination

Figure 6a is to examine the effect of gibberellin (GA ₃ ) in the Cv20ox gene expression control process of the present invention after the treatment of GA ₃ to the fruit on the third day after a few minutes after extracting RNA from the harvested 24 hours Gel photo showing Northern blot analysis.

+: Fruits 3 days after pollination with GA ₃

-: Fruits on day 3 after moisture not treated with GA ₃

Figure 6b shows the harvested seeds after normal moisture and N- (2-chloro-4-pyridyl) -N'-phenylurea (N- (2-chloro-4-pyridyl) -N'-phenylurea; (B) abbreviated Northern blot analysis of Cv20ox gene expression between harvested seeds.

Lane 1: 2 days after pollination Lane 2: 3 days after pollination

Lane 3: Day 4 After Moisture Lane 4: Day 2 After CPPU Treatment

Lane 5: 3 days after CPPU treatment Lane 6: 4 days after CPPU treatment

Figure 6c is a result of Northern blot analysis comparing the expression pattern of the Cv20ox gene of the present invention in the outer skin and internal seed tissue 10 days after CPPU treatment,

Lane 1: Jacketed Lane 2: Internal Seed Tissue

Figure 7 shows a cleavage map of the recombinant expression vector pGA2118 cloned GUS (β-glucuronidase) gene as a reporter gene downstream of the promoter in order to measure the activity of the Cv20ox gene promoter of the present invention,

8 is a result of histochemical detection of the expression of the GUS reporter gene after transient expression of the pGA2118 recombinant vector in various organs of watermelon using particle bombardment.

A: developing seeds on day 8 after pollination

B: Inside the seed at 8 days after pollination cut in parallel

C: Leaf D: Root

F: Axle G: Cotyledon

9 is a cleavage map of a binary vector (KCCM-10216) in which the Cv20ox gene is reversely linked downstream of the Cv20ox gene promoter and tetracycline and kanamycin resistance genes are inserted as selection marker genes.

The present invention relates to an endothelial specific Cv20ox gene promoter isolated from a developing seed of watermelon ( Citrullus lanatus ). Specifically, the present invention is strongly expressed in the integumental tissue and weakly expressed in the seed internal tissue during the generation of watermelon seeds. The present invention relates to a Cv20ox gene encoding a GA 20-oxidase showing a tissue-specific expression pattern, a Cv20ox gene promoter for regulating its endothelial specific expression, and a method for producing a seedless plant using the same.

The present invention provides an endothelial specific Cv20ox gene isolated from the developing seed of watermelon ( Citrullus lanatus ) and a promoter regulating the endothelial specific expression of the gene.

The cDNA of the Cv20ox gene consists of 1420 nucleotides (see SEQ ID NO: 3), encodes a Gibberellin 20-oxidase protein consisting of 379 amino acid residues (see SEQ ID NO: 4), His-239 , Fe ²⁺ binding site (Fe ²⁺ binding motif) and 2-oxoglutarate (2-oxoglutarate) 2-oxoglutarate comprising a binding site consisting of Asp-241, and His-295 - dependent dioxide It has a conservative region typical of siogenases (2-oxoglutarate-dependent dioxygenases). In addition, the GA 20-oxidase encoded by the gene has an amino acid sequence that is very similar to the amino acid sequence of the GA 20-oxidase of other plants previously known, specifically, GA of Marah macrocarpus ( Marah macrocarpus) 76% homologous to 20-oxidase and 63% homologous to GA 20-oxidase in amber, and 50% or more homologous to GA 20-oxidases in peas, beans, arabidopsis, spinach and lettuce (See FIGS. 1A and 1B).

DNA and RNA blot analysis of watermelon seeds revealed that the Cv20ox gene is present in one or two copy numbers in the watermelon genomic DNA (see FIG. 2 ), and is absent in other reproductive or growing organs other than the developing seed. In particular, the gene exhibits a spatial expression pattern that is very strongly expressed in the outer tissue of the various various tissues of the seed, while weakly expressed in the inner tissue of the seed (see FIGS. 3A and 3B).

In addition, according to the results of in situ hybridization, the transcript of the Cv20ox gene shows a distribution pattern mostly present in the inner layer of the seed envelope tissue when watermelon seeds are generated (see FIGS. 4B, E, and G). ). In terms of temporal expression of the Cv20ox gene, it is expressed from seeds at 3 days after pollination and is very strongly expressed at 15 days after pollination (see FIG. 5).

On the other hand, results of gibberellin is compared to the level of expression of sucrose gene synthase (sucrose synthase) as a control to examine the effect of the level of expression of the Cv20ox gene examined the expression pattern of the process the presence of GA _3, GA ₃ In the case of treatment, there was no effect on the expression of sucrose synthase , but the expression of Cv20ox gene was greatly suppressed (see FIG. 6A). This shows that, as in the case of other plants reported previously, the expression of the Cv20ox gene of watermelon seeds is also subject to feedback regulation by excessive gibberellin.

In another embodiment of the present invention, after processing CPPU, a chemical that promotes plant parthenocarpy, gene expression patterns were investigated by comparing with normal pollinated seeds by Northern blot analysis. The Cv20ox gene was early expressed in Parthenocarpic fruits (see FIG. 6B). This is believed to be due to the early occurrence of seeds and fruits promoted by CPPU treatment. On the other hand, the transcript of the gene is strongly expressed in the seed envelope and weakly expressed in the interior of the seed (see Figure 6c), CPPU-treated seed is not observed in the seed as described above because it does not develop into embryos or endosperm. The transcript of GA 20-oxidase, which is expressed, is expressed in parental tissue such as nucellus tissue or metastatic cells.

The present invention also provides a promoter that regulates the endothelial specific expression of the Cv20ox gene isolated from the developing seed of watermelon.

The Cv20ox promoter region consists of the nucleotide sequence set forth in SEQ ID NO: 5, a region presumed to be the TATAAATC box at the 132 nucleotides upstream from the translation start codon, and an A / T-rich sequence region As more than 19 bp of A / T nucleotides. The distribution of this base sequence is already high in A / T content and is located in the 5 'upstream region of the ConA gene, one of the major seed storage protein genes of Canavalia, which is known to contain several long A / T rich sequences. Similar to base sequence distribution. Further, A / T rich sequences in the 5 'upstream region of the gene, so known to play an important role in promoting the transfer (such as Yamamoto, Plant Mol Biol 27: 729-741, 1995) the promoter region of Cv20ox gene expression of genes Cv20ox Not only useful for revealing the mediation signal to control the plant, but also by connecting the genes involved in plant growth regulation to the promoter and introduced into the plant can control the overexpression of plant growth regulators in the seedling, so useful for the production method of making seedless fruit Can be.

In order to investigate the endothelial specific activity of the Cv20ox gene promoter, the present inventors construct a transient expression system by connecting the promoter to a reporter gene which is easy to express and can easily identify its expression without complex relationships in plants. . That in a preferred embodiment, the Cv20ox gene promoter as a reporter gene at the downstream was prepared by placing them in various organs of watermelon an expression vector that combines the GUS (β-glucuronidase) coding region results confirmed the expression pattern of the GUS staining, Cv20ox The activity of the gene promoter was detected only in the outer tissue of the developing seed, especially very strongly in the layers adjacent to the nucleus, while not at all in non-reproductive organs such as leaves, roots, hypocotyls and cotyledon (see FIG. 8).

Also within the scope of the present invention are promoters having the same nucleotide sequence as the 23 or more consecutive sequences in the nucleotide sequence of the Cv20ox gene promoter set forth in SEQ ID NO: 5. That is, more than 23 nucleotide contiguous, as the same as the nucleotide sequence of Cv20ox gene promoter, and replacing some sequences do not affect the control of the promoter, modified, deleted or added in the promoter also Cv20ox gene of the present invention As with the promoter, it is possible to regulate the expression of the endothelial specific gene, which can be used for the production of seedless watermelon and the like. Theoretically, the probability of identical 23 consecutive sequencing sequences is very low, one of 4 ²³ (7.03 x 10 ¹³ ), and also found in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) There are no more than 23 contiguous nucleotide sequences in a particular gene that show identity with at least 23 contiguous nucleotide sequences of the Cv20ox gene promoter of the invention. Therefore, it can be seen that the base sequence itself described in SEQ ID NO: 5 as well as having the same base sequence as 23 or more consecutive base sequences in the base sequence belong to the category of identity with the Cv20ox gene promoter of the present invention.

In addition, the present invention provides a method for producing seedless fruit using the Cv20ox gene promoter.

Specifically, a method for producing seedless fruit using the Cv20ox gene promoter of the present invention includes 1) preparing an expression vector functionally linked to a gene encoding a plant growth regulator downstream of the Cv20ox gene promoter, and 2) Transform the desired plant with an expression vector, then 3) select the transgenic plant to induce regeneration, 4) purify it to soil, and 5) inhibit natural pollination of the flower of the transgenic plant. Maintaining the state and inducing fruit deletion and growth.

In this regard, known methods (see Giuseppe et al., Nature Biotechnology 15: 1398-1402, 1997) on obtaining seedless fruits from plants transformed with plant growth regulator genes functionally linked to a room-specific promoter are described . For reference.

In the above, a seed plant growth regulator gene for the production of fruit without the iaaM, iaaH, or may use the ipt gene and the like, preferably tryptophan mono oxide gene coding for the (tryptophan mono-oxigenase) dioxygenase iaaM It is good to use.

The IaaM gene encodes tryptophan monooxygenase, which oxidizes tryptophan to indolacetamide, and the indoleacetide produced thereby does not have auxin activity, which is one of the plant growth hormones, Chemically or by the action of enzymes such as plant hydrolase, it is slowly converted to IAA, a representative form of auxin. Therefore, as the iaaM gene is overexpressed by the Cv20ox gene promoter that regulates the endothelial specific expression, high plant growth hormone concentrations are expressed as if water or fertilization was performed, even in a state in which water was not normally pollinated. Seedless fruit will grow and grow. In particular, when the seedless fruit is manufactured using the iaaM gene, there is an advantage in that the seedless fruit can be made without the treatment of a separate plant hormone from the outside even under conditions of moisture and fertilization. Compared to the method, more favorable results can be obtained in terms of the labor required for the production of seedless fruits and the cost of controlling the greenhouse temperature for cultivation.

In addition, in the method of preparing the seedless fruit, the Cv20ox gene promoter has a nucleotide sequence as set out in SEQ ID NO: 5, and the sequence is expressed in 23 or more consecutive bases, as well as in a gene promoter specifically expressed in the seed of watermelon. As a nucleotide having identity with the promoter of 5, one isolated from watermelon or other plants can also be used. In the present invention, a promoter is a meaning generally used, that is, a base sequence upstream of a transcription start position, to directly or indirectly recognize or bind a DNA-dependent RNA polymerase to initiate transcription. It may be used to mean a site or an upstream transcriptional regulatory site comprising the same, and may be preferably used to include untranslated signal regions including introns with a promoter site. That is, as a Cv20ox gene promoter for the production of seedless fruit, a non-coding sequence of the Cv20ox gene may be used together in addition to the promoter region of SEQ ID NO: 5.

On the other hand, in the method for producing the seed-free fruit, a process of preparing a transgenic plant, re-differentiating, inducing roots and purifying soil, etc. may generally use known methods used for plant transformation.

In the process of preparing the seedless fruit from the transformed plant prepared through the above process, fruit is deleted and grown in accordance with the overexpression of iaaM while suppressing the moisture of the flower of the transgenic plant. Before anther is opened (before dehiscence of anthers), flowers are wrapped in a paper bag when the length of the flower reaches about 2 ~ 3 cm.

The method for producing seedless fruit using the Cv20ox gene promoter of the present invention can be applied to various plants, and preferably to watermelon and plants such as watermelon or melon, but the plant can be functionally expressed. All of the ramen is applicable and not limited to those mentioned above.

On the other hand, as another method for producing a seedless fruit using the Cv20ox gene promoter of the present invention, there is a method of inhibiting seed generation by inhibiting the expression of normal Cv20ox gene by connecting the Cv20ox gene to the promoter in a reverse direction. Is an enzyme that oxidizes GA ₁₂ or GA ₅₃ synthesized by monooxygenase to synthesize C ₁₉ -GAs with physiological activity and is involved in the early and late stages of seed development. Since the expression of gibberellin is essential for seed development as confirmed in gibberellin-deficient mutant strains, seedless fruit can be produced by inhibiting seed generation by connecting Cv20ox gene to the promoter in the reverse direction and inhibiting its expression.

To this end, in a preferred embodiment of the present invention, the Cv20ox gene promoter is reversely connected to the Cv20ox gene, and the T7'-5 'transcription sequence is connected thereto, and the pGA810 vector (An et al., Plant Molecular Biology Manual , Kluwer Academic, PGA2257 expression vector was prepared by inserting between the left border and the right border of T-DNA of Dordrecht, Belgium, 1988). The expression vector contains tetracycline and kanamycin resistance genes as selection marker genes, and transforms E. coli JM83 with the expression vector pGA2257, thereby preserving the transformed E. coli microorganisms on September 29, 2000. Deposited to the center (Accession No .: KCCM-10216)

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only for exemplifying the present invention, and the contents of the present invention are not limited to the following examples, which will be apparent to those skilled in the art.

Example

Example 1 : GA 20-oxidase gene Cv20ox CDNA Isolation

In order to isolate the GA 20-oxidase gene involved in the biosynthesis of gibberellin in watermelon, cDNA library was constructed from the watermelon seeds that were being generated. Specifically, the first generation (F1) hybrid of watermelon ( Citrullus lanatus (Thunb.) Var., COUNTRY HOME) was cultured in a greenhouse at 30 to 35 ° C. during the day and 15 to 20 ° C. at night. It was. Poly (A) ⁺ mRNA was extracted from the watermelon seeds 6 to 10 days after pollination using oligo (dT) -cellulose chromatography. cDNA library was prepared according to the method provided by the company using Stratagene's ZAPII cDNA cloning kit and Gigapack III Gold. The cDNA library was amplified in Escherichia coli ( E. coli XL1-Blue, Stratagene) and was an independent recombinant with an initial plaque forming unit of 4 × 10 ⁶ , sufficient to contain low-frequency clones. It was.

In order to separate only the cDNA of GA 20-oxidase gene from the cDNA library, polymerase chain reaction (PCR) was performed using a degenerate primer for the consensus sequence in GA-20 oxidase. . Specifically, a forward primer described in SEQ ID NO: 1 and a reverse primer described in SEQ ID NO: 2 were used, and PCR was performed using DNAs isolated from the seed cDNA library 6 to 10 days after the obtained minutes, and PCR. The reaction was denatured by heating at 94 ° C. for 5 minutes, then repeated 40 times under conditions of 1 minute at 94 ° C., 1 minute at 40 ° C. or 50 ° C., and 1 minute at 72 ° C., followed by amplification at 72 ° C. for 10 minutes. The reaction was terminated. The 300 bp DNA fragment obtained by the PCR reaction was isolated by agarose gel electrophoresis and cloned into pGEM-T Easy vector (Promega). The clones were sequenced using a DNA sequence analysis kit (Big DyeTM Terminator Cycle Sequencing Ready Reaction, PE Applied Biosystems) and a sequence analyzer (model ABI 373 DNA Sequencer, Applied Biosystems).

The partial Cv20ox cDNA obtained in this way was searched for the cDNA library according to the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press) using a probe to isolate the Cv20ox full-length gene (full-length cDNA).

Example 2 : Cv20ox Sequence analysis of genes

The results of the above sequencing analysis were compared with the nucleotide sequence of the GA 20-oxidase gene previously known using the BLAST program of the DNA Data Bank of Japan.

As a result, the amino acid sequence estimated from the GA 20-oxidase gene showed high homology with the amino acid sequence of GA 20-oxidase of various plant species. However, since the clone contains only a portion of the GA 20-oxidase gene, a cDNA clone containing a full-length ORF estimated to be GA 20-oxidase was searched for a cDNA library of watermelon seeds using a probe of 300 bp obtained above. Secured. The clone encoded a protein consisting of 1420 base sequences set forth in SEQ ID NO: 3 and consisting of 379 amino acid residues (GenBank Accession No. AF074709). In addition, the peptide is a 2-oxoglutarate-dependent comprising a 2-oxoglutarate binding site and a Fe ²⁺ binding site consisting of His-239, Asp-241, and His-295 It has a consensus sequence typical of 2-oxoglutarate-dependent dioxygenase.

Sequence analysis of the peptide showed that the Cv20ox peptide sequence set forth in SEQ ID NO: 4 was very similar to the amino acid sequence of the known GA 20-oxidase (see FIGS. 1A and 1B). Specifically, the Cv20ox protein isolated from the watermelon seeds showed amino acid sequence homology of 76% with GA 20-oxidase of Marah macrocarpus and 63% with GA 20-oxidase of amber. It was. In addition, the proteins are peas, beans, Arabidopsis (Arabidopsis), exhibited the spinach, and the GA 20- oxidase of lettuce and also at least 50% amino acid sequence homology.

Example 3 : Genomic DNA Cv20ox Gene distribution pattern

Southern blot analysis of genomic DNA was performed to confirm the copy number of the Cv20ox gene in watermelon genomic DNA. Specifically, total genomic DNA was isolated from the leaves of two-week-old watermelons using CTAB (cetyltrimethylammonium bromide, Rogers SO & Bendich AJ, Extraction of DNA from plant tissue A6: 1-10, 1988) method. . The isolated genomic DNA was digested with Hin dIII or Eco RI restriction enzymes and subjected to electrophoresis on a 0.8% agarose gel, after which the DNA in the gel was transferred to a nylon membrane (Hybond-N, Amersham,). The nylon membrane was heated at 80 ° C. for 1 hour to fix DNA to the membrane. The 300 bp cDNA clone obtained in Example 1 was labeled with ³² P through a random priming method (Sambrook et al ., Molecular Cloning, 1989), and then hybridized to a membrane bound to genomic DNA using the probe as a probe. .

Prehybridization solution (7% SDS, 0.5M NaPO ₄ , 1mM EDTA, 1% BSA) was added to nylon-bonded nylon membrane for 2 hours in a 65 ° C reaction oven. After denaturation for 5 minutes, the reaction mixture was added for 18 hours, and washed twice at 60 ° C. with 0.1 × SSC, 0.1% SDS, and then exposed to X-ray film.

As a result, the cDNA probes were labeled with ³² P was hybridized to one or two main bands (Fig. 2), indicating that the gene is present in the Cv20ox one or two copies of the number watermelon genomic DNA. In addition, the appearance of a weakly hybridized band means that there is an additional gene associated with the Cv20ox gene.

Example 4 : Cv20ox Tissue Specific Expression Patterns of Genes

<4-1> Northern blot analysis of total RNA and mRNA

To investigate the tissue-specific expression of the GA 20-oxidase gene Cv20ox , the whole seed, seed integument, internal tissue of the seed, fruit 1 day after pollination, fruit 2 to 3 days after pollination, tendril Northern blot analysis was performed by separating total RNA from various reproductive and growth organs, including young leaves, male flowers, and ovaries before pollination (Sambrook et al ., Molecular Cloning, 1989).

Specifically, total RNA was extracted from various organs as described above using a TRI reagent (Molecular Research Center). Samples of leaves and roots were taken from plants of 2 weeks of age, and samples of reproductive organs (floral organs) were obtained by cutting mature flowers using an anatomical microscope. 10-25 μg of the extracted RNA was added at 10 × MOPS (0.2 M 3- (N-morpholino) propanesulfonic acid pH 7.0, 50 mM sodium acetate, 10 mM EDTA pH 8.0), formamide, formaldehyde, and 1: 1.8: Add to the ratio of 5 to make a total of 20μl, heat treatment at 65 ° C. for 15 minutes to release the secondary structure, formaldehyde gel loading buffer solution (50% glycerol, 1 mM EDTA pH 8.0, 0.25% bromophenol blue, 0.25 2 μl of% xylene cyanol FF) was mixed well and electrophoresed slowly at 4V / cm on 1% agarose gel containing formaldehyde (2.2M). The developed RNA was immersed in DEPC treated water for about 1 hour to remove formaldehyde, and then transferred to a nylon membrane by capillary transfer. Then, prehybridization and hybridization were performed by the same method as the Southern blot.

As a result, the Cv20ox transcript was detected at a band position of about 1.4 kb (FIG. 3a), which was strongly detected in the seed, in particular the outer tissue of the seed, weakly detected in the inner tissue of the seed, while other than the seed. It was not detected at all in the reproductive organs. On the other hand, Northern blot analysis using mRNA isolated from cotyledon, hypocotyl, shoot meristem, and root of 15 days after planting, Cv20ox transcript was detected in any organ in the developmental stage. Was not (Figure 3b).

<4-2> Tissue Hybridization Experiment ( in situ hybridization)

Meanwhile, in situ hybridization experiments were performed to confirm the expression distribution of the Cv20ox gene in developing seeds. Specifically, watermelon seeds were treated with FAA fixative solution (50% ethanol, 0.9 M glacial acetic acid, and 3.7% formaldehyde) and fixed at 4 ° C. for 15 hours, dehydrated with ethanol, and then filtered with xylene. And paraffin (paraffin, paraplast x-tra, OXFORD labware). Sections of 8-10 μm size were cut from the seed fixed with paraffin and transferred to a slide coated with Vectabond (Vectabond, Vector Laboratories), and dried at 45 ° C. for 12 hours to be used as a sample for tissue hybridization experiments. It was. On the other hand, DIG nucleic acid labeling kit using (DIG nucleic acid labeling kit, Roche Molecular Biochemicals) digoxigenin (digoxigenin) were the production of a labeled RNA probe (riboprobe), the anti-sense and sense RNA probes Cv20ox cDNA T7 and T3 Synthesis was carried out using RNA polymerase. The sections on the slides were treated with a solution containing 20 μg / ml of proteinase K (Roche Molecular Biochemicals) at 37 ° C. for 30 minutes, followed by 50% formamide, 0.3 M NaCl, 20 mM. Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM Na ₂ HPO ₄ , 10% dextran sulphate, 1 Denhardt's solution, 0.5 mg / ml yeast tRNA, 80 g / Acetylated and hybridized at 50 ° C. for 18 hours with a solution containing ml salmon sperm DNA and 300 ng / ml of the digoxigenin labeled RNA probe. In order to remove the remaining probe after the hybridization reaction was terminated, the slide was treated with 20 μg / ml RNA decomposing enzyme (RNase, Roche Molecular Biochemicals) and reacted at 37 ° C. for 30 minutes, followed by 1 × SSC (sodium citrate). , sodium chloride) and 0.1 × SSC were washed at 55 ° C. for 1 hour. The probe hybridized on the slide was reacted with an anti-digoxigenin alkaline phosphatase conjugate (Roche Molecular Biochemicals) conjugated with alkaline phosphatase, followed by nitro blue tetrazolium (Roche Molecular Biochemicals) and Hybridization sites were measured using 5-bromo-4-chloro-3-indole-phosphate (5-bromo-4-chloro-3-indolyl phosphate, Roche Molecular Biochemicals) as a color substrate.

As a result, the Cv20ox transcript was abundantly present in the inner layer of the outer tissue (Figs. 4B, E and G), which is consistent with the RNA blot analysis of <4-1>. However, the internal tissues of watermelon seeds did not detect the type of tissue expressing the Cv20ox gene, presumably because the Cv20ox gene is expressed at very low levels in the internal tissues of the seeds.

Example 5 : Cv20ox Temporal Expression of Genes

To study the temporal expression patterns of the Cv20ox gene during watermelon seed generation, RNA was isolated from seven seeds at 3, 4, 6, 8, 10, 12 and 15 days after pollination, and the northern method was performed in the same manner as in Example 4. Blot analysis was performed.

As a result, Cv20ox transcripts began to be detected from seeds 3 days after pollination, increased significantly on day 4, and remained at high levels until day 15 (FIG. 5).

Example 6 : Cv20ox Gene expression regulation

<6-1> Gibberellin Cv20ox Effect on gene expression

In order to investigate factors influencing the expression of the Cv20ox gene isolated from watermelon seeds, the present inventors treated GA ₃ and CPPU to identify expression patterns.

First, in order to confirm what nutrients GA ₃ affects the expression level of Cv20ox transcripts, 300 μl of GA ₃ dissolved in 0.1% Tween 20 was treated in the fruit on day 3 after several minutes, and after 24 hours, Seeds were harvested and RNA extracted, followed by Northern blot analysis with RNA extracted from seeds not treated with GA ₃ . At this time, the transcript expression level of sucrose synthase was measured together as a control. As a result, the expression of Cv20ox the expression of sucrose synthase was inhibited in seeds treated with GA ₃ than seeds not treated with GA ₃ did not notice any change (Fig. 6a). This means that the expression of the GA 20-oxidase gene of watermelon seeds, as already reported in several plants, is also subject to feedback regulation by excess active gibberellin.

<6-2> CPPU Cv20ox Effect on gene expression

In addition, the expression pattern of the gene in the unit Cv20ox deletion fruit (Parthenocarpic) induced by CPPU treatment was examined to confirm the expression of the CPPU how the affect of Cv20ox gene. In order to induce the generation of unit fruit, 100 mg / l of CPPU dissolved in 0.1% Tween 20 aqueous solution was treated in ovaries during flowering. Subsequent analysis was performed using the same Northern blot analysis method as in <6-1>.

As a result, Cv20ox genes were expressed early in CPPU treated seeds as compared to the control group (FIG. 6B). Early expression of these Cv20ox genes was probably due to early development of seeds and fruits promoted by CPPU treatment. I think it was. In addition, the expression pattern of the Cv20ox gene 10 days after CPPU treatment, the transcripts of the Cv20ox gene mainly accumulated in the outer shell and weakly accumulated inside the seed (Fig. 6c), which means that the seed treated with CPPU is embryo or endosperm As described above, the transcript of GA 20-oxidase observed inside the seed is probably expressed in nucellus tissue or transfer cells.

Example 7 : Cv20ox Cloning of Gene Promoter

To clone the Cv20ox gene promoter that regulates the endogenous specific expression, about 700 bp upstream of the Cv20ox promoter 5 ′ was isolated from the whole genomic DNA of watermelon using a PCR cloning kit ( TAKaRa ). 10 μg of the watermelon genomic DNA isolated from the above was digested with Hin dIII restriction enzyme, inserted into a Hin dIII cassette, and amplified by PCR. The PCR product was cloned into the Hin dIII cleavage site of the pGEM-T Easy vector (Promega) to complete the recombinant vector pGA2044.

As a result of analyzing the nucleotide sequence of the recombinant vector, the Cv20ox promoter 5 'upstream portion is composed of the nucleotide sequence set forth in SEQ ID NO: 5, and a region presumed as a TATAAATC box is located at 132 nucleotides upstream from the translation initiation codon. It was. In addition, the 5 'upstream portion of the gene had an A / T-rich sequence showing an A / T content of 72%, and contained more than 19 bp of A / T nucleotides. The A / T-rich sequence of this 5 'upstream region is believed to play an important role in the promotion of transcription.

Example 8 : Cv20ox Activity measurement of gene promoter

In order to measure the activity of the Cv20ox gene promoter isolated in Example 7, the inventors prepared an expression vector in which the GUS coding region, a reporter gene, was fused to the Cv20ox promoter region. First, the 0.7 kb promoter fragment of the Cv20ox gene obtained from the recombinant vector pGA2044 prepared in Example 7 in the cleavage portion of Hin dIII and Pst I of pGA1230 (Clontech), a vector derivative having a high copy number and containing the GUS reporter gene. Was inserted to prepare an expression vector pGA2118 (Fig. 7). The expression vector pGA2118 is adsorbed onto tungsten particles (M-10, BioRad), and the tungsten particles to which the expression vector DNA is adsorbed are previously prepared using a particle bomber (BioRad Biolistic PDS-1000 / He, Bio-Rad Laboratories). The particle bombers were inserted into the various organs of the watermelon plants subjected to particle bombardment. The various organs of the watermelon plant with the expression vector inserted were partially cut and immersed in X-Gluc (5-Bromo-4-Chloro-3-Indolyl-BD-Glucuronic Acid, 1mg / ml) solution, and then 30 Transient expression of the fusion gene was measured by histochemical detection, which is a method of culturing for a period of time and observing with a dissecting microscope and observing the blue cell site resulting from the activity of the GUS reporter gene.

As a result, the activity of the Cv20ox promoter was detected only in the outer tissue of the developing seed, especially very strongly in the layer adjacent to the nucleus (FIG. 8B), whereas leaves (FIG. 8C), roots (D in FIG. 8) ), Non-reproductive organs such as hypocotyl (FIG. 8E) and cotyledon (F). The expression pattern of the Cv20ox promoter fusion gene was consistent with the results of in situ localization shown in Example 4.

Example 9 Expression Vector Preparation for Seedless Plant Preparation

Reverse link the gene to Cv20ox Cv20ox gene promoter, and again this is connected to T7'-5 'transcription termination sequence, and then, vector pGA810 (An, etc., Plant Molecular Biology Manual, Kluwer Academic, Dordrecht, Belgium, 1988) of T- The pGA2257 expression vector was prepared by inserting between the left and right borders of the DNA (see FIG. 9). The expression vector includes tetratracyclin and kanamycin resistance genes as selection marker genes. E. coli JM83 was transformed with the expression vector pGA2257, and the transformed E. coli was deposited at the Korea Microbial Conservation Center on September 29, 2000 (Accession No .: KCCM-10216).

Claims (17)

Watermelons (Citrullus lanatus) seed coat from the seed specific GA 20 oxidase gene is expressed in Cv20ox. The gibberellin 20 oxidase gene Cv20ox according to claim 1, which has a nucleotide sequence set forth in SEQ ID NO: 3 . Gibberellin 20 oxidase protein encoded by the gene of claim 1. The gibberellin 20 oxidase protein according to claim 3, which has an amino acid sequence as set forth in SEQ ID NO: 4. Cv20ox promoter that regulates the endothelial specific expression of gibberellin 20 oxidase gene Cv20ox of claim 1. 6. The Cv20ox promoter according to claim 5, having a nucleotide sequence set forth in SEQ ID NO: 5 . A promoter having the same base sequence as 23 or more consecutive base sequences among the base sequences of the promoter of claim 6 and having the activity of regulating the endothelial specific expression of a gene. 1) preparing an expression vector by functionally connecting genes encoding plant growth regulators downstream of a promoter that regulates seed specific gene expression of the plant; 2) transforming a plant with the expression vector; 3) selecting transgenic plants to induce regeneration; 4) purifying to soil; And 5) The method of producing a seedless fruit, characterized in that it comprises the step of maintaining the fruit of the transgenic plant in the state of natural pollination is suppressed and fruit deletion and growth. 1) preparing a plant transformation vector by reversely connecting the GA 20-oxidase gene downstream of a promoter for controlling seed specific gene expression of the plant; 2) transforming a plant with the vector; 3) selecting transgenic plants to induce regeneration; 4) purifying to soil; And 5) The method of producing a seedless fruit, characterized in that it comprises the step of maintaining the fruit of the transgenic plant in the state of natural pollination is suppressed and fruit deletion and growth. The method for producing a seedless fruit according to claim 9, wherein the plant transformation vector is pGA2257 (Accession No .: KCCM-10216) in which Cv20ox genes are connected in a reverse direction to a promoter of the Cv20ox gene. 10. The method of claim 8 or 9, wherein the promoter of step 1 is a promoter of the gibberellin 20 oxidase gene Cv20ox , which is specifically expressed in the seed of watermelon ( Citrullus lanatus ) seeds. 12. The method of claim 11, wherein the promoter of step 1 is a promoter of Cv20ox having the nucleotide sequence set forth in SEQ ID NO: 5. 10. The method according to claim 8 or 9, wherein the promoter of step 1 is a promoter having the same nucleotide sequence as 23 or more consecutive nucleotide sequences in the nucleotide sequence of SEQ ID NO: 5 and controlling the endothelial specific gene expression, How to make fruit without seeds. The method of claim 8, wherein the gene coding for a plant growth regulator of step 1. The method of manufacturing an iaaM, iaaH or ipt gene seedless, characterized in, that the fruit. 10. The method according to claim 8 or 9, wherein fruiting and growth induction of the fruit of step 5 is carried out before the anther is opened, when the flower reaches about 2 to 3 cm in length, and the flower is turned into a paper bag or the like. A method for producing a seedless fruit, characterized in that the casing is made by inhibiting moisture and fertilization for about 1 month. The method for producing seedless fruit according to claim 8 or 9, wherein the plant is a gourd and plant including watermelon, melon and the like. An expression vector pGA2257 (accession number: KCCM-10216) in which the Cv20ox gene is reversely linked downstream of the promoter of the Cv20ox gene.