JP6990904B2 - Parthenogenetic fruit set induction method using tissue- and time-specific promoters - Google Patents

Description

The present invention relates to a parthenogenetic method of inducing fruit set using a tissue- and time-specific promoter.

In the production of transformed plants, the 35S promoter derived from CaMV, which is a constitutive promoter, is often used to induce the expression of the target gene in the plant body. On the other hand, promoters that induce specific developmental stages of plants and specific tissues have also been isolated, and have been utilized for the production of transformed plants for which limited expression is desirable. For example, it is known that the promoter of the E8 gene isolated from tomato induces the expression of the target gene specifically at the ripening time of the fruit (Non-Patent Document 1). In addition, fruit-specific promoters having promoter activity have been isolated not only in red-ripened fruits but also in green-ripened fruits (Patent Document 1).

On the other hand, tomato is a self-pollinating plant, but it is known that in facility cultivation, the wind and insects that assist pollination are eliminated, so that the pollination rate decreases and the fruit set rate decreases. Therefore, a method of promoting parthenogenetic results and fruit enlargement by phytohormonal treatment of flower clusters is widely used. In addition, pollination promotion methods using bumblebees and vibrators are also often used. However, plant hormone treatment and pollination promotion treatment with a vibrator require enormous labor, and workability is significantly reduced. The method using bumblebees has good workability, but since the activity temperature range of bumblebees is limited, there is a problem that the cost and labor of temperature control in the facility increase in summer and winter. In addition, in fruit set based on pollination and fertilization, there is also a problem that it is difficult to secure a stable production amount throughout the year due to a decrease in pollen fertility in summer and winter. Therefore, in order to reduce the influence of environmental factors such as seasons and enable stable cultivation with less labor and cost, it is required to develop a technique for inducing parthenogenetic results with higher work efficiency in tomato plants. ing.

Relatively recent techniques for inducing parthenocarpy in tomato plants include the use of non-phytohormonal fruit set promoters and the introduction of parthenocarpy genes into tomato plants (eg, patents). Document 2) and so on. However, the method of using a fruit set accelerator still has a problem in terms of workability. In addition, many of the conventional parthenogenetic tomato varieties produced by introducing a parthenogenetic gene have problems in terms of fruit quality. Therefore, it is desired to develop tomato varieties having favorable fruit characteristics as well as parthenogenicity. Patent Document 3 discloses the production of tomatoes showing parthenocarpy by introducing a predetermined amino acid mutation, but in the mutant tomato plant, a clear change in leaf type occurs. There is also a need for a method that can induce parthenogenetic results in tomato plants while suppressing unwanted changes in phenotype, including appearance.

Furthermore, it is desired to develop a parthenogenetic method that enables stable fruit production not only in tomato plants but also in many other cultivated plants with lower labor and cost.

International release WO 2012/131919 International release WO 2009/005343 International release WO 2017/022859

Deikman et al., EMBO J., 7: 3315-3320 (1988)

An object of the present invention is to provide a promoter suitable for parthenogenetic fruit set induction. It is also an object of the present invention to provide parthenogenetic plants.

As a result of diligent studies to solve the above problems, the present inventors have found a promoter suitable for parthenocarpy fruit set induction that induces time and tissue-specific expression in plant reproductive organs including female buds. We found that it can be used to induce the expression of parthenocarpy-inducible genes and to produce parthenocarpy plants, resulting in the suppression of undesired changes in the phenotype of nutritional organs in parthenocarpy plants. , The present invention has been completed.

That is, the present invention includes the following.
[1] A promoter DNA specific to a plant reproductive organ, comprising a base sequence having 80% or more identity with respect to the base sequence represented by SEQ ID NO: 1 or 2.
[2] The promoter DNA according to the above [1], which exhibits promoter activity in the pistil.
[3] A DNA construct comprising the promoter DNA according to the above [1] or [2] and a gene arranged under the control thereof.
[4] The DNA construct according to [3] above, wherein the gene is a parthenogenetic inducible gene.
[5] The DNA construct according to [4] above, wherein the parthenogenetic inducible gene encodes an RNAi construct that suppresses the expression of the IAA9 gene.
[6] An expression vector containing the promoter DNA according to the above [1] or [2] or the DNA construct according to the above [3].
[7] An expression vector comprising the DNA construct according to [4] or [5] above.
[8] A transformed cell containing the DNA construct according to any one of the above [3] to [5] or the expression vector according to the above [6] or [7].
[9] A transformed plant into which the DNA construct according to the above [3] or an expression vector containing the DNA construct has been introduced.
[10] A parthenogenetic transformed plant into which the DNA construct described in [4] or [5] above or the expression vector described in [7] above has been introduced.
[11] The transformed plant according to the above [9] or [10], wherein the plant is a Solanaceae plant.
[12] The transformed plant according to the above [11], wherein the Solanaceae plant is tomato.
[13] The DNA construct described in the above [4] or [5] or the expression vector described in the above [7] is introduced into a plant cell, and the plant body is regenerated and grown from the obtained transformed cell. A method for producing a parthenocarpy transformed plant, which comprises confirming the result.
[14] The DNA construct according to any one of the above [3] to [5] or the expression vector according to the above [6] or [7] is introduced into a plant cell, and the plant body is obtained from the obtained transformed cell. A method of inducing gene expression specific to a plant reproductive organ, including regeneration and flowering.
[15] The DNA construct according to any one of the above [3] to [5] or the expression vector according to the above [6] or [7] is introduced into a plant cell, and the plant body is obtained from the obtained transformed cell. A method for recombinant production of an organic compound, which comprises regenerating, flowering and fruiting, and obtaining an organic compound produced by a gene product derived from a gene in the DNA construct or expression vector or an activity thereof from a fruit.

By using the promoter of the present invention, it becomes possible to specifically induce the expression of a foreign gene in a reproductive organ such as a pistil (for example, an ovary). Further, by the method using the promoter of the present invention, parthenogenetic results of plants can be induced, and parthenogenetic plants can be produced.

FIG. 1 is a diagram showing the results of RT-PCR analysis on the expression of the Solyc03g007780 gene (FIG. 1A) and the Solyc02g067760 gene (FIG. 1B) in vegetative or reproductive organs at various stages of development. In the figure, 1 DBA represents 1 Day Before Anthesis. DAA represents the number of days after flowering (Day After Anthesis). 0 DAA is the flowering date. The number of days for cotyledons, leaves, stems, and roots is the number of days from the germination day (day 1). FIG. 2 is a diagram schematically showing the T-DNA structure of the vector used for the promoter activity analysis by GUS staining. A promoter derived from Solyc03g007780 or Solyc02g067760, or the 35S promoter was used as the promoter. FIG. 3 is a photograph showing the results of promoter activity analysis by GUS staining in various organs. pSolyc03g007780-GUS shows the Solyc03g007780-derived promoter, and pSolyc02g067760-GUS shows the transformant into which the GUS gene is introduced under the control of the Solyc02g067760-derived promoter. p35S-GUS represents a transformant into which the GUS gene has been introduced under the control of the 35S promoter. WT shows wild type (non-transformant). WT (untreated) is a wild-type organ that has not been stained. Leaves and roots are 30 days after germination, 3-4 mm buds are 5 days before flowering, flowers are on the day of flowering (0 days after flowering), and fruits are 18 and 40 days after flowering. be. The fruits after 18 days are hypertrophic (middle) fruits, and the fruits after 40 days are red ripening fruits. FIG. 4 is a photograph showing the results of promoter activity analysis by GUS staining in anthers on flowering days and ovaries at different developmental stages. FIG. 5 is a diagram schematically showing the T-DNA structure of a tissue- and time-specific SlIAA9 expression-suppressing vector. FIG. 6 is a photograph of parthenocarpy fruits obtained in transformed tomato plants (Solyc03g007780-IAA9 RNAi and Solyc02g067760-IAA9 RNAi) using the Solyc03g007780-derived promoter or the Solyc02g067760-derived promoter for RNAi induction. FIG. 7 is a photograph showing the plant type of a transformed tomato plant. FIG. 7A shows transformed tomato plants # 4-17, # 4-23, and # 4-25, as well as wild type (WT) and iaa9-3, using Solyc 03g007780-derived promoters for RNAi induction. FIG. 7B shows transformed tomato plants # 6-10 and # 6-18 using a promoter derived from Solyc02g067760 for RNAi induction, as well as wild type (WT) and iaa9-3. FIG. 8 is a photograph showing a leaf type of a transformed tomato plant. FIG. 9 is a diagram showing the number of leaves (FIG. 9A) and the number of flower clusters (FIG. 9B) of the transformed tomato plant. FIG. 10 is a photograph showing the developmental state of fruits in a transformed tomato plant. "WT pol" represents a wild-type plant that has been artificially pollinated, and "un pol" represents a plant that has been sterilized without artificial pollination. DAA represents the number of days after flowering (Day After Anthesis). FIG. 11 is a diagram showing changes over time in the diameter of the ovary of a transformed tomato plant. FIG. 12 is a diagram showing suppression of SlIAA9 expression in the ovary. FIG. 13 is a diagram showing suppression of SlIAA9 expression in ovules.

Hereinafter, the present invention will be described in detail.

The present invention relates to a promoter capable of inducing gene expression specific to a plant reproductive organ, specifically a pistil and its neighboring organs. The promoter of the present invention has tissue- and time-specific promoter activity, and specifically exhibits promoter activity specific to plant reproductive organs including pistil. The promoter of the present invention can induce gene expression before and after flowering, specifically from the flower bud maturation stage to the early fruit hypertrophy stage (flower bud maturation stage, flowering stage, and early fruit hypertrophy stage). In the present invention, the "flower bud maturation stage" refers to the progress of flower bud maturation (physiological development) after the flower bud morphology (petals, pistils, stamens, etc.) is formed for each flower and before flowering. Refers to the time. The "flowering period" refers to the period from the time when the buds begin to open to the time when the fruits set and the fruit begins to grow. "Early fruit hypertrophy period" refers to the initial stage of fruit enlargement (early fruit development) after flowering and fruit set for each flower and fruit, and in the present invention, the first one-third of the fruit hypertrophy period. Say the period. In the early stage of fruit enlargement, for example, in tomatoes, it generally refers to up to 10 days after flowering, but it may vary depending on cultivation conditions and varieties. Here, in order to induce parthenogenesis, it is important that the parthenogenetic gene is expressed in the plant reproductive organs at the time of fruit set. "Fruit-bearing time" refers to the time when individual flowers can be pollinated, and in the case of tomatoes, it is not limited to the following, but is typically 5 days after flowering (120 hours after flowering). Until later). The promoter of the present invention is excellent in that it can induce gene expression in plant reproductive organs from the flower bud maturation stage to the early stage of fruit hypertrophy, particularly in the plant reproductive organs at the fruit set stage. The promoter according to the present invention can exhibit promoter activity in pistils (anthers, etc.), particularly in pistils (ovules, ovules, placenta, etc.) at the time of fruit set.

More specifically, the promoter according to the present invention is a promoter DNA consisting of a base sequence having 80% or more identity with respect to the base sequence represented by SEQ ID NO: 1 or 2. In a preferred embodiment, the promoter according to the present invention is a promoter DNA specific for a plant reproductive organ and exhibits promoter activity particularly in a plant reproductive organ including a pistil. The promoter according to the present invention is preferably capable of exhibiting promoter activity from the flower bud maturation stage to the early stage of fruit hypertrophy.

In the present invention, the "base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 1 or 2" is the base sequence represented by SEQ ID NO: 1 or 2, when aligned with the base sequence. Or, the full-length sequence is 80% or more, more preferably 90% or more, further preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more (for example, 99.5% or more) or more than the full-length sequence of 2. Refers to a base sequence having 99.9% or more and 100% or less) identity. The base sequence alignment is, for example, the multiple alignment program Clustal W (Thompson, JD et al, (1994) Nucleic Acids Res. 22, p.4673-4680; DNA Data Bank of Japan (DDBJ) and European Bioinformatics Institute (EBI). ) Can be used with the default settings), but it may also be done manually.

Alternatively, the promoter according to the present invention is deleted, substituted and / or deleted from, for example, 1 to 50, more preferably 1 to 10, and even more preferably 1 to 5 bases in the base sequence represented by SEQ ID NO: 1 or 2. It may be a promoter DNA consisting of an added base sequence and specific to a plant reproductive organ.

In the present invention, the "plant reproductive organ" or "reproductive organ" includes flowers and organs contained therein (for example, stamens and anthers and pollen contained therein, pistils and ovules contained therein (ovules, placenta, etc.). ) Etc., as well as petals, calyxes, etc.) and fruits (eg, flesh). The promoter specific to the plant reproductive organ according to the present invention has significantly stronger promoter activity in the plant reproductive organ as compared with the nutritional organ in fruit plants. The promoter specific to the plant reproductive organ according to the present invention is typically more than twice, preferably more than twice, in the plant reproductive organ as compared with other plant tissues (for example, nutritional organs such as leaves, stems and roots). It results in a gene expression level that is 10-fold or more, more preferably 100-fold or more. The promoter of the present invention preferably has strong promoter activity in plant reproductive organs (for example, female buds), while it preferably has little or no promoter activity in vegetative organs, and during periods other than the flower bud maturation stage to the early stage of fruit hypertrophy. It is preferable that the reproductive organ also has little or no promoter activity. The "promoter specific to a plant reproductive organ" is not limited to a promoter having promoter activity in all reproductive organs, and includes a promoter having promoter activity in at least some reproductive organs.

The promoter according to the present invention preferably has promoter activity in the pistil. The promoter according to the present invention also preferably has promoter activity not only in the pistil but also in the reproductive organ in the vicinity thereof. In one embodiment, a promoter according to the present invention, for example, a promoter specific to a plant reproductive organ consisting of a base sequence having 80% or more identity with respect to the base sequence shown in SEQ ID NO: 1, is a promoter in a pistil. It is preferable to exhibit activity, and it is more preferable to have promoter activity in pistils (particularly ovary), fruits (particularly fruits in the early stage of hypertrophy, such as flesh), and stamens (eg, pollen and anthers). It is preferable that the promoter consisting of a base sequence having 80% or more identity with respect to the base sequence shown in SEQ ID NO: 1 exhibits promoter activity specifically for plant reproductive organs from the flower bud maturation stage to the early stage of fruit hypertrophy. In one embodiment, a promoter according to the present invention, for example, a promoter specific to a plant reproductive organ consisting of a promoter having 80% or more identity with respect to the promoter set forth in SEQ ID NO: 2, is a promoter in a pistil. It is preferable to exhibit activity, and it is more preferable to have promoter activity in pistils (particularly ovary), stamens (eg, anthers or pollen), petals, and calyxes. It is preferable that a promoter consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 2 exhibits promoter activity specifically for plant reproductive organs from the flower bud maturation stage to the early stage of fruit enlargement. It is preferable to show promoter activity in plant reproductive organs mainly during the flower bud maturation stage and the flowering stage. In the present invention, "promoter activity" means the ability to induce the expression of a gene under its control (typically located immediately downstream of the promoter). By "inducing gene expression" is meant inducing transcript (mRNA) production from the gene of interest.

The promoter according to the present invention has a strong promoter activity specifically for a reproductive organ including a pistil and its neighboring organs, but in a typical example, a strong promoter activity specifically for a reproductive organ including a pistil and its neighboring organs of tomato. Has. The tomato in which the promoter according to the present invention is active may be any tomato line / variety as described later or a derivative strain thereof, and is not limited, but for example, a tomato variety Microtom or a money maker. Can be mentioned.

A particularly preferable promoter according to the present invention is a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2. The DNA consisting of each of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 is a promoter derived from tomato, and is a pistil before and after flowering (including the fruit set time, from the flower bud maturation stage to the early stage of fruit enlargement) and its vicinity. Gene expression can be strongly induced in an organ (reproductive organ).

The promoter according to the present invention may be a synthetic nucleic acid or a nucleic acid isolated from a plant. The promoter according to the present invention may be derived from angiosperms, for example, fruit plants, and may be preferably derived from Solanaceae (eg, tomato) plants. The tomatoes from which the tomatoes are derived include, but are not limited to, tomato varieties such as microtoms and money makers. The promoter according to the present invention may be a nucleic acid containing an artificial base.

The promoter according to the present invention can be isolated by PCR amplification using the genome of any fruit plant such as tomato as a template, as in the examples described later, or for restriction enzyme-treated fragments of the genome. It can also be obtained by hybridizing the promoter DNA or a part thereof as a probe. The obtained promoter DNA is preferably extracted and purified by a conventional method. Further, the promoter according to the present invention can be constructed by joining DNA fragments designed and chemically synthesized based on the base sequence information of the promoter (for example, SEQ ID NO: 1 or 2).

The promoter according to the present invention may also be prepared by modifying the base sequence of the promoter DNA once obtained by using a mutagenesis method such as a site-specific mutagenesis method. For the introduction of the mutation, a known method such as the Kunkel method or the Gapped duplex method or a method similar thereto can be adopted. These mutagenesis can be, for example, commercially available site-directed mutagenesis kits (for example, but not limited to, LA PCR in vitro Mutagenesis Kit, PrimeSTAR ^(R) Mutagenesis Basal Kit, Transformer ^TM Site-Directed Mutagenesis Kit ( Both can be easily performed by those skilled in the art using TAKARA BIO INC.)).

It is preferable to sequence the obtained promoter DNA in order to confirm whether or not the desired promoter can be obtained. Nucleotide sequence determination can be performed by any method such as Maxam-Gilbert method, dideoxy method, next-generation sequencing method (NGS), etc., but usually it may be performed using an automatic base sequence determination device.

In the present invention, an expression vector specific to a plant reproductive organ can be prepared by incorporating the promoter according to the present invention into an arbitrary vector. Therefore, the present invention also provides a vector containing the promoter according to the present invention, particularly an expression vector.

The present invention also provides a DNA construct containing the promoter DNA according to the present invention and a gene to be expressed under the control thereof. Such a DNA construct is typically a DNA construct containing the promoter DNA according to the invention and a gene linked downstream thereof. In the present invention, the "DNA construct" refers to a DNA having two or more functional units (gene, promoter, terminator, LB, RB, etc.) and having no autonomous replication ability, for example, a gene expression cassette or T-DNA. The DNA construct according to the present invention may further contain a terminator, LB, RB, restriction enzyme site, etc., in addition to the promoter DNA and the gene arranged under the control thereof. T-DNA is DNA sandwiched between the RB sequence at the 5'end and the LB sequence at the 3'end, and can be integrated into the plant genome by the Agrobacterium-mediated transformation method. The present invention also provides an expression vector containing a DNA construct containing the promoter DNA according to the present invention and a gene arranged under the control thereof. The promoter according to the present invention may be used in an expression vector or DNA construct in combination with various terminators (preferably, but not limited to, plant terminators). The expression vector and DNA construct according to the present invention may contain an inverted repeat sequence.

The vector into which the promoter or DNA construct according to the present invention is incorporated is not particularly limited as long as it can be replicated in a host cell, and for example, plasmid DNA, phage DNA, viral vector (retroviral vector, adenoviral vector, etc.) and the like. Can be mentioned. The vector incorporating the promoter or DNA construct according to the present invention is preferably a vector for plant expression. Vectors for plant expression include Agro, such as binary vectors that can replicate in both Agrobacterium and other host cells (such as E. tumef) when transforming plants using Agrobacterium-mediated transformation. It is preferable to use an expression vector suitable for the bacterium-mediated transformation method. The vector used in the present invention may be a vector for making RNAi constructs (for example, a Gateway ^(R) binary vector for making RNAi constructs). Examples of such a vector for plant expression include pBI101, pBI121, pBIN19, pSMAB704, pCAMBIA, pGreen, pBI-sense, antisense and the like.

The expression vector containing the promoter or DNA construct according to the present invention can be prepared by a conventional method. For example, an expression vector containing the promoter or DNA construct according to the present invention may be prepared by substituting the promoter in the existing vector for plant expression with the promoter according to the present invention. Alternatively, for example, the end of a DNA fragment (eg, a DNA construct) containing the promoter is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or multicloning site of the vector DNA and ligated to form the present invention in the vector. Such promoters can also be incorporated. The expression vector according to the present invention may be prepared by incorporating the promoter or DNA construct according to the present invention into the vector by the Gateway ^(R) cloning method.

The DNA construct and expression vector according to the present invention can be suitably used as a tool for specifically expressing a gene product (RNA or protein) encoded by a gene of interest in a plant reproductive organ (for example, pistil or ovary). .. The DNA construct and expression vector according to the present invention may be for inducing gene expression specific to a plant reproductive organ. The expression vector according to the present invention may contain a gene to be expressed under the control of the promoter according to the present invention. The expression vector according to the present invention may typically contain a gene to be expressed (foreign gene) linked downstream of the promoter DNA according to the present invention.

The gene to be expressed contained in the expression vector or DNA construct according to the present invention is not particularly limited, and is limited to translocated RNA (tRNA), ribosome RNA (rRNA), intranuclear small molecule RNA (snRNA), and nuclear body small molecule RNA ( It may be a gene encoding untranslated RNA such as snoRNA), shRNA (short hairpin RNA), short interfering RNA (siRNA), microRNA (miRNA), guide RNA (gRNA), or any protein. It may be the encoding gene. The gene to be expressed may be a gene encoding a fusion protein of two or more kinds of proteins. The gene to be expressed may be one or two or more. Since the expression vector according to the present invention induces gene expression in a plant reproductive organ, for example, a female bud, the gene to be expressed is preferably a reproductive organ (for example, female bud, ovary, fruit, stamen, pollen, anther, petal, etc. A nucleic acid encoding a gene product (RNA or protein) whose expression, accumulation, and / or production in stamens, etc. is desired.

The gene to be expressed in the present invention may be derived from any organism (for example, plant, animal, fungus or bacterium) or virus, or may be artificially produced. The gene to be expressed according to the present invention may be a gene obtained from the same plant line as the host plant or another line of the same plant species, or a gene derived from another species, and in any case. It is included in the range of foreign genes. In the present invention, the "gene (of expression)" is not limited to a naturally occurring gene, but means any nucleic acid encoding a peptide, polypeptide, protein, or RNA.

The gene to be expressed in the present invention is, for example, a carotenoid synthase (phytoen synthesis) involved in the biosynthesis of carotenoids such as lycopene (lycopene), β-carotene, α-carotene, lutein, astaxanthin, zeaxanthin, and β-cryptoxanthin. Enzyme, Phytoen Desaturation Enzyme, ζ-Caroten Desaturation Enzyme, Caroten Isomelase, Ricopen β-Cyclolase, Ricopen ε-Cyclase, β-Caroten Hydroxylase, α-Caroten Hydroxylase, ε-Caroten Hydroxide, Zeinoxanthin hydroxylase, carotenoid oxygenase, carotenoid hydroxylase, etc.), Vitamin synthase (L-glonolactone (L-GulL) oxidase, aldonolactonase, L-galactonolactone (L-GalL) dehydrogenase , L-galactose dehydrogenase, riboflavin synthase, etc.), Amino acid synthase (glutamine synthase, glutamic acid synthase, amino group transfer enzyme, GABA synthase (glutamic acid decarboxylase), amino acid racemase, etc.), Polyphenol synthase (polyquetide) It may be a gene encoding a peptide or protein involved in the synthesis of a nutritional component, such as a synthase, curcumin synthase, etc.).

The genes to be expressed used in the present invention are also, for example, various human or domestic vaccine antigens, cytokines (eg, interferon, interleukin, etc.), enzymes (eg, artificial nucleases, DNA polymerases, RNA polymerases, amyloglycosidases, amylases, etc.). Invertase, isoamylase, protease, papaine, pepsin, rennin, cellulase, pectinase, lipase, lactase, glucose oxidase, lysoteam, glucose isomerase, chymotrypsin, trypsin, cytochrome, seaprose, seratiopeptidase, hyaluronidase, bromeline, urokinase, hemocoagulase. , Urease, etc.), Hormonal proteins (eg insulin, glucagon, secretin, gastrin, cholecystkinin, oxytocin, vasopressin, growth hormone, thyroid stimulating hormone, prolactin, luteinizing hormone, follicular stimulating hormone, adrenal cortex stimulating hormone, thyroid stimulating hormone Release hormone, luteinizing hormone-releasing hormone, adrenal cortex stimulating hormone-releasing hormone, growth hormone-releasing hormone, somatostatin, etc.), opioid peptide (endolphin, enkephalin, dynorfin, etc.), blood coagulation factor (fibrinogen, prothrombin, etc.), protease inhibitor ( It may be a gene encoding any peptide or protein having pharmacological activity such as SSI).

The gene to be expressed as used in the present invention may also encode an RNAi construct for inducing RNA interference. The RNAi construct may be interfering RNA such as long double-stranded RNA or shRNA (small hairpin RNA) that contains inverted repeat sequences that have hairpin loops and are capable of forming double-stranded RNA. Long-chain double-stranded RNA and shRNA can be cleaved intracellularly by Dicer to produce siRNA. In the present invention, the RNAi construct is a sequence of genes expressed in the reproductive organ of the target plant into which the gene is introduced (for example, pistil), typically the reproductive organ from the flower bud maturation stage to the early stage of fruit hypertrophy (for example, fruit set time). It is preferable to have a sequence homologous to at least a part.

In one embodiment, the gene to be expressed used in the present invention is preferably a parthenogenetic gene. Parthenogenetic inducible genes will be described later.

The expression vector or DNA construct according to the present invention easily inserts a gene into a selection marker gene or reporter gene indicating that the vector is retained in a cell, a restriction enzyme site or a multicloning site, or a vector in the correct orientation. Additional useful sequences such as a polylinker for, a polyA addition sequence, a secretory signal sequence, a histidine tag sequence for purification, an additional promoter, etc. may be further included as needed. Examples of the selectable marker gene include a dihydrofolate reductase gene, a hyglomycin resistance gene, an ampicillin resistance gene, a canamycin resistance gene, a neomycin resistance gene, and a chloramphenicole resistance gene (CAT gene).

In the present invention, a transformant (transformed cell, transformed plant, etc.) can be prepared by introducing the expression vector or DNA construct according to the present invention into a host cell. The expression vector according to the present invention may be introduced into a host cell so that the expression vector is maintained extrachromosomally (cytoplasm, etc.) in the transformant. Alternatively, the expression vector or DNA construct according to the present invention is introduced into a host cell (preferably a plant cell), and a DNA containing the promoter and the gene to be expressed under its control (for example, a DNA construct such as T-DNA) is introduced. , May be integrated into the genome of the host cell. As the host cell, any of bacteria such as Escherichia coli, bacillus and agrobacterium, yeast cells, insect cells, animal cells (for example, mammalian cells), plant cells and the like may be used. In the present invention, plant cells, particularly angiosperms, more preferably fruit plant cells, more preferably eggplant family plants, and particularly preferably tomato cells can be used as host cells. The host cell may be derived from any tissue and may be a cell derived from leaves or fruits. The present invention also provides transformed cells containing the expression vector or DNA construct according to the present invention.

In the present invention, a transformed plant can be prepared by introducing the expression vector or DNA construct according to the present invention into a plant.

The plant into which the expression vector or DNA construct according to the present invention is introduced is not particularly limited, but angiosperms are preferable, and fruit plants are more preferable. Specifically, but not limited to, for example, tomato (Solanum lycopersicum), apple (Malus), banana (Musa), pear (Pyrus communis), strawberry (Fragaria L.), melon (Cucumis melo), etc. Citrus such as kiwi fruit (Actinidia deliciosa), peach (Amygdalus persica), blueberry (Vaccinium myrtillus), grape (Vitis), grapefruit (Citrus X paradisi), orange (Citrus sinensis), and Wenzhou citrus (Citrus unshiu). And other plants.

In the present invention, it is more preferable to introduce the expression vector or DNA construct according to the present invention into Solanaceae [Tomato (Solanum lycopersicum), Eggplant (Solanum melongena), Peanuts (Capsicum annuum var. Grosum), especially tomato plants. Preferred tomato plants include, but are not limited to, Solanum lycopersicum, Solanum cerasiforme (also known as Lycopersicon cerasiforme), and Solanum pimpinellifolium; Lycopersicon pimp. Solanum cheesmanii (also known as Lycopersicon cheesmanii), Solanum parviflorum (also known as Lycopersicon parviflorum), Solanum chmielewskii (also known as Lycopersicon chmielew) , Solanum hirsutum (also known as Lycopersicon hirsutum), Solanum Lycopersicon pennellii (also known as Solanum Lycopersicon pennellii), Solanum pennellii (also known as Lycopersicon peruvianum), Solanum chilense (also known as Lycopersicon peruvianum) ; Lycopersicon chilense), Solanum lycopersicoides, Solanum habrochaites, etc. include, but are not limited to, tomato strains / varieties or derivatives thereof. Solanum lycopersicum cv. Micro-Tom (Scott JW, Harbaugh BK (1989) Micro-Tom A miniature dwarf tomato. Florida Agr. Expt. Sta. Circ. 370, p. 1-6) can also be used. Microtoms are commercially available and are also available from the Tomato Genetics Resource Center (TGRC) (USA) under accession number LA3911. The wild-type tomato variety Microtom is dwarf (about 10 to 20 cm), has small leaves and fruits, and can be crossed with conventional tomato varieties. The entire genome sequence of the wild-type tomato variety Microtom has been determined (Kobayashi M, et al., (2014) Plant Cell Physiol. 2014 Feb; 55 (2): 445-454).

In the present specification, the derivative strain refers to a progeny plant obtained by one or more mating of the original plant with another plant line / variety, or by mutagenesis or mutagenesis.

As a method for introducing the expression vector or DNA construct according to the present invention into a plant, a method generally used for plant transformation, for example, an Agrobacterium-mediated transformation method, a whisker method, a particle gun method, or an electroporation method. , Polyethylene glycol (PEG) method, microinjection method, protoplast fusion method and the like can be used. For details on plant transformation methods, see general textbooks such as Yutaka Tabei, Transformation Protocol [Plants], (2012) Kagaku-Dojin, and Sun, HJ, et al., (2006) A highly efficient. References should be made to documents such as transformation protocol for Micro-Tom, a model cultivar of tomato functional genomics. Plant Cell Physiol. 47, 426-431.

When the Agrobacterium method is used, the expression vector according to the present invention prepared by using a vector suitable for the Agrobacterium method is electroporated into a suitable Agrobacterium, for example, Agrobacterium tumefaciens. It may be introduced by a method or the like, and the obtained recombinant Agrobacterium may be infected with a plant cell, callus, leaflet section or the like by a conventional method. Suitable Agrobacterium includes, but is not limited to, strains such as GV2260, GV3101, C58, C58C1Rif ^(R) , EHA101, EHA105, AGL1 and LBA4404. Infection with recombinant Agrobacterium allows T-DNA contained in expression vectors or DNA constructs to be integrated into the genome of plant cells.

In the particle gun method and the electroporation method, the expression vector and DNA construct according to the present invention can be directly introduced into plant cells. For the introduction of the expression vector or DNA construct according to the present invention into cells, tissue sections derived from plant cells, callus, leaves, leaflets, etc., or protoplasts may be used (Christou P, et al., Bio / technology (1991) 9: 957-962). For example, in the particle gun method, a nucleic acid delivery device (eg, PDS-1000 (BIO-RAD), etc.) is used according to the manufacturer's instructions to obtain metal particles sprinkled with the expression vector or DNA construct according to the present invention. By driving into a simple sample, it can be introduced into plant cells to obtain transformed plant cells. The operating conditions can be, for example, a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

Then, the plant can be regenerated from the transformed plant cells (including tissues such as cotyledon sections) obtained by introducing the expression vector or DNA construct according to the present invention. Transformed plant cells are cultured in a selective medium according to, for example, a conventionally known plant tissue culture method, and surviving callus is subjected to a redifferentiation medium (appropriate concentrations of plant hormones (auxin, cytokinin, diberelin, absidiic acid, ethylene, brush). Plants may be regenerated from transformed cells into which the expression vector or DNA construct according to the present invention has been introduced by culturing in (including nolide etc.). In this way, the transformed plant can be obtained. The present invention also provides a method for producing such a transformed plant.

The PCR method, Southern hybridization method, etc. can be used to confirm whether or not the promoter and the gene under its control according to the present invention have been reliably introduced into cells or plants by introducing the expression vector or DNA construct according to the present invention. It can be carried out by using a Northern hybridization method, a Western blotting method, or the like. For example, DNA or genomic DNA extracted from transformant cells, organs or tissues (eg, leaves of transformed plants) is specific to the promoter or integrated gene to be expressed in the expression vector or DNA construct according to the present invention. PCR amplification may be performed using a typical primer. The amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a clear band to detect the desired primer. And the introduction of genes can be confirmed. Alternatively, the PCR amplification product can be bound to a solid phase such as a microplate, and the amplification product can be confirmed by fluorescence, enzymatic reaction, or the like. The introduction may be confirmed by determining the base sequence of the amplified product. It is also preferable to confirm the activity of the prepared transformant with respect to the gene product of the introduced gene to be expressed.

In a preferred embodiment, by using the promoter according to the present invention in a plant for inducing gene expression, the expression of the gene to be expressed is strongly induced specifically in a reproductive organ (for example, a female bud or a male bud such as an anther), and the gene is strongly induced. Products (RNAs such as interfering RNA, mRNA, rRNA, and / or proteins) are highly produced. In the present invention, the promoter according to the present invention induces tissue and time-specific gene expression in at least some reproductive organs. In the transformed plant according to the present invention, it is preferable that the expression of the gene to be expressed under the control of the promoter according to the present invention is substantially or not induced in the nutrient tissues such as leaves, stems and roots. For example, even in the transformed tomato according to the present invention, it is preferable that the expression of the gene under the control of the promoter according to the present invention is substantially or not induced in the nutritional tissues such as leaves, stems and roots.

Accordingly, the present invention is specific for plant reproductive organs (particularly female buds), which comprises introducing the expression vector or DNA construct according to the present invention into plant cells and regenerating and flowering the plant body from the obtained transformed cells. Also provided are methods of inducing gene expression. The method of the invention may induce tissue and time-specific gene expression in at least some reproductive organs. In a preferred embodiment, a promoter consisting of the base sequence represented by SEQ ID NO: 1 or a promoter consisting of the above-mentioned mutant sequence (for example, a base sequence having 80% or more identity with respect to the base sequence) is used for inducing gene expression in a plant. In the case, expression specific to female buds and male buds is induced. In another preferred embodiment, a promoter consisting of the base sequence represented by SEQ ID NO: 2 or a promoter consisting of the above-mentioned mutant sequence (for example, a base sequence having 80% or more identity with respect to the base sequence) is used to induce gene expression in a plant. When used in, specific expression is induced in the promoter, as well as in neighboring organs including promoters, petals, and promoters.

In the present invention, by introducing an expression vector or DNA construct containing a parthenocarpy-inducing gene under the control of the promoter according to the present invention into a plant, germ cells, preferably reproductive organs from the flower bud maturation stage to the early stage of fruit hypertrophy. The expression of a parthenocarpy-inducible gene can be specifically induced, and as a result, the parthenocarpy can be induced. The parthenogenetic gene is a gene having a function of inducing parthenogenetic results in fruit plants. The parthenogenetic gene is a nucleic acid or polynucleotide, preferably DNA. The parthenogenetic gene may encode RNA such as a protein or RNAi construct. The parthenogenetic gene may be, for example, one that suppresses the function of the gene for suppressing fruit set. A preferred example of a parthenogenetic inducible gene is a gene that suppresses the function of the IAA9 (Indole acetic acid 9) gene. As another example of a participatory result-inducing gene, a gene that suppresses the function of IAA family genes other than IAA9, a gene that suppresses the function of the DELLA gene, and a gene that suppresses the function of the SlETR1 (Solanum lycopersicum ETHYLEN RECEPTOR 1; Solyc12g011330) gene. Gene, SlARF7 (Solanum lycopersicum AUXIN RESPONSE FACTOR 7; Solyc07g042260) Gene that suppresses gene function, SlAGL6 (Solanum lycopersicum AGAMOUS LIKE 6; Solyc01g093960) Gene that suppresses gene function, SlAGL11 (Solanum) A gene that suppresses the function of SlVAS1-like (Solanum lycopersicum VAS1 like; Solyc03g120450) gene that suppresses the function of the gene can also be mentioned.

The parthenocarpy-inducible gene that suppresses the function of the gene of interest may encode an RNA construct that can suppress the expression of the gene of interest. Such RNA constructs may be long double-stranded RNAs or interfering RNAs such as shRNAs, siRNAs, miRNAs, etc. for the gene of interest. In a preferred embodiment, the gene that suppresses the function of the IAA9 gene encodes an RNA construct that suppresses the expression of the IAA9 gene. An example of the base sequence (CDS sequence) of the IAA9 gene is shown in SEQ ID NO: 23, and the amino acid sequence of the IAA9 protein encoded by the base sequence is shown in SEQ ID NO: 24. The IAA9 gene may be, for example, a gene consisting of the base sequence represented by SEQ ID NO: 23 or a homologous gene thereof. The IAA9 gene typically has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99%, for example, 99.5% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 23. It may be a gene consisting of a base sequence having. The IAA9 gene of the present invention also typically has 90% or more, preferably 95% or more, more preferably 99% or more, for example 99.5% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. It may be a gene encoding an amino acid sequence having. The IAA9 gene encodes a protein with transcriptional repressive activity that acts as a negative regulator in the auxin signaling pathway. The gene encoding the RNA construct that suppresses the expression of the IAA9 gene may contain a DNA consisting of the base sequence shown by SEQ ID NO: 22 and a DNA consisting of a complementary sequence thereof, or the base shown by SEQ ID NO: 22. It may contain DNA consisting of a partial sequence of the sequence and DNA consisting of a complementary sequence thereof. The gene encoding such an RNA construct may contain a continuous 15 or more bases of the base sequence shown in SEQ ID NO: 22, for example, a base sequence of 15 to 30 bases and a complementary sequence thereof. The gene encoding the RNA construct may also contain a contiguous 30-715 base sequence of the base sequence set forth in SEQ ID NO: 22 and a complementary sequence thereof.

In the present invention, the "parthenocarpy" means that when pollination and fertilization do not occur in a plant, the ovary, the tentacles, etc. are enlarged without seed formation, and seedless fruits are produced. In the present invention, "parthenocarpy" means that when pollination and fertilization do not occur, the plant is parthenogenetic without the need for artificial parthenogenetic treatments such as phytohormonal treatment or specific physical stimuli. The property (ability) that produces the result.

Transformed plants introduced with an expression vector or DNA construct contained under the control of the promoter according to the present invention show parthenocarpy when not pollinated, and seeded fruits (fruits containing seeds) when pollinated. ) Can be formed.

A transformed plant into which an expression vector or DNA construct contained under the control of the promoter according to the present invention has been introduced is, in a preferred embodiment, by gene transfer or gene modification by causing tissue and timed gene expression in the reproductive organs. Undesirable phenotypic changes can be suppressed. In a preferred example, the transformed plant according to the present invention can suppress changes in the phenotype of the vegetative tissue (for example, plant type, leaf type, number of leaves, number of flower clusters, etc.) as compared with the wild type.

On the other hand, in a preferred embodiment, the transformed plant into which the expression vector or DNA construct contained under the control of the promoter according to the present invention is introduced forms a fruit having an increased sugar content and / or acidity as compared with the wild type. be able to.

In the present invention, an expression vector or DNA construct containing a parthenocarpy-inducing gene under the control of the promoter according to the present invention is introduced into a plant cell, and the plant body is regenerated and grown from the obtained transformed cell. Also provided are methods of producing parthenocarpy transformed plants, including confirming the results. Parthenogenetic results can be confirmed by observing the formation of seedless fruits. The method for producing a parthenocarpy transformed plant according to the present invention may include a step of self-mating the obtained transformed plant to obtain a progeny plant. The presence of the nucleic acid introduced into the genome of the progeny plant obtained by self-mating, that is, the promoter according to the present invention and the parthenocarpy-inducible gene under its control was confirmed by a conventional method, and homozygotes were selected. You may.

The present invention also introduces an expression vector or DNA construct containing the above-mentioned foreign gene under the control of the promoter according to the present invention into plant cells, and regenerates the plant body from the obtained transformed cells to flower and set fruit. Also provided is a method for recombinant production of an organic compound, which comprises obtaining a gene product derived from a gene in the DNA construct or expression vector or a product (organic compound) produced by the activity thereof from a fruit. By this method, various gene products or products (organic compounds) produced by their activities can be synthesized and accumulated in the fruits of plants to produce them. A gene product derived from a gene in a DNA construct or expression vector is a protein (eg, enzyme), polypeptide (including peptide), or RNA encoded by the gene. The product (organic compound) produced by the activity of the gene product is preferably produced in the fruit, for example, from a suitable substrate by a reaction based on the activity of the gene product (eg, an enzyme). Acquisition of the gene product or the product produced by its activity (organic compound) from the fruit can be carried out using various separation methods and isolation and purification methods of the compound (eg, centrifugation, chromatography, etc.).

Molecular biological and biochemical experimental operations such as DNA preparation, PCR, ligation into vectors, cell transformation, DNA sequencing, primer synthesis, mutagenesis, and protein extraction used in the present invention are performed. , Basically, it can be carried out according to the description of a normal experiment book. Such experimental books include, for example, Molecular Cloning: A laboratory manual (Third Edition) (2001) Eds., Sambrook, J. & Russell, DW. Cold Spring Harbor Laboratory Press and Molecular Cloning: A laboratory manual (Fourth). Edition), (2012) Eds., Michael R. Green & Sambrook, J., Cold Spring Harbor Laboratory Press.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Selection of candidate genes from RNA-seq data RNA-seqing (RNA-seq) data of various tissues of tomato (Solanum lycopersicum) varieties Micro-Tom were obtained and compared. Therefore, candidate genes that were not expressed in trophic tissues and showed specific expression of tomatoes were selected. First, samples of various tissues and developmental stages of Microtom (100 mg or less) were collected, and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, USA). As a sample, pistil and cotyledon of 2 to 2.5 mm bud; pistil and cotyledon of bud of 3 to 4 mm; pistil and cotyledon of bud 1 day before flowering; pistil, cotyledon, cotyledon and petal of flower on flowering day; 5 days after flowering Flower pistil; ovary 5 mm in diameter; flesh of green ripening fruit; flesh of red ripening fruit; seedling root, embryo axis and cotyledon 1 week after sowing; true leaf of plant 3 weeks after sowing Was used.

The "green ripe fruit" refers to a relatively immature fruit in the green ripe stage. In the agricultural field, the period when fruit enlargement is completed is called the green ripening period. Fruits in the green ripening stage are generally green, greenish white or yellowish white and have not yet been colored. After the green ripening period, the period during which the fruit ripens is called the ripening period, during which the sweetness and aroma of the fruit increase and the flesh becomes softer. In tomatoes, the ripe ripe fruit (ripe fruit) obtained by ripening (on the tree or after harvesting) through the green ripening stage and the breaker stage (yellow fruit) is called the red ripe fruit (red ripe fruit). .. Similarly, ripe fruits of other colored and mature fruits are also called red-ripened fruits or ripe fruits.

Genomic DNA (gDNA) was removed from the obtained total RNA sample using DNA-free RNA Kit ^TM (Zymo Research, USA). For the obtained RNA solution, the OD260 value was measured and the amount of RNA was calculated using a microplate reader Safire (Tecan, Switzerland). Subsequently, a cDNA library was prepared using TruSeq RNA Sample Prep Kit v2 (Illumina).

Next, RNA-seq was performed by sequencing 36 bases or 100 bases on each side using the next-generation sequencer Illumina Genome Analyzer IIx system and Illumina HiSeq 2000. A contig was created using the Denovo assembler ABySS 1.2.6 from the acquired read data. The contigs of 300 bases or more obtained were compared between the pistil and the tissues of other organs, and the contigs confirmed specifically for the pistil were selected. Subsequently, a BLAST search identified the tomato gene encoding the selected contig. The candidate genes selected were Solyc03g007780 and Solyc02g067760.

The Solyc03g007780 gene and the Solyc02g067760 gene were isolated from the tomato variety Micro-Tom.

[Example 2] Total RNA extraction and RT-qPCR expression analysis The candidate genes selected in Example 1 were subjected to RT-PCR expression analysis. First, in the same manner as in Example 1, of nutritional organs or reproductive organs (leaves, stems, roots, flower organs, hypertrophic fruits, green ripening fruits, breaker ripening fruits and red ripening buds) of different developmental stages. MRNA was extracted from the sample. Samples include: day 7 leaflets, stems and roots; day 30 plant leaves, stems and roots; 1 mm flower buds; 2 mm flower buds; 2 mm bud buds; 3-4 mm Bud buds; 5-6 mm bud buds; bud buds 1 day before flowering; flower buds on flowering day; flower buds 1 day after flowering; flower buds 2 days after flowering; flower petals 5-6 mm Petals of buds 1 day before flowering; Petals of flowering day 1; Petals 1 day after flowering; Petals 2 days after flowering; 3-4 mm buds of buds; 5-6 mm buds of buds; Flower pieces on the day of flowering; Flower pieces 1 day after flowering; Flower pieces 2 days after flowering; Flower pieces 3 days after flowering; Flower pieces 4 days after flowering; 2 mm bud ovary; 3-4 mm buds Ovary; 5-6 mm bud ovary; bud ovary 1 day before flowering; flower ovary on flowering day; flower ovary 1 day after flowering; 2 days, 3 days, 4 days, 6 days after flowering , 12 days, 18 days, 24 days, 30 days, 36 days, and 42 days later were used. 1 mm flower buds are 12 days before flowering, 2 mm flower buds (buds) are 9 days before flowering, 3-4 mm buds are 5 days before flowering, and 5-6 mm buds are 3 days before flowering. Fruits 2 to 24 days after flowering are bloated fruits (fruits 2 to 6 days after flowering are early fruits of bloating), fruits 30 days after flowering are green ripening fruits, fruits 36 days after flowering are breaker fruits, and flowering. The fruits after 42 days corresponded to the red ripe fruits.

After DNase treatment of the extracted RNA solution, cDNA was synthesized by a conventional method. Specifically, fast-strand cDNA was prepared by reverse transcription reaction using SuperScript ^(R) VILO ^TM MasterMix (Life Technologies, USA) from 2 μg of DNase-treated total RNA.

Subsequently, quantitative PCR was performed to amplify each gene using the prepared fast-strand cDNA as a template. Table 1 shows the primer set and estimated amplification size used for amplification of each gene.

As a control (reference gene), quantitative PCR was also performed to amplify the SAND gene. The PCR reaction was performed with a total volume of 25 μl. The composition of the PCR reaction solution was 1 μl of each primer (10 μM), 3 μl of cDNA, 12.5 μl of SYBR Premix Ex Taq (TAKARA), and 7.5 μl of sterile water. The PCR reaction was carried out under thermal cycle conditions in which 40 cycles of heat denaturation at 94 ° C. for 30 seconds, denaturation at 94 ° C. for 5 seconds, annealing and elongation at 60 ° C. for 30 seconds were performed as one cycle. The expression levels (relative amount of mRNA) of the genes Solyc03g007780 and Solyc02g067760 are shown in FIG.

As can be seen from FIG. 1, although there are some differences in the expression patterns of the two genes Solyc03g007780 and Solyc02g067760, expression was observed in the pistil (ovary, etc.). The Solyc03g007780 gene was particularly strongly expressed in the ovary and fruit before and after flowering. On the other hand, the Solyc02g067760 gene was strongly expressed in the pistil tissue before and after flowering as well as in the nearby organs (stamens, petals, calyxes). Expression at the RNA level was observed from about 3 days before flowering to 6 days after flowering, which corresponds to the flower bud maturation stage to the early fruit hypertrophy stage. No RNA-level expression was observed in any of the genes in organs other than the reproductive organs. That is, it was shown that the genes Solyc03g007780 and Solyc02g067760 are expressed tissue- and time-specifically in the pistil and its neighboring organs (reproductive organs).

Two selected genes, Solyc03g007780 and Solyc02g067760, were used as candidate genes for obtaining a promoter in the following examples.

[Example 3] BLAST analysis For the purpose of investigating the function of the selected candidate gene, sequence analysis was performed using the BLASTN program of NCBI (National Center for Biotechnology Information, USA).

As a result of this BLAST analysis, in Solyc03g007780, no gene having a homologous sequence was found in white inuna (Arabidopsis thaliana) and rice (Oryza sativa), while tobacco (Nicotiana emitter; GenBank accession number OIT38151, Nicotiana tabacum; A gene encoding a protein with highly homologous and conserved cysteine residues was discovered in GenBank accession number AAB18190). In addition, analysis of the amino acid sequence of the protein encoded by Solyc 03g007780 showed that it was a cysteine-rich peptide-like gene.

Solyc02g067760 showed homology to the tomato gene (Solanum lycopersicum myb-related protein 305-like) indicated by GenBank accession number XM_004233282. The Solyc02g067760 gene is also a petunia gene (Petunia x hybrida EMISSION OF BENZENOID II (PhEOBII)) indicated by GenBank accession number EU374207, and a tobacco gene (Nicotiana attenuata myb-related protein) indicated by GenBank accession number XM_019369866. It has homology with 305-like), suggesting that it may be a gene homologue of them. Others include capsicum annuum (GenBank accession number XM_016703603), asagao (Ipomoea nil; GenBank accession number XM_019305811), cotton (Gossypium hirsutum; GenBank accession number XM_016857435), cucumber (Cucumis sativus; GenBank accession number) ), Tensai (Beta vulgaris; GenBank accession number XM_010679560), Ingenmame (Phaseolus vulgaris; GenBank accession number XM_007150957) Melon (Cucumis melo; GenBank accession number XM_008445303), Tanjin (Salvia miltiorrhiza; A gene encoding a protein highly homologous to Solyc02g067760 was found in species (Silene littorea; GenBank accession number KT954912), orange (Citrus sinensis; GenBank accession number XM_006470245), and white inunazuna (Arabidopsis thaliana; GenBank accession number NM_123399). rice field.

[Example 4] Isolation of promoter In order to perform promoter analysis, we attempted to isolate the promoter region from each of the two genes Solyc03g007780 and Solyc02g067760 as follows.

(1) Extraction of genomic DNA Genomic DNA (gDNA) was extracted from the leaves of tomato (variety Microtom). Specifically, about 100 mg of leaves were first crushed into powder in an Eppendorf tube cooled under liquid nitrogen. From this powder sample, gDNA was extracted using the Maxwell ^(R) 16 System DNA Purification Kit (Promega, USA) and the Maxwell ^(R) 16 Instrument (Promega, USA). For the obtained DNA extract, the OD260 value was measured using Nanodrop 2000c (Thermo Fisher, USA), and the amount of genomic DNA was calculated.

(2) Isolation of promoter and determination of nucleotide sequence In order to clone the promoter region, a primer set and federity targeting the upstream sequence of each gene using the genomic DNA of the cultivated cultivar Microtom prepared in (1) above as a template. Approximately 2 kb upstream from the start of translation was amplified by PCR using the high KOD Plus Neo (TOYOBO, Japan). For Solyc03g007780, the primer used for this amplification was the primer pro007780-F-1 (5'-CCTAGGACGGAGTGTGCCACATAAGAC-3') to which the recognition sequence 5'-CCTAGG-3' of the restriction enzyme BlnI (AvrII) was added. Primer pro007780-R-1 (5'-GTCGACCTCGAGAGCTTTTTTTTTTTCCCTTCTCAC) with the restriction enzyme XhoI recognition sequence 5'-CTCGAG-3'and the restriction enzyme SalI recognition sequence 5'-GTCGAC-3' added to SEQ ID NO: 9) and 3'side. -3'; SEQ ID NO: 10). The primer used for amplification of Solyc02g067760 was the primer pro067760- with the restriction enzyme SalI recognition sequence 5'-GTCGAC-3'and the restriction enzyme BlnI (AvrII) recognition sequence 5'-CCTAGG-3' added to the 5'side. F-1 (5'-GTCGACCCTAGGGTTCCTAGCTTTGACACACAAGAG-3'; SEQ ID NO: 11) and restriction enzyme XhoI recognition sequence 5'-CTCGAG-3'and restriction enzyme BamHI recognition sequence 5'-GGATCC-3' were added to the 3'side. Primer pro067760-R-1 (5'-GGATCCCTCGAGGTAGAGAGGAAGATGAGAGA-3'; SEQ ID NO: 12). The PCR reaction was performed with a total volume of 50 μl. The composition of the reaction solution was 1 μl of each primer (10 μM) of the primer set, 1 μl (30 ng) of genomic DNA, 5 μl of 10x PCR buffer, 5 μl of dNTPs, ₂ 3 μl of MgCl, and 1 μl of KOD-Plus-polymerase enzyme. , And 33 μl of sterile water. The PCR reaction consists of 28 cycles of heat denaturation at 98 ° C for 5 minutes, denaturation at 98 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 68 ° C for 1 minute, and finally at 68 ° C. It was carried out under the thermal cycle condition of stretching for 3 minutes.

The PCR product after the reaction was fractionated by agarose gel electrophoresis supplemented with SYBR Safe DNA Gel Stain (Invitrogen, USA), and DNA fragments of the desired size were excised from the agarose gel and Wizard ^(R) SV Gel and PCR Clean-Up. Purified using System (Promega, USA). Deoxyriboadenosine (dA) was added to both ends of the DNA fragment by adding 10x PCR buffer, 0.2 mM dATP, and rTaq to the purified DNA and reacting at 72 ° C. for 10 minutes. The dA-added DNA fragment was subcloned into the pCR2.1 vector (Invitrogen, USA) by TA cloning. The pCR2.1 vector contains an ampicillin resistance gene and a kanamycin resistance gene as selectable markers. The base sequence of the DNA fragment cloned in the obtained plasmid vector was determined by a conventional method. The prepared plasmid vectors were named pCR2.1-pro007780 vector and pCR2.1-pro067760 vector, respectively. Promoters were excised from the pCR2.1-pro007780 vector whose sequence was confirmed using the restriction enzymes HindIII and SalI, and introduced into the pBI101 GUS vector linearized with the same enzyme using T4 DNA ligase (Invitrogen, USA). In addition, promoters were excised from the pCR2.1-pro067760 vector using the restriction enzymes SalI and BamHI, and introduced into the pBI101 GUS vector linearized with the same enzyme using T4 DNA ligase. The prepared plasmid vectors were named pBI101-pro007780 vector and pBI101-pro067760 vector, respectively.

The nucleotide sequences of the promoter regions derived from Solyc03g007780 and Solyc02g067760 cloned in these vectors are shown in SEQ ID NO: 1 (promoter sequence of Solyc03g007780) and SEQ ID NO: 2 (promoter sequence of Solyc02g067760).

The structure of T-DNA in the pBI101-pro007780 vector, the pBI101-pro067760 vector, and the expression vector pBI121 containing the GUS gene under the control of the CaMV-derived 35S promoter used as a control in Example 5 described later is schematically shown in FIG. Shown in.

[Example 5] Evaluation of activity of promoter region The activity of each promoter of Solyc03g007780 and Solyc02g067760, whose expression was confirmed specifically for reproductive organs including pistil in Example 2, was evaluated using the GUS gene expression activity. First, the pBI101-pro007780 vector and the pBI101-pro067760 vector in which each promoter was inserted upstream of the GUS gene of the pBI101 vector prepared in Example 4 were introduced into the Agrobacterium tumefaciens GV2260 strain by electroporation. did. Recombinant Agrobacterium was then selected on LB agar medium supplemented with the antibiotic kanamycin at 50 mg / L.

Using the obtained recombinant Agrobacterium, a transformant of Microtom was prepared by the method of Sun et al. (Plant Physiol., 114: 1547-1556, 2006). As a control, a recombinant Agrobacterium into which an expression vector pBI121 containing the GUS gene was introduced under the control of the 35S promoter was used to prepare a transformant of Microtom in the same manner. More specifically, first, recombinant Agrobacterium was cultured with shaking overnight in LB medium supplemented with the antibiotic kanamycin at 50 mg / L. The culture was centrifuged at 3000 rpm for 10 minutes at room temperature to remove the supernatant. After washing the collected pellets, they were suspended in MS liquid roasted land supplemented with 200 μM of acetosyringone and 10 μM of mercaptoethanol at a concentration of 0.1 OD600 value. A section of sterile microtom cotyledons 7 days after sterile seeding was immersed in this Agrobacterium solution. As a result, Agrobacterium-infected tomato cotyledon sections were co-cultured for 3 days in MS medium supplemented with 1.5 mg / L zeatin. Then, the culture section was transferred to a selective MS medium supplemented with 1 mg / L zeatin and 100 mg / L kanamycin, cultured while changing the medium every two weeks, and then the elongated shoots were 50 mg / L kanamycin. Was transferred to the rooted MS medium to which the above was added, and roots were formed.

Genomic DNA was extracted from the leaves of the redifferentiated individuals rooted in the rooted MS medium according to the method described in Example 4. Using this genomic DNA as a template, the GUS gene was amplified by PCR. The primers used were GUS-F (5'-CCGGGTGAAGGTTATCTCTATG-3'; SEQ ID NO: 13) and GUS-R (5'-CATGACGACCAAAGCCAGTA-3'; SEQ ID NO: 14). The PCR reaction was performed with a total volume of 50 μl. The composition of the reaction solution was 1 μl of each primer (10 μM), 1 μl of genomic DNA, 5 μl of 10x PCR buffer, 4.0 μl of 2.5 mM dNTPs, 0.4 μl of polymerase enzyme Ex-Taq (5U / μl), and 38.6 μl of sterile water. (10x PCR buffer, dNTPs, polymerase enzyme used from TAKARA Taq Hot Start Version). The PCR reaction consists of 35 cycles of heat denaturation at 95 ° C for 3 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute, and finally at 72 ° C. It was carried out under the thermal cycle condition of 5 minutes extension. 10 μl of the obtained PCR product was electrophoresed on an agarose gel, the amplified product was confirmed, and the redifferentiated individual in which the amplification of the GUS gene was confirmed was selected as a transformant.

Furthermore, a line into which one copy of the recombinant construct was introduced was selected by Southern blotting analysis using an NPTII probe for use in the growth survey in the examples described later. The NPTII probe was synthesized using the DIG DNA Labeling Kit (Roche, Switzerland) and the NTPII amplification primer set (primers of SEQ ID NO: 20 and SEQ ID NO: 21).

For the transformant generation thus prepared, the tissue-specific expression-inducing activity of the promoter by GUS staining was further analyzed. The expression analysis based on this GUS staining was performed by Jefferson et al. (EMBO J., 6: 3901-3907) using X-Gluc (5-bromo-4-chloro-3-indrill-β-D-glucuronic acid) as a substrate. , 1987) with some changes. Specifically, in order to reduce the background of staining, the reaction solution (1 mM X-Gluc, 0.5 mM) in which the 50 mM phosphate buffer (pH 7.0) of the reaction solution was changed to 100 mM phosphate buffer (pH 8.0) was used. Potassium ferricyanide, 0.5 mM potassium ferricyanide, 100 mM phosphate buffer (pH 8.0)) was used. Since the background signal was detected in the red-ripe fruits even when the above reaction solution was used, the method of Kosugi et al. (Plant Sci., 70: 133-140) was used for the red-ripe fruits in order to further reduce the background. , 1990), and staining with the above reaction solution to which 20% methanol was added at the final concentration was also performed. Staining is performed by immersing each tissue collected from the transformed tomato in the above reaction solution, reducing the pressure for 15 minutes to allow the stain solution to permeate the tissue, and at 37 ° C. overnight (16 hours) or 6 hours (20% methanol). Only the added reaction solution) was performed by incubation. After incubation, the reaction was stopped by washing the sample with 70% ethanol.

Examples of the results are shown in FIGS. 3 and 4. Promoters derived from Solyc03g007780 and Solyc02g067760 showed activity in transformed plants. No GUS staining was observed in the vegetative organs (leaves, roots) of the transformants using the Solyc03g007780 and Solyc02g067760-derived promoters, and no GUS staining was observed in the 3-4 mm buds (Fig. 3). When the promoter derived from Solyc03g007780 was used, GUS staining was observed specifically in the reproductive organs, and strong GUS staining was observed especially in the pistil of the flower (Fig. 3). In FIG. 4, as a result of expression induction using the promoter derived from Solyc03g007780, the stamens (pollen) on the flowering day, and the ovule in the ovule from the flowering date to 4 days after the flowering, and the placenta and the ovule wall near the ovule. GUS staining is shown. On the other hand, when the Solyc02g067760-derived promoter was used, GUS staining was observed specifically in the reproductive organs, and strong GUS staining was observed especially in the pistil (ovary) and its neighboring organs (stamens, petals, and calyxes) (Fig. 3). 4). FIG. 4 shows GUS staining of the entire stamen containing pollen on the flowering day and the entire ovary from the flowering day to 4 days after flowering as a result of expression induction using the Solyc02g067760-derived promoter. In contrast, when the 35S promoter was used, GUS staining was observed in all tissues tested. No GUS staining was observed in wild type (WT). This result supports the result of RT-qPCR expression analysis of Example 2.

Therefore, it was shown that the promoters derived from the two genes Solyc03g007780 and Solyc02g067760 induce gene expression specifically in the reproductive organs including the pistil and have strong promoter activity.

The conventional plant promoter E8 is known to function only in the late stage of fruit development (ripening stage). However, from the above results, unlike the E8 promoter, the promoters derived from Solyc03g007780 and Solyc02g067760 are from the flower bud maturation stage to the early stage of fruit hypertrophy, including the fruit set time in the pistil, especially the ovary or fruit, and the plant reproductive organs such as stamen. It became clear that it can function.

[Example 6] Preparation of tomato transformed plant using isolated promoter IAA9 (Indole acetic acid 9), which is a gene related to auxin signaling, is a parthenocarpy gene, and its downregulation brings parthenocarpy results. It is known. In this example, RNAi constructs capable of suppressing the expression of the IAA9 gene were expressed under the control of each promoter derived from Solyc03g007780 and Solyc02g067760 isolated in Example 4, and their effects were investigated.

First, a DNA fragment containing promoter regions derived from Solyc03g007780 and Solyc02g067760 was introduced into pBI-sense, antisense-GW (INPLANTA INOVATIONS, Japan), which is a Gateway ^(R) binary vector for RNAi, as follows. From the pBI101-pro007780 vector and pBI101-pro067760 vector prepared in Example 4, promoter regions derived from Solyc03g007780 and Solyc02g067760 were excised by restriction enzyme treatment using BlnI and XhoI, and the excised DNA fragments were excised from pBI from which the 35S promoter was removed. -Incorporated into the upstream of the GATEWAY cassette of the sense and antisense vectors by ligation reaction.

Subsequently, a DNA fragment targeting IAA9 was introduced into the entry vector pCR8 / GW / TOPO vector for Gateway ^(R) by the following method. First, a 715 bp DNA fragment (SEQ ID NO: 22) of tomato (Solanum lycopersicum) IAA9 (SlIAA9) was amplified by PCR reaction. Primers SlIAA9-F-1 (5'-TGGCCACCCATTCGATCTTTTAG-3'; SEQ ID NO: 15) and SlIAA9-R-1 (5'-ACAAACTCCAATATCAAACGG-3'; SEQ ID NO: 16) were used for PCR amplification. The reaction was carried out with a total volume of 50 μl, and the composition of the reaction solution was 1 μl of each primer (10 μM) of the primer set, 2 μl of cDNA derived from tomato ovary, 5 μl of 10x PCR buffer, 5 μl of dNTPs, ₂ 3 μl of MgCl, 1 μl of KOD-Plus-Neo polymerase enzyme and 32 μl of sterile water were used. The PCR reaction consists of 28 cycles of heat denaturation at 98 ° C for 5 minutes, denaturation at 98 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 68 ° C for 1 minute, and finally at 68 ° C. It was carried out under the thermal cycle condition of extending for 3 minutes. The PCR product after the reaction was separated by agarose gel electrophoresis supplemented with SYBR Safe DNA Gel Stain (Invitrogen, USA), and DNA fragments of the desired size were excised from the agarose gel and Wizard ^(R) SV Gel and PCR Clean-Up System. Purified using (Promega, USA). 10x PCR buffer, 0.2 mM dATP, and rTaq were added to the purified DNA sample and reacted at 72 ° C. for 10 minutes to add dA to both ends of the DNA fragment. The dA-added DNA fragment was introduced into the pCR8 / GW / TOPO vector by TA cloning using the pCR ^(R) 8 / GW / TOPO ^(R) Vector Kit (Invitrogen, USA).

Next, the DNA fragment targeting SlIAA9 cloned into the pCR8 / GW / TOPO vector was transferred to the pBI-sense, antisense vector into which the Solyc03g007780 and Solyc02g067760 promoters prepared above were introduced, and LR ^chronaseTM (Invitrogen, Invitrogen, It was introduced by an LR reaction using USA). The obtained vector was used as a tissue- and time-specific SlIAA9 expression-suppressing vector as described below. FIG. 5 schematically shows the structure of T-DNA in this vector.

The prepared SlIAA9 expression-suppressing vector was introduced into the Agrobacterium tumefaciens GV2260 strain by electroporation, and recombinant Agrobacterium was selected on LB agar medium supplemented with the antibiotic kanamycin at 50 mg / L.

Using the obtained recombinant Agrobacterium, a transformant of Microtom was prepared by the method of Sun et al. (Plant Physiol., 114: 1547-1556, 2006). Specifically, first, recombinant Agrobacterium was cultured with shaking overnight in LB medium supplemented with the antibiotic kanamycin at 50 mg / L. The culture was centrifuged at 3000 rpm for 10 minutes at room temperature to remove the supernatant. After washing the collected pellets, they were suspended in MS liquid roasted land supplemented with 200 μM of acetosyringone and 10 μM of mercaptoethanol at a concentration of 0.1 OD600 value. A section of sterile microtom cotyledons 7 days after sterile seeding was immersed in this Agrobacterium solution. As a result, Agrobacterium-infected tomato cotyledon sections were co-cultured for 3 days in MS medium supplemented with 1.5 mg / L zeatin. Then, the culture section was transferred to a selective MS medium supplemented with 1 mg / L zeatin and 100 mg / L kanamycin and cultured while changing the medium every two weeks, and then the elongated shoots were cultivated with 50 mg / L kanamycin. Was transferred to the rooted MS medium to which the above was added, and roots were formed.

Genomic DNA was extracted from the leaves of the redifferentiated individuals rooted in the rooted MS medium according to the method described in Example 4. Using this genomic DNA as a template, the presence or absence of vector introduction was confirmed by PCR. The primers used were SlIAA9-R-1 (5'-ACAAACTCCAATATCAAACGG-3'; SEQ ID NO: 16) and NOST-R-1 (5'-CAAGACCGGCAACAGGATT-3'; SEQ ID NO: 19). The PCR reaction was performed with a total volume of 50 μl. The composition of the reaction solution was 1 μl of each primer (10 μM), 1 μl of genomic DNA, 5 μl of 10x PCR buffer, 4.0 μl of 2.5 mM dNTPs, 0.4 μl of polymerase enzyme Ex-Taq (5U / μl), and 38.6 μl of sterile water. (10x PCR buffer, dNTPs, polymerase enzyme used from TAKARA Taq Hot Start Version). The PCR reaction consists of 35 cycles of heat denaturation at 95 ° C for 3 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute, and finally at 72 ° C. It was carried out under the thermal cycle condition of 5 minutes extension. 10 μl of the obtained PCR product was electrophoresed on a 1.0% agarose gel, the amplified product was confirmed, and the redifferentiated individual in which the amplification of the vector was confirmed was selected as a transformed (transgenic) plant. More than 40 lines of transformed plants were obtained for each nucleic acid construct. Furthermore, by Southern blotting analysis using an NPTII probe, a strain into which one copy of the nucleic acid construct (T-DNA derived from the above-mentioned SlIAA9 expression-suppressing vector) was introduced was selected. The DNA probe was synthesized by PCR amplification using a DIG DNA Labeling Kit (Roche, Switzerland) and a primer set for NTPII amplification (primers of SEQ ID NO: 20 and SEQ ID NO: 21).

These 1-copy lines obtained were self-mated (self-fertilized) to obtain homozygous lines (T2 generation).

In the homozygous line (T2 generation), suppression of SlIAA9 expression was confirmed using quantitative RT-PCR method. First, using the same method as in Example 2, the flowering date of the transformants (4-17 line, 4-23 line, 4-25 line, 6-10 line, 6-18 line), 2 days after flowering, Total RNA was extracted from the ovary after 4 days. In addition, total RNA was extracted from the ovules of wild-type and Solyc 03g007780-derived promoter lines (4-17 line, 4-23 line, 4-25 line) 2 days after flowering. The ovules were isolated using tweezers under a stereomicroscope. The extracted total RNA was treated with DNase and then cDNA was synthesized by a conventional method. Specifically, the PrimeScript II 1st strand cDNA Synthesis Kit (Takara bio, Japan) was used to prepare fast-strand cDNA by reverse transcription reaction.

Using the prepared fast-strand cDNA, suppression of the expression of the SlIAA9 gene in the transformant was confirmed. The primers used were SlIAA9-F-2 (5'-CTCAGGCTCGGTCTACCTG-3'; SEQ ID NO: 17) and SlIAA9-R-2 (5'-CCTCTGAGAATCCATCCATAGC-3'; SEQ ID NO: 18). The SAND gene was used as an endogenous control. Primers for the SAND gene are SAND-F-1 (5'-TTGCTTGGAGGAACAGACG-3'; SEQ ID NO: 3) and SAND-F-1 (5'-GCAAACAGAACCCCTGAATC-3'; SEQ ID NO: 4). The PCR reaction was performed with a total volume of 25 μl. The composition of the reaction solution was 1 μl of each primer (10 μM), 3 μl of cDNA, 12.5 μl SYBR Premix Ex Taq II mix, and 7.5 μl of sterile water. The PCR reaction was carried out under thermal cycle conditions in which 40 cycles of heat denaturation at 94 ° C. for 30 seconds, denaturation at 95 ° C. for 5 seconds, annealing and elongation at 60 ° C. for 30 seconds were performed as one cycle.

As a result of expression analysis, two promoter lines derived from Solyc03g007780 (4-23 line, 4-25 line) and Solyc02g0677602 line (6-10 line, 6-18 line) suppressed SlIAA9 expression in ovary compared with wild type. A tendency was confirmed (Fig. 12). In addition, suppression of expression in ovules, which is important for fruit set control by SlIAA9, was also confirmed for the 4-17 strain (Fig. 13).

[Example 7] Investigation of growth, parthenogeneticity, and fruit quality of transformed plants A growth investigation was conducted on the transformed homozygous strain (T2 generation) prepared in Example 6. As the transformed homozygous line (T2 generation), a transformed line using a promoter derived from Solyc03g007780 (4-17, 4-23 and 4-25 lines) and a transformed line using a promoter of Solyc02g067760 (6-10). And 6-18 strains) were used.

As a control, wild-type microtoms (WT) and iaa9-3 mutant strains were similarly investigated for growth. The iaa9-3 mutant strain is a microtom mutant strain having a single mutation in which the 237th base T is replaced with A in the endogenous IAA9 gene, which was created by EMS mutagenesis. The IAA9 gene has a stop codon that produces a protein with only 79 amino acids, resulting in a loss of IAA9 gene function (Saito et al., Plant Cell Physiol. (2011) 52 (2): 283). -296). The iaa9-3 mutant line and other IAA9 downregulated lines show parthenocarpy as well as leaf and petiole fusion (Wang et al., The Plant Cell, Vol.17, p.2676-2692; Saito et. al., Plant Cell Physiol. (2011) 52 (2): p.283-296). In addition, the Azygous strain (AZ) was used as a comparative example for eliminating the effects of the tomato transformation method itself on gene expression and plant phenotype. This AZ strain is a strain in which the transgene was removed after the transformant self-fertilized. Furthermore, a vector for suppressing SlIAA9 expression was prepared by incorporating a DNA fragment targeting IAA9 under the control of the 35S promoter that induces constitutive expression, and the micro was introduced by the Agrobacterium method in the same manner as in Example 6. The Tom transformed line was also investigated in the same manner.

These plants were cultivated under the following conditions. First, plant seeds were sown on a petri dish lined with filter paper, grown at 25 ° C for 1 week, and then the seedlings were transplanted into 75 x 75 x 65 mm sized rock wool (Grodan, The Netherlands). For the transplanted plants, liquid fertilizer mixed with Otsuka House No. 1 and Otsuka House No. 2 (OTSUKA, Japan) was applied, and the electrical conductivity (EC) was 1.8 dS / m or less, 16 hours / 8 hours (light / dark period). ), Light intensity 300 μmol m ^-2 s ^-1 , cultivated under the condition of 25 ℃.

Each plant was self-mated by artificial insemination, and the obtained fruits were examined. As a result of the growth survey, all the transformed lines had the same fruit set rate (ratio of the number of flowers that formed fruits to the number of pollinated flowers) and fruit set as in the wild type (non-transformed plants) by self-fertilization. The rate (the ratio of the number of seeded fruits, that is, the fruits containing seeds) to the number of formed fruits was shown (Table 2). On the other hand, in the iaa9-3 mutant strain lacking the function of IAA9, the fruit set rate and the fruit set rate after pollination were considerably lower than those of the wild type and the transformed strain.

On the other hand, the flowers of the transformed line and the iaa9-3 mutant line 1 day before flowering were selected and sterilized, and the fruits obtained 48 days later (parthenocarpy fruits) were harvested without artificial pollination to obtain fruit quality. It was tested for analysis. As a control, for the wild type, only male removal (WT) or artificial pollination and male removal (WT pol) was performed, and the fruits were analyzed in the same manner.

Both the transformed line using the Solyc03g007780-derived promoter and the transformed line using the Solyc02g067760-derived promoter showed parthenogenetic properties similar to the iaa9-3 mutant line (Fig. 6). The wild type formed seeded fruits by artificial pollination. As shown in FIG. 6, the fruits of the transformed line showed the same morphology as the wild-type fruits.

In addition, the transformed lines 4-17, 4-23 and 4-25 using the Solyc03g007780-derived promoter showed the same high parthenogenetic rate as the iaa9-3 mutant line (Table 3). On the other hand, the parthenogenetic rate of the transformed lines 6-10 and 6-18 using the Solyc02g067760-derived promoter was lower than that of the iaa9-3 mutant line, but it was still about 40% (Table 3). .. The parthenocarpy result rate was calculated as the ratio (%) of the number of flowers that formed fruits to the number of flowers that had been subjected to male removal treatment without artificial pollination.

Furthermore, the morphology of plants was compared. Both transformed lines using the Solyc03g007780-derived promoter or the Solyc02g067760-derived promoter showed the same plant type (Fig. 7) and leaf type (Fig. 8) as the wild type. On the other hand, the iaa9-3 mutant line and the transformed line using the 35S promoter (p35S-IAA9 RNAi; right end of Fig. 8) showed a slightly elongated plant type (Fig. 7) and a potato type leaf type (Fig. 8). rice field. The potato-type leaf type is specifically a leaf in which the leaf blades are not separated and are single leaves.

In addition, all the transformed lines showed the same number of leaves and flower clusters as the wild type (Fig. 9). On the other hand, the number of leaves and the number of flower clusters of the iaa9-3 mutant line were significantly reduced as compared with the transformed line and the wild type (Fig. 9). From these results, it was shown that the use of the above promoter can induce parthenogenetic results with almost no effect on traits such as plant type, leaf type, number of leaves, and number of flower clusters.

Table 4 shows the results of comparing the fruit yields. Transformed strains (4-17, 4-23 and 4-25 strains) using the Solyc03g007780-derived promoter showed a yield that was about 20% lower than that of the wild type, but similar to that of the iaa9-3 mutant strain. The yield was shown. The number of harvested fruits was higher in the transformed lines (4-17, 4-23 and 4-25 lines) using the promoter derived from Solyc03g007780 than in the wild type. The number of fruits of the transformed lines (6-10 and 6-18 lines) using the promoter derived from Solyc02g067760 was lower than that of the wild type, but higher than that of the iaa9-3 mutant line. Fruit size was reduced in both transformants, while the iaa9-3 mutant had larger fruits.

The size of the ovary was measured over time to investigate its effect on fruit development. In FIG. 10, 2 days before flowering date (-2 DAA), 1 day before flowering date (-1 DAA), flowering date (0 DAA), 2 days after flowering (2 DAA), 4 days after flowering (4 DAA), 6 days after flowering. (6 DAA), and 12 days after flowering (12 DAA), photographs of ovaries or fruits are shown. FIG. 11 shows the change over time in the diameter of the ovary (fruit) of each line. All transformed lines showed an increase in ovary (fruit) size over time, but the transformed lines using the Solyc02g067760-derived promoter were compared with the transformed lines using the Solyc03g007780-derived promoter and the iaa9-3 mutant line. Then, the rate of increase in the initial stage was slow.

The sugar content, acidity, β-carotene content, and lycopene content of the harvested fruits were measured and compared in order to evaluate the quality (Table 5). The sugar content (here, Total Soluble Solids; TSS) was measured by grinding a fruit sample cryopreserved at -80 ° C in a mortar and using a refracting sugar content meter (PAL-J, Atago).

Acidity (titration acidity; TA) was measured according to the method of DALAL (1965). Specifically, the fruit sample that had been cryopreserved at -80 ° C was mashed in a mortar, 1 g of the mashed sample was dissolved in 10 ml of distilled water, and neutralization titration was performed using a 0.1N sodium hydroxide aqueous solution up to pH 8.1. went. An oxalic acid aqueous solution was used as the standard solution.

β-carotene and lycopene were measured according to the method of Nagata & Yamashita (1992). Specifically, fruit samples that had been cryopreserved at -80 ° C were mashed in a mortar, and β-carotene and lycopene were extracted from 300 mg of the mashed sample using 3 ml of acetone-hexane (4: 6, v / v) extraction solvent. did. After allowing the sample to stand, 1 ml of the supernatant was separated, and the absorbance was measured at four wavelengths of 663 nm, 645 nm, 505 nm, and 453 nm using a spectrophotometer (Beckman Coulter DU 640 Spectrophotometer). The measured values of each wavelength were substituted into the following equations to calculate the amounts of β-carotene and lycopene.

β-carotene (mg / 100ml) = 0.216A ₆₆₃ --1.22A ₆₄₅ --0.304A ₅₀₅ + 0.452A ₄₅₃
Lycopene (mg / 100ml) = -0.0458A ₆₆₃ + 0.204A ₆₄₅ + 0.372A ₅₀₅ --0.0806A ₄₅₃
Note that A ₆₆₃ , A ₆₄₅ , A ₅₀₅ and A ₄₅₃ refer to the absorbance at wavelengths of 663 nm, 645 nm, 505 nm and 453 nm, respectively.

As a result, the iaa9-3 mutant line and all transformed lines showed significantly higher sugar content than the wild type. Specifically, the values were higher in the order of wild type <iaa9-3 mutant <Solyc03g007780-derived promoter-using transformation line <Solyc02g067760-derived promoter-using transformation line. A similar tendency was seen for acidity. On the other hand, regarding the β-carotene and lycopene contents, no significant difference was observed between the wild type, the transformed line, and the iaa9-3 mutant line. From these results, it was judged that in the transformed fruit, both the sugar content and the acidity were increased, and the taste was improved.

From the above examples, the promoters derived from Solyc03g007780 and Solyc02g067760 can induce tissue- and time-specific gene expression in reproductive organs, especially female buds (ovary, etc.) and their neighboring organs, and further, the IAA9 gene can be expressed using this promoter. By specifically suppressing expression in reproductive organs, especially female buds (ovary, etc.) and its neighboring organs at that time, parthenocarpy can be induced with little effect on the development of other nutritional organs, and sugar content. It was shown that high-quality parthenocarpy fruits can be produced, both in terms of acidity and acidity compared to the wild type.

Unlike the conventional ripening stage-specific promoters, the promoter of the present invention has a strong expression-inducing activity in the reproductive organs, especially the female buds and their neighboring organs, from the flower bud maturation stage to the early stage of fruit hypertrophy. It can be suitably used for inducing the expression of a foreign gene that is different from the conventional one and is time-specific. By using the promoter of the present invention, for example, it becomes possible to pinpoint the expression of a foreign gene in the early stage of development in a fruit. In the present invention, by controlling the expression of a parthenocarpy-inducing gene in a tissue- and time-specific manner using the promoter of the present invention, parthenocarpy can be induced while avoiding adverse effects on other organs. Therefore, the present invention can be advantageously utilized for the production of parthenogenetic plants in which unwanted phenotypic changes in nutritional organs are suppressed.

SEQ ID NO: 1: Solyc03g007780-derived promoter SEQ ID NO: 2: Solyc02g067760-derived promoter SEQ ID NO: 3-21: Primer SEQ ID NO: 22: IAA9 targeting RNAi construct SEQ ID NO: 23: IAA9 gene SEQ ID NO: 24: IAA9 protein

Claims (9)

A promoter DNA specific to a plant reproductive organ, consisting of a base sequence having 95% or more identity with respect to the base sequence shown in SEQ ID NO: 1, and a participatory result-inducing gene arranged under the control thereof. DNA constructs, including. The DNA construct according to claim 1, wherein the promoter exhibits promoter activity in the pistil. The DNA construct according to claim 1 or 2, wherein the parthenocarpy-inducible gene encodes an RNAi construct that suppresses the expression of the IAA9 gene. An expression vector comprising the DNA construct according to any one of claims 1 to 3. A parthenocarpy transformed plant into which the DNA construct according to any one of claims 1 to 3 or the expression vector according to claim 4 has been introduced. The transformed plant according to claim 5, wherein the plant is a Solanaceae plant. The transformed plant according to claim 6, wherein the Solanaceae plant is a tomato. The DNA construct according to any one of claims 1 to 3 or the expression vector according to claim 4 is introduced into a plant cell, and the plant body is regenerated and grown from the obtained transformed cell, resulting in parthenocarpy. A method for producing a parthenocarpy transformed plant, which comprises confirming. The DNA construct according to any one of claims 1 to 3 or the expression vector according to claim 4 is introduced into a plant cell, and the plant body is regenerated and flowered from the obtained transformed cell. A method of inducing gene expression specific to plant reproductive organs.

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