JP2011115164A - Receptacle promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a receptacle promoter having transcriptive activity in a receptacle as a constituent of a fruit (pseudocarp) and expressing an aimed gene in the fruit, and to provide a method for creating a recombinant plant that produces a recombinant protein in the receptacle. <P>SOLUTION: A gene encoding a protein highly accumulated in the receptacle constituting a pseudocarp and/or a gene highly expressed in the receptacle is(are) identified; the 5' transcriptional control region that controls the expression of the gene(s) is analyzed; and the base sequence of a promoter region is determined; subsequently, the activity of a reporter gene is measured using transitory expression analysis or a gene-recombinant plant, and the activity of the promoter is examined, thus obtaining the receptacle promoter. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a floret promoter, and the promoter is used for the development of a new variety of plants using a gene recombination technique and the development of a plant for producing a recombinant protein.

  Plant cultivar improvement has already been put into practical use by gene recombination technology in which foreign genes are expressed in existing varieties and traits such as herbicide resistance and pest resistance are added. In addition, new varieties that have been manipulated with foreign genes into which metabolic systems such as pigment synthesis have been introduced and that could not be produced by ordinary cross breeding have been developed. Furthermore, attention has been focused on attempts to express useful proteins and peptides (hereinafter simply referred to as “useful proteins”) in recombinant plants. Useful proteins to be expressed in recombinant plants are not limited to plant proteins, and animal proteins can also be produced. In particular, the application to the production of medical proteins such as vaccines, antibodies and cytokines is progressing.

  In the production of useful proteins by recombinant plants, the production cost is lower than in production using microorganisms and cultured cells, the production scale is easily controlled by adjusting the cultivation environment, and contamination with animal-derived pathogens such as viruses There are many advantages such as no safety and high safety. In addition, if a useful protein is expressed in the edible part of a plant, it can be orally administered without directly purifying the edible part from the plant body. In addition to reducing purification costs, it can be expected to avoid stress due to invasive administration of purified products.

  There are various edible parts depending on the plant species, but typical examples include leaves, fruits, seeds, roots (tuberous roots, bulbs), stems (tubers), and the like. In order to produce useful proteins suitable for oral administration in recombinant plants, it is necessary to express an appropriate amount of useful proteins in the edible part. Furthermore, from the viewpoint of avoiding thermal denaturation of proteins, it is also required to express useful proteins in tissues and organs that can be eaten raw.

  In order to produce a useful protein in a specific tissue / organ, it is necessary to express a gene encoding the useful protein in the target tissue / organ. A promoter plays an important role in controlling the gene expression. Promoters can be broadly classified into constitutive, inducible, tissue / organ-specific, or temporal depending on the mode of expression control. In particular, it is effective to use a constitutive promoter that controls the high expression of a recombinant gene or a promoter that specifically controls the expression of a tissue or organ in a tissue or organ that can be eaten live.

  By the way, a leaf, a fruit, or a seed is mentioned as what can be eaten raw among the edible parts of a plant. Among them, fruits and seeds are excellent in storability, and are suitable for production and accumulation of useful proteins. Furthermore, since many fruits are edible, it can be said to be the most suitable tissue / organ for oral administration of useful proteins. Seeds are formed as a result of sexual reproduction in flowers, and in angiosperms, various organs that wrap seeds mature to form fruits. Fruits can be broadly classified into fruit that has matured pistil ovary and fruit that has matured and enlarged other than ovary. Examples of fruits classified as fruits include tomatoes, grapes, oysters, citrus fruits and legumes. On the other hand, fruits classified as false fruits include main crops such as strawberries, apples, and pears. Although fruits and false fruits are generally treated as fruits, they are different tissues and organs in plant histology.

  Many fruit-specific or seed-specific promoters have been reported so far. However, most of the fruit-specific promoters are classified as fruit-specific promoters (Patent Document 1). Since fruits and false fruits are composed of different tissues in plant histology as described above, even if a true fruit-specific promoter is applied to the false fruits, the accumulation of the recombinant protein in the desired tissue / organ is not effective. I wo n’t. Furthermore, with a seed-specific promoter, the recombinant protein does not accumulate in the edible part in the fruit of a plant species whose seed is not edible, such as pear fruits such as apples and pears. In addition, if the seeds are edible and difficult to digest, such as strawberries with characteristic small-grain seeds such as strawberries on the surface, it is difficult or very difficult to consume the protein in the seeds. ineffective. Therefore, in order to produce a recombinant protein in a fruit classified as a false fruit, it is important to use a promoter having transcription activity in a flower bud constituting the false fruit.

  In strawberry, which can be said to be a representative example of false fruits, analysis of genes that are specifically expressed in fruits or florets and promoters that control the expression of these genes has been conducted. There are reports that comprehensively analyze genes expressed in strawberry buds and fruits with microarrays, and in particular, identify genes that are highly expressed in florets, but only comparative analysis with fruits expressed genes (Non-patent Document 1). Since strawberry end-β-1,4-glucanase is highly expressed in fruits, there is a report on expression analysis of the promoter (FaEG1, 2) of the gene. However, the transcriptional activity was comparable to that of the commonly used cauliflower mosaic virus (hereinafter CaMV) 35S promoter (Non-patent Document 2). It has been reported that a D-galacturonase promoter (GgalUR promoter) derived from strawberry fruit functions in a fruit-specific manner. However, it has been reported that it is photorequiring and that its transcriptional activity is at most similar to that of the control CaMV35S promoter. In addition, it has been verified that the transcription activity of the tomato fruit promoter is extremely low in strawberry fruit (Non-patent Document 3). In addition, although there is a report evaluating the transcriptional activity of the FBP promoter, a petunia-derived flower bud high expression promoter, in the strawberry fruit, the transcriptional activity of the promoter in the transformed strawberry fruit is inferior to the control CaMV35S promoter ( Non-patent document 4). This report does not apply to flower buds that constitute false fruits, such as strawberries, even if they are promoters with high expression of flower buds, even if they can be applied. This suggests that transcriptional activity higher than that of the promoter cannot be expected. Therefore, prior art of promoters that function in floral tissues and organs including flower buds that have not been verified for transcriptional activity in flower buds that are enlarged and constitute false fruits disclose promoters that can be applied to that organ (Patent Documents 2 and 3).

  Based on such a technical background, the present inventors examined strawberry (Fragaria × ananassa) to isolate a promoter having high transcriptional activity in the floret from its endogenous gene. First, proteomic analysis of strawberry florets proteins has led to the separation and identification of multiple types of strawberry florets highly accumulated proteins. If a gene encoding a strawberry flower bud high accumulation protein is identified and the 5 'transcriptional regulation of the gene is analyzed, it is expected to isolate a flower bud high expression promoter. It was found that one of the identified strawberry florets highly accumulating proteins was chalcone synthase (CHS) (Non-patent Document 5). So far, research and development of CHS and CHS promoters are diverse in plant species other than strawberries. In recent years, co-suppression of the CHS gene in epigenetics research of plants is mentioned (Non-patent Document 6). Regarding strawberry CHS, there are reports of isolation of CHS genes from seasonal varieties (Non-patent Document 7) and RNAi reports of strawberry CHS in fruits using a transient expression system (Non-patent Documents). 8). In addition, strawberry CHS is involved in pigment synthesis of strawberry fruits, so it has been reported that strawberry CHS is highly expressed in flower buds, especially at the later stage of fruit ripening. Has been suggested to be limited (Non-Patent Document 1). That is, in these research results, promoter analysis of strawberry CHS, evaluation of transcriptional activity in the flower bud, etc. have not been studied. Therefore, the present inventor conducted an evaluation study as a flower bud promoter of a strawberry CHS promoter.

Japanese Patent No. 35399961 Japanese Patent No. 4113952 Japanese Patent No. 4134281

Aharoni et al. (2002) Journal of Experimental Botany, Vol. 53, no. 377, pp. 2073-2087 “Gene expression analysis of strawberry achene and receptor maturation using DNA microarrays” Spoleore et al. (2003) Journal of Experimental Botany, Vol. 54, no. 381, pp. 271-277 “Isolation and promoter analysis of two genes encoding differential endo-b-1, 4-glucoses in the non-clinical strawberry” Agius et al. (2005) Journal of Experimental Botany, Vol. 56, no. 409, pp. 37-46 “Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression” Schaart et al. (2002) Plant Cell Rep, 21, pp 313-319 “Tissue-specific expression of the β-glyconidase reporter in transgenic strawberry (Fragalia x ananasas)” Furuta et al. (2008) "Isolation of Strawberry Flower Highly Expressed Gene and Analysis of Transcriptional Regulatory Region", BMB2008 (31st Annual Meeting of the Molecular Biology Society of Japan and 81st Annual Meeting of the Biochemical Society of Japan) 2P- 1359 Supervised by Isao Shimamoto et al. (2008) Cell Engineering Separate Volume Plant Cell Engineering Series 24 “Plant Epigenetics” Sakurai et al. (1997) Plant Biotechnology, 14 (3), 183-185, “Isolation and charac- terization of a cDNA for charcone synthase cultivated cells of the stur- bers of fragrance. Hoffman et al. (2006) The Plant Journal 48, 818-826 "RNAi-induced silencing of genera ration of affiliation: agroinfiltration:

  The present invention provides a floret promoter that has transcriptional activity in a floret constituting a false fruit and expresses a target gene in the false fruit, and a method for producing a recombinant plant that produces a recombinant protein in the flower bud. It is.

The present inventors have advanced research to solve the above problems, and found that the strawberry CHS promoter has transcription activity several times higher than the CaMV35S promoter that is already widely used in florets, and has completed the present invention.
That is, the present invention relates to a flower promoter (a) comprising the DNA described in (a), (b) or (c) below,
(B) DNA having a base sequence having insertion, deletion or substitution of one or several nucleic acids in the base sequence described in SEQ ID NO: 1 and having a flower promoter function
(C) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 1 and has a flower promoter function
It is about.

  Furthermore, the present invention provides a continuous partial sequence selected from the base sequence represented by SEQ ID NO: 2, and the nucleotide sequence (SEQ ID NO: 1) at positions 4509 to 4648 of SEQ ID NO: 2 is used as the core promoter region. It is related with the floret promoter which consists of DNA characterized by containing as follows.

  The present invention also relates to a floret promoter composed of DNA having the above-mentioned base sequence or having several nucleic acid insertions, deletions or substitutions and having a floret promoter function. In addition, the present invention relates to a floret promoter composed of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the above base sequence and has a floret promoter function.

The present invention also relates to a flower bud promoter comprising the DNA of the following (a), (b) or (c) (a) a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 25 to 27
(B) DNA comprising a base sequence having insertion, deletion or substitution of one or several nucleic acids in the base sequence of SEQ ID NO: 25 to 27 and having a flower promoter function
(C) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of SEQ ID NO: 25 to 27 and has a flower promoter function
About.

  Furthermore, the present invention relates to a plant expression cassette comprising the promoter and an arbitrary gene under the transcriptional control of the promoter. The present invention also relates to a plant expression vector comprising the plant expression cassette. Furthermore, this invention relates to the transformed plant cell holding the said plant expression cassette. The present invention also relates to a transformed plant cell into which the plant expression vector has been introduced. Furthermore, this invention relates to the transformed plant with which the said cell was hold | maintained. The present invention also relates to a transformed plant that is a descendant or clone of the transformed plant.

Furthermore, the present invention provides a method for producing a transformed plant by introducing an arbitrary gene into a plant, comprising the following steps (a), (b) and (c): (a) the plant expression cassette described above or the above A step of introducing a plant expression vector into a plant cell; (b) a step of regenerating a plant from the plant cell; and (c) a step of selecting the plant in which the introduced gene is expressed in the floret.

  Furthermore, the present invention relates to the transformed plant cell, wherein the cell is a cell from strawberry. Moreover, this invention relates to the said transformed plant whose plant is a strawberry. The present invention also relates to the above method wherein the plant is a strawberry.

  ADVANTAGE OF THE INVENTION According to this invention, the promoter which has transcription activity in a flower bud can be provided, and it becomes possible to express a recombinant gene in the flower bud which comprises false fruits, such as a strawberry. Furthermore, a useful protein can be produced by a plant by using the flower bud promoter and the gene of the useful protein of the present invention.

  In useful protein production by recombinant plants, production costs are lower than production using microorganisms and cultured cells, and the production scale can be easily controlled by adjusting the cultivation environment. Moreover, since contamination with pathogens derived from animals such as viruses can be avoided, safety can be ensured. Since useful proteins can be produced in the edible part of plants such as spices, it is possible to ingest the effective protein as it is without requiring purification from the plant body.

  Conventionally, useful proteins that have been invasively administered by injection or the like can be noninvasively administered and can avoid invasive stress. By selecting plant species that can be eaten raw such as strawberries that do not require heat treatment, it is possible to avoid inactivation of useful proteins due to heat denaturation.

  In addition, according to the present invention, since any gene expression can be performed in the floret, it can be utilized as an effective tool for analyzing metabolic engineering in the floret or false fruit.

A two-dimensional electrophoresis image of strawberry flower protein and a protein spot of CHS are shown. The floret promoter delation fragment is shown. The outline of the structure of the vector for plant expression is shown. The evaluation result of the promoter activity in the strawberry fruit by a transient expression analysis is shown. The GUS dyeing | staining image in the fruit of a flower bud promoter transformed strawberry is shown. The GUS dyeing | staining image in the fruit of a flower bud promoter transformed strawberry is shown. The GUS activity for every growth stage of the fruit of the strawberry promoter-transformed strawberry FACP21 line is shown. The quantitative analysis result of the GUS activity in the leaf and fruit of a strawberry promoter transformed strawberry is shown. The quantitative analysis result of the GUS activity in the leaf and fruit of a strawberry promoter transformed strawberry is shown. The quantitative analysis result of GUS activity in the fruit of a flower bud promoter transformed strawberry is shown.

  In the present specification, “plant” or “plant species” refers to all organisms belonging to the plant kingdom, and representative ones refer to flowering plants. Includes both monocotyledonous and dicotyledonous plants. A plant having an edible part is preferable. More preferably, the edible part is a plant that can be eaten raw. Specific examples include rose family plants. Other examples include, but are not limited to, strawberries, apples, pears, citrus, peaches, bananas, pineapples and figs.

  In the present specification, the “edible part” means a part that can be eaten mainly by animals such as humans when ingesting the plant, and the edible part differs depending on the plant species, but is representative. Examples thereof include leaves, flowers (including flower buds), fruits, seeds, roots (including tuberous roots and bulbs), stems (including tubers), and the like. The term “can be eaten raw” means that when an edible part of a plant is ingested, it can be ingested as it is without being subjected to heat treatment or the like.

  In this specification, “having a promoter function” means having a transcription activity as a promoter. The transcriptional activity of a promoter can be evaluated by fusing a promoter and a reporter gene, introducing the gene into a plant, producing a transformant, and testing the activity of the reporter. As the reporter gene, β-gluconidase (GUS) gene, luciferase (LUC) gene, Green fluorescein protein (GFP) gene and the like can be used, and reporter activity evaluation methods according to each gene are well known in the art. It is.

  In the present specification, the “floral promoter” refers to a promoter composed of DNA having transcriptional activity or promoter function in a floral bud which is one of the flower formation sites in a plant. In addition, a promoter that controls expression of a gene encoding a protein highly accumulated in the floret or a promoter that controls expression of a highly expressed gene of the floret is desirable. “Protein highly accumulated in florets” can be selected from proteins accumulated in high concentrations in the composition of florets by proteomic analysis of proteins expressed in florets. Also called “accumulated protein”. Further, a gene encoding a flower bud highly accumulating protein is referred to as a “flower bud highly expressing gene”, and the flower bud highly accumulated protein cDNA is obtained from the cDNA expressed in the flower bud, and further analysis of its 5 ′ non-transcribed region, etc. This refers to those that can be selected by selecting flower buds with high expression frequency.

  The floret promoter includes a constitutive promoter having activity in addition to the floret-specific promoter. For a constitutive promoter, it is desirable that, for example, the transcriptional activity of florets is higher than that of leaf tissue, or the transcriptional activity of florets is higher than that of, for example, the existing CaMV35S promoter. In addition, the florets are not particularly limited, but the flowers that are observed in plant species such as strawberries and apples have transcriptional activity in the florets that constitute false fruits through development such as enlargement after pollination. desirable.

  One embodiment of the floret promoter of the present invention is a floret promoter comprising the DNA represented by the nucleotide sequence of SEQ ID NO: 1. The base sequence represented by SEQ ID NO: 1 is derived from the 5 'upstream region of the strawberry CHS gene and is a region that controls the strawberry CHS gene. In another embodiment, as long as it has a floret promoter function, it may be a floret promoter composed of DNA having insertion, deletion or substitution of one or several nucleic acids in the nucleotide sequence set forth in SEQ ID NO: 1. Moreover, as long as it has the floret promoter function, it may be a floret promoter composed of DNA that hybridizes under stringent conditions with DNA composed of a base sequence complementary to the base sequence shown in SEQ ID NO: 1.

In another embodiment, the flower promoter of the present invention consists of the following DNA. That is, it is a continuous partial sequence selected from the base sequence shown in SEQ ID NO: 2 and contains the base sequence from position 4509 to 4648 of SEQ ID NO: 2 (SEQ ID NO: 1) as a core promoter region. Flower bud promoter comprising DNA

  The base sequence represented by SEQ ID NO: 2 is derived from the 5 'upstream region of the strawberry CHS gene and is a region that controls the strawberry CHS gene. The core promoter region consisting of the sequence from the 4509th position to the 4648th position matches the base sequence shown in SEQ ID NO: 1.

  Moreover, it is a continuous partial sequence selected from the base sequence represented by SEQ ID NO: 2 and contains the base sequence from position 4509 to 4648 of SEQ ID NO: 2 (SEQ ID NO: 1) as a core promoter region. The DNA characterized by the above may have insertion, deletion or substitution of one or several nucleic acids as long as it has a flower promoter function. Similarly, as long as it has the flower promoter function, it is a continuous partial sequence selected from the base sequence represented by SEQ ID NO: 2, and the base sequence from position 4509 to 4648 of SEQ ID NO: 2 (SEQ ID NO: 2 1) as a core promoter region, it may be a region consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence.

  The “continuous partial sequence selected from the base sequence represented by SEQ ID NO: 2” herein is a continuous sequence selected from SEQ ID NO: 2, and the sequence length can be freely selected by those skilled in the art. This is a base sequence that can be produced, and the maximum sequence length is 4767 bases. When the core promoter region is contained and the chain length is selected with 140 bases, it is synonymous with the case where only the core promoter region is selected.

  The “core promoter region” in the promoter of the present invention is a minimum region necessary for transcriptional activity, and is a region containing TATA-box as an essential cis-element in the region. The core promoter region of the floret promoter of the present invention is specifically a region consisting of the nucleotide sequence from position 4509 to position 4648 of SEQ ID NO: 1 or SEQ ID NO: 2. In this region, these nucleotide sequences are also included in the core promoter region as long as they have a promoter function even if one or several nucleic acids are inserted, deleted or substituted.

  In addition, as long as a region that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence from position 4509 to position 4648 of SEQ ID NO: 1 or SEQ ID NO: 2 is also a core, Contained in the promoter region.

In another embodiment, the flower promoter of the present invention consists of the following DNA.
That is,
A continuous partial sequence selected from the nucleotide sequence represented by SEQ ID NO: 2 and containing the nucleotide sequence (SEQ ID NO: 1) at positions 4509 to 4648 of SEQ ID NO: 2 as a core promoter region A DNA comprising a nucleotide sequence having hot, insertion, deletion or substitution of one or several nucleic acids, and having a flower promoter function.

In another embodiment, the flower promoter of the present invention consists of the following DNA.
That is,
A continuous partial sequence selected from the nucleotide sequence represented by SEQ ID NO: 2 and containing the nucleotide sequence (SEQ ID NO: 1) at positions 4509 to 4648 of SEQ ID NO: 2 as a core promoter region A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a characteristic base sequence and has a flower promoter function.

In still another embodiment, the flower promoter of the present invention consists of the following DNA.
That is,
The floret promoter comprising the DNA of the following (a), (b) or (c) (a) the DNA comprising the base sequence of any one of SEQ ID NOs: 25 to 27
(B) DNA comprising a base sequence having insertion, deletion or substitution of one or several nucleic acids in the base sequence of SEQ ID NO: 25 to 27 and having a flower promoter function
(C) DNA that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of any one of SEQ ID NOs: 25 to 27 and has a flower promoter function.

  The DNA comprising the above base sequence is synthesized, for example, based on the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and this is used as a primer for Polymerase Chain Reaction method. For example, PCR is performed using genomic DNA as a template. You can earn at. Furthermore, it is possible to introduce mutations such as insertion, deletion or substitution of nucleic acids by a point mutation method or the like. It can also be obtained from a genomic DNA library of a desired plant by chemical synthesis based on SEQ ID NO: 1 and SEQ ID NO: 2, or by hybridizing a nucleic acid fragment having the sequence as a probe.

  The stringent condition in the present invention refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Such conditions can be determined by the chain length and base composition of the base sequence and will be understood by those skilled in the art. For example, a hybridization temperature condition is 50 ° C., and a nucleic acid having a complementary sequence and a nucleic acid to be tested are subjected to a washing treatment with a buffer solution (0.1 × SSC, 0.1% SDS, etc.) at 50 ° C. And the reaction conditions.

  The “expression cassette” of the present invention is composed of at least a promoter that controls transcription initiation, a gene under the transcription control, and a terminator that controls transcription termination. In addition to a promoter, gene, and terminator, a region such as an enhancer that enhances transcription efficiency or a translation enhancer that promotes translation of a transcribed gene may be included.

  For the construction of the “plant expression vector” of the present invention, vectors used as plant expression vectors such as pBI and pUC vectors can be suitably used.

  The plant expression cassette and the plant expression vector may contain a gene encoding a selection marker and a promoter or terminator for controlling the expression in order to efficiently select transformants. As a selection marker, in addition to the reporter gene described above, a drug resistance gene is preferably used. Specific examples of drug resistance genes include neomycin phosphotransferase II (npt II) gene for imparting kanamycin resistance, hygromycin phosphotransferase gene (hpt) for imparting hygromycin resistance, and the like.

  In the present invention, in a plant expression cassette or a plant expression vector, “under transcriptional control” means a state in which an arbitrary gene is linked to the downstream side of a promoter so that the gene is expressed under the control of the promoter. A method for linking an arbitrary gene to the downstream side of a promoter is a well-known technique in the art.

  In the present invention, any gene under the transcriptional control of the floret promoter is not particularly limited as long as it is a known gene. For example, in addition to a gene encoding a protein, its antisense strand is also included. Genes that encode useful proteins encode proteins that serve as pharmaceuticals, such as vaccine genes, genes encoding antibodies, genes encoding antigenic peptides, genes encoding various cytokines, genes encoding various adipocytokines, etc. The thing etc. can be mentioned.

  In the present invention, a “transformed cell” refers to a cell among transformants produced by transformation, and a “transformed plant” is a plant that holds a transformed cell produced by transformation. . “Regeneration” means that the whole individual is restored from a part of the individual. In a plant body, a part of an individual includes cells, and in addition, tissue pieces such as leaves, petiole, roots, and callus are also included. The method of regeneration is a well-known technique in the art, although the optimum conditions differ depending on the plant species.

A method for producing a transformed plant and a method for introducing a plant expression cassette or a plant expression vector into plant cells are not particularly limited. An electroporation method, a particle gun method, an Agrobacterium method, and the like can be mentioned. Various transformation methods can be selected according to plant species and cells, and these methods are well known in the art.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these embodiments.

1. Isolation and identification of highly accumulated protein of florets A proteome analysis was performed to isolate highly accumulated proteins of strawberry florets. Strawberry cultivar 'H-ES-138' was cultivated, and strawberry fruits were harvested for each growth stage divided from flowering to ripeness into 6 stages. The strawberries and fruits are removed from the harvested strawberry fruits, the protein is extracted from the flower buds, and this is separated by two-dimensional electrophoresis using isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. did. The gel after electrophoresis was stained by a silver staining method to obtain a protein spot pattern. The protein spot analysis was performed using analysis software ImageMaster (GE Healthcare). Proteins accumulated at high concentrations in the florets were detected by matching analysis of protein spots at each growth stage of the florets and comparative analysis of accumulated amounts of leaf and cocoon proteins collected at the same time (FIG. 1). When a flower bud highly accumulated protein spot was separated by two-dimensional electrophoresis and the N-terminal amino acid sequence was analyzed, chalcone synthase (CHS) was identified (SEQ ID NO: 3).

2. CDNA Cloning of Highly Accumulated Proteins of Flowers The degenerate primers were synthesized based on the determined amino acid sequence for the identified CHS (SEQ ID NO: 4, SEQ ID NO: 5). Total RNA was extracted from strawberry florets and leaves, and using GeneRacer Kit (Invitrogen), 5 'adapter sequence (SEQ ID NO: 6) and 3' adapter sequence (SEQ ID NO: 7) added cDNA (Race-ready) cDNA) synthesis was performed. Using this cDNA as a template nucleic acid, nested PCR was performed using a degenerate primer, 3 ′ primer (SEQ ID NO: 8) (Invitrogen) and 3 ′ nested primer (SEQ ID NO: 9) (Invitrogen), and 3 ′ RACE. The cDNA was amplified by the method. As the PCR enzyme, AccuPrime Taq (Invitrogen) was used. The PCR product was introduced into the plasmid vector pCR2.1 using TA cloning kit (Invitrogen), and transformation of E. coli (INVαF ′) was performed according to the method described in TA cloning kit (Invitrogen), and the obtained transformant. Was extracted with QIAquick miniprep kit (QIAGEN). The obtained plasmid clone was subjected to a sequencing reaction, and the base sequences of CHS cDNAs for florets and leaves were determined. Sequencing reaction was performed using fluorescent dye (IRD700 and IRD800) -labeled M13 primer and SequiTherm Excel II DNA sequencing Kit (EPICENTRE), and the reaction product was analyzed with DNA sequencer LONGREADIR 4200 (LI-COR).

3. Identification of flower bud highly expressed gene In order to determine the CHS transcription start site and to identify the flower bud highly expressed CHS gene, we further attempted to determine the 5 ′ untranslated region (5′UTR) of CHS mRNA by 5 ′ RACE. Based on the base sequence determined by 3 ′ RACE, primers for 5 ′ RACE were designed (SEQ ID NOs: 10 and 11). RNA was extracted from florets and leaves, and Race-ready cDNA was synthesized using this as a template. Using this cDNA as a template nucleic acid, 5 ′ primer (SEQ ID NO: 12) (Invitrogen), 5 ′ nested-primer (SEQ ID NO: 13) (Invitrogen) and the strawberry CHS-derived 5 ′ RACE primer Nested PCR was performed, and cDNA was amplified by the 5 ′ RACE method. As the PCR enzyme, AccuPrime Taq (Invitrogen) was used. TA cloning & sequencing of the PCR product obtained by 5′RACE was performed. The base sequence of a total of 80 cDNA clones derived from flower buds and leaves was determined and subjected to comparative analysis by multiple alignment to determine the CHS 5′UTR sequence with the highest expression frequency in flower buds (SEQ ID NO: 14). Based on the determined CHS 5′UTR, a cDNA probe was prepared and used for the next genomic clone selection.

4). Preparation of a strawberry genomic library and selection of clones of high expression genes for florets Genomic DNA was extracted from the leaves of a strawberry (variety 'H-ES-138'), and about 1 mg of genomic DNA was used for preparation of a λ phage library. Library preparation was performed using Lambda DASH II / BamHI Vector kit (STRATGENE). 1 μl of the prepared library was used to inoculate MRA (P2) as a host E. coli, and the titer was measured. The result was 8.8 × 10 ² pfu / μl, and the total titer of the library was 1.2 × 10 ⁶ . In addition, LA-PCR (TAKARA) was performed from 36 independent plaques, and the PCR product was confirmed by agarose electrophoresis. Amplification was observed in 31 clones, and the average insert size was about 13 Kb. A genomic library (λ phage) is diluted to prepare 10 phages of approximately 6 × 104 pfu, and this is cultured and plaque-formed with 10 14 × 10 cm square petri dishes together with host E. coli, and a phage plate for screening Was made. Each plate was subjected to a plastic lift to 2 Hybond-N + (GE Healthcare), followed by alkali denaturation treatment and drying at 80 ° C. for 2 hours. A cDNA probe was prepared using Gene Images AlkPhos Direct Labeling and Detection System (GE Healthcare), and hybridization was performed. The signal was detected by chemiluminescence using VersaDoc5000 (BIO-RAD). Plaques in which signal was detected were identified, single plaques were isolated, and floret highly expressing CHS genomic clones were isolated.

5. Analysis of transcriptional regulatory region of flower bud accumulating protein and selection of promoter region An isolated CHS genomic clone (λ phage) was propagated by the plate lysate method, and DNA was extracted using QIAGEN Lambda Kit (QIAGEN). PCR was performed with a primer designed on the 5 ′ side of the CHS gene highly expressing flower buds and T7 (SEQ ID NO: 15) or T3 primer (SEQ ID NO: 16), and the 5 ′ upstream region was subcloned. PCR PrimeTaq High-fidelity (Invitrogen) was used as the PCR enzyme, and the amplified fragment was cloned using TA cloning kit (Invitrogen). The nucleotide sequence of the 5 ′ upstream region from the obtained subclone was analyzed, and the sequence of the 5 ′ transcription regulatory region 4767nt containing the 5 ′ UTR of the highly expressed CHS gene was determined (SEQ ID NO: 2).

5. Cloning of fragrance fragment of highly expressed CHS promoter Primers were designed based on the base sequence of the 5 'transcriptional regulatory region of the highly expressed CHS gene (Table 1). A restriction enzyme recognition site was inserted into the 5 'end of each primer. Torus high expression CHS gene genomic clones (lambda-DNA) as a template, dilation fragment of the promoter-like sequence by PCR (F ragarina × a nanassa C HS P romoter: FACP12,13,21 and 61) was amplified (Figure 2). These fragments were cloned into the plasmid vector pCR2.1 using TA cloning kit (Invitrogen) to obtain respective plasmid clones (referred to as pCR2.1-FACP12, 13, 21, and 61, respectively).

6). Preparation of β-glucuronidase gene (GUS) expression vector pCR2.1-FACP12, 13, 21 and 61 were treated with the enzyme corresponding to the restriction enzyme recognition site added to each primer site, and the dilation fragment was recovered. This was mixed with pBI101 (Jefferson et al. (1987) The EMBO Journal vol. 6 no13.pp3901-3907) treated with the same restriction enzyme as the end of each dilation fragment to perform GUS expression for plant expression. Plasmid vectors pBI-FACP12, 13, 21, and 61 were prepared (FIG. 3). The dilation fragment of each CHS promoter inserted into pBI-FACP is located 5 ′ upstream of the GUS gene, pBI-FACP12 is SEQ ID NO: 1, pBI-FACP13 is SEQ ID NO: 25, and pBI-FACP21 is a sequence. No. 26, and pBI-FACP61 has the base sequence shown in SEQ ID NO: 27 as a promoter sequence. In addition, as a transient expression vector, an intron sequence derived from strawberry quinone oxidoreductase was inserted into the SnaB I site in each of the GUS genes of pBI-FACP12, 13, 21, and 61. The intron sequence was amplified by the PCR method of FW primer (SEQ ID NO: 23) and RV primer (SEQ ID NO: 24) primer synthesized based on the strawberry quinone oxidoreductase sequence (Genbank AY1588836) using strawberry genomic DNA as a template. This is for suppressing the expression of the GUS gene in Agrobacterium during the transient expression analysis by the Agoroinfiltration method. That is, by inserting an intron sequence derived from the strawberry gene into the GUS gene, it inhibits the formation of mature mRNA of the GUS gene in the body of Agrobacterium which does not have a plant splicing mechanism, and transcription activity in plant tissues (Vancanyet et al. (1990) Mol Gen Genet 220: 245-250, Hoffman et al. (2006) The Plant Journal 48, 818-826). The pBI-FACP vectors into which intron sequences were inserted were designated as pBI-FACP12int, pBI-FACP13int, pBI-FACP21int and pBI-FACP61int, respectively (FIG. 3).

7). Transient expression analysis by Agroinfiltration method Four types of pBI-FACPint vectors for transient expression prepared in the above 6 were introduced into Agrobacterium (strain LBA4404) by the direct introduction method. Using the acquired kanamycin resistant strain, a transient expression analysis of the fruit by the Agroinfiltration method was performed. The Agroinfiltration method was carried out according to the method of Spoleore et al. (Spoleore et al. (2001) Journal of Experimental Botany, vol. 52, No. 357 pp. 845-850). Strains of Agrobacterium carrying the pBI-FACPint vector were inoculated on strawberries and allowed to stand under light conditions at 22 ° C. for 16 hours. A section of a cross section was cut out from the strawberry fruit after standing, and this was cut into a GUS staining solution ((1 mM X-Gluc, 100 mM phosphate buffer (pH 7.0), 0.1% Triton X-100, 10 mM K ₃ ( CN) ₆ , 10 mM K ₄ (CN) ₆ , 10 mM EDTA, 20% methanol) and allowed to stand in the dark for 12 hours at 37 ° C. After standing, the staining solution was replaced with 70% ethanol, The color reaction was stopped, and GUS activity was confirmed by the degree of staining of the fruit slices, and color development showing GUS activity was observed from any of FACP12, 13, 21, and 61. Especially in FACP12, CaMV35S used as a control was tested. PBI121 with promoter: GUS (Jefferson et al. (1987) The EMBO Journal vol. 6 no 13. Stronger color was observed than in the Agrobacterium-inoculated section into which pp3901-3907) was introduced (the strawberry gene-derived intron sequence was inserted into the GUS gene for transient expression: pBI121int) (FIG. 4). From the results, it was confirmed that the 140-base sequence contained in FACP12 has flower bud promoter activity.

8). Production of Transformed Strawberry FACP Line Four pBI-FACP vectors produced in the above 6 were introduced into Agrobacterium (strain LBA4404) by the direct introduction method. The acquired kanamycin resistant strain was used to transform a strawberry (variety 'HSS-138'). Callus induction and redifferentiation were performed using a kanamycin-added medium to obtain a transformed strawberry resistant to kanamycin. DNA was extracted from the leaves of the transformed strawberry, and introduction of the GUS gene was confirmed by PCR. As a control, a GUS-expressing transformed strawberry that was transcriptionally controlled by the CaMV35S promoter, into which pBI121: GUS ((Jefferson et al. (1987) The EMBO Journal vol. 6 no13.pp3901-3907) was introduced was also prepared.

9. Confirmation of Promoter Activity by GUS Staining of Transformed Strawberry Fruit The cultured seedlings of each FACP line obtained in 7 above were acclimated in a specific net room and transplanted to a culture soil for fruit cultivation. The fruits of FACP21 and FACP61 were collected with red ripe fruits as the target for harvesting. A section was cut out from the harvested fruit, immersed in a GUS stain, and allowed to stand in the dark at 37 ° C. for 12 hours. After standing, the staining solution was replaced with 70% ethanol to stop the color reaction. GUS activity was confirmed by the degree of staining of the fruit slices. Strawberry fruits and non-recombinant strawberry fruits collected from a transformant (35S: GUS strain) introduced with cauliflower mosaic virus 35S promoter and GUS gene were used as comparative controls. Color development was observed in the fruits of FACP21 and FACP61. In particular, strong color development was observed in the FACP21 line. Color development was observed in the cortex, vascular bundle, pith, central column and all areas of the floret tissue, but there was a tendency to develop strong color especially in the vascular bundle. In addition, coloring was recognized also in a part of strawberry fruit skin. The FACP61 line showed the same color development as the 35S: GUS line (FIG. 5).

  Subsequently, the fruits of FACP12 and FACP13 were collected and GUS staining of the fruits was performed in the same manner as described above. In both fruits, the color development was inferior to that of the FACP21 strain, but a color showing GUS activity was observed (FIG. 6).

  For the FACP21 line that showed the strongest color development, GUS staining was performed for red fruits and green fruits at the early stage of fruit ripening and white fruits at the middle stage of ripening. As a result, GUS staining was observed for both green and white fruits. This indicates that the highly flowering CHS promoter has transcriptional activity in florets throughout the fruit ripening process (FIG. 7).

10. Measurement of quantitative GUS activity and promoter evaluation of transformed strawberry fruit-1
In order to quantitatively evaluate GUS activity in flower buds, measurement by a fluorescence method using 4-MU production reaction as an index according to the method of Jefferson et al. (Jefferson et al. (1987) The EMBO Journal vol. 6no13.pp3901-3907) went. Strawberries are removed from the strawberry fruit collected from the FACP21 strain (flower buds containing berries), and this is 5 times (w / v) GUS extraction buffer (50 mM sodium phosphate buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% sarkosyl, 10 mM β-mercaptoethanol, Complete protease inhibitor cooktail (Roche)) using a pestle and mortar, centrifuging the milled liquid, The supernatant was recovered and used as a protein extract. 10 μl of the supernatant was collected, added to and mixed with 500 ul of a fluorescent substrate solution (1 mM MUG), and reacted at 37 ° C. for 15 minutes. 100 ul of the reaction solution was collected and added to 900 ul of 0.2 M sodium carbonate to stop the reaction. The fluorescence of 4-MU contained in the reaction solution after stopping was measured (excitation at 365 nm, emission at 455 nm), and GUS activity was calculated. The protein concentration of the protein extract was quantified with a protein assay kit (BIO-RAD). The measurement results of GUS activity in the fruits of the FACP21 line and the 35S: GUS line are shown in FIG. The highest GUS activity was observed in the leaves of the 35S: GUS line, but the GUS activity of the fruits was significantly reduced compared to the leaves. In the fruit of FACP21 line, GUS activity 5 times less than that of the fruit of 35S: GUS line was observed. In the 35S: GUS line, the GUS activity of the leaf was higher than that of the fruit, but in the FACP21 line, the GUS activity was reversed and the fruit was higher than the leaf.

  The same experiment was performed using a sample collected separately. The measurement results of GUS activity in the fruits and leaves of the FACP21 line and the 35S: GUS line are shown in FIG. Similar results to those described above were obtained. In the FACP21 line, the GUS activity of the fruit was higher than that of the leaf, and an activity almost twice as high as that of the fruit of the 35S: GUS line was observed. From the above, it was found that the promoter derived from CHS highly expressing CHS has higher transcriptional activity than the 35S promoter that is already widely used in florets.

11. Measurement of quantitative GUS activity and promoter evaluation of transformed strawberry fruit-2
The maturation stage of strawberry fruit was divided into three according to the fruit color, and the transition of the promoter activity was evaluated by measuring the GUS activity at each stage. The early stage of fruit ripening was green fruit “Green”, the middle ripening stage was white fruit “White”, and the late ripening stage was red ripened fruit “Red”. The method for measuring GUS activity is the same as the previous method. 35S: Comparison was made between the GUS line, the FACP21 line, and the FACP61 line. The results are shown in FIG. 35S: GUS activity and FACP21 strain showed high GUS activity, and the activity at the early stage of maturation was high. Since the FACP21 line generally showed higher activity than the 35S: GUS line, it was found that the high school-expressed CHS-derived promoter had higher transcriptional activity than the existing 35S promoter in florets.

  ADVANTAGE OF THE INVENTION According to this invention, the promoter which has transcription activity in a flower bud can be provided, and it becomes possible to express a recombinant gene in the flower bud which comprises false fruits, such as a strawberry. Furthermore, a useful protein can be produced by a plant by using the flower bud promoter and the gene of the useful protein of the present invention. In useful protein production by recombinant plants, production costs are lower than production using microorganisms and cultured cells, and the production scale can be easily controlled by adjusting the cultivation environment. Further, since contamination with animal-derived pathogens such as viruses can be avoided, safety can be ensured. Since useful protein can be produced in the fruit, it is possible to ingest the effective protein as it is, without requiring purification from the plant body. Conventionally, useful proteins that have been invasively administered by injection or the like can be noninvasively administered and can avoid invasive stress. By selecting plant species that can be eaten raw such as strawberries that do not require heat treatment, it is possible to avoid inactivation of useful proteins due to heat denaturation.

Claims (15)

The DNA comprising the DNA sequence described in (a), (b) or (c) below, and the DNA sequence comprising the base sequence described in SEQ ID NO: 1
(B) DNA having a base sequence having insertion, deletion or substitution of one or several nucleic acids in the base sequence described in SEQ ID NO: 1 and having a flower promoter function
(C) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence described in SEQ ID NO: 1 and has a flower promoter function A continuous partial sequence selected from the nucleotide sequence represented by SEQ ID NO: 2 and containing the nucleotide sequence (SEQ ID NO: 1) at positions 4509 to 4648 of SEQ ID NO: 2 as a core promoter region Flower bud promoter consisting of characteristic DNA The floret promoter comprising DNA having insertion, deletion or substitution of one or several nucleic acids and having a floret promoter function in the base sequence according to claim 2. A flower bud promoter comprising a DNA hybridized under stringent conditions with a DNA comprising a base sequence complementary to the base sequence of claim 2 and having a flower bud promoter function. The floret promoter comprising the DNA of the following (a), (b) or (c) (a) the DNA comprising the base sequence of any one of SEQ ID NOs: 25 to 27
(B) DNA comprising a base sequence having insertion, deletion or substitution of one or several nucleic acids in the base sequence of SEQ ID NO: 25 to 27 and having a flower promoter function
(C) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence of SEQ ID NO: 25 to 27 and has a flower promoter function A plant expression cassette comprising the floret promoter according to claim 1, 2, 3, 4, or 5, and an arbitrary gene under transcriptional control of the promoter. A plant expression vector comprising the plant expression cassette according to claim 6. A transformed plant cell holding the plant expression cassette according to claim 6. A transformed plant cell into which the plant expression vector according to claim 7 has been introduced. A transformed plant in which the cell according to claim 8 or 9 is retained. A transformed plant which is a descendant or clone of the transformed plant of claim 10. A method for producing a transformed plant by introducing an arbitrary gene into a plant, comprising the following steps (a), (b) and (c):
(A) a step of introducing the plant expression cassette according to claim 6 or a plant expression vector according to claim 7 into a plant cell (b) a step of regenerating a plant from the plant cell (c) any gene introduced Selecting the plant that is expressed in the floret The transformed plant cell according to claim 8 or 9, wherein the cell is a cell from strawberry. The transformed plant according to claim 10 or 11, wherein the plant is a strawberry. The method according to claim 12, wherein the plant is a strawberry.