JP4794093B2 - Gentian antibacterial protein and its gene - Google Patents

Description

【００９３】
【発明の効果】
本発明の抗菌性タンパク質及びその遺伝子を用いれば、植物の病害抵抗性を向上させることができる。また、本発明の抗菌性タンパク質は遺伝子組換え技術により大量生産可能であり、抗菌剤として利用しうる。
【００９４】
【配列表】

【００９５】
【配列表フリーテキスト】
配列番号3−人工配列の説明：合成ＤＮＡ
配列番号4−人工配列の説明：合成ＤＮＡ
配列番号5−人工配列の説明：合成ＤＮＡ
配列番号6−人工配列の説明：合成ＤＮＡ
配列番号7−人工配列の説明：合成ＤＮＡ
配列番号8−人工配列の説明：合成ＤＮＡ
配列番号9−人工配列の説明：合成ＤＮＡ
【図面の簡単な説明】
【図１】図１は、リンドウ抗菌性タンパク質のSDS-ポリアクリルアミドゲル電気泳動の結果を示す写真である。
【図２】図２は、リンドウ抗菌性タンパク質の抗糸状菌活性を示すグラフである。
【図３】図３は、リンドウペルオキシレドキシン遺伝子のサザンブロット解析結果を示す写真である。
【図４】図４は、大腸菌で発現させたリンドウペルオキシレドキシンの抗菌活性を示すグラフである。
【図５】図５は、植物形質転換体作成用ベクターの構築を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel antibacterial protein derived from gentian, its gene, and methods for using them.
[0002]
[Prior art]
In recent years, attempts have been made to introduce useful genes into plants using genetic engineering techniques to improve the productivity of useful substances and create disease-resistant plants that are resistant to specific diseases. For example, phytopathogenic fungi such as Botrytis cinerea, Phytophthora infestans, powdery mildew (Erysiphe cichoracearum), Ralstonia solanacearum, Xanthomonas oryzae, etc. In order to strengthen the defense mechanism against phytopathogenic bacteria, there have been reports of the introduction of antibacterial protein genes into plant cells and increased resistance to diseases (Nishizawa et al .: Chemistry and Biology 37: 295-305 (1999) etc.).
[0003]
Plants have acquired a defense mechanism against attacks from other organisms in the course of evolution. Plant resistance is divided into static resistance that plants potentially have, regardless of the presence or absence of stress, and dynamic resistance that quickly develops against the attack of microorganisms including filamentous fungi, bacteria, and viruses. It is done. Examples of static resistance include leaf cuticle layer, wax layer, cell wall firmness, potential antibacterial substances and antibacterial proteins. Examples of dynamic resistance include hypersensitive cell death, phytoalexin accumulation, and expression of PR-proteins.
Focusing on these defense mechanisms, there are reports that the above-mentioned antibacterial proteins and phytoalexin synthesis genes have been introduced into plants to acquire disease resistance (Nishizawa et al .: Chemistry and Biology 37: 295-305 (1999)).
[0004]
[Problems to be solved by the invention]
However, the stress tolerance mechanisms of plants including disease resistance have not yet been fully elucidated, and the differences between species are large, so gene recombination techniques do not always provide the expected effects. Absent. Therefore, it is desired to search for and identify genes that can function effectively for a wide variety of plants.
[0005]
[Means for Solving the Invention]
As a result of intensive studies to solve the above problems, the present inventors have succeeded in isolating a novel protein having a very strong antibacterial activity derived from gentian (Gentiana triflora) and its gene. Identified as one species. The inventors have also succeeded in producing the protein by microorganisms. Furthermore, the present inventors have found that the disease resistance of plants can be enhanced by introducing and transforming the gene into plants such as gentian, rose, rice and eustoma.
[0006]
That is, the present invention provides the following (1) to (9).
(1) The following antibacterial protein (a) or (b).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having antibacterial activity.
(2) A gene encoding the following antibacterial protein (a) or (b).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having antibacterial activity.
(3) A gene comprising the following DNA (c) or (d):
(C) DNA consisting of the base sequence represented by SEQ ID NO: 1.
(D) DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 under a stringent condition and encodes a protein having antibacterial activity.
(4) A recombinant vector containing the gene according to (2) or (3).
(5) A transformant obtained by introducing the gene according to (2) or (3).
(6) The transformant according to (5), wherein the transformant is a plant.
(7) A method for producing an antibacterial protein, wherein the transformant according to (5) or (6) is cultured, and the antibacterial protein is collected from the obtained culture.
(8) A transformed plant imparted with disease resistance by introducing the gene according to (2) or (3).
(9) An antibacterial agent comprising the antibacterial protein according to (1) as an active ingredient.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0008]
The present inventors tried to isolate various antibacterial peptides exhibiting antibacterial activity from gentian leaves using liquid chromatography. As a result, we have succeeded in isolating and purifying a specific protein having an antibacterial property with a molecular weight of about 20 kDa. As a result of examining the antibacterial activity of the protein by the method of Terras et al. (Terras et al.: J. Biol. Chem. 267: 15301-15309), gray mold fungus (Botrytis cinerea), apple spotted fungus (Alternaria alternata), It has been found to inhibit the growth of Fusarium solani.
[0009]
Furthermore, based on the N-terminal and internal amino acid sequence of the antibacterial protein, a denatured primer is designed, and the antibacterial protein gene (including the entire coding region of the protein) expressed in Gentian by RT-PCR is used. I tried to leave. As a result, we succeeded in cloning one antibacterial protein gene and identified it as a gentian peroxiredoxin gene.
[0010]
That is, the antibacterial protein of the present invention is gentian-derived peroxiredoxin. Peroxiredoxin is one of the antioxidant proteins and is known to be widely preserved from bacteria to higher plants. However, peroxiredoxin has not been known for specific actions other than antioxidant action so far, and there is no report that it has been used for recombinant plants. Moreover, there is no report that it was isolated from a plant of Gentianaceae.
[0011]
The isolation / purification / antibacterial activity evaluation of the antibacterial protein of the present invention can be performed, for example, as follows.
1. Protein isolation and purification
Examples of protein sources include gentian leaves, stems, petiole, flowers and roots. For protein purification, the above plant tissue is ground in a phosphate buffer containing various protease inhibitors such as PMSF (phenylmethanesulfonyl fluoride), and then ion exchange column (SP-Sepharose: Pharmacia), reverse phase Fractionation is performed by using a column generally used for protein purification, such as a system column (phenyl 5PWRP: manufactured by TOSO), a gel filtration column (Superose 12 column: manufactured by Pharmacia), and the like.
[0012]
2． Antibacterial activity of proteins
Using various microorganisms grown in potato dextrose medium (PDA), the antibacterial activity of the isolated gentian peroxiredoxin protein is assayed using growth inhibition as an indicator (Terras et al.: J. Biol. Chem. 267 : 15301-15309). The antibacterial activity can be measured, for example, by measuring absorbance, observing the inhibition rate of spore germination and the elongation inhibition rate under a microscope. Alternatively, observation may be performed by placing an antibiotic test paper disk (ADVANTEC) containing the protein solution on a medium such as PDA.
[0013]
The antibacterial protein gene of the present invention can be cloned, for example, as follows.
3． Cloning of Gentian peroxiredoxin gene
1) cDNA synthesis and PCR
Examples of mRNA sources include gentian leaves, stems, petiole, flowers, roots and the like. mRNA can be prepared by a conventional method. For example, after treating the above plant tissue with a guanidine reagent, a phenol reagent, etc. to obtain total RNA, except for a gene with a particularly low expression level, total RNA is removed. Perform PCR reaction on the template. If necessary, poly (A) can be obtained by affinity column method using poly-Sepharose with oligo-dT cellulose or Sepharose 2B as a carrier, or batch method.⁺RNA (mRNA) may be obtained. Furthermore, if necessary, poly (A) can be obtained by sucrose density gradient centrifugation.⁺RNA may be further finely fractionated.
[0014]
Single-stranded cDNA is synthesized using the total RNA thus obtained as a template and a primer obtained by adding M13 primer M4 (SEQ ID NO: 3) to oligo dT and reverse transcriptase. Using this single-stranded cDNA as a template, PCR was performed using a primer (SEQ ID NO: 4, 5, 6, 7) synthesized based on the amino acid sequence of gentian peroxiredoxin protein (SEQ ID NO: 2) and M13 primer M4. The gene of the invention can be obtained, but the primer is not limited to these.
[0015]
2) Determination of nucleotide sequence
The obtained PCR fragment is subcloned into an appropriate vector such as pCR2.1 (manufactured by Invitrogen), pBlueScriptSK (+) (manufactured by Stratagene), and then the nucleotide sequence is determined. The nucleotide sequence can be determined by a known technique such as a chemical modification method of Maxam-Gilbert or a dideoxynucleotide chain termination method using M13 phage, but an automatic nucleotide sequence analyzer (manufactured by PERKIN-ELMER: ABI PRISM 377). A method using DNA Sequence System etc. is simple and preferable.
[0016]
SEQ ID NO: 1 shows the nucleotide sequence of the gentian peroxiredoxin gene of the present invention thus identified. The gene encoding the antibacterial protein according to the present invention is not limited to the above sequence, and includes other genes that can hybridize with these (SEQ ID NO: 1) genes under stringent conditions.
[0017]
The stringent condition refers to, for example, a condition where the sodium concentration is 300-2000 mM, preferably 600-900 mM, and the temperature is 40-75 ° C., preferably 65 ° C. In addition, when introducing a mutation of a gene, for example, a mutation introduction kit (for example, Mutant-K (Takara) using a site-directed mutagenesis method) by a known method such as the Kunkel method or the Grapped duplex method or a method similar thereto. Or Mutant-G (Takara)), or LA PCR in vitro Mutagenesis series kit (Takara).
[0018]
SEQ ID NO: 2 shows the amino acid sequence of the gentian peroxiredoxin protein of the present invention. The amino acid sequence of the antibacterial protein of the present invention is not limited to this. As long as the product protein has antibacterial activity, one or several amino acids in the amino acid sequence are mutated such as deletion, substitution, addition, etc. It may be. The number of mutations such as deletion, substitution and addition is preferably 1 to 10, more preferably 1 to 5, with respect to the total number of amino acids.
[0019]
Once the base sequence of the gene of the present invention has been determined, then hybridization is performed by chemical synthesis, by PCR using the cDNA or genomic DNA of the gene as a template, or by using a DNA fragment having a nucleobase sequence as a probe. Can further obtain the gene of the present invention.
[0020]
Next, a method for producing a transformant according to the present invention and the resistance of the transformant to phytopathogenic bacteria will be described. In addition, the following transformed plants and plant pathogens are examples, and the present invention is not limited to these.
[0021]
Four. Production of transgenic plants introduced with gentian peroxyredoxin gene
By using a known genetic engineering technique, a DNA encoding the antibacterial protein of the present invention is introduced into a plant host to produce a transgenic plant having resistance to phytopathogenic fungi and phytopathogenic bacteria. be able to. That is, introduction of the antibacterial protein gene of the present invention into cultivated plants by genetic engineering techniques is an effective means for protecting plants from phytopathogenic fungi and phytopathogenic bacteria.
[0022]
1) Construction of vector and transformation of Agrobacterium
The DNA obtained in the above 1 can be used as it is or after digestion with an appropriate restriction enzyme or by linking an appropriate linker. Examples of the vector into which DNA is introduced include pUC vectors such as pUC18, pUC19, pUC118, and pUC119, and binary vectors such as pBI101, pBI121, and pGA482. In particular, when an Agrobacterium binary vector is used, a foreign gene is inserted between the boundary sequences (LB, RB) of the binary vector, and this recombinant vector is amplified in E. coli. Next, the amplified recombinant vector was Agrobacterium tumefaciens LBA4404, EHA101, EHA105, C58C1Rif^R And the like by freeze-thawing method, electroporation method, etc., and used for producing transformants of plants.
[0023]
In order to express a foreign gene or the like in a plant body, it is necessary to place a plant promoter and terminator before and after the structural gene, respectively. The promoter and terminator are not particularly limited, and any one known to function in plants can be used. For example, 35S transcripts derived from cauliflower mosaic virus (CaMV) [The EMBO J. 6: 3901-3907 (1987), corn ubiquitin [Plant Mol. Biol. 18: 675-689 (1992)], Examples include promoters of nopaline synthase (NOS) gene and octopine (OCT) synthesis gene. Examples of the terminator sequence include a terminator derived from cauliflower mosaic virus or a nopaline synthase gene.
[0024]
In addition, an intron sequence having a function of enhancing gene expression, such as an intron sequence of corn alcohol dehydrogenase (Adh 1) [Genes & Development 1: 1183- 1200 (1987)] can be introduced.
[0025]
In order to select target transformed cells more efficiently, it is preferable to use an effective selection marker gene in combination with the above-mentioned gentian peroxiredoxin gene. Examples of the selection marker include a hygromycin phosphotransferase (htp) gene that imparts resistance to the antibiotic hygromycin to plants and a phosphinothricin acetyltransferase (bar) gene that imparts resistance to bialaphos to plants. Can do.
[0026]
The gentian peroxiredoxin gene and the selectable marker gene may be incorporated together in a single vector, or two types of recombinant DNAs each incorporated in a separate vector may be used. The gentian peroxiredoxin gene has a prepro structure as described above, and in order to produce active gentian peroxiredoxin, the signal peptide that is a pre-sequence must be cleaved when passing through the endoplasmic reticulum. There is. Therefore, if the signal peptide of the gentian peroxiredoxin gene does not function well in other plant cells, a sequence that allows these genes to be successfully cleaved in the plant is placed upstream of the gentian peroxyredoxin gene, Can be incorporated into DNA.
[0027]
2) Introduction of the gentian peroxiredoxin gene of the present invention into a host plant
In the present invention, a host plant refers to a plant cultured cell, a whole plant of a cultivated plant, a plant organ (eg, leaf, petal, stem, root, rhizome, seed, etc.), or a plant tissue (eg, epidermis, phloem, soft tissue, tree). Part, vascular bundle, etc.). When plant cultured cells, plant bodies, plant organs, or plant tissues are used as hosts, the DNA encoding the protein of the present invention is collected from the collected plant sections using the Agrobacterium binary vector method, particle gun method, or polyethylene glycol method. Etc. can be used to transform host plants. Or you may introduce | transduce into a protoplast by the electroporation method, and you may produce a transformed plant.
[0028]
The direct gene transfer method such as the particle gun method can be performed by a so-called co-transformation method in which a vector containing a selection marker gene and a vector containing a gentian peroxiredoxin gene are mixed and simultaneously shot into a plant cell. Shoots, hairy roots, etc. obtained as a result of transformation can be used for cell culture, tissue culture or organ culture, and by using a conventionally known plant tissue culture method, an appropriate concentration of a plant hormone can be obtained. It can be regenerated into a plant body by administration or the like.
[0029]
Plant cells into which the gentian peroxiredoxin gene has been introduced can be selected for transformed cells carrying the gentian peroxiredoxin gene by screening with a selectable marker or by analyzing the expression of the gentian peroxiredoxin gene or its expression product. It is. The obtained plant can be cultivated in a pot filled with soil or vermiculite and can be proliferated by straining. The gentian peroxiredoxin transgenic plant thus grown is also included in the scope of the transgenic plant of the present invention.
Plants introduced with the gentian peroxiredoxin gene according to the present invention are expected to be resistant to stress including various plant diseases caused by pathogenic filamentous fungi and pathogenic bacteria.
[0030]
3) Analysis of the site of expression of the gene of the present invention in plant tissues
Confirmation that the target gentian peroxiredoxin gene has been incorporated into the resulting transformed plant and its next generation plant is obtained by extracting DNA from these cells and tissues according to a conventional method, It can be carried out by detecting the transgene by PCR or Southern analysis. Moreover, the expression site in the tissue of the antibacterial protein gene of the present invention can be confirmed, for example, by analyzing mRNA expression or protein expression in each tissue by a known method. For example, the expression of the antibacterial protein gene of the present invention can be confirmed by RT-PCR method, Northern analysis method and the like. Examples of the method for confirming the expression of the antibacterial protein of the present invention include a Western analysis method using an antibody against the protein.
[0031]
Next, a genetic engineering production method of a novel antibacterial protein using the above gene will be described.
Five. Production of the antibacterial protein of the present invention by genetic engineering techniques
The antibacterial protein of the present invention can be produced by genetic engineering as follows, for example.
1) Production of recombinant vector
The recombinant vector of the present invention can be obtained by linking (inserting) the gene of the present invention to a known vector. The vector is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA.
[0032]
Examples of the plasmid DNA include plasmids derived from E. coli (eg, pBR322, pBR325, pUC18, pUC119, pTrcHis, pBlueBacHis, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and yeast-derived plasmids (eg, YEp13, YEp24, YCp50). , pYE52, etc.), and phage DNA includes λ phage. Furthermore, animal viruses such as retrovirus or vaccinia virus, insect virus vectors such as baculovirus, plant virus vectors such as potato ex virus, and the like may be used.
[0033]
For insertion of the gene of the present invention into the vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate restriction enzyme site or a multicloning site of the vector DNA, and then ligated to the vector. Is done. The gene of the present invention needs to be incorporated into a vector so that the function of the gene can be suitably exhibited. Therefore, the vector can contain a promoter, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like in addition to the gene of the present invention. Examples of selectable markers include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, and the like.
[0034]
2) Production of transformants
The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. For example, Escherichia such as Escherichia coli, Bacillus subtilis such as Bacillus subtilis, Pseudomonas・ Pseudomonas putida and other Pseudomonas genus, Rhizobium melilotei and other Rhizobium genus bacteria, and Saccharomyces cervisiae and Saccharomyces pombe yeast and monkey cells (S. pombe) COS cells), animal cells such as Chinese hamster ovary cells (CHO cells), and insect cells such as Sf19 and Sf21.
[0035]
When a bacterium such as Escherichia coli is used as a host, it is desirable that the recombinant vector of the present invention is capable of autonomous replication in each bacterium and is composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Moreover, the gene which controls a promoter may be contained. Examples of E. coli include Escherichia coli K12, DH1, and TOP10F. Examples of Bacillus subtilis include B. subtilis MI114 and 207-21.
[0036]
The promoter is not particularly limited as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli such as trp promoter, lac promoter, PL promoter, and PR promoter can be used. An artificially designed promoter such as a tac promoter may be used.
[0037]
The method for introducing a recombinant vector into bacteria is not particularly limited as long as it can introduce DNA into bacteria. For example, a method using calcium ions (Cohen, SN et al. Proc. Natl. Acad. Sci. USA, 69 : 2110 (1972)), electroporation method and the like.
[0038]
When yeast is used as a host, for example, S. cervisiae, S. pombe, Pichia pastoris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast.For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX promoter, etc. Can be mentioned.
[0039]
The method for introducing a recombinant vector into yeast is not particularly limited as long as it can introduce DNA into yeast. For example, the electroporation method (Becker, DM et al .: Methods. Enzymol., 194: 180 (1990 )), Spheroplast method (Hinnen, A. et al .: Proc Natl. Acad. Sci. USA, 75: 1929 (1978)), lithium acetate method (Itoh, H .: J. Bacteriol., 153: 163) (1983)).
[0040]
When animal cells are used as hosts, monkey cells (COS-7), Vero, Chinese hamster ovary cells, mouse L cells, rat GH3 cells, human FL cells and the like are used. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like are used. In addition, an early gene promoter of human cytomegalovirus may be used. Examples of methods for introducing a recombinant vector into animal cells include electroporation, calcium phosphate, and lipofection.
When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into an insect cell, for example, an electroporation method, a calcium phosphate method, a lipofection method, or the like is used.
[0041]
3) Production of antibacterial protein of the present invention
The antibacterial protein of the present invention can be obtained by culturing the transformant and collecting the protein from the culture. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host. As a medium for cultivating transformants obtained using microorganisms such as Escherichia coli and yeast as a host, it contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, and can efficiently culture transformants. As long as it is a medium, either a natural medium or a synthetic medium may be used.
[0042]
As the carbon source, carbohydrates such as glucose, fructose, sucrose, starch, maltose and dextrin, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic or organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, casamino acid, NZ amine Etc. are used.
[0043]
Examples of inorganic substances that can be used include monopotassium phosphate, dipotassium phosphate, calcium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, zinc sulfate, and cobalt chloride. The culture is usually performed at about 30 ° C. for 24 to 96 hours under aerobic conditions such as shaking culture or aeration and agitation culture. During the cultivation period, the pH is maintained at 5.0 to 8.0. The pH is adjusted with an inorganic or organic acid or alkaline solution.
[0044]
During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the Lac promoter, cultivate a microorganism transformed with an expression vector using isopropyl-β-thiogalactopyranoside (IPTG) or the like using the trp promoter. In this case, indole acrylic acid (IAA) or the like may be added to the medium.
[0045]
Examples of a medium for culturing a transformant obtained using animal cells as a host include generally used RPMl 1640 medium, DMEN medium, or a medium obtained by adding fetal calf serum or the like to these mediums. Culture is usually 5% CO₂Perform in the presence at 20-30 ° C. for 1-7 days. During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
[0046]
Examples of a medium for culturing a transformant obtained using insect cells as a host include a medium obtained by adding fetal calf serum or the like to Grace's Insect Medium (Grace, T.C.C .: Nature, 195: 788 (1962)). The culture is usually performed at 25 ° C. for 1 to 7 days. During the culture period, maintain the pH at 6.0 to 7.0, and add aeration or agitation as necessary.
[0047]
After culturing, when the protein of the present invention is produced in cells or cells, the cells or cells are crushed. On the other hand, when the protein of the present invention is secreted outside the cells or cells, the culture solution is used as it is or after removing the cells or cells by centrifugation or the like, a supernatant is obtained. For protein isolation / purification, generally, for example, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, or the like is used alone or in appropriate combination to obtain the above culture (cell disruption solution). , Culture solution, or supernatant thereof), the antibacterial protein of the present invention or a salt thereof can be isolated and purified.
[0048]
6． Chemical synthesis of the antimicrobial protein of the present invention
Chemical synthesis of the protein of the present invention can be carried out by ordinary peptide synthesis means. Examples thereof include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, and a carboimidazole method. In addition, for the synthesis, either a solid phase synthesis method or a liquid phase synthesis method may be applied. That is, when the amino acid capable of constituting the protein of the present invention and the remaining part are condensed and the product has a protecting group, the target protein is synthesized by removing the protecting group. Any known method may be used as the condensation method and desorption method.
[0049]
After the reaction, the protein of the present invention can be purified by a combination of usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. The protein of the present invention is usually carboxyl group (—COOH) or carboxylate (—COO—) at the C-terminus, but amide (—COONH) at the C-terminus.₂), Ester (-COOR). Here, R 1 in the ester includes an aralkyl group having 7 to 12 carbon atoms, a cycloaralkyl group having 3 to 10 carbon atoms, an aryl group having 6 to 12 carbon atoms, an alkyl group having 1 to 12 carbon atoms, and the like. Furthermore, the proteins of the present invention include those in which the N-terminal amino acid residue is protected, or complex peptides such as glycopeptides to which sugar chains are bound.
[0050]
7． Use of the antimicrobial protein of the present invention as an antimicrobial agent
Since the antibacterial protein of the present invention has an antibacterial action against phytopathogenic filamentous fungi and has a wide antibacterial spectrum, it can be used particularly as an antibacterial agent. In addition, the antibacterial protein is combined with other pesticides, for example, in combination with an insecticide (such as an insecticide for rice pests), in combination with an insecticide or a plant growth regulator (such as a rice dwarfing agent). It can be used as a sterilizing plant growth regulator. That is, the antibacterial protein can be used alone or in combination with an appropriate liquid, solid or gas simple substance. If necessary, liquefied gas, propellant (Freon etc.), surfactant (emulsifier, dispersant, antifoaming agent, etc.) etc. are added, emulsion, oil, wettable powder, powder, granule, liquid etc. It can also be used as a formulation.
[0051]
Examples of liquid carriers used in the preparation include aromatic hydrocarbons such as xylene, toluene, benzene, and alkylnaphthalene, chlorinated aromatic hydrocarbons such as chlorobenzene, chloroethylene, and methylene chloride, and aliphatic carbonization such as cyclohexane and paraffin. Examples include hydrogen, mineral oil components, alcohols such as ethanol, butanol, glycol and ethers thereof, and esters, ketones such as acetone and methyl ethyl ketone, polar solvents such as dimethylformamide, dimethyl sulfoxide, acetonitrile, and water. One kind or a mixture of two or more kinds can be used. When water is used as a solvent, pure water or an aqueous solution of inorganic salts (such as sodium chloride and potassium chloride), sugar (such as glucose and sucrose) or sugar alcohol (such as D-sorbitol and D-mannitol) can be used. .
[0052]
Examples of the solid carrier used in the preparation include natural mineral powders such as kaolin, clay, talc, chalk, quartz, attapulgite, montmorillonite, and diatomaceous earth, synthetic mineral powders such as silicic acid, alumina, and silicate, and polymers. Natural products (crystalline cellulose, corn starch, gelatin, alginic acid, etc.) can be used, and one or more of these can be used in combination.
[0053]
Surfactants used as emulsifiers, dispersants, antifoaming agents, etc. include polyoxyethylene-fatty acid esters, polyoxyethylene fatty alcohol esters, alkylaryl polyglycol esters, alkyl sulfonates, alkyl sulfates, aryl sulfates. Nate, albumin hydrolyzate, methylcellulose, gum arabic and the like.
[0054]
The antibacterial protein of the present invention, which is an active ingredient, is suitably 0.01 to 50% by weight for emulsions, 0.01 to 50% by weight for wettable powders and 0.01 to 10% by weight for powders. May be changed as appropriate. In the case of emulsions and wettable powders, they can be diluted with water at the time of use and used at 10 to 5000 times the product weight, preferably 500 to 1000 times.
[0055]
The antibacterial agent of the present invention may be directly administered to plants affected by phytopathogenic fungi using spraying method, mist method, dust method, spraying method, injection method or the like, or directly to soil contaminated with pathogenic fungi. It may be administered. The method of use is appropriately selected depending on the purpose of use. In any case, it is desirable that the antibacterial protein of the present invention be dispersed as uniformly as possible. Although the usage-amount of the antibacterial agent of this invention changes with the usage methods, in the case of the spraying method, for example, it is preferable to spray by 1-1000g in an active ingredient amount per 10a.
[0056]
Examples of plant pathogens to which the antibacterial agent containing the antibacterial protein of the present invention should be used include gray mold fungus, Alternaria fungus, blast fungus, powdery mildew, and seedling blight fungus. Specific examples of these pathogens include gray cucumber (Cucumis sativus), tomato (Lycopersicon esculentum), green pepper (Capsicum annum), lettuce (Lactuca sativa), strawberry (Fragaria ananassa), grape (Vitis vinifera), and statice. (Limonium Mill), Eustoma grandiflorum, Gentiana triflora, Gentiana triflora, tobacco (Nicotiana tabacum), and hops (Humulus luplus) are pathogenic fungi that invade cultivated plants such as Altanaria, apple (Malus domestica), pear ( Pyrus L.), pathogenic bacteria that invade cultivated plants such as strawberries and tobacco, blast fungus is a pathogenic fungus that infects cultivated plants such as rice (Oryza sativa), and powdery mildew fungi are cultivated plants such as apples, grapes and peppers It is a pathogenic fungus that invades, and the seedling blight fungus is a pathogenic fungus that invade cultivated plants such as rice and lettuce. Therefore, for the purpose of controlling or preventing diseases caused by these pathogenic bacteria, it is preferable to use an antibacterial agent containing the antibacterial protein of the present invention in the aforementioned plant.
[0057]
Furthermore, the antibacterial protein of the present invention can also be used as a pharmaceutical composition. The pharmaceutical composition containing the protein of the present invention as an active ingredient may contain a pharmaceutically acceptable carrier or additive together. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, Examples include gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and surfactants acceptable as pharmaceutical additives. It is done. The additive to be used is appropriately selected from the above in accordance with the dosage form of the present invention.
[0058]
When the antibacterial protein of the present invention is used as an antibacterial agent or a pharmaceutical composition, its use target is not particularly limited, and for example, it can be used for the purpose of diagnosis, treatment or prevention of mycosis. These diseases may be those singly or in combination, or in combination with other diseases other than those described above.
The pharmaceutical composition containing the antibacterial protein of the present invention can be administered orally or parenterally.
[0059]
When the antibacterial protein of the present invention is administered orally, it may be a solid preparation such as a tablet, granule, powder or pill applied thereto, or a liquid preparation such as a liquid or syrup. In particular, granules and powders can be made into unit dosage forms as capsules, and in the case of liquid preparations, they may be dried products that are redissolved when used. Of these dosage forms, oral solid preparations usually contain additives such as binders, excipients, lubricants, disintegrants, and moistening agents that are commonly used in pharmaceutical compositions. Oral liquid preparations usually contain additives such as stabilizers, buffering agents, corrigents, preservatives, fragrances, coloring agents and the like that are generally used in the formulation.
[0060]
When the antibacterial protein of the present invention is administered parenterally, it may be an injection, a suppository or the like. In the case of injection, it may be a powder usually provided in a unit dose ampoule or a multi-dose container and redissolved in a suitable carrier, for example, pyrogen-free sterilized water. These dosage forms usually contain additives such as emulsifiers and suspending agents that are generally used in pharmaceutical compositions. Examples of the injection technique include intravenous drip injection, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intradermal injection. Further, the dose varies depending on the age of administration subject, administration route, and number of administrations, and can be varied over a wide range.
[0061]
In this case, the dosage form and dosage thereof can be arbitrarily selected as long as the antibacterial effect according to the present invention is recognized in consideration of the subject (including humans and animals), the type of disease, and symptoms. . For example, the dosage is about 0.001 to 10 mg / kg body weight, preferably about 0.025 to 0.5 mg / kg body weight.
[0062]
8． Use as food and feed additive
Furthermore, since the antibacterial protein of the present invention is easily degraded by proteolytic enzymes present in the stomach and intestines, at least as a result, it is expected that there is almost no toxicity. Therefore, the antibacterial protein of the present invention can be used as a food or feed additive. For example, the antibacterial protein can be dissolved as a solid or in a liquid, preferably water, to a suitable concentration and added to food or feed, for example, by mixing, dipping, coating, spraying, etc. . As a result, the present protein can prevent the occurrence of fresh foods such as meat, fish and vegetables, processed foods, and fungi such as soybean meal and fish meal feed. For example, when the antibacterial protein of the present invention, which is an active ingredient, is used as an aqueous solution, it can be 0.00005 to 0.05% by weight, preferably 0.0001 to 0.01% by weight.
[0063]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited thereto.
[Example 1] Purification of gentian novel antibacterial protein (gentian peroxiredoxin)
1) Purification of antibacterial protein
About 100 g of gentian line (arrow width) leaves 10 mM NaH₂PO_Four, 15 mM Na₂HPO_FourAfter grinding in a 1 liter solution containing 100 mM KCl, 2 mM EDTA, 1.5% PVP, 2 mM thiourea, 1 mM PMSF, a fiber part (cell wall fraction) was obtained with gauze. 2M LiCl from the fiber part (cell wall fraction)₂Extract the cell wall-bound protein by dialyzing the protein fraction against sterile water overnight (excluding molecular weight 1000 or less), adjusting to 50 mM MES (pH 6.0), and equilibrating with 50 mM MES (pH 6.0) This was applied to a modified SP-sepharose column. After washing the non-adsorbed fraction with 50 mM MES (pH 6.0), the 0.5 M NaCl-adsorbed fraction was eluted. This phase was dialyzed overnight against sterile water (with a molecular weight of 1000 or less excluded), TFA was added to this fraction to a final concentration of 0.1%, and reversed-phase HPLC equilibrated with sterile water containing 0.1% TFA. And eluted with a gradient of 0-50% acetonitrile.
[0064]
2) Measurement of antibacterial activity
Suspensions of filamentous fungi grown on potato dextrose agar medium (PDA medium) are suspended in a low ionic strength liquid medium described below, and the number of spores is 1 × 10^FiveAnd 1 × 10⁶The solution was prepared at a dose / ml. After culturing at 25 ° C. for filamentous fungi and at 30 ° C. or 37 ° C. for 24-48 hours for bacteria, the growth of the bacteria was assayed for antibacterial activity by measuring the absorbance at OD 595 nm.
(1) Test bacteria
The antibacterial activity of the antibacterial protein of the present invention includes, as representative examples of filamentous fungi, gray mold (B. cinerea (strain S1)), apple spotted leaf (B. cinerea (strain S1)), apple leaf spot (B. cineani) (Martius) Saccardo).
(2) Preparation of low ionic strength medium
According to the method of Terras et al. (J. Biol. Chem. 267: 15301-15309), a medium having the following composition and concentration was prepared. K₂HPO_Four (2.5 mM), MgSO_Four (50μM), CaCl₂ (50μM), FeSO_Four (5 μM), CuSO_Four (0.1μM), Na₂MoO_Four (2μM), H_ThreeBO_Three (0.5μM), KI (0.1μM), ZnSO_Four (0.5μM), MnSO_Four (0.1μM), Glucose (10g / l), Asparagine (1g / l), Methionine (20mg / ml), Myo-inositol (2mg / l), biotin (0.2mg / l), Thiamine-HCl (1mg / l) ), Pyridoxine-HCl (0.2mg / l)
[0065]
3) Antibacterial evaluation
A fraction having antibacterial properties was obtained by the above purification method. When this fraction was analyzed by SDS-PAGE, it was a single protein of 20 kDa (FIG. 1). When the antibacterial activity of this fraction was analyzed in detail using gray mold fungus, apple spotted leaf fungus, and Eustoma japonica, it had antibacterial activity against these pathogenic fungi (FIG. 2). The concentration that inhibits the growth of each filamentous fungus by 50% (IC₅₀) Were 15.0, 22.0, and 37.0 μg / ml for the above bacteria, respectively.
[0066]
[Example 2] Determination of amino acid sequence of novel antibacterial protein of gentian
1) Determination of N-terminal amino acid sequence
The purified antibacterial protein (gentian peroxiredoxin) was fractionated by SDS-PAGE by a conventional method, and then electroblotted onto a PVDF membrane. After detecting the protein with a 2% acetic acid solution containing 0.1% Ponceau S, which is a protein staining reagent, the blotted body was cut out with a knife. Regarding the N-terminal amino acid sequence, the amino acid sequence of the excised blot was directly determined by the Edman method (Takara; custom service).
[0067]
[Example 3] Cloning of gene
1) Preparation of total RNA
Total RNA was extracted from gentian leaves grown and grown in a greenhouse using Fast green RNA isolation Kit (Funakoshi).
2) Single-stranded cDNA production
Single-stranded cDNA was synthesized by RT-PCR kit (Takara) using 1 μg of total RNA (including polyA mRNA) obtained in 1) above. As a primer used for this reaction, a primer having a known sequence 5′-AGTTTTCCCAGTCACGAC-3 ′ (SEQ ID NO: 3) linked to the 5 ′ side of oligo (dT) 20 was used. The PCR reaction composition is as follows.
PCR Reaction composition:
RNA 1μl (1μg)
Primer 1μl (50pmol)
10 x PCR buffer 2 μl
25 mM MgCl2 4 μl
RNAse inhibitor 0.5μl
10 mM dNTP mix 2 μl
1 μl of AMV-derived reverse transcriptase
DEPC Treated water 8.5 μ l
Total volume 20μl
The mixture was incubated at 42 ° C., 50 ° C., 55 ° C., and 60 ° C. for 10 minutes each, and then incubated at 99 ° C. for 5 minutes to stop the reaction.
[0068]
3) Preparation of primer
The primers (Sawaddy Technology Inc .; Custom Service) used for amplification of a novel antibacterial protein (gentian peroxiredoxin) gene are as follows.
Sense primer:
5'-ACA ATT GAC GAC CAC CAT CGT TCC AAG AAC-3 '(SEQ ID NO: 4)
5'-TTC CAA GAA CTT TCA GTG AAA CAA ATC TCC-3 '(SEQ ID NO: 5)
Antisense primer:
5'-CTA AGC CTG ATT TCT CAT TAT ACA GTA TTC-3 '(SEQ ID NO: 6)
5'-TAC AGT ATT CTT ACA AAA CCT GGT TTC TTC-3 '(SEQ ID NO: 7)
[0069]
4) Amplification of novel antibacterial protein genes by RT-PCR
Using the synthesized single-stranded cDNA as a template, a PCR reaction was performed with the sense primer and the antisense primer described in 3) above. The PCR reaction composition is as follows.
PCR Reaction composition:
cDNA solution 1μl (1μg)
20 μM sense primer 2 μl
20 μM antisense primer 2 μl
10 x PCR buffer 5 μl
25 mM dNTP mix 4 μl
ExTaq 1μl
DEPC Treated water 37.5 μ l
Total volume 50μl
The PCR reaction was performed for 30 cycles, with heat denaturation at 95 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute as one cycle.
This PCR reaction product was subjected to 1.6% agarose gel electrophoresis, and an approximately 800 bp DNA fragment was excised from the gel. DNA fragments were extracted from this gel piece using Quia Gel Extration Kit (Qiagen).
[0070]
Next, this PCR product was TA cloned into the vector pCR2.1 (Invitrogen). That is, a PCR product having a weight ratio of about 5 to 10 times was mixed with the pCR2.1 vector, and ligation was performed using Ligation kit ver. 2 (Takara). Escherichia coli (TOP10F ′) was transformed with this recombinant plasmid. Single colonies were cultured in LB medium, and the plasmid was purified using QUIA Miniprep Kit (Qiagen).
[0071]
5) Determination of nucleotide sequence
Using the obtained plasmid as a template, perform sequencing reaction according to the method of Big Dye Cycle sequence Kit (ABI) using M13RV and T7 primer, and determine the base sequence using ABI PRISM 377 DNA Sequence System (ABI) It was. The obtained base sequence was analyzed using analysis software DNASIS and GENETIX, and homology search was performed using search software BLAST. As a result of homology search, it showed high homology with peroxiredoxin reported in several plants. Based on the above results, this gene was identified as a gentian peroxiredoxin gene.
[0072]
[Example 4] Southern blot analysis of gentian genomic DNA
1) Preparation of genomic DNA
Genomic DNA was prepared from gentian leaves by the CTAB method (Focus 12: 13-15 (1990)). That is, 5 g of gentian leaves were frozen in liquid nitrogen, then ground with a mortar and pestle, and 2 × CTAB solution (2% Cetyl trimethyl ammonium bromide (CTAB), 0.1 M Tris-HCl, pH 8.0, 1.4 M NaCl) , 1% PVP) 10 ml. After incubating at 55 ° C. for 10 minutes, 5 ml of chloroform / isoamyl alcohol (24: 1) was added and shaken at room temperature for 30 minutes. After centrifugation at 3500 rpm, collect the aqueous layer (supernatant), add 1/10 volume of 10% CTAB solution (10% CTAB, 0.7M NaCl), mix by inversion, and then add an equal amount of precipitation buffer (1% CTAB, 5 mM Tris-HCl, pH 8.0, 10 mM EDTA) was added, and the mixture was further mixed by inverting at room temperature for 30 minutes. After centrifugation at 7000 rpm, the precipitate was dissolved in 5 ml of 1M NaCl-TE (1M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at 55 ° C., and 5 ml of isopropanol was added. After mixing by inversion, the mixture was centrifuged at 3500 rpm, and the precipitate was ethanol precipitated with 5 ml of 70% ethanol. The precipitate was dried and dissolved in 500 μl of TE. After that, RNAse (1 μg / ml) treatment is performed to remove the contaminated RNA, followed by extraction with phenol / chloroform / isoamyl alcohol (25: 24: 1), ethanol precipitation, drying of the precipitate, and 500 μl of TE. To obtain genomic DNA.
[0073]
2) Hybridization
The genomic DNA obtained above was digested with restriction enzymes BamHI and EcoRI, and the degradation product was subjected to 1% agarose gel electrophoresis. After electrophoresis, transfer the DNA fragment to High Bond N + membrane (Amersham), and transfer the membrane to high SDS hybridization buffer (7% SDS, 50% formamide, 5 × SSC, 2% blocking reagent, 50 mM sodium phosphate, pH 7 0.0, 0.1% N-lauryl sarcosine) at 42 ° C. for 1 hour or longer.
[0074]
Next, the full-length cDNA of the gentian peroxiredoxin gene as a probe was DIG-labeled using a DIG (digoxigenin) luminescence detection kit (Roche Diagnostics) and then hybridized. That is, hybridization was performed by immersing in a high SDS hybridization buffer containing a labeled probe at 42 ° C. for 16 hours. The membrane was then washed twice at 50 ° C. in 2 × SSC, 0.1% SDS and twice at 68 ° C. in 0.1 × SSC, 0.1% SDS. Thereafter, the resultant was treated with an alkaline phosphatase-labeled anti-DIG antibody (Roche Diagnostics), and an autoradiogram was taken to examine the band hybridized with the probe.
As a result, since one band was observed in any restriction enzyme, it was considered that this gene was present in the gene genome.
[0075]
[Example 5] Expression of gentian peroxiredoxin by E. coli
1) Primer preparation
Primers used for amplification of the gentian peroxiredoxin gene (Sawadee Technology, Inc .; Custom Service) are as follows.
Sense primer:
5'-CGC GGA TCC GAT GGC TGC AAT TTG TCT TCC TGT TGC AAA A-3 '(SEQ ID NO: 8)
Antisense primer:
5'-CGG GGT ACC TCA AGC ACT TTG AAG GAA CTT CAA GGT TTC-3 '(SEQ ID NO: 9)
[0076]
2) Cloning into expression vector (pTrcHisB)
Using the primers prepared in 1), PCR was performed using a plasmid containing the gentian peroxiredoxin gene as a template. After digesting the amplified product of about 600 bp with 10 units of BamHI and KpnI, the DNA fragment was fractionated by agarose gel electrophoresis, and a band of about 600 bP was cut out from the agarose gel. A DNA fragment containing a 600 bp gentian peroxiredoxin gene was obtained from this gel using Quiaprep. Gel extraction kit (Qiagen).
[0077]
Mix 5 μl each of the histidine tag fusion protein expression vector (pTrcHisB, manufactured by Invitrogen) cleaved with 10 units of the same enzyme with the DNA fragment containing the gentian peroxiredoxin gene obtained as described above. Ligation solution (DNA ligation kit (manufactured by Takara)) equivalent to the DNA mixture was added for ligation. 2 μL of this ligation reaction solution was mixed with 100 μL of commercially available E. coli competent cell DH5α (manufactured by TOYOBO), and left in ice for 30 minutes, at 42 ° C. for 45 seconds, and further in ice for 1 minute. LB medium (1% Bacto trypton, 0.5% Bact yeast extract, 0.5% NaCl, 0.1% glucose pH 7.5) (100 mL) was added to the solution containing the Escherichia coli transformant thus obtained. After culturing for an hour, it was plated on LB agar medium containing 100 mg / L of ampicillin. On the next day, the colonies that appeared on the medium were cultured with shaking in LB liquid medium containing 100 mg / L of ampicillin at 37 ° C. for about 6 to 12 hours. From this culture, a plasmid is prepared using Quiaprep. Miniprep. Kit (manufactured by Qiagen), digested with BamHI and KpnI, and analyzed by agarose gel electrophoresis. Plasmid was selected (pTrcHisBGt3).
[0078]
pTrcHisBGt3 was mixed with 100 μL of Escherichia coli competent cell BL21 (manufactured by Amersham) for protein expression, and left in ice for 30 minutes, at 42 ° C. for 45 seconds, and then in ice for 1 minute. LB medium (1% Bacto trypton, 0.5% Bact yeast extract, 0.5% NaCl, 0.1% glucose pH 7.5) 100 mL was added to the solution containing the Escherichia coli transformant thus obtained. After culturing for a long time, it was plated on an LB agar medium containing ampicillin 100 mg / L to prepare Escherichia coli for expressing gentian peroxiredoxin.
[0079]
3) Expression of gentian peroxiredoxin in E. coli
The E. coli for gentian peroxiredoxin expression obtained as described above was pre-cultured at 37 ° C. for about 6 to 12 hours in an LB liquid medium containing 100 mg / L of ampicillin. The pre-cultured Escherichia coli was again added to an LB liquid medium containing 100 mg / L of ampicillin so as to have an OD of about 0.1-0.2, and cultured at 37 ° C. until it reached an OD of about 0.6. Thereafter, protein expression was induced by adding 1 mM IPTG, and the cells were cultured for 3 hours.
[0080]
4) Extraction of gentian peroxiredoxin
After inducing protein expression, E. coli cultured for 3 hours was collected by centrifugation, and the cells were lysed using a surfactant (B-PER: manufactured by Pierce). Thereafter, soluble protein was obtained by centrifugation.
[0081]
5) Purification of gentian peroxiredoxin
Since gentian peroxyredoxin was expressed as a fusion protein with a histidine tag, affinity purification using a nickel column was performed using a HisTrap protein purification kit (Amersham).
[0082]
Since the purified protein has a histidine tag that is not present in the original gentian peroxiredoxin, it was cleaved and removed using 100 units of enterokinase (manufactured by Invitrogen). The cleaved histidine tag portion was removed using HisTrap, and the added enterokinase was removed by an enterokinase removal column (EK-MAX AWAY: manufactured by Invitrogen) to obtain a purified recombinant gentian peroxiredoxin.
[0083]
6) Recombinant gentian peroxiredoxin antibacterial activity
(1) Test bacteria
The antibacterial activity of the antibacterial protein of the present invention was evaluated using the gray mold fungus B. cinerea (strain S1) and the apple spotted leaf fungus A. alternata apple pathotype as representative examples of filamentous fungi. Moreover, about the influence with respect to the growth of bacteria, it evaluated using colon_bacillus | E._coli, a lettuce rot bacterium, a rice wilt disease bacterium, and a rice seedling wilt disease bacterium.
(2) Composition of low ionic strength medium for measuring anti-filament fungal activity (Terras et al.: J. Biol. Chem. 267: 15301-15309)
K₂HPO_Four (2.5 mM), MgSO_Four (50 μM), CaCl₂ (50 μM), FeSO_Four (5 μM), CuSO_Four (0.1 μM), Na₂MoO_Four (2 μM), H_ThreeBO_Three (0.5 μM), KI (0.1 μM), ZnSO_Four (0.5 μM), MnSO_Four (0.1 μM), Glucose (10 g / l), Asparagine (1 g / l), Methionine (20 mg / ml), Myo-inositol (2 mg / l), Biotin (0.2 mg / l), Thiamine-HCl (1 mg / l), Pyridoxine-HCl (0.2 mg / l)
(3) Medium for measuring antibacterial activity
Nutrient broth (Difco)
[0084]
7) Antibacterial evaluation
Recombinant gentian peroxiredoxin showed antibacterial activity against gray mold and apple spotted leaf blight (FIG. 4). On the other hand, the growth of bacteria such as Escherichia coli, lettuce rot, rice blast, and rice seedling bacterium was not suppressed.
[0085]
[Example 6] Construction of a recombinant vector for introducing a plant
1) Construction of recombinant vector p35SGt3 for plant introduction
After digesting 10 μg of the plasmid containing the gentian peroxiredoxin gene with restriction enzymes SalI and BamHI 10 units, the fragment was fractionated by agarose gel electrophoresis, and a band of about 800 bP was cut out from the agarose gel. A DNA fragment containing an 800 bp gentian peroxiredoxin gene was obtained from this gel using a Quiaprep. Gel extraction kit (Qiagen).
[0086]
On the other hand, after digesting 10 µg of DNA of plasmid vector containing CaMV35S (cauliflower mosaic virus) promoter and NOS (nopaline synthase) terminator with 10 units of restriction enzymes BamHI and Sal I in TE buffer, the fragment was agarose gel. Fractionation was performed by electrophoresis, and a band of about 4.5 kb was cut out from the agarose gel. The digested plasmid was recovered from this gel using Quiaprep. Gel extraction kit (Qiagen).
[0087]
Mix 5 µl each of the DNA fragment containing the gentian peroxiredoxin gene and the vector DNA fragment obtained as described above, and use the same amount of ligation solution (DNA ligation kit (manufactured by Takara)) as this DNA mixture. Linked by adding. 2 μL of this ligation reaction solution was mixed with 100 μL of commercially available E. coli competent cell DH5α (manufactured by TOYOBO), and left in ice for 30 minutes, at 42 ° C. for 45 seconds, and further in ice for 1 minute. LB medium (1% Bactotrypton, 0.5% Bact yeast extract, 0.5% NaCl, 0.1% glucose pH 7.5) (100 mL) was added to the solution containing the Escherichia coli transformant thus obtained. After culturing for an hour, it was plated on an LB agar medium containing 50 mg / L of spectinomycin. On the next day, one of the colonies that appeared on the medium was selected from white colonies, and cultured with shaking in an LB liquid medium containing 50 mg / L of spectinomycin at 37 ° C. for about 6 to 12 hours. A plasmid is prepared from this culture solution using Quiaprep. Miniprep kit (Qiagen), digested with various restriction enzymes, and analyzed by agarose gel electrophoresis. A plasmid in which the peroxiredoxin gene was normally linked was selected and used as a plant introduction recombinant vector p35SGt3 (see FIG. 5).
[0088]
2) Construction of binary vector pEbisGt3KB
After digesting 10 μg of the binary plasmid vector pEBisKB and DNA of the plasmid vector p35SGt3 (10 μg) with 10 units of restriction enzyme SSeI in TE buffer, the restriction enzyme was removed by performing saturated phenol extraction twice. To this extract, 1/100 volume of 3M sodium acetate and 2 volumes of ethanol were added and left at -20 ° C for 6 hours. This solution was centrifuged at 1,500 rpm at 4 ° C. for 10 minutes, and the resulting precipitate was dried under reduced pressure, dissolved in 10 μL of TE, and ligated with a DNA ligation kit (manufactured by Takara). 10 μL of this ligation reaction solution was mixed with 100 μL of commercially available E. coli competent cell DH5α (manufactured by TOYOBO), and left in ice for 30 minutes, at 42 ° C. for 45 seconds, and then in ice for 1 minute. LB medium (1% Bacto trypton, 0.5% Bact yeast extract, 0.5% NaCl, 0.1% glucose pH 7.5) 100 mL was added to the solution containing the Escherichia coli transformant thus obtained. After culturing for an hour, the cells were plated on LB agar medium containing 100 mg / L spectinomycin and 50 mg / L kanamycin. The next day, one of the colonies that appeared on the medium was selected from white colonies, and cultured in an LB liquid medium containing spectinomycin and kanamycin at 37 ° C. for about 6 to 12 hours. From this culture solution, a plasmid is prepared using Quiaprep. Miniprep. Kit (manufactured by Qiagen), digested with various restriction enzymes, and analyzed by agarose gel electrophoresis, thereby analyzing the gentian peroxiredoxin gene. Was selected as a recombinant vector pEbisGt3KB for plant introduction (see FIG. 5). Agrobacterium was transformed with this vector by a known method and used for transformation.
[0089]
[Example 7] Introduction of gentian peroxiredoxin gene into tobacco
1) Introduction of pEbisGt3KB into tobacco
(1) Raising tobacco plants
Ripe tobacco seeds (Nicotiana tabacum SR1) are sterilized with 1% hypochlorous acid and placed in MS medium to induce germination and elongation. MS medium is Murashige-Skoog basic medium containing 3% sucrose and 0.9% agar. After 1-2 months of cultivation, fully expanded leaves were obtained that could be infected with Agrobacterium.
(2) Introduction of binary vector pEbisGt3KB
Cut the leaves of fully developed tobacco into 1 cm square as explants, and explant the explants into an Agrobacterium solution with pEBisGt3KB cultured overnight in YEB medium containing appropriate antibiotics at 28 ° C. After immersing for a minute, the excess bacterial solution attached was wiped lightly with filter paper, transferred to MS medium, and co-cultured for 2 days under low light at 25 ° C.
(3) Selection of transformed cells and regeneration into plants
The leaf explants after co-cultivation were transferred to a regeneration medium, adventitious shoots produced on callus formed one month later were cut out and placed on a rooting medium to induce rooting. The regeneration medium is an MS medium containing 1 mg / L BAP, 0.1 mg / L NAA, 500 mg / L carbenicillin, 5 mg / L bialaphos. The rooting medium is an MS medium containing 500 mg / L carbenicillin and 5 mg / L bialaphos. As a selective drug, kanamycin or the like can be used in addition to bialaphos.
[0090]
[Example 8] Evaluation of disease resistance of transformed plants
By analyzing the redifferentiated individuals of 15 gentian peroxiredoxin transgenic tobacco lines obtained by the above method using a conventional method, a northern blot analysis was conducted, and after confirming the expression of gentian peroxiredoxin gene, a disease resistance evaluation test against gray mold was performed. went.
To cut out regenerated tobacco leaves, punch out fungal fungus grown in B. cinerea in PDA medium with a cork borer, inoculate 2 fungus fungi per leaf, and keep humidity Then, the mixture was incubated at 25 ° C. for 4 days on a filter paper sufficiently absorbing water.
[0091]
In the transformant, the growth of gray mold was suppressed, whereas in the control wild type, the growth of the fungus progressed and the entire leaf became submerged, showing severe symptoms. 11 out of 15 strains showed strong resistance against gray mold and 4 strains showed moderate resistance against gray mold (Table 1). Compared to control non-transformants, there are many strains that have a small spread of lesions when inoculated with gray mold, so in tobacco introduced with the gentian peroxiredoxin gene, It was confirmed that resistance to gray mold was imparted.
[0092]
[Table 1]

[0093]
【The invention's effect】
If the antibacterial protein of the present invention and its gene are used, the disease resistance of plants can be improved. Further, the antibacterial protein of the present invention can be mass-produced by gene recombination technology and can be used as an antibacterial agent.
[0094]
[Sequence Listing]

[0095]
[Sequence Listing Free Text]
SEQ ID NO: 3—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 4—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 5—Description of artificial sequence: synthetic DNA
SEQ ID NO: 6—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 7--Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 8—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 9—Description of Artificial Sequence: Synthetic DNA
[Brief description of the drawings]
FIG. 1 is a photograph showing the results of SDS-polyacrylamide gel electrophoresis of a gentian antibacterial protein.
FIG. 2 is a graph showing the antifungal activity of gentian antibacterial proteins.
FIG. 3 is a photograph showing the results of Southern blot analysis of the gentian peroxiredoxin gene.
FIG. 4 is a graph showing the antibacterial activity of gentian peroxiredoxin expressed in E. coli.
FIG. 5 is a diagram showing the construction of a vector for producing a plant transformant.

Claims (9)

The following antibacterial protein (a) or (b).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having antibacterial activity. A gene encoding the following antibacterial protein (a) or (b).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having antibacterial activity. A gene comprising DNA consisting of the base sequence represented by SEQ ID NO: 1 .   A recombinant vector containing the gene according to claim 2 or 3.   A transformant obtained by introducing the gene according to claim 2 or 3.   The transformant according to claim 5, wherein the transformant is a plant.   A method for producing an antibacterial protein, comprising culturing the transformant according to claim 5 or 6 and collecting the antibacterial protein from the resulting culture.   A transformed plant imparted with disease resistance by introducing the gene according to claim 2 or 3.   An antibacterial agent comprising the antibacterial protein according to claim 1 as an active ingredient.

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