JP3590832B2 - Antipathogenic filamentous fungal plants and their production methods - Google Patents

Description

【００７２】
ＰＳＰまたはＰＳＳで形質転換された形質転換植物は、Ｒｈｉｚｏｃｔｏｎｉａ ｓｏｌａｎｉ（キュウリ立ち枯れ病菌）に対して抵抗性を示した。表中の括弧内の数字は、抵抗性個体の中でも正常な緑色を示し、特に旺盛な生育を続けている個体数を示す。
【００７３】
（実施例７ Ｓａｒｃｏｔｏｘｉｎ１ａ遺伝子導入タバコにおけるＰｙｔｈｉｕｍ ａｐｈａｎｉｄｅｒｍａｔｕｍ（タバコ舞病菌）抵抗性）
ＰＳＰまたはＰＳＳを有するアグロバクテリウムで形質転換した形質転換タバコ、および対照実験として３５Ｓ−ＧＵＳを形質転換したタバコを自家受粉することにより得られた種子を、カナマイシン５０μｇ／ｍｌを含む培地に播種し、２０日経過した草丈約８ｍｍの幼植物を使用した。ＭＳ寒天培地を含む直径９ｃｍのシャーレにこの幼植物３０本を均一に植えて、１日間置いた。次いでＰｙｔｈｉｕｍ ａｐｈａｎｉｄｅｒｍａｔｕｍ（タバコ舞病菌）を前培養して得られる菌そうを３ｍｍ角に切り取り、シャーレ当たり７片を均等に寒天上に置くことにより菌を接種し、２２℃に保温した。接種後１４日目に緑色を示して生存している個体を抵抗性植物体とした。結果を表２に示す。
【００７４】
【表２】

【００７５】
ＰＳＰまたはＰＳＳで形質転換された形質転換植物は、Ｐｙｔｈｉｕｍ ａｐｈａｎｉｄｅｒｍａｔｕｍ（タバコ舞病菌）に対して抵抗性を示した。表中の括弧内の数字は、抵抗性個体の中でも正常な緑色を示し、特に旺盛な生育を続けている個体数を示す。
【００７６】
（実施例８ Ｓａｒｃｏｔｏｘｉｎ１ａ遺伝子導入タバコにおけるＰｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ（疫病菌）抵抗性）
ＰＳＰまたはＰＳＳを有する形質転換当代の植物およびコントロール植物の葉の表面の６箇所に、それぞれ小薬匙の背（幅６ｍｍ程度のステンレス板状）でこすることにより傷を付けた。前培養したＰｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ（疫病菌）の菌そうが広がった平板培地を約３ｍｍ角の大きさに切ったものを傷を付けた部位に置き、これを小薬匙でこすりつけることによって菌を接種した。一定期間後にそれぞれの接種部位の病徴（褐色病斑の大きさ）の程度を調査し、６箇所の合計をもってその植物の病徴指数とした。
【００７７】
病斑の評価を以下の数字で表した。
【００７８】
０ 病斑は見られない。
【００７９】
１ 直径が５ｍｍ以下の褐色の病斑あり。
【００８０】
２ 直径が５ｍｍ−１０ｍｍの褐色の病斑あり。
【００８１】
３ 直径が１０ｍｍ−２０ｍｍの褐色の病斑あり。
【００８２】
４ 直径が２０ｍｍ以上の褐色の病斑あり。
【００８３】
このとき高い抵抗性を示した形質転換タバコ６個体、およびコントロール植物の結果をそれぞれ図１２に示す。 Ｓａｒｃｏｔｏｘｉｎ１ａ遺伝子が導入された植物の中からＰｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ（疫病菌）による感染に高い抵抗性を示すものが得られた。特に、ＰＳＳ植物においては全く病徴を示さないものが２個体見られた。
【００８４】
【発明の効果】
植物に抗細菌性ペプチドをコードする遺伝子を導入することにより、抗細菌性のみならず、病原性糸状菌に対しても耐性を有する植物の提供が可能となった。
【００８５】
【配列表】
【００８６】
【配列番号：１】

【００８７】
【配列番号：２】

【００８８】
【配列番号：３】

【００８９】
【配列番号：４】

【００９０】
【配列番号：５】

【００９１】
【配列番号：６】

【００９２】
【配列番号：７】

【００９３】
【配列番号：８】

【図面の簡単な説明】
【図１】タバコＰＲ１ａ遺伝子のゲノミッククローンを有するプラスミドｐＰＲ−γを示す図である。
【図２】プライマーＳＰＲ１ａ３１の置換された配列を示す図である。
【図３】プライマーＴＰＲ１ａ５１の置換された配列を示す図である。
【図４】プライマーＭＳＡＲＣＯ５１の配列を示す図である。
【図５】プライマーＭＳＡＲＣＯ３１の置換された配列を示す図である。
【図６】本願発明の組換え遺伝子の作成を説明する図である。
【図７】バイナリーベクターｐＴＲＡ４１５およびｐＴＲＡ４１５（Ｒ）を示す図である。
【図８】本願発明に用いた各プラスミドＰＳＰ、ＰＳＳを示す図である。
【図９】試験管内におけるＳａｒｃｏｔｏｘｉｎ１ａの抗−Ｆｕｓａｒｉｕｍ ｏｘｙｓｐｏｒｕｍ Ｆ−３（抗ホウレンソウ萎凋菌）試験の結果を示す。
【図１０】試験管内におけるＳａｒｃｏｔｏｘｉｎ１ａの抗−Ｒｈｉｚｏｃｔｏｎｉａ ｓｏｌａｎｉ ＡＧ−４ １２７２（抗ホウレンソウ株腐病菌）試験の結果を示す。
【図１１】試験管内におけるＳａｒｃｏｔｏｘｉｎ１ａの抗−Ｒｈｉｚｏｃｔｏｎｉａ ｓｏｌａｎｉ（抗キュウリ苗立ち枯れ病菌）試験の結果を示す。
【図１２】形質転換された植物のＰｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ（疫病菌）抵抗性を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a plant having a gene encoding an antibacterial peptide derived from a dipteran insect and having resistance to pathogenic fungi.
[0002]
[Prior art]
Studies have been conducted to introduce a gene encoding an antibacterial peptide into a plant to impart resistance to bacteria. In particular, it is disclosed that Sarcotoxin1a (Japanese Unexamined Patent Publication No. 7-250686), an antibacterial peptide derived from dipteran insects, has been expressed and has become antibacterial. However, a plant having an antibacterial peptide derived from a dipteran insect having resistance to an antipathogenic filamentous fungus and having antifungal resistance has not yet been known.
[0003]
[Problems to be solved by the invention]
The present invention is intended to solve the above-mentioned problems, and an object of the present invention is to provide a plant that is resistant to pathogenic fungi by using a pathogen-resistant protein as a foreign protein. Is to do.
[0004]
[Means for Solving the Problems]
The present invention relates to a plant having a gene encoding an antibacterial peptide and having resistance to pathogenic fungi.
[0005]
In a preferred embodiment, the pathogenic fungi are Rhizoctonia solani (Cucumber blight fungus), Pythium aphanidermatur (Tobacco fungus) and Phytophthora infestans (Phytophthora).
[0006]
In a preferred embodiment, the antibacterial peptide is from a dipteran insect.
[0007]
In a preferred embodiment, the antibacterial peptide derived from the dipteran insect is Sarcotoxin1a.
[0008]
In a preferred embodiment, a gene encoding the antimicrobial peptide derived from the dipteran insect is a recombinant gene having the gene, an expression cassette in which the recombinant gene is linked to a plant gene promoter, and It has been introduced into plants in a form selected from the group consisting of an expression vector composed of the expression cassette and a drug resistance gene linked to a plant promoter that is constitutively expressed.
[0009]
In a preferred embodiment, the recombinant gene is a recombinant gene in which a gene encoding the antimicrobial peptide derived from the dipteran insect and a plant gene are linked via the hinge region of tobacco chitinase.
[0010]
In a preferred embodiment, Sarcotoxin1a is linked to a signal sequence of a plant protein.
[0011]
In a preferred embodiment, the plant gene promoter is an inducible tobacco PR1a gene promoter.
[0012]
In a preferred embodiment, the expression cassette has a terminator derived from the tobacco PR1a gene.
[0013]
In a preferred embodiment, the expression vector further has a T-DNA region and a drug resistance gene.
[0014]
In a preferred embodiment, the drug resistance gene is expressed by the cauliflower mosaic virus 35S promoter.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0016]
The term "plant" as used herein includes both monocotyledonous plants and dicotyledonous plants. Particularly preferred plants include tobacco, citrus, Chinese cabbage, lettuce, peach, rice, potato, tomato, barley, wheat and apple.
[0017]
As used herein, the term “plant gene promoter” refers to a known promoter that is expressed in plants. For example, a promoter whose expression is induced by a certain stress, such as a promoter of the tobacco infection-specific protein PR1a (hereinafter, referred to as tobacco PR1a promoter), a cauliflower mosaic virus 35S promoter (hereinafter, referred to as CaMV35S promoter), a nopaline synthase Examples include, but are not limited to, promoters. An enhancer can be used for high expression. As the enhancer, an enhancer region containing the sequence upstream of the 35S promoter is preferable. A plurality of enhancers can be used.
[0018]
As used herein, the term "plant gene" refers to a gene contained in a plant. Any gene can be used as the plant gene. Suitable examples include, but are not limited to, the tobacco infection-specific protein PR1a gene.
[0019]
As used herein, the term "hinge region of tobacco chitinase" refers to a region that separates different functional regions (chitin-binding region and catalytic region) in tobacco chitinase protein. H. Shinshi et al., Plant Mole. Biol. 14, 357-368 (1990). The presence of the hinge region of tobacco chitinase at the junction between the plant gene and the foreign gene is expected to avoid steric hindrance of each protein.
[0020]
As used herein, the term "foreign gene" refers to a gene that encodes a protein that is heterologous to the host plant. Plant genes, genes of microorganisms such as bacteria and yeasts, genes of insects, genes of animals and the like can be suitably used. For example, when breeding disease-resistant plants, an antimicrobial peptide can be used as a foreign gene.
[0021]
For example, it refers to a gene encoding an antimicrobial peptide derived from a dipteran insect, but is not limited thereto. The antibacterial peptides derived from dipteran insects are preferably, but not limited to, Sarcotoxin1a, zapesin, or antifungal proteins derived from the common fly. A signal sequence can be added to the foreign gene to be introduced into the expression cassette, and the resulting fusion protein can be secreted.
[0022]
Sarcotoxin1a gene sequence is described in Biochem. J. 239, 717 (1986).
[0023]
A known gene recombination technique can be used to create a recombinant gene of the present invention in which a foreign gene and a plant gene are linked via the hinge region of tobacco chitinase.
[0024]
As used herein, the term "expression cassette" refers to the expression of the above-mentioned recombinant gene and various regulatory elements such as promoters and enhancers that regulate its expression in a host cell so that the above-mentioned recombinant gene can be expressed. A nucleic acid sequence that is operably linked. The expression cassette can be constructed according to a conventional method. A suitable promoter, such as the tobacco PR1a promoter, is linked to the recombinant gene. The tobacco PR1a promoter is known as an inducible and high-expression promoter. More preferably, a terminator can be linked to the recombinant gene.
[0025]
As used herein, the term "terminator" is a sequence that is located downstream of a protein-encoding region of a gene and is involved in terminating transcription when DNA is transcribed into mRNA and adding a polyA sequence. It is known that a terminator is involved in the stability of mRNA and affects the expression level of a gene. Examples of the terminator include, but are not limited to, cauliflower mosaic virus 35S terminator, a nopaline synthase gene terminator, and a tobacco PR1a gene terminator. It has been confirmed that the tobacco PR1a gene terminator enhances gene expression efficiency.
[0026]
As used herein, the term "expression vector" refers to a medium that transfers the above expression cassette into a host cell. It is well known to those skilled in the art that the type of expression vector and the type of regulatory element used can vary depending on the host cell. The expression vector of the present invention may further have a T-DNA region and a drug resistance gene.
[0027]
As the drug resistance gene, it is desirable that selection of a transformed plant is easy. As the drug resistance gene, neomycin phosphotransferase (NPT) gene, hygromycin phosphotransferase gene and the like can be suitably used.
[0028]
Examples of a promoter for expressing a drug resistance gene include, but are not limited to, the above-mentioned plant gene promoters such as a tobacco PR1a promoter, a CaMV35S promoter, and a nopaline synthesis promoter. Preferably, a CaMV35S promoter that is constitutively expressed at a high level can be used. When an inducible tobacco PR1a promoter is used as a promoter for expressing a foreign gene in the expression cassette, the inducible PR1a promoter is constitutively expressed by the CaMV35S promoter, and necrotic lesions associated with salicylic acid or pathogen infection are used. Formation increases the expression of the PR1a promoter. This is probably because the CaMV 35S promoter and the PR1a promoter are adjacent to each other, so that the properties of the PR1a promoter are affected by the cis element on the 35S promoter.
[0029]
As a vector used for construction of an expression vector, a pBI-based vector, a pUC-based vector, or a pTRA-based vector can be suitably used. A pBI-based vector can introduce an expression cassette into a plant via Agrobacterium such as a binary vector system or an intermediate vector system. For example, pBI121, pBI101, pBI101.2, pBI101.3 and the like can be mentioned. A pUC-based vector can directly introduce an expression cassette into a plant. For example, pUC18, pUC19, pUC9 and the like can be mentioned. In the present application, a binary vector of the pTRA system (Ohshima et al., Plant Cell 2: 95-106 (1990)) can be suitably used. This expression vector contains an expression cassette containing a fusion gene in a region (T-region) that can be introduced into a plant, and a NPT gene expressed under the control of the CaMV35S promoter as a marker gene.
[0030]
When the expression vector of the present invention has a T-region, the structure of the T-region is as follows:
(1) When transferring from Agrobacterium to a plant, an expression cassette containing the foreign gene is arranged on the right side of the T-region so that the foreign gene to be introduced transfers first, and then the marker gene transfers. obtain. Thus, transgenic plants selected for drug resistance can contain a foreign gene with a very high probability, and
(2) The CaMV35S promoter that controls the expression of the marker gene and the PR1a gene promoter that controls the expression of the foreign gene are arranged side by side. Thus, when a foreign gene was originally expressed in a plant using the PR1a promoter, almost no expression was observed under non-inducible conditions, but it probably showed constitutive expression even in a non-inducible state due to the influence of the CaMV35S promoter, and It has the characteristic that it will show a high level of expression upon induction.
[0031]
When the PR1a promoter is used, the expression level of the above expression vector is inducibly increased by the formation of necrotic lesions associated with salicylic acid or pathogen infection.
[0032]
The expression vector of the present invention can be prepared using a gene recombination technique well known to those skilled in the art.
[0033]
Plasmid PST10 expressing Sarcotoxin1a, described in JP-A-7-250685, can also be used in the present invention.
[0034]
For introducing a recombinant gene, an expression cassette, or an expression vector into a plant cell, a method well known to those skilled in the art can be used.
[0035]
In order to introduce the obtained expression cassette or expression vector into cells, there are a method via Agrobacterium as described above and a method directly into cells.
[0036]
As a method via Agrobacterium, for example, the method of Nagel et al. (Microbiol. Lett., 67, 325 (1990)) can be used. In this method, first, for example, an expression vector is transformed into Agrobacterium by electroporation, and then the transformed Agrobacterium is transformed into a plant Molecular Biology Manual (SB Gelvin et al., Academic Press Publishers). And a method of introducing into plant cells by the method described in 1. Methods for directly introducing an expression cassette and an expression vector into cells include an electroporation method and a gene gun method.
[0037]
Cells into which the expression cassette and the expression vector have been introduced can be first selected for drug resistance such as kanamycin resistance, and then regenerated into a plant by a conventional method.
[0038]
For confirmation of the expression of a foreign gene in a transformed plant, a method well known to those skilled in the art can be used. For example, a soluble protein is extracted from a transformed plant, and the protein extracted by SDS-polyacrylamide gel electrophoresis is separated and transferred to a PVDF membrane, as described in Analytical Biochemistry 166, 368-379. Expression can be confirmed by reacting with an antibody against the target protein and immunochemically detecting the resulting band.
[0039]
Whether the plant transformed with the gene encoding the antibacterial peptide derived from the dipteran insect of the present invention can have resistance to pathogenic fungi depends on whether the plant was regenerated into a transformed plant and a control. The plants can be determined using seedlings, each of which is self-pollinated and germinated under drug-resistant selection. Pathogens were inoculated on the agar medium on which the young plants grew, and after an appropriate period of time had elapsed after inoculation, individuals that showed a green color and survived were determined to be resistant plants. Examples of the pathogenic bacteria include Fusarium oxysporum F-3 (spinach wilt fungus), Rhizoctonia solani (cucumber seedling wilt), Rhizoctonia solani AG-41272 (spinach rot fungi), and Phythia germii (Phithia seri) and Phythia germii (Phythia sulphia) and Phythia m.
[0040]
The transformed plants are tested for resistance to pathogenic fungi by injuring the green leaves of the transformed plants and control plants, which have been regenerated into plants, and inoculating them by rubbing the pathogens. It can also be done by assessing the size of the plaque. Examples of pathogenic bacteria include Phytophthora infestans (Blight fungus), Botrytis cinerea (Grey mold fungus), and the like.
[0041]
In a test tube, an antifungal experiment of Sarcotoxin1a was performed by inoculating a test medium containing Sarcotoxin1a with a bacterial path obtained by pre-culturing the pathogenic bacterium and measuring the size of hyphae that elongate under transmitted light at regular intervals. And calculating the area together with the standard deviation.
[0042]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples. Restriction enzymes, plasmids, and the like used in this example can be obtained from Takara Shuzo Co., Ltd., Toyobo Co., Ltd., or the like.
[0043]
(Example 1 Preparation of recombinant gene and expression cassette)
A recombinant gene in which a foreign gene and a plant gene are linked via the hinge region of tobacco chitinase was prepared as follows. A description will be given based on FIG.
[0044]
(1) Preparation of P + S PR1a Fragment (DNA Fragment Having Part of PR1a Promoter and Sequence Encoding Signal Peptide of PR1a Protein)
Using the plasmid pPR-γ having the genomic clone of the tobacco PR1a gene shown in FIG. 1 (M. Oshima, et al., FEBS Letters 255, 243-246 (1987)) as a template, a signal peptide was transferred from the XhoI site in the PR1a promoter region. The region up to the coding region was amplified to obtain a DNA fragment (P + SPR1a fragment). The P + SPR1a fragment is a fragment obtained by PCR using Pfu polymerase (manufactured by Stratagene) using primer PPR1a51 having the sequence of SEQ ID NO: 1 and primer SPR1a31 having the sequence of SEQ ID NO: 2 as primers. Among the primers used in this PCR, SPR1a31 has a sequence complementary to the sequence encoding the signal peptide of the PR1a gene, but as shown in FIG. 2, bases are partially substituted. Although the restriction enzyme site Pst1 was newly introduced by this base substitution, the encoded amino acid sequence was not changed.
[0045]
(2) Preparation of TPR1a fragment (DNA fragment having a terminator region of PR1a gene)
Using the plasmid pPR-γ having the genomic clone of the tobacco PR1a gene in FIG. 1 as a template, a DNA fragment (TPR1a fragment) having a terminator region of the PR1a gene was obtained. The TPR1a fragment is a DNA fragment obtained by amplifying the terminator region of PR1a by PCR using the primer TPR1a51 of SEQ ID NO: 3 and the primer BKSBE of SEQ ID NO: 4. Among the primers used in this PCR, TPR1a51 has a sequence homologous to the 3 ′ end of cDNA encoding Sarcotoxin 1a at the 5 ′ end, and has a SacI recognition sequence immediately after its termination codon. Immediately before the stop codon, a base is partially substituted as shown in FIG. Although the restriction enzyme recognition site PmaCI was newly introduced by this base substitution, the encoded amino acid sequence was not changed. On the other hand, the primer BKSBE has a sequence homologous from the EcolI site to the BamHI site in the vector portion of pPR-γ. This site is adjacent to the 3 ′ end of the portion corresponding to the terminator of the PR1a gene of pPR-γ.
[0046]
(3) Preparation of an HMPR1a fragment (a DNA fragment having a hinge region of tobacco chitinase and a region encoding a mature PR1a protein)
Using the plasmid pPR-γ having the genomic clone of the tobacco PR1a gene in FIG. 1 as a template, a DNA fragment (HMPR1a fragment) having a region encoding the mature PR1a protein was obtained. The H MPR1a fragment is a fragment that has been amplified by PCR using the primer HMPPR1a51 of SEQ ID NO: 5 and the primer MPR1a31 of SEQ ID NO: 6. Among the primers used for this PCR, HMPR1a51 contains, at the 5 ′ end, the same sequence as the PmaCI site of TPR1a51 and the sequence of 12 bases around it, and further, on the 3 ′ side, a region encoding the hinge region of tobacco chitinase. Has the same sequence as
[0047]
MPR1a31 has a recognition site for the restriction enzyme SacI added immediately after the 3 ′ stop codon.
[0048]
(4) Preparation of MSARCO Fragment (DNA Fragment Having Region Encoding Sarcotoxin Mature Peptide)
The known plasmid pTO19 containing the cDNA of Sarcotoxin1a (N. Matsumoto et al., BioChem. J., 239 717 (1986)) as a template to obtain a DNA fragment (MSARCO fragment) having a region encoding Sarcotoxin mature peptide. This MSARCO fragment is a DNA fragment obtained by amplifying the region encoding the mature peptide of Sarcotoxin by PCR using the primer MSARCO51 of SEQ ID NO: 7 and the primer MSARCO31 of SEQ ID NO: 8. The sequences of primers MSARCO51 and MSARCO31 are shown in FIGS. 4 and 5, respectively. The primer MSARCO51 has a sequence complementary to the PstI site of SPR1a31 and its surrounding 14 bases near the 5 ′ end. As shown in FIG. 5, MSARCO31 has recognition sites for restriction enzymes PmaCI and SacI introduced around a sequence complementary to a region encoding a stop codon. This restriction enzyme site was introduced in the same manner as in TPR1a51.
[0049]
(5) Preparation of MSARCO-TPR Fragment (DNA Fragment Linking MSARCO Fragment and TPR1a Fragment) Using MSARCO DNA fragment obtained in (4) above and TPR1a fragment obtained in (2) as templates, primers MSARCO31 and Amplification was performed by a recombinant PCR method using BKSBE to obtain a MSARCO-TPR fragment.
[0050]
(6) Preparation of recombinant gene (connection of P + S PR1a fragment, H MPR1a fragment and MSARCO-TPR1a fragment)
The P + SPR1a fragment obtained in (1) above, the HMPR1a fragment obtained in (3), and the MSARCO-TPR1a fragment which is a binding fragment of (2) and (4) obtained in (5) were obtained, respectively. And cloned into plasmid pUC18 and named pUC P + SPR1a, pUC H MPR1a and pUC MSARCO-TPR1a, respectively (see FIG. 6). Correct insertion of the target sequence into these plasmids was confirmed using a base sequence determination kit and a base sequence analyzer manufactured by Applied Biosystems. These DNAs cloned into pUC18 and confirmed for the nucleotide sequence were used as materials for subsequent genetic recombination. SEQ ID NO: 7 shows the nucleotide sequence of the sequenced PR1a terminator.
[0051]
Hereinafter, the production of the recombinant gene of the present invention will be described with reference to FIG.
[0052]
First, the upstream (5 ′ side) portion of the XhoI site of the promoter region of the PR1a gene of the plasmid pPR-γ was cut with restriction enzymes EcoRI and XhoI, and the obtained about 1.6 kb fragment was used as a Bio 101 Gene product. Purified by Clean kit. On the other hand, the plasmid pUCP + SPR1a in which the P + SPR1a fragment was cloned was cut with EcoRI-XhoI, and the above purified fragment of about 1.6 kb was inserted into the cut site. The resulting plasmid (pUCP + SPR (2.4)) contains as inserts a full-length PR1a gene promoter and a DNA region encoding the signal sequence of the PR1a gene. This plasmid has a cassette of the tobacco PR1a promoter, which is excised with EcoRI.
[0053]
pUC MSARCO-TPR1a was digested with restriction enzyme PstI, and a DNA fragment corresponding to MSARCO-TPR1a was purified. This fragment was inserted into the PstI site of pUC P + SPR (2.4). A plasmid (pUCP + SPR (2.4) MSARCO-TPR1a) in which the inserted DNA sequence was correctly inserted in the direction of P + SPR1a-MSARCO-TPR1a was selected from the obtained plasmids. This plasmid contains a cassette (cassette PSS) of a gene encoding an expressible non-fused Saracotoxin, excised with EcoRI and driven by the tobacco PR1a promoter.
[0054]
Next, the H MPR1a portion was cut out from the plasmid pUC H MPR1a with the restriction enzymes PmaCI and SacI. On the other hand, pUC P + SPR (2.4) MSARCO-TPR1a was cut with PmaCI-SacI, and the excised HMPR1a portion was inserted into the site (pUCP + SPR1a (2.4) MSARCO HMPR1a TPR). This plasmid comprises a recombinant gene of the present invention in which a gene of Sarcotoxin1a which is an antimicrobial peptide derived from a dipteran insect as a foreign gene and a tobacco PR1a protein gene as a plant gene are linked via a hinge region of tobacco chitinase. It contains. Furthermore, this plasmid has a recombinant gene encoding a Sarcotoxin1a-PR1a fusion protein controlled by the tobacco PR1a promoter. When this plasmid is cut with EcoRI, the expressible Sarcotoxin1a controlled by the tobacco PR1a promoter is obtained. -Hinge-PR1a has a recombinant gene encoding a fusion protein, P + S PR1a (2.4) MSARCO H MPR1a TPR, that is, an expression cassette (cassette PSP) for expressing the recombinant gene of the present invention.
[0055]
Example 2 Construction of Expression Vector
The binary vector pTRA415 (FIG. 7) (Ohshima et al., Plant Cell, Vol. 2, pp. 95-106 (1990)) has a BglII-BglII fragment having a sequence in which an NPT gene is bound downstream of the CaMV35S promoter. According to the method described in Japanese Patent Application No. 8-68809, this pTRA415 was cut with BglII, religated with ligase, introduced into Escherichia coli JM109, and the BglII-BglII fragment of pTRA415 was inserted in the opposite direction. The prepared plasmid (pTRA415 (R)) was prepared. pTRA415 (R) has an NPT gene governed by the CaMV35S promoter as a marker gene in the T-region, and EcoRI as a (unique) restriction enzyme cleavage site that cleaves only one site in pTRA415 (R) on the right side thereof. Has a part. When a foreign gene is introduced into the EcoRI site, the marker gene is transferred after the transfer of the foreign gene into a plant.
[0056]
The above plasmid pUC P + SPR1a (2.4) MSARCO TPR (plasmid expressing non-fused Sarcotoxin) was cut with EcoRI, the cassette PSS was cut out, and inserted into the EcoRI site of pTRA415 (R). From the obtained plasmids, those in which the PR1a promoter of the inserted expression cassette was inserted so as to be adjacent to the CaMV35S promoter of the marker gene were selected (plasmid PSS).
[0057]
Similarly, the plasmid pUC P + S PR1a (2.4) MSARCO H MPR1a TPR (plasmid expressing the fusion Sarcotoxin-PR1a protein) was cut with EcoRI, the cassette PSP was cut out and inserted into the EcoRI site of pTRA415 (R). From the obtained plasmids, those in which the PR1a promoter of the inserted expression cassette was inserted so as to be adjacent to the CaMV35S promoter of the marker gene were selected (plasmid PSP). FIG. 8 shows a schematic diagram of the PSP and PSS plasmids.
[0058]
(Example 3 Introduction of expression vector into tobacco)
(1) Transformation of Agrobacterium tumefaciens
Agrobacterium tumefaciens was cultured at 28 ° C. in a medium containing 250 μg / ml streptomycin and 50 μg / ml rifampicin to prepare a cell culture solution according to the method of Nagel et al. (Microbiol. Lett., 67, 325 (1990)). Expression vectors (plasmid PSP and plasmid PSS) were introduced into the bacteria by electroporation. FIG. 8 shows a schematic diagram of the PSP and PSS plasmids used.
[0059]
(2) Transformation of tobacco
Agrobacterium transformed with the plasmid PSP and the plasmid PSS was obtained by the above method, shake-cultured in a YEB medium (DNA cloning, volume 2, p. 78), diluted 20-fold with sterile water, and tobacco (Nicotiana tabacum Samsun). NN) leaf pieces were co-cultured. Two to three days later, the above bacteria are removed with a medium containing antibiotics, subcultured with a selection medium every two weeks, transformed tobacco cells are selected, and as a result of redifferentiation by a standard method, independent transformed individuals are obtained. Obtained.
[0060]
Similarly, a transformed individual transformed with the plasmid PSS was obtained.
[0061]
Example 4 Detection of Sarcotoxin1a Expression in Transformed Plants by Western Blotting
Soluble protein extracts were respectively prepared from the obtained tobacco-transformed plants transformed with PSP and PSS. Four leaf pieces of about 7 mm in diameter were cut out from tobacco leaves, and cultured for 2 days in a suspended state on a 0.5 mM aqueous solution of salicylic acid and pure water as a control, and then 20 μl of 50 mM Na-PO4 (pH 7.0). ), 1 mM Na-EDTA, 10 μM A-PMSF, 0.5 μg / ml Leupeptin, 2 mM DTT, and centrifuged at 10,000 rpm for 10 minutes to recover the supernatant to obtain a protein extract. 2.5 μl of this extract was separated by SDS-polyacrylamide gel electrophoresis represented by Analytical Biochemistry 166, 368-379, transferred to a PVDF membrane, and the band reacting with the anti-Sarcotoxin antibody was subjected to immunochemical analysis. Was detected. This confirmed that the Sarcotoxin gene was expressed in the transformed plant and that Sarcotoxin was synthesized.
[0062]
Example 5 Measurement of Sarcotoxin1a Antifungal Activity in Test Tube
Measurement of Sarcotoxin1 activity of Sarcotoxin fungi against Fusarium oxysporum F-3 (spinach wilt fungus), Rhizoctonia solani (cucumber seedling wilt fungus) and Rhizoctonia solani AG-41272 (spinach rot fungus).
[0063]
Since Sarcotoxin1a has an antifungal activity and its measuring method is not known, first, various preculture media and assay media were examined. As the preculture medium, a medium in which the bacteria were uniformly expanded in a short period of time was selected, and as the test medium, a medium that suppressed mycelial growth and was easy to confirm was selected.
[0064]
(1) Preparation of assay plate
The following three types of culture media were prepared as assay media.
[0065]
(I) Czapek modified medium (per liter: 10 g sucrose, 0.5 g MgSO ₄ ・ 7H ₂ O, 2.0g NaNO ₃ , 0.01g Fe ₂ (SO ₄ ) ₃ , 1.0g K ₂ HPO ₄ 0.5 g KCl, 15 g agarose, pH 6.8-7.0; used in Rhizoctonia solani AG-4 1272 (spinach rot fungus), (ii) 5-fold diluted PDA medium (per liter: 7.8 g Bacto) (Registered Trademark) Poteto Dextrose Agar, 12 g agarose; used in Fusarium oxysporum F-3 (spinach wilt fungus), (iii) Na ₂ HPO ₄ -NaH ₂ PO ₄ Buffer (15 g / l agarose, pH 5.8; Rhizoctonia solani (used in cucumber seedling blight fungus)).
[0066]
The assay medium was autoclaved, and when the temperature dropped to 50 ° C. or lower, Sarcotoxin1a (a sample synthesized by peptide synthesis and purified to 99% or more by HPLC) was added, and 400 μl per well was injected into a 12-well microplate. And solidified. The final concentration of Sarcotoxin1a is as described in FIGS.
[0067]
(2) Inoculation of pathogenic fungi
(A) Pre-culture
Fusarium oxysporum F-3 (spinach wilt fungus), Rhizoctonia solani (cucumber seedling wilt fungus), and Rhizoctonia solani AG-41272 (spinach rot fungus) in a PDA medium (39 g / g / g to AxeroBacTog). Pre-cultured at 22 ° C for days.
[0068]
(B) Inoculation of pathogenic fungi
The outermost mycelia of each hypha spread almost entirely on the medium surface of a 9 cm petri dish were punched out with a 4 mm diameter cork borer, and the mycelial surface was placed down in the center of the well of the assay plate. The assay plate was incubated at 22 ° C. in the dark.
[0069]
(C) Assay for antifungal fungi
The size of the mycelium that elongates under the transmitted light was measured at regular intervals on the assay plate inoculated with the above pathogenic filamentous fungi, and the area thereof was calculated together with the standard deviation (FIGS. 9, 10 and 10). 11). As shown in these figures, the antifungal activity of Sarcotoxin1a was concentration-dependent. Therefore, it was presumed that Sarcotoxin1a was effective for filamentous fungi.
[0070]
(Example 6 Rhizoctonia solani (Cucumber blight resistance) in Sarcotoxin1a transgenic tobacco)
Seeds obtained by self-pollinating transgenic tobacco transformed with Agrobacterium having PSP or PSS, and tobacco transformed with 35S-GUS as a control experiment, were sown in a medium containing 50 μg / ml of kanamycin, Young plants having a plant height of about 8 mm after 20 days were used. MS agar medium (Murashige, T. and Skoog, F. (1962) A reversed medium for rapid growth and bioassays with tobacco tissocculature. Thirty plants were evenly planted and left for 3 days. Then, a fungus obtained by pre-culturing Rhizoctonia solani (cucumber blight fungus) was cut into 3 mm squares, and the bacteria were inoculated by uniformly placing 7 pieces per agar dish on agar, and keeping the temperature at 22 ° C. On the 6th day after the inoculation, surviving individuals showing green color were defined as resistant plants. Table 1 shows the results.
[0071]
[Table 1]

[0072]
Transgenic plants transformed with PSP or PSS showed resistance to Rhizoctonia solani (cucumber blight fungus). The numbers in parentheses in the table indicate normal green color among the resistant individuals, and particularly indicate the number of individuals that continue to grow vigorously.
[0073]
(Example 7 Pythium aphanidermatum (tobacco fungus) resistance in Sarcotoxin1a transgenic tobacco)
Seeds obtained by self-pollinating transgenic tobacco transformed with Agrobacterium having PSP or PSS and, as a control experiment, tobacco transformed with 35S-GUS were sown in a medium containing 50 μg / ml of kanamycin. A young plant having a plant height of about 8 mm after 20 days was used. 30 young plants were uniformly planted in a 9 cm diameter petri dish containing MS agar medium and left for 1 day. Then, the bacteria obtained by pre-culturing Pythium aphanidermatum (tobacco fungus fungus) were cut into 3 mm squares, and the bacteria were inoculated by placing evenly 7 pieces per petri dish on agar, and kept at 22 ° C. On the 14th day after the inoculation, the surviving individuals, which showed a green color, were defined as resistant plants. Table 2 shows the results.
[0074]
[Table 2]

[0075]
Transformed plants transformed with PSP or PSS showed resistance to Pythium aphanidermatum. The numbers in parentheses in the table indicate normal green color among the resistant individuals, and particularly indicate the number of individuals that continue to grow vigorously.
[0076]
(Example 8 Phytophthora infestans (Phytophthora) resistance in Sarcotoxin1a transgenic tobacco)
At six places on the surface of the leaves of the transgenic plants having PSP or PSS and the control plant, scratches were made by rubbing each with the spine of a small spoon (stainless steel plate having a width of about 6 mm). Pre-cultured Phytophthora infestans (Phytophthora infestans) is spread on a plate medium with a spread of about 3 mm square, placed on the damaged site, and rubbed with a small spoon to inoculate the bacteria did. After a certain period of time, the degree of the symptom (the size of the brown lesion) at each inoculation site was examined, and the total of the six points was used as the symptom index of the plant.
[0077]
The evaluation of lesions was represented by the following numbers.
[0078]
0 No lesion is seen.
[0079]
1 There are brown lesions with a diameter of 5 mm or less.
[0080]
2 There are brown lesions with a diameter of 5 mm to 10 mm.
[0081]
3 There are brown lesions with a diameter of 10 mm to 20 mm.
[0082]
4 There are brown lesions with a diameter of 20 mm or more.
[0083]
FIG. 12 shows the results of 6 transformed tobacco plants showing high resistance and control plants. Among the plants into which Sarcotoxin1a gene had been introduced, those having high resistance to infection by Phytophthora infestans were obtained. In particular, two PSS plants showed no symptom at all.
[0084]
【The invention's effect】
By introducing a gene encoding an antibacterial peptide into a plant, it has become possible to provide a plant that has not only antibacterial properties but also resistance to pathogenic fungi.
[0085]
[Sequence list]
[0086]
[SEQ ID NO: 1]

[0087]
[SEQ ID NO: 2]

[0088]
[SEQ ID NO: 3]

[0089]
[SEQ ID NO: 4]

[0090]
[SEQ ID NO: 5]

[0091]
[SEQ ID NO: 6]

[0092]
[SEQ ID NO: 7]

[0093]
[SEQ ID NO: 8]

[Brief description of the drawings]
FIG. 1 shows a plasmid pPR-γ having a genomic clone of the tobacco PR1a gene.
FIG. 2 is a view showing a substituted sequence of a primer SPR1a31.
FIG. 3 is a view showing a sequence in which a primer TPR1a51 is substituted.
FIG. 4 is a view showing a sequence of a primer MSARCO51.
FIG. 5 is a view showing a substituted sequence of a primer MSARCO31.
FIG. 6 is a diagram illustrating the creation of a recombinant gene of the present invention.
FIG. 7 shows the binary vectors pTRA415 and pTRA415 (R).
FIG. 8 is a diagram showing plasmids PSP and PSS used in the present invention.
FIG. 9 shows the results of an anti-Fusarium oxysporum F-3 (anti-spinach wilt) test of Sarcotoxin1a in a test tube.
FIG. 10 shows the results of an anti-Rhizoctonia solani AG-41272 (anti-spinach rot fungus) test of Sarcotoxin1a in a test tube.
FIG. 11 shows the results of an anti-Rhizoctonia solani (anti-cucumber seedling wilt) test of Sarcotoxin1a in a test tube.
FIG. 12 is a diagram showing Phytophthora infestans resistance of transformed plants.

Claims (5)

A dicotyledon having resistance to a pathogenic filamentous fungus, wherein a gene encoding Sarcotoxin 1a has been introduced in the form of an expression vector,
The expression vector comprises the following a) and b):
a) an expression cassette in which a recombinant gene having the gene encoding Sarcotoxin 1a and a promoter of the tobacco PR1a gene are linked via a DNA sequence encoding a signal peptide; and b) a cauliflower mosaic virus 35S promoter A drug resistance gene linked to
Including
Here, the tobacco PR1a gene promoter and the cauliflower mosaic virus 35S promoter are arranged side by side,
A plant wherein the plant has enhanced pathogenic fungal resistance compared to the corresponding untransformed plant. The plant according to claim 1, wherein the recombinant gene is a recombinant gene in which a gene encoding Sarcotoxin 1a and a plant gene are linked via a hinge region of tobacco chitinase. The expression cassette, the Sarcotoxin 1a was ligated downstream of the gene encoding, it has a terminator from the tobacco PR1a gene, plant of claim 1-2. The plant according to any one of claims 1 to 3 , wherein the expression vector further has a T-DNA region. It said signal peptide is a PR1a signal peptide Plant according to claim 1-4.

Images