JP3964701B2 - Disease resistant gramineous plant - Google Patents

Description

Ａ、Ｂ：キチナーゼＣ遺伝子導入形質転換イネ系統
＋ ：ウェスタン分析によるキチナーゼＣ発現有り
− ：ウェスタン分析によるキチナーゼＣ発現無し
【００５９】
〔配列表の説明〕
配列番号１：キチナーゼＣ遺伝子の塩基配列
配列番号２：キチナーゼＣのアミノ酸配列
配列番号３：イネ由来キチナーゼのシグナルペプチドをコードするＤＮＡの塩基配列
配列番号４：イネ由来キチナーゼのシグナルペプチドのアミノ酸配列
配列番号５：シグナルペプチドとキチナーゼＣとの融合タンパク質のアミノ酸配列
配列番号６：イネ由来キチナーゼ遺伝子のシグナル配列増幅用プライマー
配列番号７：イネ由来キチナーゼ遺伝子のシグナル配列増幅用プライマー
配列番号８：導入遺伝子確認用プライマー
配列番号９：導入遺伝子確認用プライマー
【発明の効果】
本発明の病害抵抗性イネ科植物は、キチナーゼＣ遺伝子を導入されて形質転換されたことにより、イネいもち病等の菌類病に対する抵抗性が上昇し、農業生産上重要なイネ科植物を提供することが出来る。
【００６０】
【配列表】

【００６２】

【００６３】

【００６４】

【００６５】

【００６６】

【００６７】

【００６８】

【００６９】

【図面の簡単な説明】
【図１】 キチナーゼＣ遺伝子導入用プラスミドｐＢＩ−ＥＮ４−ＲＣ２ｓｓ／ＣｈｉＣの構造の模式図。
【図２】 形質転換植物のイネ葉いもち病に対する抵抗性の試験結果を表すグラフ。
【符号の説明】
３５Ｓ：ＣａＭＶ ３５Ｓプロモーター
ＨＰＴ：ハイグロマイシン耐性遺伝子
ＮＯＳ３’：ＮＯＳ遺伝子ターミネーター
ＥＮ４：ＣａＭＶ ３５Ｓプロモーター（エンハンサー４反復）
ＲＣＣ２ＳＳ／ＣｈｉＣ：イネ・キチナーゼ遺伝子のシグナル配列を持つキチナーゼＣキメラ遺伝子
ＲＢ， ＬＢ：Ｔ−ＤＮＡ右側ボーダー配列、左側ボーダー配列
ＮＰＴＩＩＩ：カナマイシン耐性遺伝子
ＮＴ：非形質転換体（コントロール）
Ｐ：キチナーゼＣ遺伝子を含まないプラスミドによる形質転換イネ
Ｃ：Ｎ−ＥＲ２−５５−５−１（イネキチナーゼ高発現形質転換イネ）
１、２，３，４：キチナーゼＣ発現形質転換イネ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a grass family plant that exhibits resistance to various fungal diseases important in agricultural production.
[0002]
[Prior art]
In recent years, attempts have been made to introduce useful genes into plants using genetic engineering techniques to produce fungal disease resistant plants that are resistant to plant fungal diseases. For example, the introduction of chitinase genes into plants has been promoted for the purpose of strengthening the defense mechanism against pathogenic fungi of plant fungal diseases.
[0003]
Various filamentous fungi are known as pathogenic fungi of plant fungal diseases. Filamentous fungi have chitin as one of the main components constituting their cell walls. On the other hand, the plant has an enzyme that degrades chitin, chitinase (EC 3.2.1.14), which acts on chitin in the cell wall of the filamentous fungus to infect the plant, causing infection by these pathogens.・ It is considered to be a defense mechanism to prevent the disease.
[0004]
Focusing on the defense mechanism as described above, disease-resistant plants with enhanced defense mechanisms have been created by introducing chitinase genes derived from various organisms into plants. For example, as an example of introducing a plant-derived chitinase gene: 1. Introduced chitinase gene isolated from kidney beans into tobacco [Science, Vol. 254, pp. 1194-197 (1991)]. 2. Introduced chitinase gene isolated from tobacco into tobacco, carrot, sunflower, tomato, cotton, corn, and camouflage [JP-A-3-35783, JP-A-3-112488] 3. A chitinase gene isolated from the bean Arachis hypogaea introduced into tobacco [Japanese Patent Laid-Open No. 5-76873]; Chitinase gene isolated from tomato introduced into corn, soybean, peat, wheat, barley, poppy, rape, sunflower, alfalfa, rose, carnation, gerbera, tomato, carrot, lettuce, chicory, melon, cabbage, tobacco [ JP-A-6-38762], recombinant rice in which rice class I chitinase (Cht-2) is highly expressed [Theoretical Applied Genetics (Theor. Appl. Genet.), Vol. 99, pp. 383- 390, 1999].
[0005]
On the other hand, examples of introducing a chitinase gene derived from a microorganism include: 1. A chitinase gene isolated from the bacterium Serratia marcescens introduced into tobacco [Transgenic Research, Vol. 3, pp. 90-98 (1994)]. Known is one in which chitinase derived from Rhizopus-oligosporus is introduced into bent glass [Japanese Patent Laid-Open No. 8-172957].
[0006]
However, there has been reported an example in which the disease resistance of the plant body has not been improved despite overexpression of chitinase exhibiting high antifungal activity in the plant cell. For example, even when overexpressing tobacco class I chitinase, the resistance to tobacco white spot disease (Cercospora nicotiana) did not increase (Plant Mol. Biol., Vol. 16, pages 141-151, 1991], an example in which the over-expression of sugar beet class III chitinase in tobacco did not increase the disease resistance of Cercospora nicotiana [Mole. Plant-Microbe Interaction, Vol. 6, 494] Page 506, 1993]. It is also known that when a chitinase gene is expressed in a plant cell, a positive correlation is not always obtained between the chitinase activity in the plant and the disease resistance newly acquired by the plant. ing. [Theoretical Applied Genetics (Theor. Appl. Genet.), 99, 383-390, 1999]. In this way, when trying to enhance disease resistance by introducing a chitinase gene into a plant, expression of a large amount of chitinase exhibiting a high antifungal activity in the plant necessarily increases plant disease resistance. It was speculated that it was not connected.
[0007]
Accordingly, the present inventors have conducted intensive studies to produce disease-resistant gramineous plants by paying attention to the grouping of chitinases apart from the intensity of antifungal activity possessed by chitinases. That is, natural chitinases are roughly divided into two groups, one is a group that can be called family 19 of plant-derived chitinases due to the difference in the conserved region of amino acid sequence, and the other is a chitinase that can be called family 18 including microorganisms. [Takeshi Watanabe Chemistry and Biology, Vol. 35, No. 6, 408, 1997].
[0008]
However, in the process of searching for various chitinase base sequences derived from microorganisms, the chitinase named chitinase C derived from Streptomyces griseus was found to be the only chitinase having a domain structure common to family 19 except for plants. [TSUYOSHI OHNO, TAKESHI WATANABE, J. et al. BACTERIOLOGY, 178, 17, p. 5065, 1996]. Furthermore, as a result of investigating the properties of the chitinase, it was revealed that the chitinase has high antifungal activity [Japanese Patent Laid-Open No. 2000-93182].
[0009]
Thus, microbially derived chitinase C, of course, has a high antifungal activity, but belongs to family 19 which has a conserved region of amino acid sequence closer to plants, unlike microbially derived chitinases that have been used so far. However, an attempt to impart disease resistance by introducing and expressing this chitinase C gene in gramineous plants has not been known yet.
[0010]
[Problems to be solved by the invention]
This invention is made | formed from this viewpoint, and makes it a subject to produce the Gramineae plant which shows resistance with respect to a fungal disease.
[0011]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have intensively studied. As a result, the disease resistance is enhanced by introducing a chitinase C gene belonging to family 19 derived from Streptomyces griseus into a grass family plant. It has succeeded for the first time, and it has become possible to produce a grass family plant that exhibits extremely higher disease resistance than transformed rice plants overexpressed with chitinase produced so far, thereby completing the present invention.
That is, the gist of the present invention is as follows.
[0012]
(1) Gramineae plants that are transformed with DNA encoding the following proteins and have disease resistance:
(A) a protein having the amino acid sequence represented by amino acid numbers 31 to 294 of SEQ ID NO: 2, or
(B) The amino acid sequence represented by amino acid numbers 31 to 294 of SEQ ID NO: 2 has an amino acid sequence including substitution, deletion, or insertion of one or several amino acid residues, and has chitinase activity protein.
(2) The rice protein according to (1), wherein the DNA encoding the protein further encodes a plant extracellular secretion signal peptide having the amino acid sequence shown in SEQ ID NO: 4, and expresses a fusion protein of the signal peptide and the protein. Family plant.
(3) The gramineous plant according to (1) or (2), wherein the gramineous plant is rice.
(4) The gramineous plant according to any one of (1) to (3), wherein the disease is a fungal disease.
(5) The grass family plant according to (4), wherein the fungal disease is rice blast.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The production of the disease resistant gramineous plant of the present invention can be obtained by introducing and transforming DNA encoding chitinase C derived from Streptomyces griseus. The base sequence of DNA encoding the chitinase C is described in Japanese Patent Application Laid-Open No. 2000-93182. The DNA encoding chitinase C is, for example, a PCR method (Am. J. Hum. Genet., 37, 172) using a plasmid pGC01 (Japanese Patent Laid-Open No. 2000-93182) containing the same DNA in pUC119 as a template. 1985)) and can be prepared from the same plasmid. As a primer used for PCR, the oligonucleotide shown to sequence number 6 and 7 is mentioned, for example.
[0014]
In the present invention, chitinase C may be a part thereof as long as it has chitinase activity and can impart disease resistance to rice. For example, in the amino acid sequence shown in SEQ ID NO: 2, the region represented by amino acid numbers 1 to 30 may be deleted. Specifically, examples of the DNA used in the present invention include DNAs encoding the following proteins.
(A) a protein having the amino acid sequence represented by amino acid numbers 31 to 294 of SEQ ID NO: 2, or
(B) The amino acid sequence represented by amino acid numbers 31 to 294 of SEQ ID NO: 2 has an amino acid sequence including substitution, deletion, or insertion of one or several amino acid residues, and has chitinase activity protein.
[0015]
The “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically 2 to 35, preferably 2 to 20, more preferably 2 to 10 is there.
In the amino acid sequence of chitinase C shown in SEQ ID NO: 2, amino acid numbers 1 to 29 are presumed to be signal peptides.
[0016]
In addition to chitinase C or a part thereof, the DNA used in the present invention further includes a sequence encoding a signal peptide that functions as a secretion signal in plant cells described later, and encodes a fusion protein of the signal peptide and the protein. It may be DNA. Furthermore, it may be a fusion protein in which an artificial sequence such as a sequence derived from another protein or a linker is inserted between the signal peptide and the protein. In the present invention, such a fusion protein is sometimes simply referred to as “chitinase C”.
[0017]
Plants to which the present invention can be applied are gramineous plants, and are not particularly limited as long as they belong to the gramineous family. Specific examples include rice, wheat (wheat, barley, rye, etc.), Japanese millet, millet, buckwheat, and corn. Rice is particularly preferred in the present invention.
[0018]
In the present invention, DNA encoding chitinase C is introduced into gramineous plant cells using an appropriate vector. At that time, DNA encoding chitinase C is introduced by linking a promoter functioning in grasses at the 5 ′ end and a terminator functioning in grasses at the 3 ′ end. In addition to DNA encoding chitinase C, such a DNA containing a sequence necessary for expression thereof, such as a promoter and a terminator, is sometimes referred to as “chitinase C gene”.
[0019]
Specifically, for example, DNA encoding chitinase C is inserted into a plasmid having a promoter that functions in gramineous plants and a terminator that functions in gramineous plants to prepare a chitinase C expression plasmid. Can be introduced. For example, a promoter of 35S RNA gene (CaMV35S) derived from cauliflower mosaic virus can be used as the promoter. As the terminator, a terminator derived from an Agrobacterium-derived nopaline synthase (NOS) gene can be used. Of course, it is not limited to these.
[0020]
In order for the chitinase C protein to accumulate stably in plants, for example, a secretory signal peptide may be bound in front of the sequence encoding chitinase C to secrete chitinase C extracellularly. The secretory signal peptide is not particularly limited as long as it functions in a gramineous plant cell. For example, a rice-derived chitinase signal peptide having the amino acid sequence described in SEQ ID NO: 4 can be used, but the present invention is not limited thereto. The signal peptide can be encoded by the base sequence shown in SEQ ID NO: 3, for example. An example of the amino acid sequence of the fusion protein of the signal peptide and chitinase C is shown in SEQ ID NO: 5.
In the rice chitinase, the N-terminus of the mature protein is a glutamic acid residue. Therefore, in designing the fusion protein shown in SEQ ID NO: 5, in order to ensure cleavage of the signal peptide and the mature protein, the amino acid sequence shown in SEQ ID NO: 3 includes C A glutamic acid residue is added to the terminal. On the other hand, the alanine residue (amino acid number 30 in SEQ ID NO: 2) presumed to be N-terminal has been removed from the mature protein of chitinase C. Thus, in this invention, the signal peptide which has an amino acid sequence shown to sequence number 4 is a peptide which consists of a signal peptide and the glutamic acid residue added to the C terminal. In the present invention, this glutamic acid residue is not essential, and the sequence before and after the cleavage site is particularly limited as long as cleavage between the signal peptide and chitinase C occurs and active chitinase C can be generated. Not.
[0021]
In order to facilitate selection of disease resistant gramineous plants, a technique of introducing a drug resistance gene as a selection marker gene simultaneously with the chitinase C gene may be used. When this technique is used, the genes introduced into the transformed gramineous plant are the chitinase C gene and the drug resistance gene. As a drug resistance gene, it is desirable that a transformant can be easily selected. For example, neomycin phosphotransferase (NPT) gene, hygromycin phosphotransferase (HPT) gene and the like can be preferably used, but are not limited thereto.
[0022]
Examples of promoters for expressing drug resistance genes include, but are not limited to, the above plant gene promoters such as the CaMV35S promoter and the NOS promoter.
[0023]
Nucleic acid sequences in which a chitinase C gene and various regulatory elements such as a promoter, terminator, signal peptide, etc. that regulate the expression thereof are operably linked in a host cell so that the recombinant chitinase C gene can be expressed. Is called an expression cassette. This expression cassette can be constructed by standard methods.
[0024]
As a vector used for constructing an expression vector, a pBI vector, a pUC vector, or the like can be used. A pBI-based vector which is a binary vector can introduce an expression cassette into a plant via Agrobacterium. 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 etc. are mentioned. In the present invention, pBI-EN4 can be preferably used. This vector contains an expression cassette containing 4 repeats of the enhancer region of the CaMV 35S promoter and an HPT gene expressed as a marker gene under the control of the CaMV 35S promoter.
[0025]
There are two methods for introducing an expression cassette or expression vector into a gramineous plant cell, as described above, via Agrobacterium, or directly into the cell.
As a method using 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 using an electroporation method or the like, and then the transformed Agrobacterium is transformed into Plant Molecular Biology Manual (SB Gelvin et al.,). (Academic Press Publishers). Examples of the method for directly introducing an expression cassette and an expression vector include an electroporation method and a gene gun method.
[0026]
Gramineae plant cells into which an expression cassette or expression vector has been introduced are first selected for drug resistance such as hygromycin resistance, and then can be regenerated into plants by conventional methods.
[0027]
A well-known method can be used for confirmation of a foreign gene in a transformed gramineous plant. For example, DNA is extracted from a plant by a method according to the method of Walbot et al. (Walbot and Warren, Mol. Gen. Genet., 211, 27-34, (1988)), and the PCR method (Am. J. Hum. Genet). , 37, 172 (1985)) or the Southern method (J. Mol. Biol., 98, 505 (1980)).
[0028]
Moreover, the expression of the chitinase C gene into which the transformed gramineous plant has been incorporated is, for example, the Northern method (Thomas, Proc. Natl. Acad. Sci.) Using the chitinase C gene or a part thereof as a probe. USA, 77, 5201-5205 (1980)) or a Western method using antiserum against chitinase C (Towbin et al., Proc. Natl. Acad. Sci. USA, 76). , 4350-4354 (1979)) and can be clarified by a method for confirming the chitinase C gene product.
[0029]
The obtained transformant is used for a resistance test against various pathogens. The pathogen may vary depending on the target plant, but in the case of rice, for example, rice blast disease, rice blight disease and the like are typical fungal diseases, and the effect on rice blast disease is particularly remarkable . In this resistance test, individuals in the current generation (R0 generation) may be used, or individuals in the breeding progeny (R1 generation and later) may be used.
[0030]
Rice blast fungus spores for inoculation can be obtained by growing rice blast fungus on an oatmeal agar medium at 25 ° C. for about 2 weeks according to a conventional method, and then irradiating with a fluorescent lamp or a black light lamp for several days. Resistance to rice blast can be easily determined by inoculating a plant body with such a rice blast spore suspension. For example, 4 to 5 leaf stage seedlings of rice cultivated by a conventional method are spray-inoculated with a spore suspension of rice blast fungus to which 0.05% of a spreading agent such as Tween 20 is added. 20-24 hours after inoculation, placed in a 25 ° C. chamber with a humidity of almost 100%, and then cultivated in a greenhouse for 7-8 days. Resistance can be determined by the degree of appearance of lesions on the inoculated leaves.
[0031]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples as long as the gist thereof is not exceeded.
[0032]
[Example 1]
<Construction of chitinase C gene expression vector>
The construction of a chitinase C gene expression vector will be described with reference to FIG. As a vector having a promoter, a selectable marker gene, and a terminator, a cauliflower mosaic virus 35S promoter and an artificial promoter EN4 in which its enhancer region is repeated four times, an Escherichia coli hygromycin phosphotransferase gene, and an Agrobacterium nopaline synthase Plasmid pUC-EN4 (obtained from Dr. Hirohiko Hirochika, National Institute for Agrobiological Sciences) having a gene-derived terminator was used.
[0033]
As a vector having a chitinase C gene, pUC-RC2ss / ChiC was used. This vector comprises a fragment excised with SacI and KpnI from a plasmid pGC01 (Japanese Patent Laid-Open No. 2000-93182) containing DNA encoding chitinase C on pUC119 and a cDNA clone of rice-derived chitinase gene (EMBL Acc. # X56787). 5) -CACGGATCCGCATGTCGACGCCCGAGACGCG-3 'primer (SEQ ID NO: 6) and 5'-AGGAGCTCCCAGGCCGTGGCCGCGGCCGCGCCGCCCGT-3' primer (SEQ ID NO: 7) A fragment obtained by treating the encoded fragment with BamHI and SacI was prepared by using a DNA ligation kit (manufactured by Toyobo). It was prepared by coupling between amHI and KpnI sites.
[0034]
By ligating a large fragment obtained by cleaving pUC-EN4 with restriction enzymes XbaI and KpnI and a fragment containing the chitinase C gene obtained by cleaving pUC-RC2ss / ChiC with XbaI and KpnI, pEN4- RC2ss / ChiC was constructed.
[0035]
A fragment obtained by cleaving this pEN4-RC2ss / ChiC with EcoRI, and in addition to this, in the T-DNA region of the binary vector pBI121 (Clontech), a 35S promoter, a hygromycin phosphotransferase (HPT) gene and A plasmid pBI-HPT having a nopaline synthase gene-derived terminator (obtained from Dr. Hiroshi Kamada, University of Tsukuba) was ligated with a large fragment, and the chitinase C gene introduction plasmid pBI-EN4-RC2ss / ChiC Built. The structure of this plasmid is shown in FIG.
[0036]
According to the method of Nagel et al. (Microbiol. Lett., 67, 325 (1990)), transformed Agrobacterium was transformed into Agrobacterium tumefaciens by electroporation using the expression vector pBI-EN4-RC2ss / ChiC. And then cultured at 28 ° C. on a medium containing 50 μg / ml kanamycin and 25 μg / ml chloramphenicol.
[0037]
[Example 2]
<Introduction of expression vector into rice>
Rice transformation was performed according to an ultra-rapid transformation method (Japanese Patent No. 3141084). That is, ripe rice seeds from which rice husks were removed were sterilized intact in a 2.5% sodium hypochlorite solution, washed thoroughly with sterile water, and then 2,4-dichlorophenoxyacetic acid (2,4- D) and N6D medium (30 g / L sucrose, 0.3 g / L casamino acid, 2.8 g / L proline, 2 mg / L 2,4-D, 4 g / L gellite, pH 5.8). It was allowed to stand at 28 ° C. for a day.
[0038]
After immersing the seeds previously cultured in the transformed Agrobacterium suspension, 2N6 · AS medium (30 g / L sucrose, 10 g / L glucose, 0.3 g / L casamino acid, 2 mg / L 2) , 4-D, 10 mg / L acetosyringone, 4 g / L gellite, pH 5.2) and co-cultured in the dark at 28 ° C. for 3 days.
[0039]
After thoroughly washing Agrobacterium from seeds using a washing solution obtained by adding 500 mg / L of carbenicillin, which is an antibiotic for Agrobacterium, to N6D medium excluding gellite, 500 mg / L of carbenicillin and hygromycin as a selection marker are used. The washed seeds were placed on N6D medium supplemented with 50 mg / L and selectively cultured at 28 ° C. for about 2 weeks.
[0040]
The selected callus was regenerated into a regeneration medium (500 mg / L carbenicillin, 30 mg / L hygromycin, 30 g / L sucrose, 30 g / L sorbitol, 2 g / L casamino acid, 2 mg / L kinetin, 0.002 mg / L α-naphthylacetic acid. (NAA) 4 g / L gellite, MS medium added to pH 5.8) and continued to culture at 28 ° C. until redifferentiation.
[0041]
The redifferentiated solid was placed on a rooting medium (hormone-free MS medium added to give hygromycin 30 mg / L) and assayed for hygromycin resistance. In the non-transformant, new roots did not grow and died in about one week, whereas the transformant considered to have a gene introduced showed resistance and exhibited growth similar to that of the wild strain. When the transformed plant became large, it was acclimated and transplanted into a pot and grown in a greenhouse. As a result, a mature and completely transformed rice plant body was obtained.
[0042]
[Example 3]
<Confirmation of expression of chitinase C in transformed rice plant>
Extraction of water-soluble protein from the leaves of the regenerated transformed rice plant, and detection of chitinase C protein by Western method (Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350, (1979)) Tried.
[0043]
Rice leaves were extracted with a citrate-phosphate buffer adjusted to pH 6.0, and then centrifuged at 15000 rpm, 4 ° C. for 20 minutes. The supernatant was used as a sample and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by a conventional method. The gel after electrophoresis was brought into close contact with Immobilon membrane (Millipore), and with a blotting buffer (25 mM trishydroxyaminomethane, 192 mM glycine, 15% methanol) using a semi-driving apparatus (manufactured by Nippon Aido), 180 mA, 6 V, 1 The protein was transferred from the gel to the membrane by energizing under time conditions.
[0044]
Detection of chitinase C protein on the membrane was carried out by using goat IgG (ICN manufactured by ICN) with antiserum against chitinase C protein obtained by immunizing rabbits with chitinase C as the primary antibody and secondary phosphatase binding to rabbit IgG as the secondary antibody. ), A membrane was sequentially immersed in a solution containing these and a solution containing nitroblue tetrazolium (NBT) and bromochloroindole phosphate (BCIP), which are substrates for alkaline phosphatase, to develop the color of the substrate.
[0045]
As a result, a protein mainly having a band of about 31.5 kDa that reacts with antiserum against chitinase C protein was detected.
[0046]
[Example 4]
<Transmission of chitinase C gene to the next generation of self breeding>
The presence of the transgene (HPT gene) in the progeny of transformation was confirmed by PCR.
The regenerated plant body obtained in Example 2 was grown in a greenhouse in a conventional manner to obtain self-propagating seeds (R1 generation) after about 4 months.
[0047]
After removing the rice husks of these self-grown seeds, sterilized with a 1% sodium hypochlorite solution and cultivated on a 1% agar plate containing 25 mg / L hygromycin for about 10 days. It was raised to Bonsol culture soil (Sumitomo Chemical) one by one.
[0048]
Total DNA was extracted from about 1 cm of leaf blade of each line containing non-transformant according to the method of Tsutomu Kawasaki (“Simple method for isolating rice genomic DNA for PCR analysis”, plant PCR experiment protocol Shujunsha).
[0049]
PCR was performed at an annealing temperature of 60 ° C. using 1 μl of 100 μl of the extracted DNA solution of each strain as a template and using primer 1: ATGAAAAACCCTGAACTCACCGCGA (SEQ ID NO: 10) and primer 2: TCCATCAGATTTGCCAGTGATACA (SEQ ID NO: 11).
[0050]
After PCR, a part of the reaction solution was electrophoresed on a 1.2% agarose gel. The presence or absence of a band and the PCR product using the plasmid pBI-EN4-RC2ss / ChiC constructed in Example 1 as a template as a control were used. Comparison with length was made. As a result, it was confirmed that the transgene was inherited in the self breeding next generation.
[0051]
[Example 5]
<Disease resistance of transformed plants>
The resistance of the transformed rice obtained in Example 2 to rice leaf blast was tested. As rice for inoculation, rice seeds (R1 generation) of regenerated plants were removed and then sterilized with 1% sodium hypochlorite on a 1/4 × MS medium containing 25 mg / L hygromycin. Cultivated for about 10 days, 10 individual hygromycin-resistant individuals were planted in Bonsol culture soil (Sumitomo Chemical Co., Ltd.), and individuals cultivated in the greenhouse until the 4th to 5th leaf stage were used.
As a control, seeds from which rice husks of non-transformed rice (Nipponbare) were removed were sterilized in the same manner as described above, and then grown in the same manner on 1/4 × MS medium not containing hygromycin. Plants cultivated in the above were used. As a comparison with rice into which a chitinase gene other than chitinase C was introduced, recombinant rice in which rice class I chitinase (Cht-2) was highly expressed [Theoretical Applied Genetics (Theor. Appl. Genet.)]. 99, 383-390, 1999] was used as a control.
[0052]
The rice blast fungus spore suspension was grown on an oatmeal agar medium at 25 ° C. for about 2 weeks, and then the spores formed by irradiating with a black light lamp for 4 days were scraped off with a sterilized paint brush. 5 × 10 ^Five / Ml to prepare a suspension in 0.05% Tween 20 aqueous solution. The plant body sprayed with this rice blast fungus spore suspension was allowed to stand for 24 hours in an inoculation box at 100% humidity and 25 ° C. in the dark, and then placed in an isolated greenhouse at 28-30 ° C. (23 ° C. at night). Cultivated for ~ 8 days.
[0053]
The resistance test was performed according to the leaf blast resistance determination chart based on the diseased area developed by Furuta Laboratory of the Ministry of Agriculture, Forestry and Fisheries (former) China Agricultural Experiment Station. In other words, the extent of glutinous lesions formed on the inoculated leaves is expressed in terms of lesion area ratio in the light of the judgment diagram, and the control value is calculated in comparison with the lesion area ratio of the original variety, and chitinase C is not expressed. The control value of the transformed rice was compared. Compared to non-transformants, the number and spread of lesions were significantly suppressed in transformed rice that constantly expresses the chitinase C gene. In addition, even when compared to transformed rice that highly expresses rice chitinase, the number and spread of lesions were significantly suppressed in transformed rice that constantly expressed chitinase C, and the control value was increased. The results are shown in FIG.
[0054]
The control value was calculated by the following formula.
Control value (%) = (average lesion area ratio of control−average lesion area ratio of test strain) / average lesion area ratio of control × 100
[0055]
[Example 6]
<Expression level of chitinase C and disease resistance in the same strain>
The blast resistance in the same strain was examined, and the expression level of each chitinase C was analyzed by Western method, and the correlation between the expression level of chitinase C and blast resistance was investigated.
[0056]
The self-propagated seeds (R1 generation) of the regenerated plant of transformed rice obtained in Example 2 were sown on Bonsol culture soil (Sumitomo Chemical Co., Ltd.) at a rate of 4 to 5 leaf stages in a greenhouse. The resistance test shown in Example 5 was performed using the cultivated individuals. Thereafter, the inoculated leaves after the blast resistance test were collected, and the expression level of chitinase C was confirmed using the Western method shown in Example 3.
[0057]
As a result, as shown in Table 1, only an individual expressing chitinase C exhibits a high control value indicating blast resistance, and the expression of chitinase C is involved in imparting blast resistance. Was strongly suggested.
[0058]
[Table 1]

A, B: Chitinase C gene-introduced transformed rice line
+: With chitinase C expression by Western analysis
−: No chitinase C expression by Western analysis
[0059]
[Explanation of Sequence Listing]
SEQ ID NO: 1: Base sequence of chitinase C gene
SEQ ID NO: 2: amino acid sequence of chitinase C
SEQ ID NO: 3: Base sequence of DNA encoding signal peptide of rice-derived chitinase
SEQ ID NO: 4: Amino acid sequence of signal peptide of rice chitinase
SEQ ID NO: 5: amino acid sequence of fusion protein of signal peptide and chitinase C
SEQ ID NO: 6: Primer for signal sequence amplification of rice-derived chitinase gene
SEQ ID NO: 7: Primer for signal sequence amplification of rice-derived chitinase gene
SEQ ID NO: 8: primer for transgene confirmation
SEQ ID NO: 9: primer for transgene confirmation
【The invention's effect】
The disease-resistant gramineous plant of the present invention is transformed by introducing a chitinase C gene, thereby increasing resistance to fungal diseases such as rice blast and providing an important gramineous plant for agricultural production. I can do it.
[0060]
[Sequence Listing]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[Brief description of the drawings]
FIG. 1 is a schematic diagram of the structure of a chitinase C gene introduction plasmid pBI-EN4-RC2ss / ChiC.
FIG. 2 is a graph showing the test results of the resistance of transformed plants to rice leaf blast.
[Explanation of symbols]
35S: CaMV 35S promoter
HPT: Hygromycin resistance gene
NOS3 ': NOS gene terminator
EN4: CaMV 35S promoter (enhancer 4 repeats)
RCC2SS / ChiC: chitinase C chimeric gene with signal sequence of rice chitinase gene
RB, LB: T-DNA right border sequence, left border sequence
NPTIII: kanamycin resistance gene
NT: Non-transformant (control)
P: Rice transformed with a plasmid not containing the chitinase C gene
C: N-ER2-55-5-1 (Rice chitinase high expression transformed rice)
1,2,3,4: Chitinase C expression transformed rice

Claims (2)

DNA encoding a protein shown in the following (A) or (B), wherein a DNA encoding a plant extracellular secretion signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is further bound before the sequence A rice plant resistant to rice blast, which is transformed with the protein and expresses a fusion protein of the signal peptide and the protein :
(A) a protein comprising the amino acid sequence represented by amino acid numbers 31 to 294 of SEQ ID NO: 2, or (B) one or several amino acid residues in the amino acid sequence represented by amino acid numbers 31 to 294 of SEQ ID NO: 2 A protein comprising an amino acid sequence containing a group substitution, deletion or insertion, and having chitinase activity. The gramineous plant according to claim 1 , wherein the gramineous plant is rice.

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