CN103175809A - Method for inducing rice plant system to generate defensive reaction - Google Patents
Method for inducing rice plant system to generate defensive reaction Download PDFInfo
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- CN103175809A CN103175809A CN201210554679XA CN201210554679A CN103175809A CN 103175809 A CN103175809 A CN 103175809A CN 201210554679X A CN201210554679X A CN 201210554679XA CN 201210554679 A CN201210554679 A CN 201210554679A CN 103175809 A CN103175809 A CN 103175809A
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Abstract
本发明公开了一种稻瘟病菌基因的原核表达产物处理水稻根尖诱导水稻系统防御反应的方法，利用一定浓度的融合蛋白处理水稻根尖0.5cm处24h，苯胺蓝和DAB染色后观察除去0.5cm根尖以外的根组织的其他部位的胼胝质形成及活性氧产生情况。本发明的有益效果是利用稻瘟病菌基因的原核表达产物处理水稻根尖0.5cm处，通过观察根尖0.5cm以外根组织其他部分胼胝质和活性氧产生，可明确水稻系统防御反应产生的情况，克服了直接利用病原菌接种水稻叶片观察胼胝质和活性氧产生周期长且易受到叶片组织大量自发荧光干扰等弊端，最终达到为今后鉴定病原菌效应蛋白诱导植物系统防御反应产生提供简便有效的方法的目的。The invention discloses a method for treating rice root tip with a prokaryotic expression product of rice blast fungus gene to induce rice systemic defense response, using a certain concentration of fusion protein to treat rice root tip at 0.5 cm for 24 hours, and observing and removing 0.5 cm after aniline blue and DAB staining. The callose formation and active oxygen generation in other parts of the root tissue other than the root tip in cm. The beneficial effect of the present invention is that the prokaryotic expression product of the rice blast fungus gene is used to treat the root tip of rice at 0.5 cm, and the generation of callose and active oxygen in other parts of the root tissue beyond the root tip of 0.5 cm can be observed to clarify the situation of the rice system defense response It overcomes the disadvantages of directly using pathogenic bacteria to inoculate rice leaves to observe the long callose and reactive oxygen species generation cycle and is easily interfered by a large amount of autofluorescence in leaf tissue, and finally achieves the goal of providing a simple and effective method for identifying pathogenic bacteria effector proteins inducing plant system defense responses in the future Purpose.
Description
Technical field
The prokaryotic expression product that the present invention relates to a kind of Pyricularia oryzae gene is processed the method for paddy rice root tip inducing paddy rice system defense reaction, belongs to plant protection and biological technical field.
Background technology
Plant and its pathogen were evolved up to a million years jointly, and natural selection impels plant to produce various identifications and resistance mechanism prevents and limit pathogen infection.Also impelling pathogenic bacteria gene to evolve to adapt to or resist plant defense response in the time of plant evolution makes it infect the host to reach pathogenic final purpose.System's defense reaction refers to that the activation of defense reaction is not confined to the infection court that pathogen is invaded plant, and plant self has not been produced defense reaction by the part of pathogen infection yet.System's defense reaction plays a very important role at tool aspect enhancing plant opposing pathogen.Therefore, research botanical system defense reaction can be further research plant and pathogen provides theoretical foundation as mechanism mutually, can be simultaneously the effective control of plant disease from now on important reference frame is provided.
Summary of the invention
Fundamental purpose of the present invention is to produce for plant identification system defense reaction a kind of quick, easy and method accurately that provides.
The embodiment of the present invention is to realize like this, a kind of prokaryotic expression product of Pyricularia oryzae gene is processed the method for paddy rice root tip inducing paddy rice system defense reaction, utilize certain density fusion to process the paddy rice root tip 0.5cm 24h of place, the callose of observing other positions of removing the abapical root tissue of 0.5cm root after aniline blue and DAB dyeing forms and the active oxygen production.
Further, the prokaryotic expression product of this gene carries out aniline blue and DAB dyeing after processing black paddy root tissue 24h according to 5.0mg/ml concentration, callose directly observed by fluorescent microscope and active oxygen produces, and produces definite rice system defense reaction generation according to callose and active oxygen.
Further, with Pyricularia oryzae effect protein gene expression product in 4 ℃ of centrifugal collection thalline of hydro-extractor, with freshly prepared damping fluid suspension thalline; Centrifugal collection supernatant after having children outside the state plan smudge cells on ice-water bath; Processing is to process the disease resisting rice kind Lijiang xintuanheigu tip of a root 0.5cm 24h of place according to the concentration of 5.0mg/ml after the prokaryotic expression product purifying of this gene, and callose is carried out in aniline blue and DAB direct staining and active oxygen is observed.
Further, freshly prepared damping fluid is 20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA; The 10mM beta-mercaptoethanol.
The invention has the beneficial effects as follows the prokaryotic expression product processing paddy rice root tip 0.5cm place that utilizes the Pyricularia oryzae gene, by observing tip of a root 0.5cm other part calloses of root tissue and active oxygen generation in addition, can be clear and definite rice system defense reaction situation about producing, overcome directly utilize pathogen Inoculated Rice blade observe callose and the active oxygen generation cycle long and be vulnerable to the drawback such as a large amount of autofluorescence interference of leaf tissue, finally be reached for the purpose that pathogen identification effect protein inducing plant from now on system defense reaction produces the method that provides simple and effective.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
The embodiment of the present invention is to keep present prokaryotic expression product expression and purification, callose and active oxygen observational technique constant, comprise that prokaryotic expression technology, protein purification technology, DAB dyeing, aniline blue colouring method are all with conventional identical, it is characterized in that utilizing certain density fusion to process the paddy rice root tip 0.5cm 24h of place, the callose of observing other positions of removing the abapical root tissue of 0.5cm root after aniline blue and DAB dyeing forms and the active oxygen production.
The prokaryotic expression product of this gene carries out aniline blue and DAB dyeing after processing rice varieties Lijiang xintuanheigu root tissue 24h according to 5.0mg/ml concentration, callose directly observed by fluorescent microscope and active oxygen produces, produce according to callose and the definite rice system defense reaction of active oxygen generation, this other part root tissue calloses of the abapical root tissue of root and the active oxygen generation of passing through to observe without the albumen processing determines that the method for the protein induced botanical system defense reaction generation of pathogen is comparatively easy, accurate, quick.
The fusion of embodiment: 5.0mg/ml is processed the paddy rice root tip 0.5cm 24h of place, directly observes callose and the active oxygen production of other parts of tip of a root 0.5cm root tissue in addition after aniline blue and DAB dyeing.
With Pyricularia oryzae effect protein gene expression product in 4 ℃ of centrifugal collection thalline of hydro-extractor, with freshly prepared damping fluid (20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA10mM beta-mercaptoethanol) suspension thalline.Centrifugal collection supernatant after having children outside the state plan smudge cells on ice-water bath.Processing is to process the disease resisting rice kind Lijiang xintuanheigu tip of a root 0.5cm 24h of place according to the concentration of 5.0mg/ml after the prokaryotic expression product purifying of this gene, and callose is carried out in aniline blue and DAB direct staining and active oxygen is observed.Contrast is to process the Lijiang xintuanheigu tip of a root 0.5cm 24h of place with PBS, and callose is carried out in aniline blue and DAB direct staining and active oxygen is observed.Other parts that found that Lijiang xintuanheigu tip of a root 0.5cm root tissue have in addition all been observed callose and active oxygen generation.
The above is only preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., within all should being included in protection scope of the present invention.
Claims (4)
1. the prokaryotic expression product of a Pyricularia oryzae gene is processed the method for paddy rice root tip inducing paddy rice system defense reaction, it is characterized in that, utilize certain density fusion to process the paddy rice root tip 0.5cm 24h of place, the callose of observing other positions of removing the abapical root tissue of 0.5cm root after aniline blue and DAB dyeing forms and the active oxygen production.
2. the method for inducing paddy rice as claimed in claim 1 system defense reaction, it is characterized in that, the prokaryotic expression product of this gene carries out aniline blue and DAB dyeing after processing black paddy root tissue 24h according to 5.0mg/ml concentration, callose directly observed by fluorescent microscope and active oxygen produces, and produces definite rice system defense reaction generation according to callose and active oxygen.
3. the method for inducing paddy rice as claimed in claim 2 system defense reaction, is characterized in that, with Pyricularia oryzae effect protein gene expression product in 4 ℃ of centrifugal collection thalline of hydro-extractor, with freshly prepared damping fluid suspension thalline; Centrifugal collection supernatant after having children outside the state plan smudge cells on ice-water bath; Processing is to process the disease resisting rice kind Lijiang xintuanheigu tip of a root 0.5cm 24h of place according to the concentration of 5.0mg/ml after the prokaryotic expression product purifying of this gene, and callose is carried out in aniline blue and DAB direct staining and active oxygen is observed.
4. the method for inducing paddy rice as claimed in claim 3 system defense reaction, is characterized in that, freshly prepared damping fluid is 20mM Tris-HCl, pH7.4; 200mM NaCl; 1mM EDTA; The 10mM beta-mercaptoethanol.
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| CN201210554679XA CN103175809A (en) | 2012-12-05 | 2012-12-05 | Method for inducing rice plant system to generate defensive reaction |
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| CN201210554679XA CN103175809A (en) | 2012-12-05 | 2012-12-05 | Method for inducing rice plant system to generate defensive reaction |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2327307A1 (en) * | 2008-09-16 | 2011-06-01 | Kitakyushu Foundation for the Advancement of Industry, Science and Technology | Water that expresses pathogen-resistance genes (pr gene clusters) to encode plant immunoproteins, a method of preventing plant diseases using the water, and a device for producing the water |
| CN102559575A (en) * | 2011-12-31 | 2012-07-11 | 云南农业大学 | Method for treating paddy rice tissues by using prokaryotic expression product and inducing generation of callose |
| CN102675434A (en) * | 2012-02-16 | 2012-09-19 | 中国农业科学院植物保护研究所 | Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein |
| CN102749308A (en) * | 2012-06-11 | 2012-10-24 | 云南农业大学 | Method for in vitro detection of transport of Magnaporthe grisea effector proteins in root tissues of rice |
-
2012
- 2012-12-05 CN CN201210554679XA patent/CN103175809A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2327307A1 (en) * | 2008-09-16 | 2011-06-01 | Kitakyushu Foundation for the Advancement of Industry, Science and Technology | Water that expresses pathogen-resistance genes (pr gene clusters) to encode plant immunoproteins, a method of preventing plant diseases using the water, and a device for producing the water |
| CN102559575A (en) * | 2011-12-31 | 2012-07-11 | 云南农业大学 | Method for treating paddy rice tissues by using prokaryotic expression product and inducing generation of callose |
| CN102675434A (en) * | 2012-02-16 | 2012-09-19 | 中国农业科学院植物保护研究所 | Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein |
| CN102749308A (en) * | 2012-06-11 | 2012-10-24 | 云南农业大学 | Method for in vitro detection of transport of Magnaporthe grisea effector proteins in root tissues of rice |
Non-Patent Citations (2)
| Title |
|---|
| 任鄄胜等: "水稻稻瘟病病菌研究进展", 《现代农业科学》, vol. 15, no. 1, 30 January 2008 (2008-01-30) * |
| 张哲等: "稻瘟菌无毒基因研究进展", 《遗传》, vol. 33, no. 6, 30 June 2011 (2011-06-30) * |
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Application publication date: 20130626 |