KR20060031524A - A pathogenesis-related gene ogpr1 isolated from wild rice, the sequences of amino acid and the transgenic plant using the same - Google Patents

Abstract

The present invention relates to a plant disease resistance gene OgPR1 isolated from wild rice ( Oryza grandiglumis ) , its amino acid sequence and transformant plants using the same, and more specifically, it is isolated from wild rice by wound and yeast extract stress treatment. , Its expression is increased by hormonal treatment such as jasmonic acid, cantharidin, and endothall, and Pseudomonas syringe and gray mold fungus, which cause tomato foot blight The present invention relates to a novel plant disease resistance gene OgPR1 that exhibits resistance to fungal pathogens such as Botrytis cinera and the like, its amino acid sequence and transformant plants using the same. The present invention therefore, by providing the gene, external environmental stress and plant pathogens, in particular Pseudomonas syringe (Pseudomonas syringe) and Botrytis cine la newly developed plant disease-resistant crops to (Botrytis cinera), able to six kinds of agro-industrial and It is a very useful invention in the plant breeding industry.

Wild rice (Oryza grandiglumis), plant disease resistance gene OgPR1, Pseudomonas syringe, Botrytis cinera

Description

A pathogenesis-related gene OgPR1 isolated from wild rice, the sequences of amino acid and the transgenic plant using the same}

Figure 1 shows the cDNA sequence of the plant disease resistance gene OgPR1 of the present invention isolated from wild rice ( Oryza grandiglumis ).

Figure 2 shows the amino acid sequence deduced from the cDNA sequence of the plant disease resistance gene OgPR1 of the present invention isolated from wild rice ( Oryza grandiglumis ).

Figure 3 is to examine the expression pattern of the plant disease resistance gene OgPR1 of the present invention isolated from wild rice ( Oryza grandiglumis ), jasmonic acid (JA), cantharidin (CN) and endosal in wild rice After processing (endothall: EN), total RNA was isolated and Northern blot analysis was performed.

Figure 4 is a cyclone with cantharidin (CN) and endothall (EN) treatment in wild rice in order to determine the expression of the plant disease resistance gene OgPR1 of the present invention isolated from wild rice ( Oryza grandiglumis ) Hexamide (cycloheximide: CHX) and tetracycline (Tetracycline: TET) treatment of the total RNA was isolated and the result of Northern blot analysis.

5 is a result of the Northern blot analysis of each of the F1 generation by transforming the plant disease resistance gene OgPR1 of the present invention isolated from wild rice ( Oryza grandiglumis ) with a model plant Arabidopsis thalina .

6 is a result of observation of the symptoms after treatment with wild rice present invention Pseudomonas syringe an Arabidopsis plant transformed with a plant disease resistance gene, respectively OgPR1 (Pseudomonas syringe) isolated from (Oryza grandiglumis).

Figure 7 is a result of observing the disease after treatment of Arabidopsis plants transformed with the plant disease resistance gene OgPR1 of the present invention isolated from wild rice ( Oryza grandiglumis ) with Botrytis cinera , respectively.

The present invention relates to a plant disease resistance gene OgPR1 isolated from wild rice ( Oryza grandiglumis ) , its amino acid sequence and transformant plants using the same, and more specifically, as isolated from wild rice by stress treatment of wound and yeast extract. , Its expression is increased by hormonal treatment such as jasmonic acid, cantharidin, and endothall, and Pseudomonas syringe and gray mold fungus, which cause tomato foot blight It relates to a novel plant disease resistance gene OgPR1, its amino acid sequence, and a transformant plant using the same, which are resistant to fungal pathogens such as Botrytis cinera .

The world population is expected to reach 11 billion by 2050, and food shortages will become a bigger problem in the future due to the rapid increase in population. Therefore, in order to solve the above problems, the fostering of insect-resistant and multiplicity of food crops is most urgent.

Currently, the production of food crops around the world is greatly affected by extreme weather. In extreme cases, domestic food sowing occurred in 1972 and 1973, when grain production declined by 3% at the time, causing the international price of rice to soar more than three times, causing serious problems. This phenomenon confirms the specificity of food resources and their importance as a national infrastructure.

In the case of domestic water cultivation, many harvest reduction factors occur every year, such as damage to farmland due to heavy rains in summer, abnormal low temperature, lack of average sunshine time, and rapid increase of water pests. As the reduction factor is more likely to occur in the future, it is necessary to secure stable insect resistant varieties as a countermeasure. However, observations of the rice and major crop yield indexes of major countries such as Korea, Japan, Thailand, and the United States over the last 10 years show that there is little change. Means that the limit has already been reached.

Although wild rice has not been studied in full scale yet, it is expected to be a major research subject to secure various kinds of useful genes that are gradually lacking in cultivated rice. In this regard, wild rice has been reported to have a multiplicity of related genes by genetic linkage mapping or Southern blot assay. However, there have been no reports of phytopathogenic genes expressed in wild species. .

Accordingly, the present inventors have first isolated the plant disease resistance gene OgPR1 induced by a large amount of insects from the wild rice ( Oryza grandiglumis ), not the general rice varieties, and tried to apply it to the development of plant disease resistant plants.

Accordingly, an object of the present invention is to provide a plant disease resistance gene OgPR1 and its amino acid sequence isolated from wild rice ( Oryza grandiglumis ).

Another object of the present invention to provide a transformant plant prepared using the plant disease resistance gene OgPR1 isolated from wild rice ( Oryza grandiglumis ).

The object of the present invention is to isolate the plant disease resistance gene OgPR1 from the cDNA gene bank of fragments induced by treating stress (wound, yeast extract, Fungal elicitor) on the leaves of wild rice ( Oryza grandiglumis ) After analyzing the nucleotide sequence and amino acid sequence of the gene, assaying the expression according to the hormonal treatment of the gene, preparing and selecting a transformant using Arabidopsis, the pseudomonas syringe of the Arabidopsis transformant and This was achieved by examining disease resistance to Botrytis cinera and confirming the function of the gene.

Hereinafter, the configuration and operation of the present invention will be described in detail.

The present invention provides a plant disease resistance gene OgPR1 of SEQ ID NO: 1 isolated from wild rice ( Oryza grandiglumis ) according to the above object.

The gene OgPR1 of the present invention is isolated from the cDNA gene bank of fragments induced by treating wound and yeast extract stress on the leaves of wild rice ( Oryza grandiglumis ) by RACE PCR technique, a total of 504 bp sequences and 168 amino acid sequences. It is a new gene consisting of.

In addition, the expression is increased in time and light conditions by hormonal treatment such as jasmonic acid, cantharidin, and endothall, and when transformed with Arabidopsis, Arabidopsis transformants are particularly tomato Increasing the expression of the gene of the present invention against fungal pathogens, such as Pseudomonas syringe ( Pseudomonas syringe ) that causes foot blight disease and Botrytis cinera that causes gray mold fungus disease.

Thus, the gene OgPR1 of the present invention is found to be a new plant disease resistance gene of the wild rice-derived unknown to the existing, as this not only for research external environmental stress and plant pathogens, in particular Pseudomonas syringe (Pseudomonas syringe) and Botrytis cine La ( Botrytis cinera ) can be used to newly develop and breed phytopathogenic crops.

The present invention also provides an amino acid sequence of SEQ ID NO: 2 encoded in the plant disease resistance gene OgPR1 isolated from wild rice ( Oryza grandiglumis ).

In another aspect, the present invention provides a transformant plant prepared using the plant disease resistance gene OgPR1 isolated from wild rice ( Oryza grandiglumis ) according to the other object.

The transformant plant of the present invention is the OgPR1 gene represented by the sequence of SEQ ID NO: 1 according to a conventional method, or the gene of interest other than the OgPR1 gene, for example β-glucuronidase (GUS) A recombinant vector was prepared by fusion of a reporter gene such as an appropriate plant expression vector, and then introduced into a plant callus according to a conventional plant transformation method. It can be obtained by transferring it.

In this case, as a method of transforming the plant, an Agrobacterium mediated transformation method, a particle injection method, etc. may be mentioned, and an Agrobacterium mediated transformation method is preferable.

Transgenic plants provided by the present invention by containing a plant disease resistance gene OgPR1 of the present invention isolated from wild rice (Oryza grandiglumis) external environmental stress and plant pathogens, in particular Pseudomonas syringe (Pseudomonas syringe) and Botrytis cine La ( Botrytis cinera ) is a plant disease resistant plant that exhibits excellent resistance to it.

Thus the present invention together with the novel plant disease resistance genes OgPR1 and the amino acid sequence of the wild rice-derived, transformed with the gene being external environmental stress and Pseudomonas syringe (Pseudomonas syringe) and Botrytis cine la (Botrytis cinera) It is a very useful invention in the agricultural industry and plant breeding industry because it provides a transformant plant exhibiting resistance to plant pathogens, including.

Meanwhile, in the present invention, standard single characters are used to represent nucleotide sequences and amino acid sequences, and the meanings of these abbreviations are standard biochemistry textbooks and molecular biology experiments, for example, Leninginger, principle of Biochemistry, Worth Publishers Inc., p. .96, 789, 1984; Sambrook et al., Molecular cloning: A laboratory manuals, Cold Spring Harbor Laboratory Press, pp. 174-184, 1989.

Hereinafter, the specific configuration of the present invention will be described in detail by way of examples, but it will be apparent to those skilled in the art that the scope of the present invention is not limited only to these examples.

Example 1 Wild Rice Oryza grandiglumis Plant Disease Resistance Genes from OgPR1 Isolation, Sequence and Amino Acid Sequence Analysis]

Oryza grandiglumis , grown for more than four weeks in growth , used a paper punch to punch holes in the leaf veins and wounds using a knife. In addition, the yeast extract was dissolved in water and then sprayed on the whole leaf using a spray. Poly (A +) mRNA was extracted from the stressed and non-stressed leaves, respectively, and cDNA of fragments expressed only by the stress according to the manufacturer's recommendation method for PCR-Select cDNA Subtraction (purchased by Clontech). Secured.

The obtained cDNA was cloned into pCR2.1 (purchased by Invitrogen), a subcloning vector, and plasmid DNA was isolated using a Plasmid purification kit (purchased by NucleoGen). Based on the sequencing results, the RACE PCR was performed to secure the entire sequencing.                     

As a result, as shown in FIG. 1, the cDNA of the plant disease resistance gene OgPR1 of the present invention was composed of a total of 504 bp, and the amino acid sequence was deduced by the nucleotide sequence of FIG. 1, as shown in FIG. 2. It consisted of 168 amino acids. In addition, the nucleotide sequence and the deduced amino acid sequence was searched in the database using the Blast search program of the National Center for Biological Information (NCBI), and showed a high similarity of 93.5% to the pathogenic gene (PR1) known from ordinary rice. In rice, the gene has not yet been found. Thus, the gene OgPR1 was found to be a new gene for plant disease resistance derived from wild rice.

Example 2: Plant Disease Resistance Gene OgPR1 Expression Test by Hormone Treatment of

Oryza grandiglumis cultivated for more than 4 weeks in growth, various hormones of jasmonic acid (JA), cantharidin (CN), and endothall (EN) for 3 hours, 6 hours, The treatment was carried out for 12 hours and 21 hours, and cycloheximide (CHX) and tetracycline (TET) were treated with cantharidin (CN) and endosal (EN), respectively. Total RNA was isolated from the hormone-treated leaves using Tri-Reagent (purchased by Molecular Research Center). The whole RNA was electrophoresed on a 1.0% formaldehyde gel, and then the gel was transferred to a nylon membrane (Amersham Bioscience, Inc.) with 20 × SSC solution (3M NaCl, 0.3M Sodium citrate), and radioactive. Northern blot analysis was performed using the plant disease resistance gene 504 bp cDNA in Example 1 labeled with isotope [α- ³² P] dCTP as a probe.

As a result, as shown in Figure 3, the expression of the plant disease resistance gene OgPR1 increased in light conditions at each time period in each hormone (jasmonic acid, candaridine , endosal ). Meanwhile, as shown in FIG. 4, the experimental group treated with cyclohexamide with the hormones candaridine (CN) and endosal (EN) decreased the expression of the plant disease resistance gene OgPR1 while the tetracycline was treated together. It was confirmed that the expression increased in the experimental zone.

Example 3 Preparation and Selection of Transformant Using Arabidopsis

In Example 2, the plant disease resistance gene OgPR1 of the present invention was confirmed to be a gene whose expression is increased by time and condition under hormonal treatment.

Then, transformation of the model plant Arabidopsis was performed to investigate the function of the plant disease resistance gene. the gene OgPR1 the pBI121 plasmid DNA (Clontech, USA) at a site for cutting with BamHⅠ SacⅠ with restriction enzymes was inserted was amplified using the PCR. The completed pBI121-PR1 plasmid was introduced into Agrobacterium (LBA4404) by electroshock. Arabidopsis transformed Arabidopsis firearms grown for 4 weeks for 1 minute in a solution containing 0.8% Agrobacterium and 0.05% Silwet L-77 added to the solution, placed in a plastic bag and left for 3 days, then removed from the plastic bag. Grows for weeks (Bent Method, 1998).

Later, seeds were collected from later F1 generations and germinated again in selective medium (MS medium + hygromycin) to select only surviving individuals and planted in soil. Total RNA was isolated from 20 leaves of the selected Arabidopsis transformants using Tri-Reagent (purchased by Molecualr Research Center). The whole RNA was electrophoresed on 1.0% formaldehyde gel, and then the gel was transferred to a nylon membrane (Amersham Bioscience Co., Ltd.) with 20 × SSC solution (3M NaCl, 0.3M Sodium citrate), and radioactive. Northern blot analysis was performed using a plant disease resistant gene 504 bp cDNA labeled with isotope [α- ³² P] dCTP as a probe.

As a result, as shown in Figure 5, it was confirmed that the expression of the plant disease resistance gene OgPR1 significantly increased in most Arabidopsis transformants.

Example 4 Pseudomonas Syringe of Arabidopsis Transformant Pseudomonas syringe Confirmation of plant disease resistance to

Tomatoes were selected from 6, 10, 11, 12, and 16 transformants randomly selected from the 20 Arabidopsis transformants prepared and selected in Example 3 and the untransformed Arabidopsis (control). Pseudomonas syringe (Pseudomonas syringe) to cause foot blight 1 × 10 ⁵ cfu / ml fold dilution was then inoculated with a syringe on the back of each leaf. The leaves of each inoculated Arabidopsis transformants were harvested and crushed with 10 mM MgCl ₂ , and then aliquoted into selective medium (KB + kanamycin) and incubated at 30 ° C. for 3 days. The number of Pseudomonas bacteria after the incubation was calculated by observing under a microscope.

As a result, as shown in Figure 6, it was confirmed that the number of Pseudomonas syringe bacteria ( Pseudomonas syringe ) bacteria in all of the Arabidopsis transformants transformed with the gene OgPR1 of the present invention than the control.

Example 5 Botrytis cinera of Arabidopsis Transformants Botrytis cinera Confirmation of plant disease resistance to

Among the 20 Arabidopsis transformants prepared and selected in Example 3, 6, 10, 11, 12, and 16 randomly selected transformants were grayed out for the untransformed Arabidopsis (control). Botrytis cinera, a causative fungal disease, was sprayed on the whole plant at a concentration of 5 × 10 ⁵ conidia / ml, and the disease progress was visually observed.

As a result, as shown in FIG. 7, 14 days after the infection, the Arabidopsis transformants transformed with the gene OgPR1 of the present invention, unlike the control group, visually confirmed that the infection of the gray-colored fungal disease was reduced. Could.

As is clearly described through the above examples, the present invention provides a novel plant disease resistance gene OgPR1 and its amino acid sequence first isolated from wild rice ( Oryza grandiglumis ) by wound and yeast extract stress treatment, thereby reducing external environmental stress and plant pathogens, in particular Pseudomonas syringe (Pseudomonas syringe) and Botrytis cine LA (Botrytis cinera) because it allows you to develop new disease-resistant crops and breeding plants for the agro-industrial and plant breeding industry is a very useful invention.

<110> CHUNG, Young-Soo <120> A pathogenesis-related gene OgPR1 isolated from wild rice, the          sequences of amino acid and the transgenic plant using the same <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 507 <212> DNA <213> Oryza grandiglumis <220> <221> CDS (222) (1) .. (504) <400> 1 atg gcg agt tcg tcg agc agg tta tcc tgc tgc ttg cta gtg ctc gcg 48 Met Ala Ser Ser Ser Ser Arg Leu Ser Cys Cys Leu Leu Val Leu Ala   1 5 10 15 gcg gca gcc atg gcg gcg acg gcg cag aac tcg gcg cag gac ttc gtg 96 Ala Ala Ala Met Ala Ala Thr Ala Gln Asn Ser Ala Gln Asp Phe Val              20 25 30 gac ccg cac aac gcg gcg agg gcg gag gtc ggg gtg ggg ccg gtg agc 144 Asp Pro His Asn Ala Ala Arg Ala Glu Val Gly Val Gly Pro Val Ser          35 40 45 tgg gac gac acg gtg gcg gcg tac gcg cag agc tac gcg gag cag cgg 192 Trp Asp Asp Thr Val Ala Ala Tyr Ala Gln Ser Tyr Ala Glu Gln Arg      50 55 60 cgc ggc gac tgc gcg ctg cag cac tcg gac tcc ggc ggg aag tac ggc 240 Arg Gly Asp Cys Ala Leu Gln His Ser Asp Ser Gly Gly Lys Tyr Gly  65 70 75 80 gag aac atc ttc tgg ggc tcc gcc ggc ggc gac tgg acg gcg gcg agc 288 Glu Asn Ile Phe Trp Gly Ser Ala Gly Gly Asp Trp Thr Ala Ala Ser                  85 90 95 gcc gtg tcg tcg tgg gtg tcg gag aag cag tgg tac gac cac ggc agc 336 Ala Val Ser Ser Trp Val Ser Glu Lys Gln Trp Tyr Asp His Gly Ser             100 105 110 aac agc tgc tcg gcg ccg gag ggg cag tcg tgc ggc cac tac acg cag 384 Asn Ser Cys Ser Ala Pro Glu Gly Gln Ser Cys Gly His Tyr Thr Gln         115 120 125 gtg gtg tgg cgc gac tcg acg gcg atc gga tgc gcc cgc gtc gtc tgc 432 Val Val Trp Arg Asp Ser Thr Ala Ile Gly Cys Ala Arg Val Val Cys     130 135 140 gac aac aac ctc ggc gtc ttc atc acc tgc aac tac tcg ccg ccg ggc 480 Asp Asn Asn Leu Gly Val Phe Ile Thr Cys Asn Tyr Ser Pro Pro Gly 145 150 155 160 aac ttc gtc ggc caa tct ccc tac tga 507 Asn Phe Val Gly Gln Ser Pro Tyr                 165 <210> 2 <211> 168 <212> PRT <213> Oryza grandiglumis <400> 2 Met Ala Ser Ser Ser Ser Arg Leu Ser Cys Cys Leu Leu Val Leu Ala   1 5 10 15 Ala Ala Ala Met Ala Ala Thr Ala Gln Asn Ser Ala Gln Asp Phe Val              20 25 30 Asp Pro His Asn Ala Ala Arg Ala Glu Val Gly Val Gly Pro Val Ser          35 40 45 Trp Asp Asp Thr Val Ala Ala Tyr Ala Gln Ser Tyr Ala Glu Gln Arg      50 55 60 Arg Gly Asp Cys Ala Leu Gln His Ser Asp Ser Gly Gly Lys Tyr Gly  65 70 75 80 Glu Asn Ile Phe Trp Gly Ser Ala Gly Gly Asp Trp Thr Ala Ala Ser                  85 90 95 Ala Val Ser Ser Trp Val Ser Glu Lys Gln Trp Tyr Asp His Gly Ser             100 105 110 Asn Ser Cys Ser Ala Pro Glu Gly Gln Ser Cys Gly His Tyr Thr Gln         115 120 125 Val Val Trp Arg Asp Ser Thr Ala Ile Gly Cys Ala Arg Val Val Cys     130 135 140 Asp Asn Asn Leu Gly Val Phe Ile Thr Cys Asn Tyr Ser Pro Pro Gly 145 150 155 160 Asn Phe Val Gly Gln Ser Pro Tyr                 165  

Claims (3)

Plant disease resistance gene OgPR1 of SEQ ID NO: 1 isolated from wild rice ( Oryza grandiglumis ). The amino acid sequence of SEQ ID NO: 2 encoded in the plant disease resistance gene OgPR1 isolated from wild rice ( Oryza grandiglumis ) described in claim 1. A plant disease resistant transformant plant, which is transformed using the plant disease resistance gene OgPR1 of SEQ ID NO: 1 isolated from wild rice ( Oryza grandiglumis ) and is asexually propagated by a tissue culture method.

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