KR20060033496A - A pathogenesis-related gene ogpr10 isolated from wild rice, the sequences of amino acid and the transgenic plant expressing the same gene - Google Patents

Abstract

The present invention relates to a plant disease resistance gene OgPR10 isolated from wild rice ( Oryza grandiglumis ) , its amino acid sequence and a transformed plant using the same, and more particularly, as isolated from wild rice by stress treatment with wound and yeast extract. Novel plant disease resistance gene that increases its expression by hormonal treatment such as, cantharidin and endothall, and is resistant to the Magnaporthe grisea pathogen causing rice blast OgPR10, its amino acid sequence and transformant plant using the same. The present invention is a very useful invention for the agricultural industry and plant breeding industry, by providing the above genes, to enable the new development and breeding of plant disease resistant crops against external environmental stress and plant pathogens, in particular Magnaporthe grisea ( Magnaporthe grisea ) .

Wild rice (Oryza grandiglumis), plant disease resistance gene OgPR10, Magnaporthe grisea

Description

A pathogenesis-related gene OgPR10 isolated from wild rice, the sequences of amino acid and the transgenic plant expressing the same gene}

Figure 1 shows the cDNA sequence of the plant disease resistance gene OgPR10 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 OgPR10 of the present invention isolated from wild rice ( Oryza grandiglumis ).

Figure 3 is to examine the expression pattern of the plant disease resistance gene OgPR10 of the present invention isolated from wild rice ( Oryza grandiglumis ), jasmonic acid (JA), salicylic acid (SA), candaridine in wild rice (Cantharidin: CN), endothall (EN), and yeast extract (fungal elicitor: F) after processing the total RNA is isolated and the result of the Northern blot analysis.

Figure 4 is a day 1, 3 and 6 days to examine the expression pattern of the plant disease resistance gene OgPR10 of the present invention isolated from wild rice ( Oryza grandiglumis ), rice blast control agent in wild rice The result is a result of Northern blot analysis after the total RNA is isolated by treatment.

5 is a result of histochemical GUS analysis on callus and transformants obtained by transforming the plant disease resistance gene OgPR10 of the present invention isolated from wild rice ( Oryza grandiglumis ) with cultivated rice ( dongjin rice). .

Figure 6 is treated with Magnaporthe grisea transformed cultivated rice ( Dongjin rice) transformed plants using the plant disease resistance gene OgPR10 of the present invention isolated from wild rice ( Oryza grandiglumis ), then the symptoms Observed.

The present invention relates to a plant disease resistance gene OgPR10 isolated from wild rice ( Oryza grandiglumis ) , its amino acid sequence and a transformed plant using the same, and more particularly, as isolated from wild rice by stress treatment with wound and yeast extract. Novel plant disease resistance gene that increases its expression by hormonal treatment such as, cantharidin and endothall, and is resistant to the Magnaporthe girsea pathogen causing rice blast OgPR10, its amino acid sequence and transformant plant using the same.

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. As an extreme example, the domestic food sowing situation in 1972 and 1973 caused a serious problem with the international price of rice soaring more than three times, despite a slight decrease of 3% in grain production at that time. 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. .

Therefore, the present inventors have first isolated the plant disease resistance gene OgPR10 induced by a large amount of insects from the wild rice ( Oryza grandiglumis ), not the general rice varieties, 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 OgPR10 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 OgPR10 isolated from wild rice ( Oryza grandiglumis ).

The object of the present invention is to isolate the plant disease resistance gene OgPR10 from the cDNA gene bank of fragments induced by treating stress (wound, 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 treatment of hormones, etc. of the gene, preparing and selecting a transformant using cultivated rice (Dongjin rice), the cultivated rice (Dongjin rice) was achieved by checking the resistance of the Magnaporthe grisea pathogen of the transformant to confirm 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 OgPR10 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 the wound and yeast extract stress on the leaves of wild rice ( Oryza grandiglumis ) by RACE PCR technique, a total sequence of 483 bp and 160 amino acids It is a novel gene consisting of sequences.

In addition, the expression is increased by treatment with hormones such as cantharidin and endothall, fungal elicitor, and probenazol, and when transformed with cultivated rice, Cultivated rice (Dongjin rice) transformants exhibit resistance by increasing the expression of the gene of the present invention against plant diseases, particularly Magnaporthe grisea pathogens causing rice blast.

Therefore, the gene OgPR10 of the present invention has been found to be a new plant disease resistance gene derived from wild rice, which is not previously known, and it is not only used for research, but also for plants against external environmental stress and plant pathogens, particularly Magnaporthe grisea pathogens. It can be used to newly develop and breed disease resistant crops.

The present invention also provides the amino acid sequence of SEQ ID NO: 2 encoded in the plant disease resistance gene OgPR10 isolated from wild rice ( Oryza grandiglumis ).

In another aspect, the present invention provides a transformant plant prepared using the plant disease resistance gene OgPR10 isolated from wild rice ( Oryza grandiglumis ) according to the other object.

The transformant plant of the present invention is the OgPR10 gene represented by the sequence of SEQ ID NO: 1 according to a conventional method, or the gene of interest other than the OgPR10 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.

The transformant plants provided by the present invention contain the plant disease resistance gene OgPR10 of the present invention isolated from wild rice ( Oryza grandiglumis ), which is excellent against external environmental stress and plant pathogens, especially Magnaporthe grisea pathogens. It is a plant disease resistant plant showing resistance.

As described above, the present invention, together with the novel plant disease resistance gene OgPR10 derived from wild rice and its amino acid sequence, is transformed into the gene to exhibit resistance to external environmental stress and plant pathogens including Magnaporthe grisea . The invention provides a transformant plant, which is a very useful invention for the agricultural and plant breeding industries.

Meanwhile, in the present invention, standard single letters 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 OgPR10 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 OgPR10 of the present invention was composed of a total of 483 bp, and the amino acid sequence was deduced by the nucleotide sequence of FIG. 1, as shown in FIG. 2. It consisted of 160 amino acids. In addition, the nucleotide sequence and the deduced amino acid sequence of the database using the Blast search program of the National Bioinformatics Center (NCBI) showed a high similarity of 90% to the pathogenic gene (PR10) known from ordinary rice, In rice, it appears as a gene not yet found, and the gene OgPR10 was found to be a new gene for plant disease resistance derived from wild rice.

Example 2: Plant Disease Resistance Gene OgPR10 Expression Test According to the Treatment of Hormones, etc.]

(1) Jasmonic acid in wild rice ( Oryza grandiglumis ) grown for more than 4 weeks in growth in order to test the expression patterns of various hormones and fungal elicitor treatment of the OgPR10 gene obtained in Example 1 : JA), salicylic acid (SA), cantharidin (CN), endothall (EN), and yeast extract (F), respectively, for 24 hours, and then treated whole leaves RNA was isolated using Tri-Reagent (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 483 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 OgPR10 increased in the treatment group (CN), endosal (EN), yeast extract (F).

(2) Also, in order to examine the expression patterns of the OgPR10 gene in more detail, Probenazol , a rice blast control agent, was used for 1 or 3 days in Oryza grandiglumis grown for more than 4 weeks in growth. And after each treatment for 6 days, Northern blot analysis was performed by separating total RNA in the same manner as in (1) above.

As a result, as can be seen in Figure 4, the expression of the plant disease resistance gene OgPR10 was the highest increase on the day 6, the expression increased from day 3 in the probevenazole treatment.

Example 3 Preparation and Selection of Transformant Using Cultivated Rice (Dongjin Rice)

In Example 2, the plant disease resistance gene OgPR10 of the present invention was confirmed to be a gene whose expression is increased by treatment with hormones, yeast extract and rice blast control agent.

Then, transformation was performed with Dongjin rice, a cultivated rice, in order to examine the function of the plant disease resistance gene. First inserted and then amplified by PCR for gene OgPR10 the pCAMBIA1301 plasmid DNA (CAMBIA, Canberra, Australia) at a site for cutting with KpnⅠ SacⅠ with restriction enzymes. The completed pCAMBIA1301-PR10 plasmid was introduced into Agrobacterium (LBA4404) by electroshock. Callus grown for 4 weeks for transformation into cultivated rice was soaked in a solution of 0.05% Silwet L-77 added to 0.8% Agrobacterium for 30 minutes and then in a medium for transformation (AAM medium) for 3 days. After culturing, the washed solution was washed three times, and selected from N6D selective medium to which cefotaxim was added. Selected callus was transferred to regeneration medium, differentiated into complete plants, and transferred to soil. As a result, all 50 transformed rice plants were obtained.

5-bromo-4-chloro-3-indolyl-glucuronide (5-bromo-4-chloro-3-indolyl-glucuronide: X-Gluc) for each of 50 callus and transformed rice plants Histochemical GUS staining for 24 hours using Duchefa's reagent showed blue color in 19 callus and plants. From the results, it was confirmed that the plant disease resistance gene OgPR10 of the present invention was properly expressed in cultivated rice ( Dongjin rice) transformants. On the other hand, Figure 5 shows a picture of some callus and plants in which expression of this GUS gene was observed.

Example 4 Magnesium Glysis of Cultivated Rice (Dongjin Rice) Transformant Magnaporthe grisea Confirmation of plant disease resistance to

Of the 50 cultivated rice (Dongjin rice) transformants prepared and selected in Example 3, 19 transformants whose expression of the GUS gene were observed (Nos. 1, 2, 19, 28, 29, 30) 32, 33, 35, 36, 37, 38, 41, 43, 45, 46, 47, 48, 49) and untransformed rice (control) Magnaporthe grisea , which causes rice blast, was diluted to 1 × 10 ⁵ cfu / ml fold and each leaf was inoculated with a spray. The inoculated cultivated rice (Dongjin rice) transformants were treated for 24 hours in a 25 ° C. wet culture, and then cultured for 7 days in a 16 hour light condition, and the symptoms of the cultured transformants were observed. Table 1 shows.

Results of confirming resistance to Magnaporthe grisea of cultivated rice transformed with the gene OgPR10 of the present invention Transgenic plant Control M1 M2 M19 M28 M29 M30 M32 M33 M35 Symptom (mm) 50 10 30 15 50 50 3 10 15 30 Resistant (R) / Byeongseong (S) S R S S S S R R S S Transgenic plant M36 M37 M38 M41 M43 M45 M46 M47 M48 M49 Symptom (mm) 20 30 5 10 5 15 10 5 10 10 Resistant (R) / Byeongseong (S) S S R R R S R R R R

As can be seen in Table 1, the pathology showed a symptom between 10 and 50, and the resistance showed a symptom between 0 and 10. In particular, transformants 30, 38, 43, and 47 exhibited 3 to 5 symptoms, showing excellent resistance to Magnaporthe grisea .

Therefore, the plant transformed with the gene OgPR10 of the present invention from the above results is superior resistance to plant pathogens, including Magnaporthe grisea that causes rice blasts than plants (control) not transformed with the gene It could be confirmed that the crop was resistant to plant diseases.

On the other hand, the growth of the four transformants were photographed in photographs, and the degree of symptom is displayed in a graph shown in FIG.

As is evident from the above examples, the present invention provides a novel plant disease resistance gene OgPR10 and its amino acid sequence first isolated from wild rice ( Oryza grandiglumis ) by wound and yeast extract stress treatment, thereby preventing external environmental stress and plant It is a very useful invention in the agricultural and plant breeding industry because it enables the new development and breeding of plant disease resistant crops against pathogens, especially Magnaporthe grisea , which causes rice blasts.

<110> CHUNG, Young Soo <120> A pathogenesis-related gene OgPR10 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> 483 <212> DNA <213> Oryza grandiglumis <220> <221> CDS (222) (1) .. (480) <400> 1 atg gct ccg gcc tgc gtc tcc gac gag cgc gcc gtc gcg gtg tcg gca 48 Met Ala Pro Ala Cys Val Ser Asp Glu Arg Ala Val Ala Val Ser Ala   1 5 10 15 gac cgg gtg tgg aag gtc ttc tcc gac gcc ccc gcg atg ccc aag gtc 96 Asp Arg Val Trp Lys Val Phe Ser Asp Ala Pro Ala Met Pro Lys Val              20 25 30 tgt gcc ggc ttc atc gac gcc att gag gtc gag ggg gat ggc gag gcg 144 Cys Ala Gly Phe Ile Asp Ala Ile Glu Val Glu Gly Asp Gly Glu Ala          35 40 45 ggc act gtc acc acc atg aag ctc aac cct gcc gtc gag gat ggg ggg 192 Gly Thr Val Thr Thr Met Lys Leu Asn Pro Ala Val Glu Asp Gly Gly      50 55 60 aca ttc aaa aca cgt gtg gtg gca cgt gac aat gca gct cat gtt atc 240 Thr Phe Lys Thr Arg Val Val Ala Arg Asp Asn Ala Ala His Val Ile  65 70 75 80 aag tca gag gtg ctg gat gtg ccg gcc gga agt aag gtg ggc aag ctc 288 Lys Ser Glu Val Leu Asp Val Pro Ala Gly Ser Lys Val Gly Lys Leu                  85 90 95 aag tcg cac gtg aca gag acg aag atc gag gcc gcc ggt gca ggc tct 336 Lys Ser His Val Thr Glu Thr Lys Ile Glu Ala Ala Gly Ala Gly Ser             100 105 110 tgc ttg gcc aag ata aag gtg gag tat gag ctt gag gac ggc ggc tcg 384 Cys Leu Ala Lys Ile Lys Val Glu Tyr Glu Leu Glu Asp Gly Gly Ser         115 120 125 ctg tca ccg gag aag gag aag ctc atc ctc gat ggc tac ttc ggc atg 432 Leu Ser Pro Glu Lys Glu Lys Leu Ile Leu Asp Gly Tyr Phe Gly Met     130 135 140 ctc aag atg atc gag gat tac ctc gtt gct cac cct acc gag tac gcc 480 Leu Lys Met Ile Glu Asp Tyr Leu Val Ala His Pro Thr Glu Tyr Ala 145 150 155 160 taa 483 <210> 2 <211> 160 <212> PRT <213> Oryza grandiglumis <400> 2 Met Ala Pro Ala Cys Val Ser Asp Glu Arg Ala Val Ala Val Ser Ala   1 5 10 15 Asp Arg Val Trp Lys Val Phe Ser Asp Ala Pro Ala Met Pro Lys Val              20 25 30 Cys Ala Gly Phe Ile Asp Ala Ile Glu Val Glu Gly Asp Gly Glu Ala          35 40 45 Gly Thr Val Thr Thr Met Lys Leu Asn Pro Ala Val Glu Asp Gly Gly      50 55 60 Thr Phe Lys Thr Arg Val Val Ala Arg Asp Asn Ala Ala His Val Ile  65 70 75 80 Lys Ser Glu Val Leu Asp Val Pro Ala Gly Ser Lys Val Gly Lys Leu                  85 90 95 Lys Ser His Val Thr Glu Thr Lys Ile Glu Ala Ala Gly Ala Gly Ser             100 105 110 Cys Leu Ala Lys Ile Lys Val Glu Tyr Glu Leu Glu Asp Gly Gly Ser         115 120 125 Leu Ser Pro Glu Lys Glu Lys Leu Ile Leu Asp Gly Tyr Phe Gly Met     130 135 140 Leu Lys Met Ile Glu Asp Tyr Leu Val Ala His Pro Thr Glu Tyr Ala 145 150 155 160  

Claims (3)

Plant disease resistance gene OgPR10 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 OgPR10 isolated from wild rice ( Oryza grandiglumis ) described in claim 1. Plant disease against Magnaporthe grisea , characterized in that it is transformed using the plant disease resistance gene OgPR10 of SEQ ID NO: 1 isolated from wild rice ( Oryza grandiglumis ) and asexually propagated by tissue culture method Resistant transformant plants.

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