KR20080050127A - Transgenic plant for increasing disease resistant and crop yield using osglu2 gene - Google Patents

Abstract

A transgenic rice using an OsGlu2 gene is provided to increase the resistance of rice against blase disease and promote the growth of the rice by over-expressing the OsGlu2 gene in the rice, thereby providing a novel species rice capable of increasing the quantity. A transgenic rice is characterized in that it shows increased resistance against rice blast disease and has increased quantity by being transformed by a recombinant vector OsCcl::OsGlu2 or recombinant vector Ai::OsGlu2, each of which includes a beta-1,3-glucanase(OsGlu2) cDNA gene having a sequence of SEQ ID : NO. 2.

Description

Transgenic Plant for Increasing Disease Resistant and Crop Yield Using OsGlu2 Gene}

1 is a protein after inoculation of the plaque from the rice leaves to confirm the protein expression by rice blast, Figure 1 (A) is 8 types of defense-related proteins through the 2-DE analysis, Figure 1 (B) Are two β-1,3-glucanase (OsGlu1, OsGlu2) genes identified by MALDI-TOF analysis.

Figure 2 is a cloning of the identified β-1,3-glucanase (OsGlu2) from the cDNA library, Figure 2 (A) is a specific primer combination of OsGlu2, Figure 2 (B) DNA fragments amplified by PCR It is shown.

FIG. 3 (A) shows a comparison of the amino acid sequences of the well-known β-1,3-glucanase from two types of β-1,3-glucanase (OsGlu1, OsGlu2) identified from rice and other plants (Arabidopsis and tobacco). , Figure 3 (B) shows their phylogenetic relationship.

Figure 4 shows the Genomic DNA gel blot analysis of the OsGlu2 gene.

Figure 5 is to confirm and produced the expression of the recombinant protein of OsGlu2 using a protein expression vector with histag.

Figure 6 shows the Northern blot analysis of the OsGlu2 gene according to plant hormones (JA, SA, ABA) and stress (CN, CHX, ST).

Figure 7 shows the change in the expression pattern of OsGlu2 gene by treating the blasts from rice leaves by time, Figure 7 (A) is Northern blot analysis confirmed from RNA samples treated with blasts by time from rice leaves, Figure 7 (B ) Is Western blot analysis from protein samples.

8 is an over-expression of the OsGlu2 gene, an RNAi rice transformed crop was produced by the Gateway method, and FIG. 8 (A) is a cloning method through the Gateway method of the OsGlu2 gene to produce a rice transformed crop. , Figure 8 (B) is to prepare a rice transformed crop using the over-expression vector OsCcl and RNAi vector P-Ai which is the basic vector of the present invention.

9 is a process of producing transformed rice by Agrobacterium transformation method.

10 is a constituent of the medium used in accordance with the various conditions and growth process used in the production of transformed rice.

Figure 11 is the expression pattern of each line of rice transformant by Northern blot analysis and Western blot analysis.

Figure 12 is a photograph of the comparison of the height of the transformed rice plants (OsCcl :: OsGlu2 and Ai :: OsGlu2) and control Nakdong rice (WT).

Figure 13 is a comparison of Stem of each transformed rice plant (OsCcl :: OsGlu2 and Ai :: OsGlu2) and control Nakdong rice (WT).

Figure 14 is a comparative photograph according to the deletion of the rice transformant of Ai :: OsGlu2 and control Nakdong rice (WT).

Figure 15 is a comparison table according to the quantity components of each transformed rice plants (OsCcl :: OsGlu2 and Ai :: OsGlu2) and control Nakdong rice (WT).

FIG. 16 is a comparison of resistance after 48 hours of rice blast treatment to Na: dong rice (WT) leaves of Ai :: OsGlu2 and control.

The present invention relates to a blast resistant and yield enhancing transgenic plant using OsGlu2 gene, and to a transgenic plant comprising a rice leaf blast resistant gene (OsGlu2) that directly protects against diseases of plants (rice). This gene, which is specifically expressed in rice, is introduced into the rice and overexpressed, thereby increasing resistance to diseases such as rice blasts, thereby promoting the growth of rice, and consequently providing new varieties of rice that can increase yield. can do.

As the plant grows, it is exposed to various environmental stresses, which have a great influence on the growth, development, and productivity of the plant. Plants actively combat environmental stress by operating various defenses to survive. A hydrolytic enzyme, β-1,3-glucanases, a member of the pathogen-related genes (PRs), a class of plant defense systems, is the cell wall of most filamentous fungi. By reducing the constituent β-1,3-glucan (glucan) is acting as a defense. In addition, β-1,3-glucanase affects the physiological or developmental stages of microsporogenesis of the crop itself, germination of pollen or seeds, and fertilization. Crazy

Control of diseases and pests is a very important factor in the cultivation of rice. Especially Pyricularia oryzae ) is an important disease that determines growth and yield. Until now, chemical fungicides (pesticides) and control methods have been developed and used, but many chemicals have to overcome such as toxicity, safety (residual problems) and control effects. It has a flaw. To date, molecular biology efforts have been attempted in some plant groups to introduce recombinant genes to make plants resistant to pests.

The present invention was able to identify two types of β-1,3-glucanase (OsGlu1, OsGlu2) during the analysis of disease-related proteins with optimal protection against rice blast. Among them, β-1,3-glucanase (OsGlu2) was cloned, and the antibody was produced to be used for its functional studies. To transduce the gene into rice, transgenic rice was prepared using an over-expression vector, Oryza sativa cytochrome C promoter (OsCc1) or an ABA-inducible vector, ABA-inducible promoter (Ai-P). By multiplying, disease resistance such as blast disease was increased, and new varieties of transformed rice were expected to be expected to increase the yields by promoting growth and yield components.

Therefore, the object of the present invention is rice ( Oryza The present invention provides a transgenic plant having an increased yield containing genes (OsGlu2) having a defensive resistance against leaf blasts of sativa ) and enhanced disease resistance of rice blasts.

Another object of the present invention to provide a recombinant vector for plant transformation comprising a gene (OsGlu2) having a protective resistance against rice leaf blast.

Another object of the present invention is to provide a new varieties of rice and a method of producing the same by significantly improving the resistance and yield to diseases by producing a transformant of rice.

In order to achieve the above object, the present invention provides an expression vector OsCcl :: OsGlu2 or Ai :: OsGlu2 that includes a rice leaf blast resistance gene (OsGlu2) that directly defends against diseases of plants (rice). Oryza sativa ). This gene, which is specifically expressed in rice, is introduced into the rice and overexpressed, thereby increasing resistance to diseases such as rice blasts, thereby promoting the growth of rice, and consequently providing new varieties of rice that can increase yield. do.

The present invention provides a recombinant vector OsCcl :: OsGlu2 or Ai :: OsGlu2 for plant transformation, comprising β-1,3-glucanase (OsGlu2) cDNA gene, and transforming with the recombinant vector for plant transformation. The result is a transgenic rice with improved yield and improved disease resistance in rice blasts.

Incidentally, rice was prepared using OsCcl :: OsGlu2 or Ai :: OsGlu2 , a recombinant vector for transformation containing a gene encoding β-1,3-glucanase (OsGlu2), which increases protein expression by rice blast. Transformation to promote the growth of rice and to improve the resistance to disease, including.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, the scope of the present invention should not be construed as being limited to these embodiments.

Example 1 Identification of β-1,3-glucanase in Defense Related Proteins against Rice Blast

After inoculating the plaque from the rice leaves, the proteins were extracted to identify defense-related proteins, mainly on the proteins with increased protein expression by the rice plaque.

Protein Extraction, 2-DE Assay, MALDI-TOF Identification

Protein Extraction: Samples of rice blast fungus (M. grisea) were inoculated for 48 hours and 72 hours on the leaves of Jinheung rice grown for 4-5 weeks in a greenhouse at 20-30 ° C. The promotion rice which did not treat blasting was used as a control. Peg (PEG) precipitation was used to separate proteins from this. This method is an effective method for analyzing relatively low-expression proteins by effectively eliminating rubisco proteins, which are significantly expressed in green plant protein analysis. The sampled rice leaves were ground with liquid nitrogen, and then 2 g of each sample was treated with Mg / NP-40 buffer (0.5 M Tris-HCl (pH 8.3), 2% v / v NP-40, 20 mM MgCl2, 2% v). / v b-mercaptoethanol, 1 mM phenyl methyl sulfonyl fluoride, and 1% w / v polyvinyl polypyrrolidone) were centrifuged at 12000xg for 15 minutes, the supernatant was extracted, and 50% w / v PEG was added to give a final sample supernatant concentration. 15% w / v PEG. The supernatant was left on ice for 30 minutes and then centrifugation (12000xg) for 15 minutes to precipitate supernatant protein extracts obtained from acetone.

2-DE Assay: 15% PEG supernatant protein extract was analyzed as 2-DE. IEF (1-DE) gel (4.5% w / v acrylamide solution, 9.5 M urea, 2% v / v NP-40, and 2.5% v / v ampholytes (pH 310: pH 58: pH 58: pH 46.5 = 1: 3.5: 2.5) is hardened for 6 hours Each sample (150ug for silver stained protein and 500ug for CBB stained protein) is dissolved in sample buffer and loaded into IEF gel for 12 hours at 600V, 6 hours at 800V, and 1000V. Place each gel in a 20 mL screw-cap tube and add 5 mL of equilibration buffer (10% v / v glycerol, 2.5% w / v SDS, 125 mM Tris-HCl, pH 6.8), 5% v Add / v β-mercaptoethanol, and 0.1 mg / mL bromophenol blue) at room temperature for 30 minutes, equilbration gel Low-Melting Agarose (low-melting agarose: 0.2g, 4 × buffer5ml, H2O: 15ml, 10% SDS : 200ul) and put it on the pre-hardened 2-DE gel, SDS-PAGE (second dimension) overnight at 40 ~ 50 V. After the dye is removed the next day, fix (50% methanol, 10% acetic acid, 40% dd H2O). ) And then silver stain (Pretreat, Impregnate, Develop). The taine protein spots are removed from the gel, washed (50% v / v ACN in 0.1 M NH4HCO3), and dried in vacuo.The gel fragments are 10 mM DTT solution (55 mM iodoacetamide in 0.1 M NH4HCO3) in 55 ° C for 45 minutes. Add 0.1 M NH4HCO3, cool on ice, wash with (50% ACN in 0.1 M NH4HCO3), add (10 mL digestion buffer containing 25 mM NH4HCO3 and 12.5 ng / mL trypsin), and dry. As shown in FIG. 1 (A), eight types of defense-related proteins were identified through 2-DE analysis.

MALDI-TOF Identification: All proteins were run using a Voyager-DE STR MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, Mass., USA). Figure 1 (B) is to identify two types of β-1,3-glucanase (OsGlu1, OsGlu2) gene by MALDI-TOF analysis.

< Example  2: Identified  β-1,3- glucanase ( Osglu2 Genetic Identification>

Identified  β-1,3- glucanase  ( Osglu2 ) cDNA library from cloning

The identified β-1,3-glucanase (OsGlu2) was identified using a database program of the National Center for Biotechnology Information (NCBI) to identify genes and specific primers were prepared. Β-1,3-glucanase (OsGlu2) was amplified by PCR using gene-specific primers from cDNA gene library obtained by reacting mRNA prepared from rice leaves derived from blasts, and then pGEM-T Easy Vector (Promega, Madison). , WI, USA) and confirmed the sequence by cloning. Figure 2 (A) shows a specific primer combination of OsGlu2.

 OsGlu2 (AB027430) Forward 5'- ATG GGT TTC GCT CCC ATG-3 '(SEQ ID NO: 3)

 OsGlu2 (AB027430) Reverse 5'-TAA GAA ACT AAT GCT GTA CGA-3 '(SEQ ID NO: 4)

DNA markers and OsGlu2 PCR products were loaded from the agarose gel and confirmed to be 1008 bp. Figure 2 (B) shows a DNA fragment amplified by PCR.

Identified  β-1,3- glucanase ( OsGlu1 , Osglu2 Β-1,3- in other plants glucanase  Amino acid sequence Homology  compare

Cloned OsGlu2 is a phylogenetic tree using Neighbor Joining methods of Clustal X program from another identified β-1,3-glucanase (OsGlu1) and other β-1,3-glucanases present in other plants. The comparison was made using (see FIG. 3 (A)). Β-1,3-glucanase genes (AtGLU1 (NCBI Acc. No AY096465) and AtGLU2 (AY096541)) and two β-1,3-glucanase genes NtGLU1 (NCBI Acc. No M20618) ) And NtGLU2 (M20619)) had high similarities to each other, and relatively low, with 62% homology between OsGlu1 (AB027428) and OsGlu2 present in rice (see FIG. 3 (B)). This confirmed that the gene is β-1,3-glucanase (OsGlu2).

Cloning Done Osglu2 of genome On copy  Can be ok

DNA fragment of OsGlu2 cloned into pGEM-T Easy Vector (10ug) was cut for 3-6 hours at 37 ° C using restriction enzymes (EcoR I, Hind III, Xba I) and loaded onto 1% DNA agarose gel. Next, transfer to nylon membrane (GeneScreen Plus, NEN). The membrane was labeled with OsGlu2 probe using [-32P] dCTP (see FIG. 4).

Protein expression and antibodies ( antibody Production

As shown in FIG. 5, expression of the recombinant protein of OsGlu2 was confirmed and produced using a protein expression vector with histag. To construct the antibody, the OsGlu2 gene was cloned using pET28 with histag, a protein expression vector. First, primers were prepared to clone OsGlu2 into pET28 vector.

  OsGlu2 Nde I Forward 5'- GCG CAT ATG ATG GGT TTC GCT CCC ATG-3 '(SEQ ID NO: 5)

  OsGlu2 BamH I Reverse 5'- GCG GGA TCC TTA GAA ACT AAT GCT GTA CGA-3 '(SEQ ID NO: 6)

After recovering the amplified insert by PCR using the prepared primers, the plasmid DNA extracted by ligation to the pGEM-T Easy Vector was cut with Nde I / BamH I restriction enzyme to recover the insert, and the DNA was extracted from the pET28 vector. The insert recovered from OsGlu2 was cut and cloned with Nde I / BamH I restriction enzyme.

To confirm protein expression, colonies generated by transforming pET28 vector and pET28 :: OsGlu2 into XL-1 blue cells were inoculated into LB broth and grown with IPTG at 37 ° C. The pellet obtained by centrifugation (6000xg) for 5 minutes was added to the protein loading butter, boiled for 10 minutes, centrifugation (6000xg) for 5 minutes, and only the supernatant was loaded on a 12% SDS-polyacrylamide gel. Soluble recombinant protein pET28 :: OsGlu2 and protein markers are also loaded. The gel was removed and stained with comssie brilliant blue R250 to confirm protein expression and OsGlu2 protein size.

To prepare an antibody from the recombinant protein pET28 :: OsGlu2, 1 ~ 2mg / mL protein and freund's complete adiuvants were emulsified 1: 1 or more for 30 minutes. In this way, the rabbit is injected first, secondly after 4 weeks, and thirdly after 2 weeks. Seven days after the third injection, blood is collected and tittered. Two weeks after the third injection, only four (4) injections of protein (antigen) were taken and a week later, blood was collected, and only serum was recovered from the blood.

For plant hormones and rice blast Osglu2 Expression Analysis of

Jasmonic acid (JA, 250uM), Salicylic acid (SA, 5mM), Abscisic acid (ABA, 200uM) and cantharidin (CN, 1uM), Cycolheximide (CHX, 1uM), Staurosporine (ST, 10uM) was treated for 48 hours (+), no treatment (-) RNA was isolated. In addition, RNA and protein were isolated by treating rice blasts (KJ401, incompatible and KJ101, compatible) with time (12, 18, 24, 36, 48, 60, 72 hours).

As shown in FIG. 6, Northern blot analysis of the OsGlu2 gene according to plant hormones (JA, SA, ABA) and stress (CN, CHX, ST) revealed that this gene was induced by JA but expressed by ABA. This was suppressed. FIG. 7 (A) shows Northern blot analysis confirmed from RNA samples treated with blasting from rice leaves by time, and FIG. 7 (B) shows Western blot analysis confirmed from protein samples. The amount of mRNA (Northern blot) and protein (Western blot) expressed was found to be higher in the incompatible reaction earlier than in the compatible reaction.

RNA isolation: Sampled rice leaves are ground with liquid nitrogen, followed by voltex with RNA extraction butter (0.2M tris (pH 9.0), 0.4M Licl, 25mM EDTA, 1% SDS) and phenol for RNA and β mercaptoethanol. (3000xg) 5 minutes to take only the supernatant, then PCI (phenol 25: chroloform 24: isoamine alcohol 1 for RNA) and add 10 minutes to the voltex, centrifugation (7500xg) to separate the supernatant. Add chroloform 1: 1 and centrifugation (7500xg) for 10 minutes, and then take only the supernatant, add isoprophanol (1: 1), add 3M sodium acetate (1/10) and settle for 1 hour at -20 ° C. . After 1 hour, discard the supernatant by centrifugation (7500xg) for 10 minutes, dry only the remaining pellet, add TE solution for RNA to dissolve, and add LiCl so that the final concentration becomes 2M LiCl. After 15 minutes of centrifugation (12000xg), wash with 70% EtOH, and after drying, add TE solution to dissolve it, add RNA in P.C.I solution, chroloform, 100% EtOH and 3M sodium acetate (1/10), and clean the RNA. Finally, store at -20 ° and dissolve in 10 mM tris-Hcl (pH7.5).

Northern blot analysis: RNA samples (20 uM) were loaded onto formaldehyde denaturing agarose gels and transferred to nylon membrane (GeneScreen Plus, NEN). The membrane was labeled with OsGlu2 probe using [-32P] dCTP.

Western blot analysis: Put protein loading butter, boil for 10 minutes, centrifugation (6000xg) for 5 minutes, load only the supernatant onto 12% SDS-polyacrylamide gel, blotting onto PVDF membrane, confirm with Ponceau S, wash with distilled water, and then wash with 5% v / v Cross-link with glutaraldehyde solution. The cross-linked membrane was blocked with 7% w / v nonfat dry milk dissolved in TTBs (50 mM Tris-HCl pH 8.2, 0.1% v / v Tween 20, and 150 mM NaCl) solution for 2 hours and then purified. Add rabbit antibody OsGlu2 (diluted 1: 1000). This antigen-anti-body interaction is for 2 hours and then washed 20 times with TTBs 3 times. Secondary goat anti-rabbit IgG antibody conjugated with horseradish peroxidase diluted 1: 5000 was added to TTBs and immunodetection was performed for 2 hours. The blots are again washed 20 times with TTBs solution three times. Finally, immunoblot signals are detected using ECL (PerkinElmer Life Sciences, Boston, MA, USA).

< Example  3: Osglu2  Gene Production and Growth of Rice Transformant>

Gateway  Through the way Osglu2 Rice transformant vector cloning

Gateway method (Invitrogen) was used to prepare rice transformants. First, primers were prepared using NCBI's database program to prepare adapter PCR primers (see FIG. 8 (A)).

   OsGlu2 12b attB1 5'- GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGA AAG CAT GGCGTTGCT -3 '(SEQ ID NO: 7)

   OsGlu2 12b attB2 5'- GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC GAA ATT GTA GGA GTA TGT CGG CGA -3 '(SEQ ID NO: 8)

   attB1 adapter primer 5'- GGG GAC AAG TTT GTA CAA AAA AGC AGG CT-3 '(SEQ ID NO: 9)

   attB2 adapter primer 5'- GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3 '(SEQ ID NO: 10)

Using a full-length cDNA of OsGlu2, amplify and recover 30 cycles of fist PCR, and then amplify again by rotating 25 cycles of second PCR using an adapter primer to recover the PCR product of OsGlu2 with an adapter. It is BP reacted with pDONR201 (Invitrogen) vector (more than 25 ° C for 16 hours). Subsequently, incubation at 25 ° C for 8 hours using an LR reaction (OsCc1 promoter (overexpression vector), a P-Ai promoter (ABA inducible vector), which is the vector to be coined with the BP reaction mixture, followed by adding proteinase K at 37 ° C for 1 hour at 37 ° C). incubation) to transform to DH 5β. The vector to be coined contains the 3 'region of the potato proteinase inhibitor II gene (3'pinII) and the bar gene expression cassette that contains a 35S promoter, bar coding region, 3' region of the nopaline synthase gene (3'nos). have.

A rice transgenic crop was prepared using OsCcl, an over-expression vector of the present invention, and P-Ai, an RNAi vector (see FIG. 8 (B)).

Preparation of Rice Transformant Using Agrobacterium

OsGlu2 gene in each of the overexpression vector and ABA-nducible vector was cultured at 28 ° C for 3 days in ABST medium by cell mix with Agrobacterium tumefaciens strain (LBA4404) and E. coli HB101 (pRK2013). transformation to callus.

Nakdong rice seeds were disinfected with 70% EtOH, washed in 3% Chlorax for 1 hour, washed, and planted in 2N6 medium to incubate at 28 ° C for 3-4 weeks in the dark. Only good callus formed was screened and cultured at 28 ° C in 2N6 medium 4 days before transformation. After mixing the cell mix Agrobacterium concentration to OD600nm to 3 at OD600nm, the selected callus was infected, and then transferred to 2N6-AS medium for 3 days at 20 ° C. After immersing the callus in a solution containing cefortaxiume in sterile water for about 1 hour, dried on filter paper and placed on a 2N6-CP medium and incubated for 3 weeks at 28 ° C. Only the callus, showing no growth, was selected and transferred to N6-70CP medium, and then cultured for 10 days at 28 ° C. Selected callus was transferred to MSCP medium and cultured in light. Root and stem-derived plants were transferred to magent medium and grown in soil (FIG. 9 shows propagation process from callus of rice transformants and FIG. 10 shows the medium and its composition used).

Osglu2 Transformation and Verification of Rice Transformant Lines

Northern blot analysis and Western blot analysis were performed to verify the To generation line of rice transformant Ai :: OsGlu2 of ABA-inducible vector grown from callus induced by Agrobacterium transformation. As shown in FIG. 11, the expression patterns of each line of rice transformants by Northern blot analysis and Western blot analysis were examined, and lines were expressed after the expression of the lines.

< Example  4: Osglu2  Functional Verification from Rice Transformants of Genes>

Osglu2 Growth of Rice Transformants in Korea

The lines of verified rice transformant Ai :: OsGlu2 were selected and propagated to T2 generations to observe their growth. Nakdong rice was used as a control. Among them, Ai :: OsGlu2 T2 generation line 2-2 and 10-3 line were observed intensively. We also observed and compared line 1 of OsCcl :: OsGlu2 using the OsCc1 promoter (overexpression vector). Yield components such as growth growth, height, flag leaf length, stem thickness, number of grains, rice grain per ear, and grain weight were investigated.

As shown in FIG. 12, the photographs of the transgenic rice plants (OsCcl :: OsGlu2 and Ai :: OsGlu2) and the control of Nakdong rice (WT) were compared to show the Ai :: OsGlu2 transformant (T2). -2-2, T2-10-3) line was found to promote growth than the control (WT) line, overexpression (OsCcl :: OsGlu2 T2-2-1) line. FIG. 13 is a Stem comparison photograph of each of the transformed rice plants (OsCcl :: OsGlu2 and Ai :: OsGlu2) and the control Nakdong rice (WT), wherein the Ai :: OsGlu2 lines of the transformants were grown more strongly. Figure 14 is a comparison picture of the deletion of the Ai :: OsGlu2 rice transformant and the control Nakdong rice (WT), it can be seen that the transformant growth is large. FIG. 15 is a comparison table according to the yield components of each transformed rice plant (OsCcl :: OsGlu2 and Ai :: OsGlu2) and a control, Nakdong rice (WT), where the transformants have a large number of heights, grains and rice grains per ear. Could confirm.

Osglu2 Of rice transformants against rice blast disease

To verify the protective function of β-1,3-glucanases, a hydrolytic enzyme belonging to pathogen-related genes (PRs), OsGlu2 rice transformants were inoculated with blasts. Ai :: OsGlu2 T2 generation, which grew well for 4 to 4 weeks, was inoculated with a blast bottle (KJ101) for each line and observed for 3 days. Among them, the number of lesions on the leaves of lines 18 and 27 of Ai :: OsGlu2 T2 generation showed a significant decrease and resistance. Then, Northern blot analysis and Western blot analysis were performed for verification. At this time, the ABA was treated before inoculation of the blast (KJ101) to express the OsGlu2 gene.

As shown in FIG. 16, a rice transformant of Ai :: OsGlu2 and a control group of Nakdong rice (WT) leaves were treated after 48 hours of rice blasting. Figure 16 (A) was treated with ABA in advance of the A :: OsGlu2 rice-type transformant and control Nakdong rice (WT) leaves, and then treated the rice blast for 48 hours to examine the lesions of these leaves. FIG. 16 (B) shows RNA and protein separation from Ai :: OsGlu2 rice transformants treated with rice blast and Nakdong rice (WT) leaves. I looked. The lines of the two transformant rice (Ai :: OsGlu2 T2-2-2, T2-10-3) were resistant to blast disease compared to the control (WT), which increased the amount of gene expression in the plant, resulting in β-1,3- Increasing expression of glucanase also suggests that blast resistance is also increased. This shows that the disease resistance in Ai :: OsGlu2 transformants is correlated with blast resistance due to the increased expression of β-1,3-glucanase.

The present invention provides a rice ( Oryza) by making the expression vector OsCcl :: OsGlu2 or Ai :: OsGlu2 containing a rice leaf blast resistance gene that directly protects against diseases of plants (rice) sativa ). This gene, which is specifically expressed in rice, is introduced into the rice and overexpressed, thereby increasing resistance to diseases such as rice blasts, thereby promoting the growth of rice, and consequently providing new varieties of rice that can increase yield. can do. As a result, while reducing the use of chemicals such as pesticides, which have many conventional problems, it is expected to significantly increase the environmental stress resistance to diseases, thereby increasing yields and producing high quality rice, thereby greatly improving the economics.

<110> Gyeongsang National University Industial & Academic Collaboration Foundation <120> OsGlu2 gene for increasing Disease Resistant and crop yield <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 336 <212> PRT <213> Oryza sativa <400> 1 Lys Met Gly Phe Ala Pro Met Leu Ser Val Ala Val Leu Leu Gly Thr   1 5 10 15 Leu Ala Ala Phe Pro Ala Ala Val His Ser Ile Gly Val Cys Tyr Gly              20 25 30 Val Val Ala Asn Asn Leu Pro Gly Pro Ser Glu Val Val Gln Leu Tyr          35 40 45 Arg Ser Lys Gly Ile Asp Ser Met Arg Ile Tyr Phe Ala Asp Ala Ala      50 55 60 Ala Leu Asn Ala Leu Ser Gly Ser Asn Ile Gly Leu Ile Met Asp Val  65 70 75 80 Gly Asn Gly Asn Leu Ser Ser Leu Ala Ser Ser Pro Ser Ala Ala Ala                  85 90 95 Gly Trp Val Arg Asp Asn Ile Gln Ala Tyr Pro Gly Val Ser Phe Arg             100 105 110 Tyr Ile Ala Val Gly Asn Glu Val Gln Gly Ser Asp Thr Ala Asn Ile         115 120 125 Leu Pro Ala Met Arg Asn Val Asn Ser Ala Leu Val Ala Ala Gly Leu     130 135 140 Gly Asn Ile Lys Val Ser Thr Ser Val Arg Phe Asp Ala Phe Ala Asp 145 150 155 160 Thr Phe Pro Pro Ser Ser Gly Arg Phe Arg Asp Asp Tyr Met Thr Pro                 165 170 175 Ile Ala Arg Phe Leu Ala Thr Thr Gly Ala Pro Leu Leu Ala Asn Val             180 185 190 Tyr Pro Tyr Phe Ala Tyr Lys Asp Asp Gln Glu Ser Gly Gln Lys Asn         195 200 205 Ile Met Leu Asn Tyr Ala Thr Phe Gln Pro Gly Thr Thr Val Val Asp     210 215 220 Asn Gly Asn Arg Leu Thr Tyr Thr Cys Leu Phe Asp Ala Met Val Asp 225 230 235 240 Ser Ile Tyr Ala Ala Leu Glu Lys Ala Gly Thr Pro Ser Val Ser Val                 245 250 255 Val Val Ser Glu Ser Gly Trp Pro Ser Ala Gly Gly Lys Val Gly Ala             260 265 270 Ser Val Asn Asn Ala Gln Thr Tyr Asn Gln Gly Leu Ile Asn His Val         275 280 285 Arg Gly Gly Thr Pro Lys Lys Arg Arg Ala Leu Glu Thr Tyr Ile Phe     290 295 300 Ala Met Phe Asp Glu Asn Gly Lys Pro Gly Asp Glu Ile Glu Lys His 305 310 315 320 Phe Gly Leu Phe Asn Pro Asn Lys Ser Pro Ser Tyr Ser Ile Ser Phe                 325 330 335 <210> 2 <211> 1159 <212> DNA <213> Oryza sativa <400> 2 ctaagatggg tttcgctccc atgctttcag tggccgtgct ccttggaacc ttggcagcat 60 ttcctgcagc ggtacactcc atcggcgtgt gttacggcgt ggtcgccaac aacctgcccg 120 ggccgagcga agtcgtgcag ctgtacagat ccaaggggat cgattcgatg cgcatctact 180 tcgcggacgc cgccgccctc aatgcactca gcggctcgaa catcggtctc atcatggacg 240 tcggcaacgg caacctctcg agcttagctt ccagcccctc cgccgcagcc ggttgggtca 300 gggacaacat ccaggcctac ccgggcgtct ccttccgcta catcgccgtc ggcaacgagg 360 ttcaaggcag cgacaccgcg aacatccttc cggccatgcg gaacgtcaac agcgcgctgg 420 tggcggccgg cctcggcaac atcaaggtat ccacgtcggt caggttcgac gcgttcgccg 480 acaccttccc gccctccagc ggcaggttca gggacgacta catgaccccg atcgcgaggt 540 tcttggccac caccggcgcg ccgctgttgg cgaacgtgta cccctacttc gcctacaaag 600 acgaccagga aagcgggcag aaaaacatca tgctcaacta cgccaccttc cagccgggca 660 cgacggtggt ggacaacggg aacaggctga cctacacgtg cctcttcgac gcgatggtcg 720 actccatcta cgccgcgctg gagaaggccg gcacaccgag cgtcagcgtc gtcgtgtccg 780 agagcgggtg gccgtccgcc ggagggaagg ttggggccag cgtgaacaac gcgcagacat 840 acaaccaggg gctgatcaac catgtccgag gcggcacgcc caagaagcgc agggcgttgg 900 agacttacat atttgctatg ttcgacgaga acggcaagcc tggggatgag atcgagaagc 960 actttgggct gttcaacccc aataagtcgc catcgtacag cattagtttc taaggggatc 1020 gtacagactg atatatattt ttatatggaa taagttgctc ccgtgatagg ccaaacgcaa 1080 gcatgggaag caagcatagt gccgtgtggt gtccatgcat ctttcccaat ttgtgaaata 1140 tatactccca gtgaatttt 1159 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of OsGlu2 <400> 3 atgggtttcg ctcccatg 18 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primr of OsGlu2 <400> 4 taagaaacta atgctgtacg a 21 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer of OsGlu2 for cloning into pET28 vector <400> 5 gcgcatatga tgggtttcgc tcccatg 27 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of OsGlu2 for cloning into pET28 vector <400> 6 gcgggatcct tagaaactaa tgctgtacga 30 <210> 7 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> OsGlu2 12b attB1 primer <400> 7 ggggacaagt ttgtacaaaa aagcaggctt catggaaagc atggcgttgc t 51 <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> OsGlu2 12b attB2 primer <400> 8 ggggaccact ttgtacaaga aagctgggtc gaaattgtag gagtatgtcg gcga 54 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> attB1 adapter primer <400> 9 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> attB2 adapter primer <400> 10 ggggaccact ttgtacaaga aagctgggt 29  

Claims (4)

Recombinant vector OsCcl :: OsGlu2 for plant transformation comprising a β-1,3-glucanase (OsGlu2) cDNA gene having the nucleotide sequence of SEQ ID NO: 2. Recombinant vector Ai :: OsGlu2 for plant transformation comprising a β-1,3-glucanase (OsGlu2) cDNA gene having the nucleotide sequence of SEQ ID NO: 2. The transformed rice is transformed with the recombinant vector for plant transformation according to claim 1 or 2, wherein the yield is increased and the disease resistance of rice blast is enhanced. OsCcl :: OsGlu2 , a recombinant vector for transformation containing a gene encoding β-1,3-glucanase (OsGlu2), which increases protein expression by rice blast Or Ai :: OsGlu2 to transform the rice to promote the growth of rice and to improve disease resistance.

Images