KR100718549B1 - Vector comprising hrp gene from erwinia pyrifoliae transgenic agrobacterium tumefaciens and pathogenic resistant transgenic plant using the same - Google Patents

Abstract

A vector comprising hrp(hypersensitive response and pathogenicity) gene encoding harpin protein, derived from Erwinia pyrifoliae, and a transgenic Agrobacterium tumefaciens and a pathogenic resistant transgenic plant by using the same vector are provided to activate the pathogenic resistant mechanisms of the plant itself without inducing hypersensitive response and replace toxic agricultural chemicals. The vector pMJC-GB-hrpEp comprises hrpEP gene derived from Erwinia pyrifoliae wild type #3 KCCM 10283. The transgenic Agrobacterium tumefaciens is produced by transforming Agrobacterium tumefaciens by the vector pMJC-GB-hrpEp. The pathogenic resistant transgenic plant is produced by transforming a plant by the vector pMJC-GB-hrpEp or the transgenic Agrobacterium tumefaciens, wherein the plant is tobacco or rice.

Description

VECTOR COMPRISING HRP GENE FROM ERWINIA PYRIFOLIAE, TRANSGENIC AGROBACTERIUM TUMEFACIENS AND PATHOGENIC RESISTANT TRANSGENGEN }

1 is a hrp Ep from the Erwinia pyrifoliae wild type # 3, which is a black blight pathogen used in the present invention The cleavage map of the plant transformation vector pMJC-GB- hrp Ep containing the gene is shown.

Figure 2 shows the result of colony PCR after transforming the plant transformation vector pMJC-GB- hrp Ep to Agrobacterium tumefaciens ( Agrobacterium tumefaciens ).

3A and 3B show the results of PCR and real-time PCR in order to confirm the introduction of the hrp Ep gene into tobacco plants.

Figure 4 is a photograph showing the resistance to gray mold of tobacco plants transformed with the hrp Ep gene. A and C represent tobacco plants to which the hrp Ep gene is not introduced, and B and D represent tobacco plants to which the hrp Ep gene is introduced.

Figure 5 shows the hypersensitivity response marker genes (hsr201, hsr203j, hsr515 and HIN1) and acquired resistance-related marker genes (PR-) in tobacco plants (W) to which the hrp Ep gene was not introduced and tobacco plants (T) to which the hrp Ep gene was introduced. 1a, PR2, PR3 and Chia5) after the RT-PCR to measure the expression level, electrophoresis picture is shown.

Figure 6 is a photograph showing the resistance to gray mold of tobacco plant T ₁ generation to which the hrp Ep gene is introduced. A, C and E represent tobacco plants to which the hrp Ep gene has been introduced, and B, D and F represent tobacco plants to which the hrp Ep gene has not been introduced.

Figures 7a to 7d are hrp Ep gene has not been introduced into tobacco plants as hrp Ep genes sensitization marker gene in the introduced tobacco plant, acquired resistance to the relevant marker genes, NPR1, GST1, PR-1b and ethylene synthesis genes (NT -EFE26, NT-1A1C, DS321, NT-ACS1 and NT-ACS2) after performing the real-time PCR, the results are shown in a graph comparing the results.

The present invention is a plant transformation vector pMJC-GB- hrp Ep comprising the hrp Ep gene derived from Erwinia pyrifoliae wild type # 3, which is a black blight pathogen, the vector pMJC-GB- hrp Ep Transformed Agrobacterium tumefaciens and pathogen resistant transgenic plants.

Harpin is a protein produced by gram-negative plant pathogenic bacteria that is acidic, glycine rich, protease sensitive and heat stable (Peng et al). , Phytopathology. 94: 1048-1055, 2004).

Harpin is known to be transferred from plant pathogens to plant cells by a hypersensitive response and pathogenicity (Hrp) system that is present in the plant when it enters the plant (Bauer et al.). al ., Mol. Plant-Microbe Interact. 8: 484-491, 1995; Charkowski et al ., J. Bact. 180: 5211-5217, 1998).

To date, the types of harpins known to be secreted from plant necrotic bacteria through plant hrp pathways are nephogenic bacteria, such as Erwinia amylovora Ea 321, Hari et al. al ., Science. 257: 85-88, 1992), Pseudomonas ache Guy (Pseudomonas syringae ) pv. (pathovar) syringae Pss61 of HrpZPss (He et al ., Cell. 73: 1255-1266, 1993), PopA1 of Pseudomonas solanacearum (Arlat et al ., EMBO J. 13: 543-553, 1994), Xanthomonas oryzae ) pv. HpaGXoo (Wen and Wang, Acta Phytopathol. Sin. 31: 296-300, 2001) and Pseudomonas syringae of oryzae pv. There are HrpPsph such phaseolicola.

Most of these have been known to cause hypersensitive reactions alone without the other proteins that make up the hrp system. hpaGxoo does not produce hypersensitivity when expressed in plants (Peng et al ., Phytopathology 94: 1048-1055, 2004) and induces resistance to a wide range of pathogens by activating resistance-related genes in different pathways in plants. (Peng et) al ., Phytopathology 94: 1048-1055, 2004).

The mechanism of action when harpin was applied to the plant has not been elucidated yet, but the results indicate that the primary action of harpin to induce hypersensitivity is in the cell wall or plasma membrane and in the plant. Time suggests that it can separate resistance-induced pathways and activate resistance-related pathways (Dayakar et. al ., Plant Molecular Biology. 51: 913-924, 2003; Peng et al ., Phytopathology 94: 1048-1055, 2004).

On the other hand, among the major pathogens that occur in plants, gray mold disease is the most terrestrial disease caused by plant cultivation, it is known to cause great damage to vegetables, flowers, fruit vegetables. The main reason why the disease is caused only by plant cultivation is derived from the ecological characteristics of pathogens, because spores, germination and host invasion occur at low temperature and close to saturated humidity.

In addition, gray mold has a large amount of sporulation and spores are formed continuously during the growth of a crop, so the disease develops rapidly at moderate humidity and temperature conditions, and mycelia or immaturity in diseased tissues when the environment is not suitable. It may overwinter in one microbial state and become an infectious agent for the following year.

Drugs used to control the disease are known to have a large number of penetrant transfer agents and a wide range of protective fungicides, but among these drugs, specialty drugs such as benomil, puroma, binzol, geopan, and e-pro are resistant to germs when they are used. have.

The object of the present invention is Erwinia pyrifoliae ) by preparing a plant transformation vector comprising the hrp Ep gene derived from wild type # 3, transforming the prepared vector into Agrobacterium tumefaciens , and then introducing it into plant cells It is to provide a transgenic plant that is resistant to a wide range of plant pathogens by inducing the mechanism of resistance of the plant itself without causing hypersensitivity reactions.

In order to achieve the above object,

The present invention Erwinia pyripoliia ( Erwinia) pyrifoliae ) It is characterized by providing a plant transformation vector pMJC-GB- hrp Ep comprising the hrp Ep gene (SEQ ID NO: 1) derived from wild type # 3.

Erwinia pyrifolia ( Erwinia) pyrifoliae ) wild type # 3 is a strain deposited with the Korean National Microbiological Conservation Center with Accession No. KCCM 10283 as of January 11, 2001.

The hrp Ep gene of SEQ ID NO: 1, US National Center for Biotechnology Information (NCBI) Accession No. Been claimed to AY237641, Ep hrp gene used in the present invention can be obtained through the library screening or PCR from El Winiah file Li poly child (Erwinia pyrifoliae).

The plant transformation vector pMJC-GB- hrp Ep including the hrp Ep gene can be cloned using a gateway system (Gateway system; Invitrogen, Carlsbad, CA, USA) as a binary vector pSB11 vector. Can be.

Is a vector pMJC-GB- hrp Ep for plant transformation containing the Ep hrp gene, a promoter is used for expression of hrp genes Ep, it can be used to all kind of acting in a plant cell, preferably an overexpression Promoter for constant expression, inducible promoter expressed only at the time of infection of the pathogen, promoter for expressing the target gene at a certain time during the plant growth period, promoter for expressing only a specific part of the plant tissue and expression of the target gene Hybrid promoters, including enhancer sequences, are included to increase the level of the molecule.

The hrp vector Ep is pMJC-GB- hrp Ep for plant transformation containing the gene, hrp Ep NPTⅡ (neomycin phosphotransferase), HPT (hygromycin phosphotransferase) gene as a selection factor for the efficient selection for the transformed plant introduced, Antibiotic resistance genes such as CAT (chloramphenicol acetyltransferase) and gentamicin; herbicide resistance genes such as bar gene; And marker genes such as GUS, GFP (green fluorescent protein), and LUX (luciferase).

In addition, the plant transformation vector pMJC-GB- hrp Ep including the hrp Ep gene includes TPin II (pin-II terminator), P35S (CaMV 35S promoter), TNos (Nos terminator), and MAR (matrix attachment region). By including the sequence of, it is possible to induce stable expression of the gene.

The present invention Agrobacterium tumefaciens transformed with the plant transformation vector pMJC-GB- hrp Ep ( Agrobacterium tumefaciens ).

Agrobacterium tumefaciens ( Agrobacterium) tumefaciens ) has a disarmed Ti or Ri plasmid, and all Agrobacterium strains, such as the octopine-type strain LBA4404 and the succinamopine-type strain EHA101 or EHA105. It can be used in the present invention.

Agrobacterium tumefaciens ( Agrobacterium) The method of introducing the hrp Ep gene into the tumefaciens ) may be performed by any known method, and preferably by tri-patental mating. Specifically, a mixture of helper strains with genes encoding proteins involved in target gene transfer, donor strains containing plasmids with target genes, and Agrobacterium for introducing target genes By this, Agrobacterium having a target gene for plant transformation can be produced.

The introduction of the hrp Ep gene into Agrobacterium tumefaciens can be confirmed by PCR (FIG. 2). Primers used for the PCR may be prepared based on the nucleotide sequences of the 5 'end and 3' end of the Harpin gene.

In addition, the present invention is characterized by providing a pathogen-resistant transformed plant transformed using Agrobacterium transformed with the plant transformation vector pMJC-GB- hrp Ep.

The plants include rice crops such as rice, beans, barley, corn, potatoes, wheat, red beans, oats and sorghum; Vegetable crops such as tomatoes, peppers, strawberries, green onions, onions, carrots, cabbages, radishes, watermelons, cucumbers, cabbages, and pumpkins; Special crops such as ginseng, sesame, perilla, peanut, rapeseed, cotton, sugar cane, sugar beet and tobacco; Fruit trees and flower plants such as apple trees, pear trees, jujube trees, grapes, citrus fruits, persimmons, apricots, bananas, leeks, roses, chrysanthemums, lilies, gerberas, carnations, freesia, gladiolus, and tulips; Fodder crops such as orchardgrass, alfalfa, tall pescue, red clover and lygras; And other plants such as Arabidopsis.

As the method for introducing the hrp Ep gene into the plant, a known method commonly used may be applied. Preferably, the plant is co-cultured with leaf fragments of the plant using Agrobacterium into which a transformation vector is introduced. After introducing the hrp Ep gene into the leaf cells, transgenic plants expressing hrp Ep from the plant leaf segments can be prepared by controlling plant hormones.

Confirmation of the transformed cells in which the hrp Ep gene has been introduced can be confirmed by the presence of transformation through PCR after separating DNA from the leaves of the plant.

Transgenic plants having resistance to pathogens of the present invention exhibit excellent resistance to any kind of plant pathogens, and specifically, may include plant pathogens of viruses, bacteria and fungi.

The present invention may be understood in more detail by the following examples, which are intended to illustrate the present invention and are not intended to limit the protection scope of the present invention.

On the other hand, molecular biology experimental techniques not specifically mentioned in the following examples refer to Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, second edition, 1989) of Sambrook et al.

[ Example ]

1. Construction of vector for transformation

The hrp Ep gene used for the present invention is a pSB11 vector (Komari et al . Using a Gateway system; Invitrogen, Carlsbad, CA, USA). al ., Plant J. 10: 165-174, 1996). The transformant carrier confirmed the introduction of the gene was named pMJC-GB- hrp Ep.

For the expression of the hrp Ep gene, the regular expression OsCc1 promoter isolated from rice (Jang et al ., Plant Physiol. 129: 1473-1481, 2002). For selection of transformed callus and plant, a bar (Bialaphos resistance) gene controlled by the CaMV 35S promoter was used, and a matrix attachment region (MAR) was added inside both borders to induce stable expression of the gene. .

The major gene sequence of the recombinant plasmid pMJC-GB- hrp Ep is shown in FIG. In Figure 1, the abbreviation of each code is as follows: left border (RB), right border (RB), PCytC (OsCc1 promoter), hrp ( hrp Ep gene), TPinll (Pin-II transcription terminator), P35S (CaMV 35S promoter), TNos (Nos transcription terminator), bar (bar coating area), matrix attachment region (MAR).

 2. pMJC - GB - hrp Ep To Agrobacterium

The transformed binary vector DNA, ie pMJC-GB- hrp Ep, was introduced into the Agrobacterium LBA4404 strain by tri-parental mating, and selection of Agrobacterium containing the recombinant plasmid was performed. Two antibiotics, spectinomycin and tetratracycline, were used. Transformation into Agrobacterium was confirmed by colony PCR.

PCR reactions were performed using 1X f-Taq buffer (Solgent, Daejeon, Korea), 50 dM of each dNTP, 1X Band Doctor (Solgent, Daejeon, Korea), 1.5 units of f-Taq DNA polymerase (Solgent, Daejeon, Korea) and each primer. 25 pM was added and a portion of the colonies transformed with each carrier was added to the PCR reaction solution with a final volume of 25 μl with sterile water. The PCR reaction was performed by MJ Research (MJ Research) PTC-200, PCR conditions were modified for 4 minutes at 94 ℃, modified for 15 seconds at 94 ℃, polymerization for 30 seconds at 55 ℃, 60 at 72 ℃ The elongation reaction was repeated 30 times for seconds, and programmed to elongate at 72 ° C. for 7 minutes. The amplified product was separated on a 0.8% agarose gel added with EtBr and confirmed by UV transilluminator.

Figure 2 is a photograph showing the results of performing a colony PCR for transforming the transforming carrier pMJC-GB- hrp Ep used in the present invention by a three-step hybridization method. All six colonies selected showed bands at the 1,280 bp position, which is the expected size of the used primers, indicating that the hrp Ep gene was stably transformed into Agrobacterium.

Agrobacterium colonies, which were confirmed to be transformed by the above method, were propagated and used for transformation of tobacco.

3. Culture and Suspension Preparation of Agrobacterium

Take out the transformed Agrobacterium transformed with recombinant plasmid stored in glycerol stock in a cryogenic freezer (-70 ℃) and streaked streaked in AB-ST medium for 4-5 days in dark conditions of 28 ℃ Incubated. After culturing, Agrobacterium was dispensed with 15-20 ml of co-culture medium (MS base medium, NAA 0.1mg / L, BAP 1mg / L) per petri dish and scraped off using sterile platinum. Transfer to falcon tube. The Agrobacterium was then voltexed for a few seconds to suspend well and the final concentration was 1.5-2.0 at OD 650 nm.

4. Tobacco Transformation

After the wound was added to the slices of tobacco leaves incubated in aseptic state, soaked in the strain solution for 5 minutes and co-culture. Then, the excess strain solution was removed from the filter paper, and the filter paper was placed on the MSO solid medium, and sections were placed thereon and incubated overnight in a 27 ° C cow. The next day, the sections were washed in MS liquid medium, followed by MS SIM (MS base medium, sucrose 30g / L cefotaxim 200mg / L, BA 1mg / L, NAA 0.1mg / L, PPT 4mg / L, Phytoagar 8g / L, pH 5.7) was transferred to the medium. MSOs were transferred to root-induced medium (MSO, sucrose 30g / L, ticarcillin 500mg / L, cefotaxim 200mg / L, IBA 1mg / L, PPT 4mg / L) and immediately after root induction, MSO Transferred to the medium.

5. In Transgenic Tobacco hrp Ep  Introduction and expression of genes

DNA was extracted from the transformed tobacco leaves, followed by PCR using a primer for amplification of the hrp Ep gene. As a result, it was confirmed that the hrp Ep gene was introduced in the transformant 4 line (FIG. 3A). Real-time PCR was performed to investigate the expression level of the hrp Ep gene in each transformant, and it was confirmed that the hrp Ep gene was expressed in all the transformants (FIG. 3B).

6. T ₀  Assay for Resistance to Gray Mold in Generation Transgenic Tobacco

The resistance to gray mold of tobacco transformants was investigated using the following method. Resistance to gray mold ( Botrytis cinerea B1035) was investigated in 4 tobacco strains, 8, 10, 11, and 12 strains, in which control tobacco plants and hrp Ep genes were introduced and expressed. Plants at similar stages of development were selected, and a 3 x 10 ⁵ cell / ml concentration of gray mold spore suspension was sprayed sufficiently on these cigarettes. After 5 days, the resistance of the control tobacco plants and the transformants was compared (FIG. 4).

Adult resistance to gray mold in plants was not known, but the incidence varies according to the developmental stage of the plant. As a result, transformants strains 8 and 11 showed the strongest resistance to gray mold (0.5% and 2% area area, respectively), and no progress was made.

Although strain 12 was more resistant than tobacco control, the disease progressed slowly, and strain 10 exhibited similar symptoms to that of the control tobacco plants, with a light brown color typical of gray mold infection as in control tobacco. A large circular lesion was formed.

7. T ₀  Expression Patterns of Resistance-Related Genes in Genetically Transgenic Tobacco

To investigate the resistance-related signaling pathways activated by hrp Ep in transgenic tobacco plants, RT-PCR expression patterns of HR-related and plant disease resistance-related genes in transformant strain 8 that were highly resistant to control tobacco plants were examined. It was confirmed through. As a result, genes such as hsr201, hsr203J, hsr515, and HIN1 related to HR response showed a slightly increased expression of transformants compared to control tobacco plants. The expression of pathogen-related genes such as PR1 (pathogen-related 1), PR2, PR3, and Chia5 was rarely expressed in control tobacco plants, but the expression of transformants was increased (FIG. 5).

8. T _One  Assay for Resistance to Gray Mold in Generation Transgenic Tobacco

To investigate the resistance to Botrytis whether genetically to T ₁ generation, 8 main, resistance to Botrytis targets T ₁ plants resistant to herbicide to germinate the seeds of the strains was performed resistance jeomjeong in the T ₀ generation The assay was performed. As a result, T I got the same results as Generation _0, the 8-3 system and the 11-4 system similar to the highly resistant, moderately resistant strains 12-2 against the gray mold, 10-3 and control tobacco plants A degree of symptom was shown. Figure 6 is a comparison of the degree of resistance of the 6, 7 and 8 leaves of tobacco control and transformant strain 8-3 after 5 days after inoculation with gray mold. The sixth leaf of the control tobacco plant had gray mold spreading throughout the leaf (FIG. 6B), while the sixth leaf of the transformant had penetrated some gray mold to form some small lesions but no longer progressed. (A in FIG. 6). The seventh leaf of the control tobacco plant is in a better condition than the eighth leaf, but the disease spreads from the edge of the lesion (FIG. 6D), while the seventh leaf of the transformant has some lesion marks but no further symptoms. Progression was not seen (FIG. 6C). The eighth leaf of the control tobacco plant was able to visually observe a number of small lesions penetrated by ash mold when one large lesion was formed and the back of the leaf was observed (FIG. 6F). On the other hand, in the case of the transformant, only a few small lesions were formed and no further development of the symptoms was shown (FIG. 6E).

9. T _One  Expression Patterns of Resistance-Related Genes in Genetically Transgenic Tobacco

To investigate the relationship between the expression of HR-associated or disease-resistant genes and the resistance to gray mold, real-time PCR was performed on control tobacco plants and hrp Ep transformant 8-3 strains. Was performed.

As a result, HR-related genes hsr201, hsr203J, hsr515, and HIN1 showed almost similar expression in control tobacco plants and transformants (FIG. 7A). On the other hand, the expression level of salicylic acid-dependent genes such as PR1a, PR2, PR3, and Chia5 was associated with the degree of resistance to gray mold (FIG. 7B). The hrp Ep transformant strain 8-3, which was highly resistant, showed the highest level of salicylic acid dependent gene expression compared to other strains as shown in FIG. 7B. PR-1b, a jasmonic acid dependent gene, was slightly expressed in transgenic plants compared to control tobacco plants (FIG. 7C), and NE-EFE26, NT1A1C, DS321, and NT-ACS1 genes related to ethylene synthesis. The degree of expression of NT-ACS2 was also higher in transformant strains 8-3 than in control tobacco plants (FIG. 7D). GST1 expression and NPR1 gene known to act as a junction between salicylic acid and jasmonic acid dependent pathways The expression of the gene was not increased in the transgenic plants compared to the control tobacco plants (FIG. 7C).

As described in detail above, the transgenic plant according to the present invention can increase the resistance to plant pathogens without the use of a control agent by activating the resistance mechanisms of the plant itself, for the control of existing crops It has the effect of developing a non-toxic biological disease-resistant transgenic crop that can prevent environmental pollution or human diseases by replacing the poisonous pesticide used.

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Claims (6)

Erwinia , the eggplant pathogen, Erwinia pyrifoliae ) plant transformation vector pMJC-GB- hrp Ep. having the cleavage map of FIG. 1, comprising hrp Ep gene from wild type # 3 KCCM 10283. Paragraph (1) plant transformation vector pMJC-GB- hrp Ep transformed Agrobacterium Tome Pacific Enschede (For Agrobacterium tumefaciens ). Claim 1 Vector pMJC GB- hrp-Ep or claim 2 Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens) a pathogen-resistant plant transformant transformed with. Claim 1 Vector pMJC GB- hrp-Ep or claim 2 Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens) transformed pathogen resistant transgenic plant seed with. Claim 1 Vector pMJC GB- hrp-Ep or claim 2 Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens) transformed pathogen resistant transgenic plants. 6. The pathogen resistant transgenic plant of claim 5, wherein the plant is tobacco or rice.

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