CN1935836A - Thinopyrum intermedium disease-resistance-related protein NPR1, an dits coding gene and use - Google Patents

Abstract

The invention discloses a middle couch grass against disease relevant protein NPR1 and its coding gene and application. The middle couch grass against disease relevant protein can be the protein with one of the following amino acid sequences that SEQ ID No: 2 amino acid residues sequence in sequence list; or its amino acid residues sequence which is replaced and/or missed and/or added with one or many amino acid residues, and relevant to plant against disease performance. The gene of the invention can be transform into plant to increase itself against disease performance, and paves good foundation for plant broad spectrum against disease breeding.

Description

Middle intermedium disease-resistance-related protein NPR1 and encoding gene and application

Technical field

The present invention relates to a plant disease resistance-related and encoding gene thereof and application, NPR1 albumen and the encoding gene thereof of couchgrass in the middle of particularly a kind of the deriving from are with the application of this gene in improving disease resistance of plant.

Background technology

Generation resistance of wide spectrum such as systemic acquired resistance (SAR) is a kind of secular defensive raction of plant, and it can be to bacterium, fungi also produce relevant albumen (PR) expression (Ryals of the course of disease, J.A., Neuenschwander, U.H., Willits, M.G., Molina, A., Steiner, H.-Y., and Hunt, M.D.1996.Systemicacquired resistance.Plant Cell, 8:1809-1819.).At dicotyledons such as Arabidopis thaliana, in the tobacco, Whitfield's ointment (SA), INA and BTH are potential inductor (Friedrich, L., the Lawton of SAR, K., Ruess, W., Masner, P, Speckner, N., Gt Rella, M., Meier, B., Dinher, S., Staub, T., Uknes, S., Metraux, J.-P., Kessman, H., and Ryals, J.1996.Abenzothiadiazole derivative induces systemic acquired resistance in tobacco.Plant is J.9:61-70.).In monocotyledon rice, also find SAR phenomenon (Smith, J.A., andMetraux, J.-P.1991.Pseudomonas syringae pathovar syringae induces systemicresistance to Pyricularia oryzae in rice.Physiol.Mol.Plant Pathol.39:451-461) and wheat (Gorlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., and Ryals, J.1996.Benzothiadiazole, a novel class of inducers of systemicacquired resistance, activates gene expression and disease resistance in wheat.Plant Cell 8:629-643), BTH can be at wheat (Gorlach simultaneously, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., and Ryals, J.1996.Benzothiadiazole, a novel classof inducers of systemic acquired resistance, activates gene expression anddisease resistance in wheat.Plant Cell 8:629-643), paddy rice (Ryals, et.al., geneencoding a protein involved in the signal transduction cascade leading tosystemic acquired in plants.2002.Patent (Arabidopsis): US 6091004; Schweizer, P., Schlagenhauf, E., Schaffrath, U., and Dudler, R.1999.Different patternsof host genes are induced in rice by Pseudomonas syringae, a biological inducerof resistance, and the chemical inducer benzothiadiazole (BTH) .Eur.J.PlantPathol.105:659-665) and corn (Morris, K., Mackerness, S.A., Page, T., John, C.F., Murphy, A.M., Carr, J.P., Buchanan-Wollaston, V.2000, Salicylic acidhas a role in regulating gene expression during leaf senescence.Plant is J.23:677-685) in induce SAR take place.

In recent years find in Arabidopis thaliana, tobacco, paddy rice, NPR1 is the important controlling gene (Cao of the systemic acquired resistance (SAR) of SA mediation, H., Bowling, S.A., Gordon, A.S., and Dong, X.1994.Characterization of an Arabidopsis mutant that is nonresponsive to inducersof systemic acquired resistance.Plant Cell 6:1583-1592; Ryals, J., Weymann, K., Lawton, K., Friedrich, L., Ellis, D., Steiner, H.-Y., Johnson, J., Delaney, T.P., Jesse, T., Vos, P., and Uknes, S.1997.The Arabidopsis NIM1 proteirshows homology to the mammalian transcription factor inhibitor IkB.PlantCell 9:425-439; Shah, J., Tsui., F., and Klessig, D.F.1997.Characterizationof a salicylic acid-insensitive mutant (sail) of Arabidopsis thaliana, identified in aselective screen utilizing the SA-inducible expression of thetms2 gene.Mol.Plant Microbe Interact.10:69-78).Under the inducing of SA, INA and BTH, the NPR1 expression amount will improve (Cao H, Glazebrook J, Clarke J D, et al.Arabidopsis NPR1gene that controls systemic acquired resistance encodes a novel proteincontaining ankyrin repeats.Cell, 1997,88:57-63; Ryals, J., Weymann, K., Lawton, K., Friedrich, L., Ellis, D., Steiner, H.-Y., Johnson, J., Delaney, T.P., Jesse, T., Vos, P., and Uknes, S.1997, The Arabidopsis NIM1 proteinshows homology to the mammalian transcription factor inhibitor IkB.PlantCell 9:425-439).NPR1 is subjected to the influence of SAR downstream SA signal, and the NPR1 mutant npr1/nim1 of Arabidopis thaliana makes its PR genetic expression and SAR be restored under SA, INA and BTH inductive situation.NPR1 also participates in improving the basic thermotolerance (Clarke of SA, S.M., Mur, L.A., Wood, J.E., and Scott, I.M.2004.Salicylic acid dependent signaling promotes basal thermotolerance but is notessential for acquired thermotolerance in Arabidopsis thaliana.Plant are J.38:432-447).The protein of NPR1 genes encoding has two protein-protein interactive domains, i.e. an ankyrin repeat structural domain ARD (Akyrin Rpeat Dmain) and a BTB/POZ structural domain (Broad-Complex, Tramtrack, and Bric-a-brac/Pox virus and Zinc Finger) (CaoH, Glazebrook J, Clarke J D, et al.Arabidopsis NPR1 gene that controls systemicacquired resistance encodes a novel protein containing ankyrin repeats.Cell, 1997,88:57-63).The position of appraising and deciding of NPR1 is that its function performance is very important.When being under the non-induction state, NPR1 exists and is positioned at tenuigenin with the polymer form.In case SAR takes place to be induced, NPR1 is oxidized to monomer and shifts, is gathered in the nucleus, could activate PR (as PR-1, PR-2, PR-5 etc.) gene, cause SAR at last, make plant produce immune response (Mou, Z., Fan, W., and Dong, X.2003.Inducers of plantsystemic acquired resistance regulate NPR1 function through redox changes.Cell 113:1-10).

Remove outside the Whitfield's ointment (SA), NPR1 is at the signal transduction path of jasmonic acid and ethene also play an important role (Spoel, S.H., Koornneef, A., Claessens, S.M.C., Korzelius, J.P., Van Pelt, J.A.Mueller, M.J., Buchala, A.J., Metraux, J.-P., Brown, R., Kazan, K., Van Loon, L.C., Dong, X., and Pieterse, C.M.J.2003.NPR1 modulatescross-talk between salicylate-and jasmonate-dependent defense pathwaysthrough a novel function in the cytosol.Plant Cell 15:760-770).Jasmonic acid and Ethylene Signal Transduction approach exist crosstalk ((Dong, X.1998.SA, JA, ethylene, and diseaseresistance in plants.Curr.Opin.Plant Biol.1:316-323; Kunkel, B.N., andBrooks, D.M.2002.Cross talk between signaling pathways in pathogen defense.CurR.Opin.Plant Biol.5:325-331).NPR1 is in jasmonic acid (JA), the conduction of ethene (ET) signal, and be independent of SA but require ethene and JA mediation inducible system resistance (ISR) in (Pieterse that plays an important role equally, C.M.J., Van Wees, S.C.M., Van Pelt, J.A., Knoester, M., Laan, R., Gerrits, H., Weisbeek, P.J., and Van Loon, L.C.1998.A novel signalingpathway controlling induced systemic resistance in Arabidopsis.Plant Cell10:1571-1580).NPR1 is necessary in the defense pathway of ISR and SAR, and to a certain extent, regulate these two kinds defence signal pathway (Hammond-Kosack KE, Parker JE.2003, Decipheringplant-pathogen communication:fresh perspectives for molecul ar resistancebreeding.Curr Opin Biotechnol.14 (2): 177-93).Arabidopis thaliana passes the defense needs ET of downright bad type fungal pathogens F.oxysporum to soil, JA, participation (the Berrocal-LoboM of SA signal pathway and NPR1 gene, Molina A.2004, Ethylene response factor 1 mediates Arabidopsis resistance tothe soilborne fungus Fusarium oxysporum.Mol Plant Microbe Interact.17:763-770).Therefore NPR1 also plays an important role at whole defensive raction network.

NPR1 gene overexpression in transgenic arabidopsis just improves the disease resistance of plant and does not have other significance harmful effects (Cao et al.1998, Generation of broad-spectrum disease resistance byoverexpression of an essential regulatory gene in systemic acquiredresistance, Proc.Nat1. Acad. Sci. USA, 95:6531-6536), the NPR1 gene day by day is subjected to people's attention as the plant broad spectrum antidisease gene.NPR1 overexpression in transgenic arabidopsis can strengthen bacterium and nematode resistance (Cao et al.1998, Generation of broad-spectrum disease resistanceby overexpression of an essential regulatory gene in systemic acquiredresistance, Proc.Nat1. Acad.Sci.USA, 95:6531-6536).Utilize the experiment of paddy rice overexpression Arabidopis thaliana NPR1 gene to determine that the NPR1 gene is in monocotyledons during institute's role, find that the resistance that transgenic rice plant shows Xoo improves, proved that first the NPR1 gene can improve monocotyledonous disease resistance (Chern MS, F itzgerald HA, Yadav RC, et al.A disease resistance pathwayin rice similar to themediated pathw ay in Arabidopsis.Plant J, 2001,27 (2): 101-113).Reported also at present that the tomato that changes Arabidopis thaliana NPR1 gene has resistance of wide spectrum (Lin WC to fungi and bacterium, Lu CF, Wu JW, Cheng ML, Lin YM, Yang NS, Black L, Green SK, WangJF, Cheng CP.Transgenic tomato plants expressing the Arabidopsis NPR1 genedisplay enhanced resistance to a spectrum of fungal and bacterial diseases.Transgenic Res.2004,13 (6): 567-581), the NPR1 gene of paddy rice can improve X.oryzae pv.oryzae resistance (Chern M in the overexpression paddy rice, Fitzgerald HA, Canlas PE, Navarre DA, RonaldPC.Overexpression of a rice NPR1 homolog leads to constitutive activationof defense response and hypersensitivity to light.Mol Plant Microbe Interact.2005 Jun; 18 (6): 511-20).

Because the NPR1 gene is with a wide range of applications, therefore this gene is caused great concern in the world in genetic engineering of plant for disease resistance.At present, publication number be US 5986082-A U.S. Patent Publication an Arabidopis thaliana NPR1 gene (Uknes, S.Joseph., Hunt, M.Denise., Steiner, H.-Y.and Ryals, J.Andrew.Altered forms of the NIM1 gene conferring disease resistance inplant (Arabidopsis) .1999.Patent:US 5986082-A), U.S. Patent Publication paddy rice, homologous sequence (Crane, E.H.III, the Rice of the Arabidopis thaliana NPR1 gene of wheat and corn, D.A., Simmons, C.R., Tossberg, J.T., Sandahl, G.A.and Zhang, L.Maize NPR1 polynucleotidesand methods of use.2000.Patent:US 6504084).Hu Fengxian Arabidopis thaliana NPR1 gene is imported in the tobacco and succeed (Hu Fengxian. change acquisition and genetic analysis .2002. Guangxi University Master's thesis thereof that the Arabidopis thaliana system obtains resistance regulatory gene (NPR1) tobacco); Sieve wipe out with Arabidopis thaliana NPR1 gene change over to paddy rice make paddy rice make resisting bacterial leaf-blight and rice blast be improved (sieve wipes out. bacterial leaf-blight and rice blast Arabidopis thaliana NPR1 gene change paddy rice over to makes paddy rice obtain broad spectrum resistance .2004. Guangxi University Master's thesis); Deng Xiaoling etc. have cloned NPR1 gene (Deng Xiaoling in the swede type rape, Kong Weiwen, Feng Zhen, Guo Ming, Li Huaping. the clone of NPR1 gene in the swede type rape, the research .2004 Chinese Plants pathology meeting nd Annual Meeting collection of vector construction and conversion thereof), the sinus dragon imports to Arabidopis thaliana NPR1 gene in the cotton, screen anti-blight, the plant of verticillium, obtained simultaneously cotton and Sunflower Receptacle NPR1 Gene Partial fragment (the sinus dragon. clone .2002. Postgraduate School, Chinese Academy of Sciences doctorate paper of genetically engineered research of cotton resistance to yellow dwarf and defense response gene).

Couchgrass in the middle of the wheat kindred plant has manyly to the useful important gene of wheat improvement, as has genes such as anti-wheat rust, Powdery Mildew, head blight, virus disease, gaeumannomyces graminis disease, banded sclerotial blight, drought resisting simultaneously, salt tolerant, characteristic such as low temperature resistant.

Summary of the invention

The purpose of this invention is to provide a kind of middle intermedium disease-resistance-related protein and encoding gene thereof.

Intermedium disease-resistance-related protein in the middle of provided by the present invention, name is called TiNPR1, and couchgrass in the middle of deriving from is the protein with one of following aminoacid sequence:

1) the SEQ ID № in the sequence table: 2 amino acid residue sequence;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement and/or disappearance and/or the interpolation and the protein relevant with plant disease-resistant of one or several amino-acid residue.

Wherein, sequence 2 is made up of 580 amino-acid residues in the sequence table.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.

The encoding gene (TiNPR1) of intermedium disease-resistance-related protein also belongs to protection scope of the present invention in the middle of above-mentioned.

The cDNA gene of intermedium disease-resistance-related protein in the middle of above-mentioned can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

The rigorous condition of above-mentioned height can be 65 ℃ of down hybridization, and 0.1 * SSPE (or 0.1 * SSC), wash film under 65 ℃ in the solution of 0.1%SDS.

Wherein sequence 1 is made up of 2003 deoxynucleotides in the sequence table, from 5 ' and the 19th to the 1761st deoxynucleotide of end is encoding sequence.

Contain expression carrier of the present invention, clone and host bacterium and all belong to protection scope of the present invention.

TiNPR1 gene of the present invention can be building up in the existing plant expression vector with existing method, can add any promotor that comprises constitutive promoter, strengthens promotor, inducible promoter, tissue-specific promoter, etap specificity promoter before it transcribes super beginning Nucleotide.For the ease of identifying and screen to changeing TiNPR1 gene plant cell or plant, can process employed carrier, as the antibiotic marker thing (gentamicin, kantlex etc.) that adds the alternative mark (BAR gene, gus gene, luciferase genes etc.) of plant or have resistance.By the plant transformed host both can be monocotyledons, also can be dicotyledons, as: paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass or lucerne place etc.Carry that TiNPR1 expression carrier of the present invention can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and plant transformed become plant through tissue cultivating, obtain the plant that disease resistance improves.

Experimental result shows, induces the expression enhancing of TiNPR1 down white powder germ and sheath blight fungus; TiNPR1 is imported Arabidopis thaliana, can improve the resistance of Arabidopis thaliana pseudomonas.Gene of the present invention can change the disease resistance that improves plant in the plant over to, also lays the good operation basis for the plant broad spectrum antidisease breeding simultaneously.

Description of drawings

Fig. 1 is the electrophorogram of amplification TiNPR1 gene fragment from the middle couchgrass of white powder germ inductive

The ANR that Fig. 2 obtains for the BLASTP by GenBank guards the position in territory

Fig. 3 is a plant TiNPR1 gene evolution tree analysis chart

Fig. 4 is TiNPR1 and AtNPR1, and NtNPR1, the aminoacid sequence of OsNPR1 are relatively

Fig. 5 is a couchgrass TiNPR1 expression analysis in the middle of the white powder germ inductive

Fig. 6 is a couchgrass TiNPR1 expression analysis in the middle of the sheath blight fungus inductive

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

The acquisition of conservative section of embodiment 1, TiNPR1 and cDNA total length thereof

With the blade inoculation white powder germ of the middle couchgrass around the growth, extract total RNA of blade after 24 hours, synthesize the 1st chain of cDNA according to the method that precious biotech firm provides, and carry out pcr amplification as template with it.According to paddy rice, Arabidopis thaliana, tobacco NPR1 gene is guarded section (QRHLLD and KAFSED) a pair of primer NPR1-F:CAGCGGCATCTCCTTGAT of design and NPR1-R:GTCCTCGCTGAACGCCTT, PCR reaction cumulative volume is 25 μ L, comprise 1 μ L (50ng) cDNA, 50 μ mol/L dNTP, 1 μ mol/L primer, 1U rTaq enzyme and damping fluid (1 *).The PCR response procedures is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of 30s then, 56 ℃ of 1min, 72 ℃ of 1min, 30 circulations; Last 72 ℃, 10min mends flat terminal.Amplified production separates through agarose gel electrophoresis, and the result shows to amplify an about 1.2kb band from the middle couchgrass cDNA of inoculation white powder germ as shown in Figure 1.Swimming lane 1 is the band of amplification among Fig. 1; Marker is a λ DNA/HindIII+EcoRI molecular weight standard; The stripe size that arrow refers to is 1.2kb.This band is reclaimed, be cloned on the pGEM-T carrier, carry out sequencing analysis.Sequential analysis shows that this sequence is the NPR1 gene fragment.According to the Auele Specific Primer that obtains conservative section the design 5 '-RACE (Invitrogen product) of NPR1 and 3 '-RACE (Invitrogen product), the specification sheets of 5 '-RACE and 3 ' that the RACE program provides according to Invitrogen company-RACE test kit carries out.Wherein, 5 '-RACE Auele Specific Primer GSP1:5 '-TTGATCTTATCCGTTGCAAACT-3 '; The AUAP primer (upstream primer, sequence: 5 '-GGCCACGCGTCGACTAGTAC-3 '), Nested-GSP:5 ' GAGAGATGCCTGGAGATGGTAG-3 '.3 '-RACE: Auele Specific Primer GSP2 (upstream primer): 5 ' ACCTGCAAGATACGCTTCTGA-3 ', the AUAP primer (downstream primer, the AUAP primer sequence: 5 '-GGCCACGCGTCGACTAGTAC-3 ').

With 5 '-the first chain cDNA of RACE test kit reverse transcription synthetic mesophase couchgrass is as template, carry out first round amplification with AUAP (upstream primer) and GSP1 (downstream primer), and then make template with 100 times first round PCR product of dilution, carry out second with AUAP (upstream primer) and Nested-GSP (downstream primer) and take turns pcr amplification, reclaim purified pcr product, connect and be cloned on the pMD18-T carrier, by bacterium colony PCR, select 5 positive colony order-checkings, The sequencing results shows, obtains the 5 ' terminal sequence (from 5 of sequence 1 ' the 1st the-the 180th deoxynucleotide of end) of TiNPR1 gene.Wherein, first round PCR reaction conditions: 94 ℃ of 5min of elder generation; 94 ℃ of 30s → 60 ℃ 30s → 72 ℃ of 4min, totally 30 circulations then; Last 72 ℃ of 10min.25 μ L reaction systems are 1 * GC damping fluid, 1 μ L cDNA (50ng), 200 μ mol/L dNTPs, every primer of 0.2 μ mol/L, 1ULA-Taq enzyme.Second takes turns the PCR reaction conditions: 94 ℃ of pre-sex change 5min of elder generation; 94 ℃ of 30s then, 60 ℃ of 30s, 72 ℃ of 4min, totally 30 circulations; Last 72 ℃ of 10min.25 μ L reaction systems are 1 * GC damping fluid, 1 μ L cDNA (50ng), 200 μ mol/L dNTPs, every primer of 0.2 μ mol/L, 1U LA-Taq enzyme.

With 3 '-the first chain cDNA of RACE test kit reverse transcription synthetic mesophase couchgrass increases with GSP2 (upstream primer) and AUAP (downstream primer), wherein the PCR reaction conditions as template: 94 ℃ of 5min earlier; 94 ℃ of 45s → 60 ℃ 45s → 72 ℃ of 4min, totally 30 circulations then; 72 ℃ of 10min again.25 μ L reaction systems are 1 * GC damping fluid, 1 μ L (50ng) cDNA, 200 μ mol/L dNTPs, every primer of 0.2 μ mol/L, 1U LA-Taq enzyme.Reclaim purified pcr product, connect and be cloned on the pMD18-T carrier,, select 5 positive colony order-checkings by bacterium colony PCR, The sequencing results shows, obtains the 3 ' terminal sequence (from 5 of sequence 1 ' the 1533rd the-the 2021st deoxynucleotide of end) of TiNPR1 gene.

5 ' terminal sequence of TiNPR1 and 3 ' terminal sequence in external splicing, are obtained the full length cDNA sequence of TiNPR1, have the nucleotide sequence of sequence 1 in the sequence table.

Exactness for the full length cDNA sequence of verifying the TiNPR1 that this splicing obtains, according to the TiNPR1 full length cDNA sequence, design 1 couple of Auele Specific Primer TiF:5 '-AGCAGTGCCAATGGAGGCT-3 ' again, TiR:5 '-CCAATATGGCAAGAATGGGC-3 ', cDNA with middle couchgrass is a template, carries out pcr amplification.This cDNA as template is with the blade inoculation white powder germ of middle couchgrass around the growth, total RNA of extraction blade after 24 hours, the 1st chain of method synthetic cDNA that provides according to precious biotech firm.Wherein the reaction system of PCR is: PCR reaction cumulative volume is 25 μ L, comprises 1 μ L (50ng) cDNA, 50 μ mol/L dNTP, 1 μ mol/L primer, 1U LA-Taq enzyme and damping fluid (1 * GC).Response procedures is: 95 ℃ of pre-sex change 5min; 95 ℃ of 30s then, 60 ℃ of 1min, 72 ℃ of 4min, 30 circulations; Last 72 ℃, 10min mends flat terminal.Amplified production carries out agarose electrophoresis to be separated, and reclaims and is cloned on the pGEM-T carrier, carries out sequencing analysis, and sequencing result shows that this pcr amplification product has the nucleotide sequence of sequence 1 in the sequence table, is TiNPR1.TiNPR1 open reading frame (encoding sequence) is the 19th to the 1761st nucleotide sequence of 5 ' end of sequence 1 in sequence table, 580 amino acid (sequence 2 in the sequence table) of encoding.With obtaining the BLASTP that full-length gene order carries out GenBank, with AtNPR1 (Arabidopis thaliana), NtNPR1 (tobacco), OsNPR1 (paddy rice), ZyNPR1 (corn) (patent No.: WO0065037), (wheat, the patent No.: WO0070069) compare of TaNPR1.Utilize DNAMAN software, above sequence generated evolutionary tree, the result as shown in Figure 3, the result shows: they and paddy rice relation is closer, homology (80%) is the highest, secondly is tobacco (54%), Arabidopis thaliana (46%), and is distant with the wheat relation.N-terminal the 272nd in sequence in sequence table 2 has ANR (Ankyrin Repeat) conserved regions (Fig. 2) between the 367th amino acids residue.By DNAMAN software, with Arabidopis thaliana, tobacco, wheat, the NPR1 sequence of corn and middle couchgrass compares, and the result is as shown in Figure 4, the result shows: the amino acid that the function of NPR1 gene is played a crucial role equally also has conservative property, as npr1-1 (H) (arrow shows), npr1-2 (C) (arrow shows), nim1-4 (R) (arrow shows).Fig. 4 is TiNPR1 and AtNPR1 (Arabidopis thaliana), NtNPR1 (tobacco), the comparison of OsNPR1 (paddy rice).

Embodiment 2, Ti-NPR1 expression of gene characteristic

With the blade of middle couchgrass around the growth, inoculate wheat powdery mildew respectively, sheath blight fungus 0,2 after 5,10,24 hours, extracts total RNA of blade with the Trizol method, is that probe carries out Northern and hybridizes with the cDNA (sequence 1) of TiNPR1.The result shows that the expression of TiNPR1 gene transcription is subjected to inducing of white powder germ, under normal operation, TiNPR1 has trace expression, after sheath blight fungus infects 2 hours, TiNPR1 expression of gene amount begins to increase, reached maximum value in 5 hours infecting, the expression amount that infects 10 hours and 24 hours is stabilized in maximum value (Fig. 5); The TiNPR1 gene transcription is expressed and is subjected to inducing of sheath blight fungus, under normal operation, TiNPR1 has trace expression, after sheath blight fungus infects 2 hours, TiNPR1 expression of gene amount begins to increase, reached maximum value in 5 hours infecting, the expression amount that infects 10 hours and 24 hours is stabilized in maximum value (Fig. 6).Explanation is induced the expression enhancing of TiNPR1 down white powder germ and sheath blight fungus.

The disease resistance analysis of embodiment 3, commentaries on classics TiNPR1 gene Arabidopis thaliana plant

T0 represents to infect the transfer-gen plant that Arabidopis thaliana obtains by Agrobacterium, and T1 represents seed that T0 produces for selfing and by plant that it grew up to.

CDNA with the TiNPR1 gene is a template, with the primer that has the SmaI/SacI enzyme recognition site: upstream primer 5 ' TTCCCGGGAGCAGTGCCAATGGAGGCT-3 ', downstream primer 5 ' CCGAGCTCCCAATATGGCAAGAATGGGC-3 ' carries out pcr amplification TiNPR1 gene ORF (the 19th of sequence 1 the to 1761 nucleotide sequence in sequence table), reclaim amplified production, its SmaI and SacI enzyme that is building up to dicotyledons expression vector pBI121 (BIODEE company) is cut between the recognition site, form carrier 35S ∷ NPR1, controlled by 35S promoter, the carrier called after pNPR1 that identifies the pBI121 that contains TiNPR1 gene ORF through order-checking.Utilize triparental mating that pNPR1 is transformed among the Agrobacterium C58C1, cultivate the bacterial strain that the screening conversion obtains by having kantlex (Kana) resistance MS substratum (containing the 50ug/ml kantlex), with the agrobacterium strains arabidopsis thaliana transformation that carries pNPR1 that obtains, collect the seed of transgenosis plant in the present age (T0), containing transgenic positive T1 that screening on the MS substratum of 50ug/ml kantlex can normal growth for plant; Simultaneously with the not negative contrast of transfer-gen plant.With Pseudomonas syringae (Pseudomonas syringae) spore suspension (10 ⁶/ ml) infect and change pNPR1 gene masculine T1 (Berroecal-Lobo etc. 2002 in the Arabidopis thaliana 6 minutes, Constitutive expression ofEHYLENE-RESPONSE-FACTORl in Arabidopsis confers resistance to severalnecrophic fungi, Plant J, 29:23-32), add up incidence after 7 days.The result shows, infects Arabidopis thaliana in the time of 7 days pseudomonas, and not genetically modified Arabidopis thaliana contrast spore is many, and morbidity is heavy.Get the commentaries on classics pNPR1 plant that pseudomonas infected 7 days, 10 blades that do not change the plant of pNPR1 respectively, by normal observation sorus number, the result shows the 1/4-1/2 of average spore count on the blade that average spore count is a not genetically modified plant on the blade that changes the pNPR1 gene plant, illustrates that commentaries on classics TiNPR1 gene plant obviously improves the resistance of pseudomonas.

Sequence table

<160>2

<210>1

<211>2021

<212>DNA

＜213〉couchgrass (Thinopyrum intermedium) in the middle of

<400>1

gccgcaacag?cagtgccaat?ggaggctccg?agcagccacg?tcaccacctc?attctcggac 60

tgcgacagcg?tctccatgga?ggacgcggcg?cctgacgcgg?acgtggaggc?gctccgccgc 120

ctctccgaca?acctcgccgc?cgccttccgc?tcgccggacg?agttcgcctt?cctcgccgac 180

gcgatcgtgg?ccgtgccggg?cgagcccgac?ctgcgcgtgc?accgctgcgt?gctgtcggcg 240

cggagcccct?tcctgcgcgc?ggtcttcaag?cgccgcgccg?ccgccgccgc?cgccggcggc 300

ggttcggccg?gcggcgcgga?gggcaaccgg?gcggagctcc?gggagcttct?cggcgaggag 360

gtggaggtcg?ggtacgaggc?gctgcagctc?gtgctcgact?acccgtacag?cggccgcgtc 420

cgcgacctcc?ccaagtcggc?gtgcgcctgc?gtcgacgtgg?acggatgccc?gcacgtcggc 480

tgccaccccg?ccgtctcctt?catggcgcag?gtcctattcg?ccgcatccac?cttccaggtc 540

ggcgagctcg?ccaacctctt?ccagcggcat?ctccttgatt?tccttgataa?agttgaagtg 600

gataaccttc?cgttgatctt?atccgttgca?aacttatgca?acaaatcttg?catgaaactt 660

ttcgagagat?gcctggagat?ggtagtccgg?tcaaatcttg?acatgattac?tcttgagaaa 720

gcattgcctg?aagatgttat?caagcaaatt?attcattcac?ggataactct?tggattagct 780

tcacccgaag?acaatggatt?tcctaacaaa?cacgtaagaa?ggatactcaa?agcacttgat 840

tctgatgatg?tggagctagt?caggatgctg?ctcaaagagg?ggcagactaa?ccttgacgat 900

gcatttgcat?tgcactatgc?tgtagaacac?tgtgactcaa?aaattacaac?agaacttctg 960

gacatcgcac?ttgcagatgt?taatcttaga?aacccaagag?gttatactgt?tcttcacatc 1020

gctgctaggc?ggagagatcc?taaaattgtt?gtctccctat?taaccaaagg?tgctaggcct 1080

tccgatttta?catttgatgg?aagaaaagca?gttcaaatct?caaagagact?cacaaaacat 1140

ggggattatt?ttgggaatac?tgaagaagga?aagccgtctc?ccaaggataa?attatgcatt 1200

gagatactgg?agcaagctga?aggaagggat?ccacagcttg?gagaagcatc?ggtttctctt 1260

gcattggctg?gtgactgtct?tcgtgggaag?ttattgtacc?ttgaaaaccg?agttgctttg 1320

gcgaggataa?tgtttccaat?tgaggcaaga?gtagcaatgg?acattgctca?agtggatggc 1380

acattggaat?ttacccttgg?ttctagtaca?actccacctc?cggagataac?aaccgtggat 1440

ctaaatgatg?cttctttcaa?aatgaaggac?gaacacttag?ctcggatgag?agccctctcc 1500

aaaacagtcg?aactcggcaa?acgtttcttc?ccgcgctgtt?caaatgtgct?ggacaagatc 1560

atggacgatg?aaccggagct?ggcttcgctc?ggaagagatg?cctcctccga?gaggaagagg 1620

aggtttcacg?acctgcaaga?tacgcttctg?aaggcgttca?gcgaggacaa?ggaggagttt 1680

aacagaacga?caaccctttc?atcttcctca?tcgtcgatgt?ccactgtagc?aaggaacttg 1740

acaggtcggc?ccaggagatg?atgagcaccc?ttgcccattc?ttgccatatt?ggtagctgat 1800

tctttttctg?tctgaaactg?cccgccggat?ttattttttt?ctgtttaacg?agtactagta 1860

gtctagcatc?atcgtcagat?atgatgaagc?tgttggcttt?ggccctgtaa?atcgcctagt 1920

tatgctaatg?tttgcttgta?cagtaacggt?tgtccataca?gtattttgtt?gatggaacag 1980

caaatggact?tcaacatgta?gtagcaaaaa?aaaaaaaaaa?a 2021

<210>2

<211>580

<212>PRT

＜213〉couchgrass (Thinopyrum intermedium) in the middle of

<400>2

Met?Glu?Ala?Pro?Ser?Ser?His?Val?Thr?Thr?Ser?Phe?Ser?Asp?Cys?Asp

1 5 10 15

Ser?Val?Ser?Met?Glu?Asp?Ala?Ala?Pro?Asp?Ala?Asp?Val?Glu?Ala?Leu

20 25 30

Arg?Arg?Leu?Ser?Asp?Asn?Leu?Ala?Ala?Ala?Phe?Arg?Ser?Pro?Asp?Glu

35 40 45

Phe?Ala?Phe?Leu?Ala?Asp?Ala?Ile?Val?Ala?Val?Pro?Gly?Glu?Pro?Asp

50 55 60

Leu?Arg?Val?His?Arg?Cys?Val?Leu?Ser?Ala?Arg?Ser?Pro?Phe?Leu?Arg

65 70 75 80

Ala?Val?Phe?Lys?Arg?Arg?Ala?Ala?Ala?Ala?Ala?Ala?Gly?Gly?Gly?Ser

85 90 95

Ala?Gly?Gly?Ala?Glu?Gly?Asn?Arg?Ala?Glu?Leu?Arg?Glu?Leu?Leu?Gly

100 105 110

Glu?Glu?Val?Glu?Val?Gly?Tyr?Glu?Ala?Leu?Gln?Leu?Val?Leu?Asp?Tyr

115 120 125

Pro?Tyr?Ser?Gly?Arg?Val?Arg?Asp?Leu?Pro?Lys?Ser?Ala?Cys?Ala?Cys

130 135 140

Val?Asp?Val?Asp?Gly?Cys?Pro?His?Val?Gly?Cys?His?Pro?Ala?Val?Ser

145 150 155 160

Phe?Met?Ala?Gln?Val?Leu?Phe?Ala?Ala?Ser?Thr?Phe?Gln?Val?Gly?Glu

165 170 175

Leu?Ala?Asn?Leu?Phe?Gln?Arg?His?Leu?Leu?Asp?Phe?Leu?Asp?Lys?Val

180 185 190

Glu?Val?Asp?Asn?Leu?Pro?Leu?Ile?Leu?Ser?Val?Ala?Asn?Leu?Cys?Asn

195 200 205

Lys?Ser?Cys?Met?Lys?Leu?Phe?Glu?Arg?Cys?Leu?Glu?Met?Val?Val?Arg

210 215 220

Ser?Asn?Leu?Asp?Met?Ile?Thr?Leu?Glu?Lys?Ala?Leu?Pro?Glu?Asp?Val

225 230 235 240

Ile?Lys?Gln?Ile?Ile?His?Ser?Arg?Ile?Thr?Leu?Gly?Leu?Ala?Ser?Pro

245 250 255

Glu?Asp?Asn?Gly?Phe?Pro?Asn?Lys?His?Val?Arg?Arg?Ile?Leu?Lys?Ala

260 265 270

Leu?Asp?Ser?Asp?Asp?Val?Glu?Leu?Val?Arg?Met?Leu?Leu?Lys?Glu?Gly

275 280 285

Gln?Thr?Asn?Leu?Asp?Asp?Ala?Phe?Ala?Leu?His?Tyr?Ala?Val?Glu?His

290 295 300

Cys?Asp?Ser?Lys?Ile?Thr?Thr?Glu?Leu?Leu?Asp?Ile?Ala?Leu?Ala?Asp

305 310 315 320

Val?Asn?Leu?Arg?Asn?Pro?Arg?Gly?Tyr?Thr?Val?Leu?His?Ile?Ala?Ala

325 330 335

Arg?Arg?Arg?Asp?Pro?Lys?Ile?Val?Val?Ser?Leu?Leu?Thr?Lys?Gly?Ala

340 345 350

Arg?Pro?Ser?Asp?Phe?Thr?Phe?Asp?Gly?Arg?Lys?Ala?Val?Gln?Ile?Ser

355 360 365

Lys?Arg?Leu?Thr?Lys?His?Gly?Asp?Tyr?Phe?Gly?Asn?Thr?Glu?Glu?Gly

370 375 380

Lys?Pro?Ser?Pro?Lys?Asp?Lys?Leu?Cys?Ile?Glu?Ile?Leu?Glu?Gln?Ala

385 390 395 400

Glu?Gly?Arg?Asp?Pro?Gln?Leu?Gly?Glu?Ala?Ser?Val?Ser?Leu?Ala?Leu

405 410 415

Ala?Gly?Asp?Cys?Leu?Arg?Gly?Lys?Leu?Leu?Tyr?Leu?Glu?Asn?Arg?Val

420 425 430

Ala?Leu?Ala?Arg?Ile?Met?Phe?Pro?Ile?Glu?Ala?Arg?Val?Ala?Met?Asp

435 440 445

Ile?Ala?Gln?Val?Asp?Gly?Thr?Leu?Glu?Phe?Thr?Leu?Gly?Ser?Ser?Thr

450 455 460

Thr?Pro?Pro?Pro?Glu?Ile?Thr?Thr?Val?Asp?Leu?Asn?Asp?Ala?Ser?Phe

465 470 475 480

Lys?Met?Lys?Asp?Glu?His?Leu?Ala?Arg?Met?Arg?Ala?Leu?Ser?Lys?Thr

485 490 495

Val?Glu?Leu?Gly?Lys?Arg?Phe?Phe?Pro?Arg?Cys?Ser?Asn?Val?Leu?Asp

500 505 510

Lys?Ile?Met?Asp?Asp?Glu?Pro?Glu?Leu?Ala?Ser?Leu?Gly?Arg?Asp?Ala

515 520 525

Ser?Ser?Glu?Arg?Lys?Arg?Arg?Phe?His?Asp?Leu?Gln?Asp?Thr?Leu?Leu

530 535 540

Lys?Ala?Phe?Ser?Glu?Asp?Lys?Glu?Glu?Phe?Asn?Arg?Thr?Thr?Thr?Leu

545 550 555 560

Ser?Ser?Ser?Ser?Ser?Ser?Met?Ser?Thr?Val?Ala?Arg?Asn?Leu?Thr?Gly

565 570 575

Arg?Pro?Arg?Arg

580

Claims (7)

1, a kind of middle intermedium disease-resistance-related protein is the protein with one of following aminoacid sequence:

1) the SEQ ID № in the sequence table: 2 amino acid residue sequence;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement and/or disappearance and/or the interpolation and the protein relevant with plant disease-resistant of one or several amino-acid residue.

2, the encoding gene of the described middle intermedium disease-resistance-related protein of claim 1.

3, gene according to claim 2 is characterized in that: the cDNA gene of intermedium disease-resistance-related protein in the middle of described has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

4, contain claim 2 or 3 described expression carrier.

5, the clone that contains claim 2 or 3 described genes.

6, the host bacterium that contains claim 2 or 3 described genes.

7, described middle intermedium disease-resistance-related protein of claim 1 and the application of encoding gene in improving disease resistance of plant thereof.

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