CN105585623A

CN105585623A - Cultivating method for disease-resistant TaMYB-KW gene-transferred wheat, related biomaterials and application

Info

Publication number: CN105585623A
Application number: CN201610133713.4A
Authority: CN
Inventors: 张增艳; 单天雷; 荣玮; 魏学宁; 杜丽璞
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2016-03-09
Filing date: 2016-03-09
Publication date: 2016-05-18
Anticipated expiration: 2036-03-09
Also published as: CN105585623B

Abstract

The invention discloses a cultivating method for disease-resistant TaMYB-KW gene-transferred wheat, related biomaterials and application. TaMYB-KW protein is one of the proteins as follows: the first protein has an amino acid sequence shown in the sequence 2, the second protein is obtained in the mode that the amino acid sequence shown in the sequence 2 in a sequence table is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and is related to the disease resistance of plants, and the third fusion protein is obtained by connecting labels to the N end or/and the C end of the first protein or the second protein. Experiments prove that the TaMYB-KW and a coding gene thereof can improve the disease resistance of the plants, and the TaMYB-KW gene is a wheat disease-resistant protein gene which is closely related to the sharp eyespot resistance and can be applied to genetic improvement of the plants.

Description

Disease-resistant breeding method and associated biomolecule material and the application that turns TaMYB-KW DNA triticum

Technical field

The present invention relates to breeding method and the associated biomolecule material of the disease-resistant TaMYB-KW of turning DNA triticum in biological technical fieldMaterial and application.

Background technology

Wheat (Triticumaaestivum) is one of four large crops of depending on for existence of the mankind, in the world more than 1/3Population is taking wheat as staple food, and the yield and quality of wheat directly affects the mankind's existence and quality of life. Along with cropping system,The change of the factor such as fertilizer and water condition and weather conditions, soil-borne disease banded sclerotial blight, root rot are and add repeating transmission at China's wheat beltEcological potential, has become one of key factor of restriction improving yield of wheat, stable yields.

Wheat sharp eyespot, also referred to as wheat point eye spot (wheatsharpeyespot), is a kind of worldwide wheat soilBorne fungus diseases, be mainly by metatrophy type disease fungus Rhizoctonia cerealis (Rhizoctoniacerealis) CAG-1 orRhizoctonia solani Kuhn (Rhizoctoniasolani) AG4, AG5 merge group and cause. China's wheat sharp eyespot the main pathogenic fungi is standing grainPhizoctonia cerealis (Rhizoctoniacerealis). Banded sclerotial blight generally can make wheat yield 10%～30%, and serious plot makesWheat yield is more than 50%. According to national agricultural technique spread station, 2005-2015 years there is area in China's wheat sharp eyespot every yearHundred million mu of about 1-1.2, more than economic loss reaches billions of units, have become the first disease of China's wheat main producing region wheat. Therefore, choosingThe wheat breed of educating and promote resistance is most economical, safety that control wheat diseases is popular and effective approach, for ensureing meState's improving yield of wheat, stable yields are extremely important. But, because wheat sharp eyespot resistance is by controlled by multiple genes, lack desirable wheatDisease-resistant germ plasm resource, conventional breeding method is slow to the progress aspect sharp eyespot resistance wheat breed in seed selection. Molecule is rawThing is learned and is engineeredly developed into plant resistance to environment stress breeding and opened up a new way. The separation clone of plant resistance to environment stress GFPWith functional analysis, to illustrating plant resistance to environment stress mechanism, effectively to carry out molecular breeding research very necessary, become domestic and international plantThe focus of scientific research.

Summary of the invention

Technical problem to be solved by this invention is how to improve the disease resistance of plant.

For solving the problems of the technologies described above, first the present invention provides name to be called the albumen of disease-resistance-related protein (TaMYB-KW)Matter, this protein is following A1), A2) or A3):

A1) amino acid sequence is the protein of sequence 2;

A2) by amino acid sequence shown in sequence in sequence table 2 through the replacement of one or several amino acid residue and/orDisappearance and/or interpolation and the protein relevant to disease resistance of plant;

A3) at A1) or N A2) end or/and C end connects the fused protein that label obtains.

Wherein, sequence 2 is made up of 243 amino acid residues.

In order to make A1) in protein be convenient to purifying, can in by sequence table, the amino acid sequence shown in sequence 2 formsThe amino terminal of protein or carboxyl terminal connect upper label as shown in table 1.

The sequence of table 1, label

Label	Residue	Sequence 1 -->
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned A2) in TaMYB-KW protein, the replacement of described one or several amino acid residue and/or disappearance and/Or be added to the replacement and/or disappearance and/or the interpolation that are no more than 10 amino acid residues.

Above-mentioned A2) in TaMYB-KW protein can manually synthesize, also can first synthesize its encoding gene, then carry out biology tableReach.

Above-mentioned A2) in the encoding gene of TaMYB-KW protein can be by by the DNA shown in the 99-830 position of sequence 1In sequence, lack the codon of one or several amino acid residue, and/or carry out the missense mutation of one or several base-pair,And/or the coded sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.

For solving the problems of the technologies described above, the present invention also provides the biomaterial relevant to TaMYB-KW, this biomaterialFor following B1) to B9) in any:

B1) nucleic acid molecules of coding TaMYB-KW;

B2) contain B1) expression cassette of described nucleic acid molecules;

B3) contain B1) recombinant vector of described nucleic acid molecules or contain B2) recombinant vector of described expression cassette;

B4) contain B1) recombinant microorganism of described nucleic acid molecules or contain B2) recombinant microorganism of described expression cassette orContain B3) recombinant microorganism of described recombinant vector;

B5) contain B1) transgenic plant cells of described nucleic acid molecules system or contain B2) transgenosis of described expression cassettePlant cell;

B6) contain B1) the genetically modified plants tissue of described nucleic acid molecules or contain B2) transgenosis of described expression cassette plantsFabric texture;

B7) contain B1) the genetically modified plants organ of described nucleic acid molecules or contain B2) transgenosis of described expression cassette plantsSundries official;

B8) reduce the nucleic acid molecules that TaMYB-KW expresses;

B9) contain B8) expression cassette of described nucleic acid molecules, recombinant vector, recombinant microorganism or transgenic plant cells system.

Above-mentionedly biological just expect B1) described nucleic acid molecules can be following b1)-b4) and in any:

B1) its coded sequence is cDNA molecule or the DNA molecular of the 99-830 position nucleotides of sequence 1 in sequence table;

B2) its coded sequence is cDNA molecule or the DNA molecular of the 1-1028 position nucleotides of sequence 1 in sequence table;

B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% above homogeneity, and the TaMYB-KW that encodesCDNA molecule or genomic DNA molecule;

B4) under stringent condition with b1) or b2) limit nucleotide sequence hybridization, and coding TaMYB-KW cDNA divideSon or genomic DNA molecule;

B8) described nucleic acid molecules be with sequence table in the DNA of arbitrary fragment reverse complemental in the DNA molecular shown in sequence 1Molecule.

Wherein, described nucleic acid molecules can be DNA, as cDNA, genomic DNA or recombinant DNA; Described nucleic acid molecules also canTo be RNA, as mRNA or hnRNA etc.

Wherein, sequence 1 is made up of 1028 nucleotides, wherein the DNA molecular shown in the 99-830 position nucleotides of sequence 1Protein shown in coded sequence 2.

Those of ordinary skill in the art can adopt known method, the side of for example orthogenesis and point mutation easilyMethod, suddenlys change to the nucleotide sequence of coding TaMYB-KW of the present invention. Those,, through manually modified, have and the present inventionThe nucleotide sequence 75% of the TaMYB-KW that separation obtains or the nucleotides of higher homogeneity, as long as coding TaMYB-KW and toolHaving TaMYB-KW function, is to be all derived from nucleotide sequence of the present invention and to be equal to sequence of the present invention.

Term used herein " homogeneity " refers to the sequence similarity with natural acid sequence. " homogeneity " comprises and thisThe nucleotide sequence of the protein of amino acid sequence shown in bright coded sequence 2 composition has 75% or higher, or 85% orHigher, or 90% or higher, or 95% or the nucleotide sequence of higher homogeneity. Homogeneity can be with the naked eye or computer softwareEvaluate. Use computer software, the homogeneity between two or more sequences can use percentage (%) to represent, it canBe used for evaluating the homogeneity between correlated series.

In above-mentioned biomaterial, described stringent condition is at 2 × SSC, and in the solution of 0.1%SDS, at 68 DEG C, hybridization alsoWash film 2 times, each 5min again in 0.5 × SSC, in the solution of 0.1%SDS, is hybridized and washes film 2 times at 68 DEG C, each15min; Or, in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS, under 65 DEG C of conditions, hybridize and wash film.

Above-mentioned 75% or 75% above homogeneity, can be more than 80%, 85%, 90% or 95% homogeneity.

In above-mentioned biomaterial, B2) expression cassette (the TaMYB-KW base of the described nucleic acid molecules that contains the TaMYB-KW that encodesBecause of expression cassette), refer to the DNA that can express TaMYB-KW in host cell, this DNA not only can comprise startup TaMYB-KW baseBecause of the promoter of transcribing, also can comprise the terminator that stops TaMYB-KW genetic transcription. Further, described expression cassette also can compriseEnhancer sequence. Can be used for promoter of the present invention includes but not limited to: constitutive promoter, tissue, organ and growth are specialPromoter, and inducible promoter. The example of promoter includes but not limited to: the constitutive promoter of cauliflower mosaic virus35S: from the wound-induced type promoter of tomato, leucine aminopeptidase (" LAP ", the people such as Chao (1999) PlantPhysiol120:979-992); From chemical inducible promoter of tobacco, pathogenesis be correlated with 1 (PR1) (by salicylic acid andBTH (diazosulfide-7-carbothioic acid S-methyl esters) induction); Tomato protease inhibitors II promoter (PIN2) or LAP openMover (all available methyl jasmonate inductions); Heat shock promoter (United States Patent (USP) 5,187,267); Tetracycline induction type startsSon (United States Patent (USP) 5,057,422); Seed specific promoters, as Millet Seed specificity promoter pF128(CN101063139B (Chinese patent 200710099169.7)), promoter (for example, the Kidney bean ball that seed storage protein matter is specialAlbumen, napin, promoter (people (1985) EMBOJ.4:3047-such as Beachy of oleosin and soybean betaconglycin3053)). They can be used alone or are combined with other plant promoter. All bibliography cited herein are all completeLiterary composition is quoted. Suitable transcription terminator includes but not limited to: Agrobacterium rouge alkali synthetase terminator (NOS terminator), flower coconut palmCauliflower mosaic virus CaMV35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and octopine synthaseTerminator (referring to, for example: the people (I such as Odell⁹⁸⁵) Nature313:810; The people such as Rosenberg (1987) Gene, 56:125;The people such as Guerineau (1991) Mol.Gen.Genet, 262:141; Proudfoot (1991) Cell, 64:671; SanfaconDeng people GenesDev., 5:141; The people such as Mogen (1990) PlantCell, 2:1261; The people such as Munroe (1990) Gene,91:151; The people such as Ballad (1989) NucleicAcidsRes.17:7891; The people such as Joshi (1987) NucleicAcidRes.,15:9627)。

The recombinant vector that available existing expression vector establishment contains described TaMYB-KW expression casette. Described plant tableReach the carrier etc. that carrier comprises double base agrobacterium vector and can be used for plant micropellet bombardment. As pAHC25, pBin438,PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa orPCAMBIA1391-Xb (CAMBIA company) etc. Described plant expression vector also can comprise 3 of foreign gene ' end non-translational regionTerritory, comprises polyadenylic acid signal and any other participates in the DNA fragmentation of mRNA processing or gene expression. Described polyadenylic acid letterNumber bootable polyadenylic acid joins 3 of mRNA precursor ' end, if Agrobacterium crown gall nodule induction (Ti) plasmid gene is (as nopalineSynthase gene Nos), plant gene (as soybean storage GFP) 3 ' end non-translational region of transcribing all has similar functions.While using gene constructed plant expression vector of the present invention, also can use enhancer, comprise translational enhancer or transcriptional enhancer,These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must with the readding of coded sequenceFrame is identical, to ensure the correct translation of whole sequence. The source of described translation control signal and initiation codon is widely,Can be natural, also can synthesize. Translation initiation region can be from transcription initiation region or structural gene. For justIn transgenic plant cells or plant are identified and screened, can process plant expression vector used, as add canThe coding of expressing in plant can produce the enzyme of change color or the gene of luminophor (gus gene, luciferase genesDeng), antibiotic marker gene (as gives the nptII gene to kanamycins and associated antibiotic resistance, gives herbicideThe bar gene of phosphinothricin resistance, gives the hph gene to antibiotic hygromycin resistance, and give methotrexate resistanceDhfr gene, gives the EPSPS gene to glyphosate resistance) or anti-chemical reagent marker gene etc. (as anti-herbicide baseBecause of), the mannose-6-phosphate isomerase gene of metabolism mannose ability is provided. From the security consideration of genetically modified plants, can be notAdd any selected marker, directly with adverse circumstance screening transformed plant.

In above-mentioned biomaterial, described carrier can be plasmid, glutinous grain, bacteriophage or viral vectors. Described plasmid specifically canFor pMD18-T or pAHC25. Described viral vectors specifically can be the carrier of BMSV virus, as γ carrier.

B3) described recombinant vector can contain the DNA order for the TaMYB-KW that encodes shown in the 99-830 position of sequence 1Row; Further B3) described recombinant vector specifically can be pA25-TaMYB-KW. Described pA25-TaMYB-KW is by pAHC25 carrierSpeI and EcolCRI recognition site between DNA sequence dna replace with the DNA sequence dna shown in the 99-830 position of sequence 1, keepOther DNA sequence dna is constant, obtains the recombinant vector of the TaMYB-KW shown in expressed sequence 2.

In above-mentioned biomaterial, described microorganism can be yeast, bacterium, algae or fungi. Wherein, bacterium can be from dust Xi ShiPseudomonas (Escherichia), Erwinia (Erwinia), Agrobacterium tumefaciems belongs to (Agrobacterium), Flavobacterium(Flavobacterium), Alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), Bacillus(Bacillus) etc.

In above-mentioned biomaterial, described transgenic plant cells system, genetically modified plants tissue and genetically modified plants organ are equalDo not comprise propagating materials.

In above-mentioned biomaterial, B8) described nucleic acid molecules specifically can be with sequence table in the 576-836 position core of sequence 1The DNA molecular of the DNA molecular reverse complemental shown in thuja acid.

For solving the problems of the technologies described above, the present invention also provides plant disease-resistant agent. Described plant disease-resistant agent contains TaMYB-KW or described biomaterial.

In above-mentioned plant disease-resistant agent, described plant disease-resistant agent can be using described TaMYB-KW as active component, all rightTaMYB-KW and other disease resistance material are combined to the composition obtaining as active component.

In above-mentioned plant disease-resistant agent, described plant can be monocotyledon or dicotyledon. Described monocotyledon toolBody can be wheat. Described wheat can be Wheat Germplasm Resources CI12633 or raises wheat 16.

In above-mentioned plant disease-resistant agent, described disease resistance can be anti-banded sclerotial blight. Described banded sclerotial blight can be by Rhizoctonia cerealis(Rhizoctoniacerealis) cause. Described Rhizoctonia cerealis (Rhizoctoniacerealis) can be Rhizoctonia cerealis(Rhizoctoniacerealis)R0301。

For solving the problems of the technologies described above, the present invention also provides TaMYB-KW or described biomaterial following 1)-3) inApplication in any:

1) regulating plant disease resistance;

2) prepare disease resistance of plant product;

3) cultivate disease resistance plant.

In above-mentioned application, described plant can be monocotyledon or dicotyledon. Described monocotyledon specifically can beWheat. Described wheat can be Wheat Germplasm Resources CI12633 or raises wheat 16.

In above-mentioned application, described disease resistance can be anti-banded sclerotial blight. Described banded sclerotial blight can be by Rhizoctonia cerealis(Rhizoctoniacerealis) cause. Described Rhizoctonia cerealis (Rhizoctoniacerealis) can be Rhizoctonia cerealis(Rhizoctoniacerealis)R0301。

For solving the problems of the technologies described above, the present invention also provides a kind of method of cultivating disease resistant transgenic plants, the partyMethod comprises that obtaining disease resistance to the encoding gene that imports TaMYB-KW in recipient plant turns higher than the disease resistance of described recipient plantGene plant.

In an embodiment of the present invention, the encoding gene of described TaMYB-KW (is the 99-830 position nucleotides institute of sequence 1The DNA molecular showing) import object plant by the TaMYB-KW DNA recombinant expression carrier that contains TaMYB-KW expression casetteIn. In described TaMYB-KW expression casette, the promoter that starts TaMYB-KW genetic transcription is that corn Ubiquitin startsSon.

In the method for above-mentioned cultivation disease resistant transgenic plants, wherein said TaMYB-KW gene can first be repaiied as followsDecorations, then import in acceptor seed plant, to reach better expression effect:

1) modify according to actual needs and optimize, so that gene efficient expression; For example, can be according to recipient plant institute partiallyThe codon of liking changes its codon and plants to meet in the amino acid sequence that keeps TaMYB-KW gene of the present inventionThing preferences; In optimizing process, preferably can make to keep certain GC content in the coded sequence after optimizing, to realize and planting bestThe high level expression of quiding gene in thing, wherein GC content can be 35%, more than 45%, more than 50% or more than approximately 60%;

2) modify the gene order of contiguous initial methionine, so that translate effectively initial; For example, utilize in plantThe effective sequence of knowing is modified;

3) be connected with the promoter of various expression of plants, be beneficial to its expression in plant; Described promoter can compriseComposing type, induction type, sequential regulate, grow adjusting, Chemical Regulation, tissue preferably and tissue-specific promoter; PromoterSelection will be along with expression time and space requirement and is changed, and depends on target species; The for example specificity of tissue or organExpress promoter, acceptor in what period of growing is determined as required; Although proved to derive from the many of dicotyledonPromoter is operational in monocotyledon, and vice versa, but ideally, selects dicotyledon promoter to be used forExpression in dicotyledon, monocotyledonous promoter is for the expression of monocotyledon;

4), with applicable tanscription termination sub-connection, also can improve the expression efficiency of gene of the present invention; For example derive fromThe tml of CaMV, derives from the E9 of rbcS; Any known available terminator working in plant can with the present inventionGene connects;

5) introduce enhancer sequence, for example, as intron sequences (deriving from Adhl and bronzel) and virus leader sequence(for example deriving from TMV, MCMV and AMV).

Described TaMYB-KW expression vector can be by using Ti-plasmids, Ri plasmid, plant viral vector, direct DNAConventional biological method transformed plant cells or the tissues such as conversion, microinjection, electricity lead, agriculture bacillus mediated, particle gun, and will turnThe plant tissue of changing is cultivated into plant.

Described in described method also comprises that screening is expressed from the plant of the encoding gene of the TaMYB-KW shown in importing sequence 2The plant of encoding gene, obtains described transgenic wheat.

In the method for above-mentioned cultivation disease resistant transgenic plants, described recipient plant can be monocotyledon or dicotyledonous plantingThing. Described monocotyledon specifically can be wheat. Described wheat can be Wheat Germplasm Resources CI12633 and raises wheat 16.

In the method for above-mentioned cultivation disease resistant transgenic plants, described disease resistance can be anti-banded sclerotial blight. Described banded sclerotial blight canCaused by Rhizoctonia cerealis (Rhizoctoniacerealis). Described Rhizoctonia cerealis (Rhizoctoniacerealis) canFor Rhizoctonia cerealis (Rhizoctoniacerealis) R0301.

In the method for above-mentioned cultivation disease resistant transgenic plants, the coded sequence of the encoding gene of described TaMYB-KW can beThe DNA molecular of the 99-830 position of sequence 1 in sequence table.

For solving the problems of the technologies described above, the present invention also provides the method for the genetically modified plants of cultivating disease resistance reduction, shouldMethod comprises the expression that reduces the encoding gene of TaMYB-KW in object plant, obtains disease resistance turning lower than described object plantGene plant.

In the method for the genetically modified plants that above-mentioned cultivation disease resistance reduces, described object plant can be monocotyledon. InstituteState monocotyledon and specifically can be wheat. Described wheat can be Wheat Germplasm Resources CI12633 or raises wheat 16.

In the method for the genetically modified plants that above-mentioned cultivation disease resistance reduces, described disease resistance can be anti-banded sclerotial blight. Described lineRot can be caused by Rhizoctonia cerealis (Rhizoctoniacerealis). Described Rhizoctonia cerealis (RhizoctoniaCerealis) can be Rhizoctonia cerealis (Rhizoctoniacerealis) R0301.

In the method for the genetically modified plants that above-mentioned cultivation disease resistance reduces, reduce the coding base of TaMYB-KW in object plantThe expression of cause be by by with sequence table in the DNA of the DNA fragmentation reverse complemental shown in the 576-836 position nucleotides of sequence 1Molecule imports described object plant realization.

In one embodiment of the invention, with sequence table in the DNA sheet shown in the 576-836 position nucleotides of sequence 1The DNA molecular of section reverse complemental imports in described object plant by the γ carrier of BMSV virus.

In the present invention, described genetically modified plants are interpreted as not only to comprise described TaMYB-KW genetic transformation object plant are obtainedThe first generation genetically modified plants that arrive, also comprise its filial generation. For genetically modified plants, this gene can be bred in these species, alsoAvailable traditional breeding method enters this transgenosis other kind of same species, in commercial variety. Described turningGene plant comprises seed, callus, whole plant and cell.

Experiment showed, after inoculation Rhizoctonia cerealis the expression of TaMYB-KW gene in anti-banded sclerotial blight wheat CI12633Amount significantly raises, and TaMYB-KW and encoding gene thereof can improve the disease resistance of plant: the TaMYB-KW gene in plant reducesAfter expression, the disease resistance of wheat sharp eyespot is declined; Turn TaMYB-KW gene by what obtain in TaMYB-KW gene transferred plantThe disease index of plant is 20.00-36.00, turns the sick level of banded sclerotial blight of TaMYB-KW gene plant for 1.00-1.67, turnsThe disease index of TaMYB-KW gene plant and sick level are all extremely significantly lower than wild type plant. Illustrate that TaMYB-KW gene is wheatAnti-banded sclerotial blight is reacted required disease-resistant gene, and forward participates in the anti-banded sclerotial blight reaction of wheat. TaMYB-KW gene is a kind of withered with lineThe closely-related disease-resistant wheat GFP of sick resistance, has great value to plant breeding. Cultivation disease resistance of the present invention is carriedThe method of high genetically modified plants has important theory and practical significance, in the genetic improvement of plant, will bring into play important workWith.

Brief description of the drawings

Fig. 1 is the expression pattern of wheat TaMYB-KW gene in anti-disease wheat CI12633. Wherein, a represents to inoculate the 4thThe expression of it and the 10th day TaMYB-KW gene is significantly higher than inoculation the 0th day; B represents to inoculate the 10th day TaMYB-KW geneExpression is significantly higher than inoculation the 4th day.

Fig. 2 inoculates the expression analysis result of TaMYB-KW gene in the wheat plant of BSMV virus. Wherein, BSMV:GFP tableShow inoculation BSMV:GFP wheat; Inoculation BSMV:TaMYB-KW-1～3 represent the wheat of three strain inoculation BSMV:TaMYB-KWKW.

The wheat CI12633 plant of Fig. 3 TaMYB-KW gene silencing and the banded sclerotial blight phenotype of contrast thereof. Wherein, BSMV:GFP represents to inoculate BSMV:GFP wheat; Inoculation BSMV:TaMYB-KW-1～3 represent that three strain inoculation BSMV:TaMYB-KW's is littleWheat.

Fig. 4 is the PCR testing result that turns TaMYB-KW DNA triticum. Wherein, a is T_OIn generation, b is T₁In generation, c is T₂Generation; P tableShow pA25-TaMYB-KW; WT represents to raise wheat 16; H₂O: empty map; MO1～MO5 all represents transfer-gen plant.

Fig. 5 is T₀-T₂In generation, turns the quantitative PCR detection result of TaMYB-KW DNA triticum. Wherein, WT represents to raise wheat 16, MO1～5 all represent transfer-gen plant. * transgenic line and WT have utmost point significant difference (P < 0.01).

The phenotype comparative result of the anti-banded sclerotial blight Transgenic plant of wheat of Fig. 6 and Yang Mai 16. Wherein, wherein, WT represents to raise wheat16, MO1～MO5 all represents transfer-gen plant.

Detailed description of the invention

Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment providing is only in order to explainBright the present invention, instead of in order to limit the scope of the invention.

Experimental technique in following embodiment, if no special instructions, is conventional method.

Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.

Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.

The anti-banded sclerotial blight of Wheat Germplasm Resources CI12633, wheat breed is raised in wheat 16 and is felt banded sclerotial blight. Anti-disease wheat material-littleWheat CI12633 comes from Jiangsu Academy of Agricultural Sciences's germplasm resource bank, and wheat breed is raised wheat 16 and come from Lixiahe region in Jiangsu's agricultureIndustry Science Institute, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment.

Wheat sharp eyespot pathogenic bacteria-Rhizoctonia cerealis (Rhizoctoniacerealis) R0301 (cold Su Feng, Zhang Aixiang,Li Wei, Chen Huaigu. the Analysis of Resistance of Wheat in Jiangsu Province new varieties (being) to banded sclerotial blight. Jiangsu agricultural journal, 2010,26 (6):1176-1180；ChenLiang,ZhangZengyan(Correspondance),LiangHongxia,LiuHongxia,DuLipu,XuHuijun,XinZhiyong.2008.OverexpressionofTiERF1enhancesresistancetosharpeyespotintransgenicwheat.JournalofExperimentalBotany.59:4195-4204), the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the applicationExperiment, not can be used as other purposes and uses.

(pAHC25 is formed by pUC8 transformation monocotyledon expression vector pAHC25, contains 2 expression cassettes, the 1st expressionBox has corn Ubiquitin promoter, Exon, Intron, GUS, Nos terminator, and GUS two ends have SmaI and SacI enzyme is cutSite, the 2nd expression cassette has corn Ubiquitin promoter, Exon, Intron, Bar, Nos terminator: (bibliography:ChristensenandQuail,1996；Ubiquitinpromoter-basedvectorsforhigh-levelexpressionofselectableand/orscreenablemarkergenesinmonocotyledonousPlants.TransgenicResearch, 5,213 – 218). The public can be from Institute of Crop Science, Chinese Academy of Agricultural ScienceObtain, to repeat the application's experiment. PAHC25 is formed by pUC8 transformation, contains 2 expression cassettes, and the 1st expression cassette has cornUbiquitin promoter, Exon, Intron, GUS, Nos terminator, GUS two ends have SmaI and SacI restriction enzyme site, the 2ndExpression cassette has corn Ubiquitin promoter, Exon, Intron, Bar, Nos terminator.

BSMV-γ (the γ carrier of BMSV virus) (HolzbergS, BrosioP, GrossC, PogueGP.2002.Barleystripemosaicvirus-inducedgenesilencinginamonocotPlant.ThePlantJournal30,315-327) public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment. BSMV-α and BSMV-β and BSMV-γ-GFP (HolzbergS, BrosioP, GrossC,PogueGP.2002.Barleystripemosaicvirus-inducedgenesilencinginamonocotPlant.ThePlantJournal30,315-327) public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment. Wherein, BSMV-γ-GFP inserts GFP (green fluorescent protein) code area full length DNAIn the polyclone restriction enzyme site of BSMV-γ carrier, obtain expressing the recombinant vector of GFP.

PMD18-T in following embodiment is precious bioengineering (Dalian) Co., Ltd product.

The clone of embodiment 1, disease-resistant wheat albumen TaMYB-KW and encoding gene thereof

Present inventor clones and isolates a disease-resistant wheat albumen, by it from anti-banded sclerotial blight wheat CI12633Called after TaMYB-KW. The concrete cloning process of TaMYB-KW gene is as follows:

The blade of taking the wheat CI12633 seedling of rhizoctonia cerealis inoculation, liquid nitrogen processing, according to InvitrogenThe method of TRIZOLReagent total RNA extraction reagent description is extracted total RNA of blade. According to Invitrogen company firstThe program of chain cDNA synthetic agent box, by synthetic the RNA sample reverse transcription of extracting the first chain cDNA, as the mould of Gene cloningPlate.

In order to obtain the cDNA sequence of TaMYB-KW full length gene, utilize two pairs of primers, adopt RACE and nested PCR methodIncrease. Utilize primer pair TaMYB-KWA-U1 (5 '-GCAGCATTTACCTTCGGAC-3 ') and AUAP (5'-GGCCACGCGTCGACTAGTAC-3') by first round pcr amplification, pcr amplification system is: 2 × GCBuffer I (TaKaRa)10μL,cDNA2.0μL(50ng),2.5mMdNTPs(TaKaRa)1.0μl,10μmol/LTaMYB-KWA-43U10.5μL,10μmol/LTaMYB-KWA-1038L10.5μL,5U/μlTaKaRaPrimeSTAR0.2μl，ddH₂O mends to 20 μL; Pcr amplification program is: first 95 DEG C of denaturations 3 minutes; Then 98 DEG C 45 seconds, 59 DEG C 7 seconds, 72 DEG C 90 seconds, totally 30 circulations; Again72 DEG C are extended 10 minutes. Then, utilize TaMYB-KWA-U2 (5 '-CGTCAACACACTGAGCAATC-3 ') and AUAP (5'-GGCCACGCGTCGACTAGTAC-3') primer and is taken turns PCR product dilution (50 times) and is done template, and carry out two and take turns pcr amplification,Amplification system is: 2 × GCBuffer I (TaKaRa), 10 μ L, one takes turns 50 times of dilutions of PCR product, 2.0 μ L, 2.5mMdNTPs(TaKaRa)1.0μl,10μmol/LTaMYB-KWA-U20.5μL,10μmol/LTaMYB-KWA-L20.5μL,5U/μlTaKaRaPrimeSTAR0.2μl，ddH₂O mends to 20 μ L; Pcr amplification program is: first 95 DEG C of denaturations 3 minutes; Then 98DEG C 45 seconds, 58 DEG C 7 seconds, 72 DEG C 90 seconds, totally 30 circulations; 72 DEG C are extended 10 minutes again. PCR product is carried out to Ago-Gel electricitySwimming, result amplification obtains the fragment of, and this PCR product is connected on pMD18-T carrier and order-checking. Sequencing result shows,The nucleotide sequence of this pcr amplification product is as shown in the sequence 1 of sequence (1-1028 position nucleotides), and its coded sequence is sequenceThe 99-830 position nucleotides of sequence 1 in table; Disease-resistant albumen TaMYB-KW shown in coded sequence 2.

Embodiment 2, TaMYB-KW gene are analyzed by the abduction delivering of wheat sharp eyespot pathogenic bacteria

Whether relevant to wheat sharp eyespot resistance in order to study TaMYB-KW gene expression amount, utilize Q-RT-PCR to analyzeThe expression of TaMYB-KW gene in the anti-banded sclerotial blight wheat CI12633 of Rhizoctonia cerealis inoculation 4 days and 10 days.

With wheat sharp eyespot pathogenic bacteria-Rhizoctonia cerealis (Rhizoctoniacerealis) R0301 mycelia toothpick, wheatBe inoculated between the leaf sheath and stem of wheat CI12633 seedling in tillering stage; After 4 days and 21 days, get wheat stalk in inoculation, after liquid nitrogen flash freezerBe stored in-80 DEG C of ultra low temperature freezers for subsequent use.

Extract respectively total RNA (the total RNA of each sample approximately 5 μ g) of each wheat stalk, according to Invitrogen company the first chainThe program of cDNA synthetic agent box, reverse transcription becomes cDNA. Utilize constructive expression's actin gene as internal reference, by sampleCDNA normalization. Then carry out real-time quantitative RT-PCR analysis with the special primer of TaMYB-KW gene, with 2^-△ΔCTMethod(LivakKJ,SchmittgenTD.2001.Analysisofrelativegeneexpressiondatausingreal-timequantitativePCRandthe2^-△ΔCTMethod.Methods.25:402-408) analyze TaMYB-The expression of KW gene under rhizoctonia cerealis is processed, every group of sample repeats 3 times.

The primer pair of reference gene actin:

WActinF：5’-CACTGGAATGGTCAAGGCTG-3’；WActinR：5’-CTCCATGTCATCCCAGTTG-3’。

The special quantitative primer pair of TaMYB-KW gene:

TaMYB-KW-QF:5’-CGACTCGTCCTCGTCCAAG-3'；TaMYB-KW-QR:5’-CGACGACGGCATCGAGTAAT-3’

Can from the TaMYB-KW gene expression amount analysis result to the CI12633 before and after inoculation Rhizoctonia cerealis of Fig. 1Find out, TaMYB-KW gene response sheath blight fungus infects, shows as up-regulated expression; In the time of inoculation Rhizoctonia cerealis 4 days and 10 days,The expression of TaMYB-KW gene in anti-banded sclerotial blight wheat CI12633 all significantly raises.

Acquisition and the Disease Resistance Identification of the reticent wheat of embodiment 3, TaMYB-KW

One, the structure of recombinant expression carrier

1, with Rhizoctonia cerealis (Rhizoctoniacerealis) R0301 inoculation wheat CI12633 stem and leaf sheath, 4 daysRear extraction RNA, reverse transcription is cDNA; Taking cDNA as template, form with TaMYB-KW-VIGS-F and TaMYB-KW-VIGS-RPrimer pair carries out pcr amplification, obtains pcr amplification product (carrying the TaMYB-KW fragment in NheI site).

TaMYB-KW-VIGS-F:5’-ACAGCTAGC(underscore is labeled as NheI enzyme to TCGTCCGCCACCGATTACT-3 'Recognition site);

TaMYB-KW-VIGS-R:5’-GGCGCTAGC(underscore is labeled as CTCTGCCTAAATCTGAGACAAAC-3 'NheI enzyme recognition site).

PCR response procedures: first 94 DEG C of denaturation 3min; Then 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 15 circulations; 94DEG C 30s, 60 DEG C of 30s, 72 DEG C of 1min, 20 circulations; Last 72 DEG C of 10min fill end.

2, reclaim pcr amplification product.

3, cut the recovery product of step 2 with restriction enzyme NheI enzyme, reclaim enzyme and cut product.

4, cut BSMV-γ-GFP vector plasmid DNA with restriction enzyme NheI enzyme, reclaim carrier framework.

5, the carrier framework that fragment step 3 being reclaimed reclaims with step 4 is connected, and obtains connecting product.

6, connection product step 5 being obtained checks order, and gets shown in the 576-836 position of sequence in sequence table 1DNA molecular oppositely inserts the clone of BSMV-γ-GFP carrier, obtains BSMV-γ-TaMYB-KW recombinant vector.

Two, the linearisation of BSMV carrier and in-vitro transcription

The BSMV-α infecting for BSMV, BSMV-β, BSMV-γ-GFP and BSMV-γ-TaMYB-KW are carried out to enzyme and cut,Wherein the BSMV-γ-TaMYB-KW of BSMV-α, BSMV-γ-GFP and step 1 carries out enzyme with restriction enzyme MluI and cuts;BSMV-β carries out enzyme with restriction enzyme SpeI and cuts. Endonuclease reaction finishes rear use 1% agarose gel electrophoresis detection carrierNo enzyme cuts entirely. If carrier, by complete degestion, reclaims, the linearizing carrier of purifying is for subsequent use.

With reference to RiboMAXLargeScaleRNAProductionSystems-T7kit (Promega) description,Carry out in-vitro transcription reaction, system is as follows:

In-vitro transcription reacts under 37 DEG C of constant temperatures carries out 2h, obtains responsive transcription liquid.

Three, BSMV inoculation wheat plant

Drawing the each 10 μ l of linearizing BSMV-α, linearizing BSMV-β and linearizing BSMV-γ-TaMYB-KW is placed inIn 1.5ml centrifuge tube, after mixing, add the RNase-freeddH that uses of 60 μ l₂O dilution, the 90 μ l liquid that obtain, then above-mentionedIn 90 μ l mixtures, add the GKP solution (50mMGlycine of 90 μ l; 30mMK₂HPO₄,pH＝9.2；1％Bentonite；1%Celite), obtain altogether the BSMV virus mixed liquor of 180 μ l.

According to the method described above, linearizing BSMV-γ-TaMYB-KW is replaced with to linearizing BSMV-γ-GFP, otherStep is all constant, obtains the contrast virus mixed liquor of 180 μ l.

According to the method described above, linearizing BSMV-γ-TaMYB-KW is replaced with to RNase-freeddH₂O, other stepsSuddenly all constant, the Mock that obtains 180 μ l contrasts mixed liquor.

In the time of one heart stage of wheat seedling growth to two leaf, draw the BSMV virus mixed liquor frictional inoculation of 10 μ l in wheat childrenOn second blade of seedling. Spray 0.1% the DEPC aqueous solution on postvaccinal wheat leaf blade surface, in 22 DEG C of-23 DEG C of conditionsLower moisturizing 2 days, obtains inoculating BSMV:TaMYB-KW wheat. Carry out with the viral mixed liquor of contrast and Mock contrast mixed liquor respectivelyExperiment, obtains respectively inoculating BSMV:GFP wheat and Mock contrast wheat.

Four, reticent effect detection and disease-resistant qualification

While growing to tillering stage respectively at wheat plant to inoculation BSMV:TaMYB-KW wheat, inoculation BSMV:GFP wheatWith Mock contrast wheat inoculation Rhizoctonia cerealis (Rhizoctoniacerealis) R0301, and in inoculation the 14th day, clip theAfter quaterfoil liquid nitrogen flash freezer, extract total RNA. Quantitative primer with TaMYB-KW gene specific:

TaMYB-KW-QF:5’-CGACTCGTCCTCGTCCAAG-3'

TaMYB-KW-QR:5’-CGACGACGGCATCGAGTAAT-3’

Utilize quantitative RT-PCR (Q-RT-RCR) to analyze the expression of TaMYB-KW gene. Result is as Fig. 2, with inoculationThe plant of BSMV:GFP virus is compared, and in the plant leaf of inoculation BSMV:TaMYB-KW virus, TaMYB-KW gene expression amount is brightAobvious downward, illustrates that inoculation BSMV:TaMYB-KW can cause the silence of TaMYB-KW gene expression in CI12633 plant.

In wheat tillering phase inoculation Rhizoctonia cerealis, inoculate latter 30 days and observe illness, result as shown in Figure 3, according to table 2The sick grade standard of wheat sharp eyespot, determines sick level. The sick level of the CI12633 plant of inoculation BSMV:GFP virus is 1.2 grades, and performance is anti-Banded sclerotial blight phenotype; The sick level of wheat of three strain inoculation BSMV:TaMYB-KW is respectively 3.8 grades, 3.2 grades, 2.8 grades, has occurred typical caseWheat sharp eyespot scab, show as the sick phenotype of sense banded sclerotial blight; These results suggest that TaMYB-KW gene is that wheat CI12633 is anti-Imperial banded sclerotial blight is reacted required gene, the defense reaction of the anti-banded sclerotial blight wheat of positive regulation CI12633 to Rhizoctonia cerealis.

Table 2, the sick grade standard of wheat sharp eyespot

Embodiment 4, TaMYB-KW cross acquisition and the Disease Resistance Identification of express transgenic wheat

One, the structure of recombinant expression carrier

1, inoculate leaf sheath and the stem of wheat CI12633 with Rhizoctonia cerealis (Rhizoctoniacerealis) R0301,4After it, extract RNA, reverse transcription is cDNA; Taking cDNA as template, form with TaMYB-KW-P25-U and TaMYB-KW-P25-LPrimer pair and high-fidelity amplification enzyme PRIMERSTAR (TAKARA company) carry out pcr amplification, obtain pcr amplification product and (carryThe TaMYB-KW gene in SpeI site).

TaMYB-KW-P25-U:5’-CAAACTAGT(underscore is labeled as SpeI enzyme to be known ATGGGGAGGTCTCCTTGC-3 'Other site);

TaMYB-KW-P25-L:5’-CTAAATCTGAGACAAACT-3’。

PCR response procedures: first 94 DEG C of denaturation 3min; Then 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 15 circulations; 94DEG C 30s, 58 DEG C of 30s, 72 DEG C of 1min, 20 circulations; Last 72 DEG C of 10min fill end.

2, reclaim pcr amplification product TaMYB-KW-P.

3, cut TaMYB-KW-P with restriction enzyme SpeI enzyme and reclaim product, recovery enzyme is cut the sheet of about 737bp in productSection.

4, with restriction enzyme SpeI and EcoICRI (cut with SacI identification enzyme the isoschizomers that sequence is consistent, butEcoICRI enzyme is cut the flat end of rear generation) enzyme cuts monocotyledon transgene expression vector pAHC25, reclaims carrier framework.

5, the carrier framework that DNA fragmentation step 3 being reclaimed reclaims with step 4 is connected, and obtains connecting product.

6, connection product step 5 being obtained checks order, and sequencing result shows that skeleton carrier is monocotyledon expressionA part of carrier pAHC25, the 99-830 position institute that has inserted SEQIDNo.2 between SpeI and EcoICRI recognition siteThe encoding gene of the TaMYB-KW albumen showing, by this recombinant vector called after pA25-TaMYB-KW. PA25-TaMYB-KW is willThe SpeI of pAHC25 carrier and EcoICRI recognition site (cutting sequence consistent with SacI identification enzyme is isoschizomers, but EcoICRI enzymeCut the flat end of rear generation) insert the 99th to the 830th nucleotides of 5 ' end of sequence 1 in sequence table between restriction enzyme siteShown TaMYB-KW gene; TaMYB-KW gene is subject to the control of Ubiquitin promoter; Plasmid also has 1 and is subject to UbiquitinThe Bar expression casette of promoter control, can be in follow-up work and utilizes herbicide bialaphos (Bialaphos) screening to transformRegeneration plant provides resistance marker.

Two, the acquisition of genetically modified plants

1, the acceptor using 1736 rataria callus of raising wheat 16 as particle gun bombardment, with particle gun by recombinant plasmidPA25-TaMYB-KW bombards callus.

2, by the post processing 16h on osmotic pressure culture medium of the callus after being bombarded by particle gun.

3, then callus is transferred to SD2 culture medium and (in the inorganic salts composition of MS culture medium, added VB₁1mg/L，Asparagus fern door acid amides 150mg/L, 2,4-D2mg/L) upper, renewal cultivation 2 weeks (26 DEG C, secretly cultivation).

4, the callus after renewal cultivation is transferred to (1/2MS culture medium+methyl α-naphthyl acetate 1mg/ in differentiation screening and culturing baseL+ kinetin 1mg/L+ bialaphos 2-5mg/L), 24-26 DEG C of illumination cultivation 14d; To after Calli Differentiation seedling, transfer toIn growth screening and culturing base (1/2MS culture medium+bialaphos 2-3mg/L), 24-26 DEG C of illumination cultivation; 44 strain regeneration are obtainedPlant.

5, regeneration plant is transferred to strong seedling culture base (1/2MS culture medium+0.5mg/L methyl α-naphthyl acetate) above, by height of seedling 7-8cmAnd the transformation seedlings of well developed root system is transplanted to flowerpot, be transplanted to greenhouse after 3 weeks, there are 44 strain plant to survive.

6, Molecular Identification

PCR detects

In 4 leaf phases, the every strain of 44 strain plant that step 5 is obtained is got 1 blade and is extracted genomic DNA, and genomic DNA is doneFor template, utilize the sequence-specific one section of sequence of TaMYB-KW gene ORF as upstream primer (TaMYB-KW-ZJF1 andTaMYB-KW-ZJF2), one section of special sequence of carrier pA25-TaMYB-KW is carried out as downstream primer (TaMYB-KW-ZJR)Nest-type PRC. With the positive contrast of recombinant expression plasmid pA25-TaMYB-KW, raise the negative contrast of genomic DNA of wheat 16, pre-Phase amplified production fragment is 375bp.

TaMYB-KW-ZJF1：5’-CGGACAACGAGATCAAGAAC-3’；

TaMYB-KW-ZJF2：5’-AGCTTTACATCGGAGGAGTTTCAG-3’；

TaMYB-KW-R-ZJR：5’-AAAACCCATCTCATAAATAACG-3’。

First round pcr amplification system (25 μ l): 2 × TaqMasterMix (health is century), 12.5 μ l, TaMYB-KW-R-ZJF1 (10 μ M) 1 μ l, TaMYB33-R-ZJR (10 μ M) 1 μ l, template DNA 100ng, mends ddH₂O to 25 μ l. Pcr amplification program:94 DEG C of 8min; 30 × (94 DEG C of 45s, 61 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of 8min; 16 DEG C of preservations.

Then, utilize TaMYB-KW-ZJF2, TaMYB-KW-ZJR primer and to take turns PCR product dilution and do template, carry outTwo take turns pcr amplification, and amplification system (25 μ l): 2 × TaqMasterMix (health is century), 12.5 μ l, TaMYB-KW-ZJF2 (10 μM) 1 μ l, TaMYB-KW-ZJR (10 μ M) 1 μ l, one takes turns 40 times of dilutions of PCR product, 1.0 μ L, mends ddH₂O to 25 μ l. Pcr amplificationProgram: 94 DEG C of 8min; 30 × (94 DEG C of 45s, 48 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of 8min; 16 DEG C of preservations.

Pcr amplification product carries out 1.5% agarose gel electrophoresis detection, and ultraviolet is taken pictures, and records result.

44 strain plant (T₀Generation) in, PCR positive plant (being transfer-gen plant) 19 strains.

7、T₁For individual plant and Molecular Identification thereof

PCR detects

To after the 19 strain PCR positive plant selfings that obtain, obtain T₁For individual plant, by T₁Carry out Molecular Identification, method for individual plantWith above-mentioned steps 6, with the positive contrast of recombinant vector pA25-TaMYB-KW, raise the negative contrast of genomic DNA of wheat 16, pre-Phase amplified production fragment is 374bp. Result shows at 149 strain transgenosis T₁In plant, detect positive plant 50 strains, adhere to separately12 strains, positive rate 33.56%. Part plant PCR testing result is shown in Fig. 4. 5 transgenic lines are at T₁For transgenic line(MO1～MO5) and T thereof₂Generation equal positive strains.

Utilize TaMYB-KW gene specific quantitative primer (TaMYB-KW-QF:5 '-CGACTCGTCCTCGTCCAAG-3',TaMYB-KW-QR:5 '-CGACGACGGCATCGAGTAAT-3 ') and qRT-PCR technical Analysis T₂For 5 transgenic linesThe relative expression quantity of TaMYB-KW gene in (MO1～MO5). Result shows the phase his-and-hers watches of TaMYB-KW gene in transfer-gen plantThe amount of reaching is apparently higher than transgenic wheat not, but expression difference to some extent between each transgenic line, TaMYB-KW gene is turning baseBecause of the expression the highest, in MO2 of the expression in strain MO1 minimum (Fig. 5).

Three, turn the acquisition of empty carrier plant

With carrier pAHC25 replacement recombinant plasmid pA25-TaMYB-KW, other same step 2, obtains turning empty carrier plant,As the contrast of transfer-gen plant.

Four, the sharp eyespot resistance of genetically modified plants qualification

Identify by the following method T₁For 5 transgenic lines (MO1～MO5) and from these 5 T₁In transgenic lineThe 10 strain offsprings that every strain is selected (are T₂Generation) sharp eyespot resistance, and with raising wheat 16 (WT) and turning empty carrier plant as rightAccording to, concrete grammar is as follows:

The Rhizoctonia cerealis inoculation method that turns TaMYB-KW DNA triticum plant adopts wheat inocalation method. Contain at wheat tilleringPhase, cover with the wheat of Rhizoctonia cerealis (Rhizoctoniacerealis) R0301 with disinfecting forceps gripping, put into lightly wheatGrain basal part of stem, 5 wheats are put in every strain, moisturizing 3 days.

In the banded sclerotial blight incidence of wheat harvest investigation wheat individual plant, according to table 2, the order of severity of banded sclerotial blight is drawnBe divided into 0～5 grade, and calculate banded sclerotial blight disease index (table 3).

Disease index (DI)=[(Σ diseased plant numbers at different levels × typical values at different levels)/(total strain number × highest typical value)] ×100

Table 3, Transgenic plant of wheat and contrast the result of banded sclerotial blight state of an illness investigation

Note: WT: transgenosis is not raised wheat 16; * is illustrated in the level of P < 0.01 each transgenic line and raises wheat 16 and have aobvious with contrastingWork difference. " the T of WT₁Generation sick level ", " T₁For disease index " represent respectively and T₁The wild type of simultaneously planting for transfer-gen plant is plantedSick level and the disease index of wheat 16 raised in strain; " the T of WT₂Generation sick level ", " T₂For disease index " represent respectively and T₂For transfer-gen plantThe wild type plant of plantation is raised sick level and the disease index of wheat 16 simultaneously; Turn " the T of empty carrier plant₁Generation sick level ", " T₁For the state of an illnessIndex " represent respectively and T₁The wild type plant of simultaneously planting for transfer-gen plant is raised sick level and the disease index of wheat 16; Turn empty" the T of carrier plant₂Generation sick level ", " T₂For disease index " represent respectively and T₂The wild type of simultaneously planting for transfer-gen plant is plantedSick level and the disease index of wheat 16 raised in strain.

Turn empty carrier contrast and the disease index and the basic indifference of sick level of raising wheat 16. Transfer-gen plant T₁The banded sclerotial blight in generationResistance improves, and disease index is 20.00-36.00, and disease index is all extremely significantly raised wheat 16 lower than acceptor wild type plant, sick levelFor 1.00-1.43, sick level is all extremely significantly raised wheat 16 lower than acceptor wild type plant; Transfer-gen plant T₂The sharp eyespot resistance in generation is carriedHeight, disease index is 20.00-33.40, and disease index is all extremely significantly raised wheat 16 lower than acceptor wild type plant, and sick level is 1.00-1.67, sick level is all extremely significantly raised wheat 16 lower than acceptor wild type plant. The positive transfer-gen plant of the anti-banded sclerotial blight of one strain and a strain open countryFig. 6 is shown in by the photo that raw type is raised wheat 16 plant. Result shows, TaMYB-KW gene overexpression has strengthened that to turn TaMYB-KW gene littleThe resistance of wheat to banded sclerotial blight.

Claims

1. protein is following A1), A2) or A3):

A1) amino acid sequence is the protein of sequence 2;

A2) replacement and/or the disappearance through one or several amino acid residue by the amino acid sequence shown in sequence in sequence table 2And/or add and the protein relevant to disease resistance of plant;

2. the biomaterial relevant to protein described in claim 1 is following B1) to B9) in any:

B1) nucleic acid molecules of protein described in coding claim 1;

B2) contain B1) expression cassette of described nucleic acid molecules;

B4) contain B1) recombinant microorganism of described nucleic acid molecules or contain B2) recombinant microorganism of described expression cassette or containB3) recombinant microorganism of described recombinant vector;

B5) contain B1) transgenic plant cells of described nucleic acid molecules system or contain B2) genetically modified plants of described expression cassetteClone;

B6) contain B1) the genetically modified plants tissue of described nucleic acid molecules or contain B2) the genetically modified plants group of described expression cassetteKnit;

B7) contain B1) the genetically modified plants organ of described nucleic acid molecules or contain B2) the genetically modified plants device of described expression cassetteOfficial;

B8) nucleic acid molecules of protein expression described in reduction claim 1;

3. associated biomolecule material according to claim 2, is characterized in that: B1) described nucleic acid molecules is following b1)-b4)In any:

B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% above homogeneity, and described in the claim 1 of encodingThe cDNA molecule of protein or genomic DNA molecule;

B4) under the stringent condition with b1) or the b2) nucleotide sequence hybridization that limits, and protein described in coding claim 1CDNA molecule or genomic DNA molecule;

B8) described nucleic acid molecules be with sequence table in the DNA molecular of arbitrary fragment reverse complemental in the DNA molecular shown in sequence 1.

4. plant disease-resistant agent, is characterized in that: described plant disease-resistant agent contains protein or claim 2 described in claim 1Or biomaterial described in 3.

Described in claim 1 described in protein or claim 2 or 3 biomaterial following 1)-3) and in any shouldWith:

1) regulating plant disease resistance;

2) prepare disease resistance of plant product;

3) cultivate disease resistance plant.

6. cultivate a method for disease resistant transgenic plants, comprise to importing protein described in claim 1 in recipient plantEncoding gene obtain the disease resistant transgenic plants of disease resistance higher than described recipient plant.

7. cultivate the method for genetically modified plants that disease resistance reduces, comprise and reduce in object plant protein described in claim 1The expression of encoding gene, obtain the genetically modified plants of disease resistance lower than described object plant.

8. antiviral agents according to claim 4, or application described in claim 5, or method described in claim 6 or 7, itsBe characterised in that: described in claim 4 or 5, described in plant, claim 6, described in recipient plant or claim 7, object plant isMonocotyledon or dicotyledon,

And/or,

Described in claim 1, the coded sequence of the encoding gene of protein is that the DNA of the 99-830 position of sequence 1 in sequence table dividesSon,

Reduce in object plant described in claim 1 expression of the encoding gene of protein and be by will with sequence in sequence table 1576-836 position nucleotides shown in the DNA molecular of DNA fragmentation reverse complemental import that described object plant realizes.

9. antiviral agents, described application or described method according to claim 8, is characterized in that: described monocotyledon isWheat.

10. antiviral agents, described application or described method according to claim 8 or claim 9, is characterized in that: described disease resistance isAnti-banded sclerotial blight, and/or,

Described banded sclerotial blight is caused by Rhizoctonia cerealis (Rhizoctoniacerealis).