CN103275202B

CN103275202B - Disease resistance-related protein RCR1 derived from wheat, related biomaterials thereof, and application for same

Info

Publication number: CN103275202B
Application number: CN201310203035.0A
Authority: CN
Inventors: 张增艳; 祝秀亮; 徐惠君; 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: 2013-05-28
Filing date: 2013-05-28
Publication date: 2015-03-25
Anticipated expiration: 2033-05-28
Also published as: CN103275202A

Abstract

The invention discloses a disease resistance-related protein RCR1 derived from wheat, the related biomaterials thereof, and an application for the same. RCR1 is the protein a) or the protein b) as follows: a) the protein composed of amino acid sequences shown in a sequence 2 in a sequence table; and b) the protein obtained by performing the substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequences shown in the sequence 2 in the sequence table, and related to plant disease resistance. Experiments proved that RCR1 and the biomaterials relates to RCR1 can be used for regulating plant disease resistance, and especially used for regulating the resistance of wheat to banded sclerotial blight.

Description

Derive from the disease resistance associated protein RCR1 of wheat and relevant biological material thereof and application

Technical field

The present invention relates to the disease resistance associated protein RCR1 and relevant biological material thereof and application that derive from wheat.

Background technology

Wheat (Triticuma aestivum) is one of mankind's Four main crop of depending on for existence, and the population of more than 1/3 take wheat as staple food in the world, and the yield and quality of wheat directly affects existence and the quality of life of the mankind.In recent years, along with the change of the factors such as cropping system, fertilizer and water condition and weather condition, the soil-borne disease such as banded sclerotial blight, root rot has become one of the important factor of restriction improving yield of wheat, stable yields.Wheat hypochnus in Anhui and Jiangsu along Huaihe River, the Huaibei, large portion, Hubei, large portion, Henan, Southern Shandong Province, Jiang-Han Area, Yangze river and Huai river, the Yellow River and Huai He River and the Southern North China Mai Qu such as the middle and south, Hebei lay particular stress on generation.Banded sclerotial blight generally can make wheat yield 10%-20%, the serious plot underproduction more than 50%.According to Jiangsu Province's plant protection unit statistics, the loss that l995 the whole province wheat causes because of banded sclerotial blight reaches 3.72 × l0 ⁶ton, wheat scab and Powdery Mildew cause loss sum then to exceed Jiangsu Province.According to national agricultural technology center, within 2004, there are 1.2 hundred million mu of harm suffering banded sclerotial blight in China, and within 2005, wheat hypochnus occurring area reaches 100,000,000 mu.Therefore, the wheat breed of selection and popularization resistance is most economical, the safe and effective approach that control wheat diseases is popular, for ensureing that China's improving yield of wheat, stable yields are extremely important.But owing to lacking desirable wheat hypochnus, Resistance To Root Rot Disease germ plasm resource, conventional breeding methods is slow to the progress in banded sclerotial blight, Resistance To Root Rot Disease wheat breed in seed selection.Molecular biology and engineeredly develop into plant resistance to environment stress breeding and open a new way.The separating clone of plant resistance proteins gene and functional analysis, to illustrate plant resistance to environment stress mechanism, effectively carry out molecular breeding research very necessary, become domestic and international plant science research focus.

Summary of the invention

The object of this invention is to provide a kind of derive from wheat disease resistance associated protein RCR1 and relevant biological material and application.

Protein provided by the invention is disease-resistant wheat associated protein, and name is called RCR1, derives from the wheat line CI12633 of anti-banded sclerotial blight, is following protein a) or b):

A) protein be made up of the aminoacid sequence shown in sequence in sequence table 2;

B) by the aminoacid sequence shown in sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and the protein relevant to disease resistance of plant.

Sequence 2 in sequence table is made up of 945 amino-acid residues.

In order to make the protein in (a) be convenient to purifying, the N-terminal of the protein that the aminoacid sequence shown in sequence 1 forms or C-terminal label as shown in table 1 can be connected in by sequence table.

The sequence of table 1 label

Label

Residue

Sequence

Poly-Arg	5-6(is generally 5)	RRRRR
			Poly-His	2-10(is generally 6)	HHHHHH
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

Protein in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the protein in above-mentioned (b) is by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.

The biomaterial relevant to RCR1 also belongs to protection scope of the present invention.

The biomaterial relevant to RCR1 provided by the present invention is following B1) to B7) in any one:

B1) nucleic acid molecule of coding RCR1;

B2) containing B1) expression cassette of described nucleic acid molecule;

B3) containing B1) recombinant vectors of described nucleic acid molecule or containing B1) recombinant vectors of described expression cassette;

B4) containing B1) recombinant microorganism of described nucleic acid molecule or containing B2) recombinant microorganism of described expression cassette or containing B4) recombinant microorganism of described recombinant vectors;

B5) containing B1) the transgenic plant cells system of described nucleic acid molecule or containing B1) the transgenic plant cells system of described expression cassette or containing B3) the transgenic plant cells system of described recombinant vectors;

B6) nucleic acid molecule that RCR1 expresses is reduced;

B7) containing B6) expression cassette of described nucleic acid molecule, recombinant vectors, recombinant microorganism or transgenic plant cells system.

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

In above-mentioned biomaterial, B1) described nucleic acid molecule specifically can be following 1) or 2) or 3) or 4) shown in gene:

1) its encoding sequence is the DNA molecular of the 270-3107 position Nucleotide of sequence 1 in sequence table;

2) nucleotide sequence is the DNA molecular of sequence 1 in sequence table;

3) under strict conditions with 1) or 2) DNA molecule hybridize that limits and the DNA molecular of the RCR1 that encodes;

4) with 1) or 2) DNA molecular that limits has the identity of more than 90% and the DNA molecular of the RCR1 that encodes.

Term used herein " identity " refers to the sequence similarity with native sequence nucleic acid." identity " can with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequence can represent with per-cent (%), and it can be used for evaluating the identity between correlated series.

In above-mentioned biomaterial, B6) described nucleic acid molecule specifically can be the DNA molecular with arbitrary fragment reverse complemental in the DNA molecular shown in the 270-3107 position Nucleotide of sequence in sequence table 1, as the DNA molecular of the DNA fragmentation reverse complemental shown in the 2716-3030 position Nucleotide of sequence in sequence table 1.

Wherein, the sequence 1 in sequence table is made up of 3451 Nucleotide, and its encoding sequence is the protein shown in sequence 2 of the 270-3107 position Nucleotide of sequence 1 in sequence table, polynucleotide.

Above-mentioned stringent condition can be at 0.1 × SSPE (or 0.1 × SSC), in the solution of 0.1%SDS, hybridizes and wash film under 65 ° of C conditions.

In above-mentioned biomaterial, the expression cassette (RCR1 expression casette) of the nucleic acid molecule containing coding RCR1 B2), refer to the DNA that can express RCR1 in host cell, this DNA not only can comprise the promotor starting RCR1 genetic transcription, also can comprise the terminator stopping RCR1 and transcribe.Further, described expression cassette also can comprise enhancer sequence.Promotor used in the present invention includes but not limited to: constitutive promoter, the promotor that tissue, organ and growth are special, and inducible promoter.The example of promotor includes but not limited to: the constitutive promoter 35S of cauliflower mosaic virus; From the wound-inducible promoter of tomato, leucine aminopeptidase (" LAP ", the people such as Chao (1999) Plant Physiol120:979-992); From tobacco chemical inducible promoter, pathogeny be correlated with 1 (PR1) (by Whitfield's ointment and BTH (diazosulfide-7-carbothioic acid S-methyl ester) induction); Tomato proteinase inhibitor II promotor (PIN2) or LAP promotor (all available jasmonic acid Yue ester induction); Heat-shock promoters (United States Patent (USP) 5,187,267); Tetracycline inducible promoter (United States Patent (USP) 5,057,422); Seed specific promoters, as Millet Seed specificity promoter pF128(CN101063139B (Chinese patent 2,007 10099169.7)), the special promotor of seed storage protein matter (such as, the promotor (people (1985) EMBO such as Beachy J.4:3047-3053) of phaseollin, napin, oleosin and soybean beta conglycin).They can be used alone or are combined with other plant promoter.All reference cited herein all quote in full.Suitable transcription terminator includes but not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, such as: the people (I such as Odell ⁹⁸⁵) Nature 313:810; 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; The people Genes Dev. such as Sanfacon, 5:141; The people such as Mogen (1990) Plant Cell, 2:1261; The people such as Munroe (1990) Gene, 91:151; The people such as Ballad (1989) Nucleic Acids Res.17:7891; The people such as Joshi (1987) Nucleic Acid Res., 15:9627).

In an embodiment of the present invention, the promotor starting described RCR1 genetic transcription in described RCR1 expression casette is corn Ubiquitin promotor, and the terminator stopping described RCR1 genetic transcription is rouge alkali synthetase gene terminator TNos.

Available existing plant expression vector construction contains the recombinant expression vector of described RCR1 expression casette.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.As pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb(CAMBIA company) etc.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as rouge alkali synthetase gene Nos), plant gene (as soybean storage protein genes) 3 ' hold the non-translational region of transcribing all to have similar functions.When using gene constructed plant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce the enzyme of colour-change or the gene (gus gene of luminophor, luciferase genes etc.), antibiotic marker gene is (as given the nptII gene to kantlex and associated antibiotic resistance, give the bar gene to herbicide phosphinothricin resistance, give the hph gene to microbiotic hygromycin resistance, with the dhfr gene given methatrexate resistance, give EPSPS gene to glyphosate) or chemical resistance reagent marker gene etc. (as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.

In above-mentioned biomaterial, B4) and B7) described in recombinant microorganism specifically can be yeast, bacterium, algae and fungi.B5) transgenic cell line and B7) does not comprise the reproductive material of plant.

Experiment proves that RCR1 or the above-mentioned biomaterial relevant to RCR1 can be used for regulating plant disease resistance.

Wherein, described disease resistance specifically can be anti-banded sclerotial blight.Described banded sclerotial blight can be caused by Rhizoctonia cerealis (Rhizoctoniacerealis), and described Rhizoctonia cerealis (Rhizoctonia cerealis) specifically can be Rhizoctonia cerealis (Rhizoctonia cerealis) R0301.Described plant can be monocotyledons or dicotyledons, as wheat.Both can be monocotyledons also can be dicotyledons.Described monocotyledons specifically can be wheat (as wheat breed raises wheat 16 or section's agriculture 199).

The invention provides two kinds of concrete methods utilizing the above-mentioned biomaterial regulating plant disease resistance relevant to RCR1, a kind of is the method for cultivating disease resistant transgenic plants, and another kind is the method for cultivating the transgenic plant that sharp eyespot resistance reduces.

The method of cultivation disease resistant transgenic plants provided by the present invention, comprises the encoding gene (RCR1 gene) importing RCR1 in recipient plant and obtains the step of disease resistance higher than the disease resistant transgenic plants of described recipient plant.

In an embodiment of the present invention, the encoding gene of described RCR1 is imported in object plant by the RCR1 gene recombinant vectors containing RCR1 expression casette.

In aforesaid method, wherein said RCR1 gene can first be modified as follows, then imports in acceptor spermatophyte, to reach better expression effect:

1) carry out according to actual needs modifying and optimizing, to make gene efficient expression; Such as, the codon can had a preference for according to recipient plant, changes its codon to meet plant-preference while the aminoacid sequence keeping RCR1 gene of the present invention; In optimizing process, keep certain GC content in the encoding sequence after preferably making optimization, to realize the high level expression of quiding gene in plant best, wherein GC content can be 35%, more than 45%, more than 50% or more than about 60%;

2) gene order of contiguous initial methionine is modified, to make translation effectively initial; Such as, effective sequence known in plant is utilized to modify;

3) be connected with the promotor of various expression of plants, be beneficial to its expression in plant; Described promotor can comprise composing type, induction type, sequential adjustment, Growth adjustment, Chemical Regulation, tissue preferably and tissue-specific promoter; The selection of promotor will change along with expression time and space requirement, and depend on target species; The such as specific expressing promoter of tissue or organ, acceptor in what period of growing is determined as required; Although it is operational for demonstrating the many promotors deriving from dicotyledons in monocotyledons, vice versa, but ideally, select dicot promoters for the expression in dicotyledons, monocotyledonous promotor is used for the expression in monocotyledons;

4) with the Transcription Termination sub-connection be applicable to, the expression efficiency of gene of the present invention can also be improved; Such as derive from the tml of CaMV, derive from the E9 of rbcS; Any known available terminator worked in plant can be connected with gene of the present invention;

5) enhancer sequence is introduced, as intron sequences (such as deriving from Adhl and bronzel) and viral leader sequence (such as deriving from TMV, MCMV and AMV).

In one embodiment of the invention, described RCR1 gene recombinant vectors be specially replace pAHC25 with the DNA molecular of the 270-3107 position Nucleotide of sequence in sequence table 1 SmaI and SacI restriction enzyme site between the recombinant expression vector that obtains of fragment.

Described RCR1 gene recombinant vectors is by using Ti-plasmids; plant virus carrying agent; directly delivered DNA; microinjection, the standard biologic technological methods such as electroporation import vegetable cell (Weissbach, 1998; Method forPlant Molecular Biology VIII; Academy Press, New York, pp.411-463; Geisersonand Corey, 1998, Plant Molecular Biology (2nd Edition).

In aforesaid method, described plant can be monocotyledons or dicotyledons, as wheat.Both can be monocotyledons also can be dicotyledons.Described monocotyledons specifically can be wheat (as wheat breed raises wheat 16, section's agriculture 199).Describedly disease-resistantly specifically can be anti-banded sclerotial blight.Described banded sclerotial blight can be caused by Rhizoctonia cerealis (Rhizoctoniacerealis), and described Rhizoctonia cerealis (Rhizoctonia cerealis) is specially Rhizoctonia cerealis (Rhizoctonia cerealis) R0301.

In aforesaid method, described transgenic plant are interpreted as the first-generation transgenic plant not only comprising and obtained by described gene transformation object plant, also comprise its filial generation.For transgenic plant, this gene can be bred in these species, also with traditional breeding method, this transgenosis can be entered other kind of same species, particularly including in commercial variety.Described transgenic plant comprise seed, callus, whole plant and cell.

The method of the transgenic plant that cultivation disease resistance provided by the present invention reduces, comprising is the expression reducing RCR1 encoding gene (RCR1 gene) in object plant, obtains the transgenic plant of disease resistance lower than described object plant.

Described object plant both can be monocotyledons also can be dicotyledons.Described monocotyledons specifically can be wheat (as wheat breed raises wheat 16, section's agriculture 199).Describedly disease-resistantly specifically can be anti-banded sclerotial blight.Described banded sclerotial blight can be caused by Rhizoctonia cerealis (Rhizoctonia cerealis), and described Rhizoctonia cerealis (Rhizoctonia cerealis) is specially Rhizoctonia cerealis (Rhizoctonia cerealis) R0301.

In one embodiment of the invention, the expression reducing RCR1 gene in object plant realizes by the DNA molecular with the DNA fragmentation reverse complemental shown in the 2716-3030 position Nucleotide of sequence in sequence table 1 is imported described object plant.

In one embodiment of the invention, with the DNA molecular of the DNA fragmentation reverse complemental shown in the 2716-3030 position Nucleotide of sequence in sequence table 1 by object plant described in the γ vector introduction of BMSV virus.

Described object plant specifically can be wheat.

The primer pair of the total length of the nucleic acid molecule of amplification coding RCR1 or its arbitrary fragment also belongs to protection scope of the present invention.

By the genetically modified Molecular Detection of RCR1 channel genes wheat and disease resistance the results show, RCR1 gene overexpression turn that RCR1 DNA triticum raises wheat 16, the resistance of section's agriculture 199 pairs of wheat hypochnuss significantly improves; Described in anti-disease wheat CI12633, RCR1 genetic expression is suppressed, and this plant is reduced sharp eyespot resistance, and illustrate that RCR1 gene is the disease-resistant gene needed for the anti-banded sclerotial blight reaction of wheat, forward participates in the reaction of anti-banded sclerotial blight.RCR1 gene is a kind of disease-resistant wheat protein gene relevant to sharp eyespot resistance, has substantial worth to plant breeding.

Accompanying drawing explanation

Fig. 1 is the line withered bacterium abduction delivering analysis of RCR1 gene.

Fig. 2 is that Fig. 3 part turns the T1 of pAHC25-RCR1 for plant PCR detection electrophoretogram.

In Fig. 2, arrow is object fragment; M is Marker II, P be pAHC25-RCR1, Y16 is the genomic dna that wild-type wheat raises wheat 16, H ₂o is water, and K199 is the genomic dna of wild-type wheat section agriculture 199, and 1-6 is the genomic dna turning pAHC25-RCR1 section agriculture 199, and 7-12 turns the genomic dna that pAHC25-RCR1 raises wheat 16.

Fig. 3 A is relative expression's component analysis of RCR1 gene in the transfer-gen plant of embodiment 2.

Fig. 3 B is relative expression's component analysis of line withered bacterium actin gene in the transfer-gen plant of embodiment 2.

Wheat 16 positive plant is raised for Y1-5, Y2-2, Y12-7, Y13-8, Y24-2, Y37-4 for turning pAHC25-RCR1 in Fig. 3 A-B, Yangmai16 is that wild-type wheat raises wheat 16, K7-4, K9-2, K15-2, K16-2, K19-3, K56-2, K57-3 are for turning pAHC25-RCR1 section agriculture 199 positive plant, and Kenong199 is wild-type wheat section agriculture 199.

Fig. 4 is relative expression's component analysis of RCR1 gene in the transfection BSMV virus plant in embodiment 3.

In Fig. 4, first three histogram is from left to right BSMV-γ: antiRCR1-CI12633 plant, and the 4th histogram is BMSV-γ-CI12633 plant, and the 5th post is wild-type wheat CI12633 plant.

Fig. 5 is relative expression's component analysis of line withered bacterium actin gene in the transfection BSMV virus plant in embodiment 3.

In Fig. 5, first three histogram is from left to right BSMV-γ: antiRCR1-CI12633 plant, and the 4th histogram is BMSV-γ-CI12633 plant, and the 5th post is wild-type wheat CI12633 plant.

Fig. 6 is the banded sclerotial blight symptom of the transfection BSMV virus plant in embodiment 3.

In Fig. 6, first three plant is from left to right BSMV-γ: antiRCR1-CI12633 plant, and the 4th plant is BSMV-γ-CI12633 plant, and the 5th plant is wild-type wheat CI12633 plant.

Embodiment

Following embodiment is convenient to understand the present invention better, but the present invention is not limited in these embodiments.Experimental technique in following embodiment, if no special instructions, is ordinary method.

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

Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.

The anti-banded sclerotial blight of Wheat Germplasm Resources CI12633, wheat breed temperature wheat 6 height sense banded sclerotial blight, wheat breed section agriculture 199 is felt banded sclerotial blight, is raised in wheat 16 and feel banded sclerotial blight.

The red wheat in wheat line mountain, warm wheat 6 and Ke Nong 199, purchased from Chinese Academy of Agricultural Sciences's germplasm resource bank, anti-disease wheat material---wheat CI12633 is purchased from Jiangsu Academy of Agricultural Sciences's germplasm resource bank.Wheat breed raises wheat 16: purchased from Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov..

Wheat hypochnus pathogenic bacterium-Rhizoctonia cerealis (Rhizoctonia cerealis) R0301(Jiangsu Province Agriculture Science Institute) (cold Su Feng, Zhang Aixiang, Li Wei, Chen Huaigu. Wheat in Jiangsu Province new variety (being) are to the Analysis of Resistance of banded sclerotial blight. Jiangsu's agriculture journal, 2010,26(6): 1176-1180).

Monocotyledon expression vector pAHC25(Christensen and Quail, 1996; Ubiquitinpromoter-based vectors for high-level expression of selectable and/orscreenable marker genes in monocotyledonous plants.Transgenic Research, 5,213 – 218), the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment.PAHC25 is transformed by pUC8 and forms, containing 2 expression cassettes, 1st expression cassette has corn Ubiquitin promotor, Exon, Intron, GUS, Nos terminator, GUS two ends have SmaI and SacI restriction enzyme site, and the 2nd expression cassette has corn Ubiquitin promotor, Exon, Intron, Bar, Nos terminator.

BSMV-γ (the γ carrier of BMSV virus) (Burch-Smith T M, Anderson J C, Martin G B, Dinesh-Kumar S P.Applications and advantages of virus-induced gene silencingfor gene function studies in plants.The Plant Journal, 2004,39:734-746) public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment.

The clone of embodiment 1, wheat resistance protein RCR1 and encoding gene thereof

The present inventor resists banded sclerotial blight wheat CI12633 by RNA-Sequencing and feels the gene differential expression data analysis that banded sclerotial blight wheat temperature wheat 6 replys sheath blight fungus, in conjunction with expression association analysis, from CI12633, clone and isolate the anti-banded sclerotial blight important gene of wheat-RCR1 gene.Concrete cloning process is as follows:

Extract the total serum IgE of wheat CI12633 leaf sheath, according to the program of Invitrogen company first chain cDNA synthetic agent box, the first chain cDNA is synthesized in the RNA sample reverse transcription of extraction, as the template of gene clone, with U:5 '-CCCAGTCAAAGTCCGTCAGT-3 ' and L:5 '-GTGGCTTACAGGCACGCAG-3 ' for primer, carry out pcr amplification.Amplification program is: first 94 DEG C of denaturations 3 minutes; Then 94 DEG C 45 seconds, 60 DEG C 45 seconds, 72 DEG C 3 minutes, totally 35 circulations; 72 DEG C extend 10 minutes.The PCR primer obtained to be connected on pMD18-T carrier and to check order.Sequencing result shows, the nucleotide sequence of this pcr amplification product is as shown in the sequence 1 (1-3235 amino acids) of sequence table, and its encoding sequence is the 270-3107 position Nucleotide of sequence 1 in sequence table; Protein RCR1 shown in encoding sequence 2.

In order to obtain the cDNA sequence of RCR1 full length gene, and design 3 ' RACE primer (3R1:5 '-CTTGACCTAAGGGAGACGAA-3 '; 3R2:5 '-GCAGGACAGCGCAAACGGA-3 '), take turns pcr amplification by 2, increase 3 ' sequence from anti-disease wheat CI12633cDNA, namely shown in sequence 1 from 5 ' end 2241-3451 position Nucleotide.

2, RCR1 gene is by the expression analysis of sheath blight fungus induction

Between the leaf sheath being inoculated in wheat CI12633 seedling in tillering phase with wheat hypochnus pathogenic bacterium-Rhizoctonia cerealis (Rhizoctonia cerealis) R0301 mycelia toothpick, wheat and stem; Not inoculate wheat leaf sheath (0h) in contrast, get wheat leaf sheath and stem respectively at inoculation after 4,7,21 days, be stored in-80 DEG C of Ultralow Temperature Freezers after liquid nitrogen flash freezer for subsequent use.

Extract the total serum IgE (each sample about 5 μ g total serum IgE) of each treatment time blade respectively, according to the program of Invitrogen company first chain cDNA synthetic agent box, reverse transcription becomes cDNA.Utilize the actin gene of constructive expression as internal reference, by sample cDNA normalization.Then real-time quantitative RT-PCR analysis is carried out, with 2 with the special primer of RCR1 gene ^{-△ △ CT}method (Livak KJ, Schmittgen TD.2001.Analysis of relative geneexpression data using real-time quantitative PCR and the2 ^{-△ △ CT}method.Methods.25:402-408) analyze the expression of RCR1 gene under rhizoctonia cerealis process, often organize sample and repeat 3 times.

The primer pair of reference gene actin:

F：5’-CACTGGAATGGTCAAGGCTG-3’；R：5’-CTCCATGTCATCCCAGTTG-3’。

The special primer pair of RCR1 gene:

RCR-U：5’-GCGACCTGCCTTTGTTGTT-3’，RCR-L：5’-TGGTTATGTCATCTGCTGTGC-3’。

The results are shown in Figure 1.The transcriptional expression of RCR1 gene in disease-resistant material C I12633 by the induction of sheath blight fungus, and reaches peak expression during 7d after inoculating banded sclerotial blight, illustrates that RCR1 may take part in the defensive raction of host to rhizoctonia cerealis.

The acquisition of embodiment 2, anti-banded sclerotial blight transgenic wheat and Disease Resistance Identification

One, the structure of recombinant expression vector

Utilize Fast PCR Clone Kit, by ORF sequence construct complete for RCR1 gene on monocotyledons efficient expression vector pAHC25, concrete steps are as follows:

1, the preparation of linearization plasmid: cut pAHC25 vector plasmid with SmaI and Sac I enzyme, 1% agarose gel electrophoresis, sepharose DNA purifying reclaims test kit and reclaims linearizing pAHC25 carrier framework.

2, the pcr amplification of target gene RCR1: according to the ORF sequences Design pair of primers RCR1-O-F:5 ' of RCR1 gene- aACTCGGTATCTAGAaTGGAGTTGGCAGTAGGTGC-3 '

And RCR1-O-R:5 '- cGATCGGGGAAATTCtCAGCTGCTGATAACATGAT-3 ', primer 5 ' holds the sequence (shown in underscore) introducing 15bp and linearized vector pAHC25 two ends complementary, high-fidelity enzyme pcr amplification amino acid encoding region full length sequence, 1% agarose gel electrophoresis amplified production, reclaims amplified production.

3, target gene RCR1 and linearized vector are recombinated: be formulated as follows reaction system (20 μ l):

Mixture is hatched 30 minutes at 22 DEG C, hatches 5 minutes on ice afterwards.

4, product will be connected heat-shock transformed in e.colistraindh5α competent cell, by bacterium colony PCR screening positive clone, to go forward side by side a step sequence verification, the recombinant expression vector called after pAHC25-RCR1 that the fragment between SmaI and the SacI restriction enzyme site being shown by sequencing result to replace pAHC25 with the DNA molecular of the 270-3107 position Nucleotide of sequence in sequence table 1 obtains.

The structure of recombinant plasmid pAHC25-RCR1: skeleton carrier is pAHC25, inserts the RCR1 gene shown in the 270th to the 3107th Nucleotide of sequence 1 in sequence table between SmaI and SacI restriction enzyme site; RCR1 gene controls by corn Ubiquitin promotor; Plasmid also has 1 Bar expression casette controlled by Ubiquitin promotor, can be in follow-up work and utilizes weedicide bialaphos (Bialaphos) to screen transformation tissue culture plant to provide resistance marker.

Two, the acquisition of transgenic plant

1, raise wheat 16 by 2000 pieces, recombinant plasmid pAHC25-RCR1, as the acceptor of biolistic bombardment, is bombarded callus with particle gun by the Immature embryo calli of 1600 pieces of section's agricultures 199.

2, by by the aftertreatment 16h on osmotic pressure substratum of the callus after biolistic bombardment.

3, then callus is transferred to SD2 substratum and (in the inorganic salts ingredients of MS substratum, add VB ₁1mg/L, asparagus fern door acid amides 150mg/L, 2,4-D2mg/L) on, renewal cultivation 2 weeks (26 DEG C, light culture).

4, the callus after renewal cultivation is transferred in differentiation screening culture medium (1/2MS substratum+naphthylacetic acid 1mg/L+ kinetin 1mg/L+ bialaphos 2-5mg/L), 24-26 DEG C of illumination cultivation 14d; To transfer in growth screening culture medium (1/2MS substratum+bialaphos 2-3mg/L) after Calli Differentiation seedling, 24-26 DEG C of illumination cultivation; Obtain 235 strain regeneration plants.

5, regeneration plant is transferred on strong seedling culture base (1/2MS substratum+0.5mg/L naphthylacetic acid), by height of seedling 7-8cm and the transformation seedlings of well developed root system is transplanted to flowerpot, be transplanted to greenhouse after 3 weeks, have 31 strains to turn pAHC25-RCR1 and raise wheat 16,37 Zhu Zhuan pAHC25-RCR1 section agriculture 199 plant survive.

6, Molecular Identification

In 4 leaf phases, the regeneration plant that every strain survives is got 1 blade and is extracted genomic dna, using genomic dna as template, utilize one section of sequence of RCR1 gene specific as upstream primer (RCR1-F:5 '-GCACCTCAGAGTCAATAGCAC-3 '), the sequence-specific one section of sequence of carrier Tnos carries out PCR as downstream primer (NOS-R:5 '-TGTATAATTGCGGGACTCTAATC-3 '), with recombinant expression plasmid pAHC25-RCR1 for positive control, raise wheat 16, the genomic dna of section's agriculture 199 is negative control, expection amplified production fragment is about 291bp.

PCR amplification system (20 μ l): 2 × GC buffer I 10.0 μ l, 2.5mM dNTP mix2.0 μ l, RCR1-F(10 μM) 0.5 μ l, NOS-R(10 μM) 0.5 μ l, rTaq(5U/ μ l) 0.2 μ l, template DNA 100ng, mend ddH ₂o to 20 μ l.

Pcr amplification program: first 94 DEG C of 3min; Then 5 circulations as follows: 94 DEG C of 45s, 54 DEG C of 45s, 72 DEG C of 25s; Then 30 circulations as follows: 94 DEG C of 45s, 52 DEG C of 45s, 72 DEG C of 25s; 72 DEG C of 10min again; 16 DEG C of preservations.

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

37 Zhu Zhuan pAHC25-RCR1 section agriculture 199 plant (T ₀generation) in, PCR positive plant (being transfer-gen plant) 8 strain; 31 strains turn pAHC25-RCR1 and raise wheat 16 plant (T ₀generation) in, PCR positive plant (being transfer-gen plant) 5 strain.

7, T ₁for individual plant and Molecular Identification thereof

199 strain T are obtained by after 13 strain positive plant selfings of step 6 ₁for individual plant, agriculture 199 plant 99 strain of its transfer pAHC25-RCR1 section, turns pAHC25-RCR1 and raises the strain of wheat 16 plant 100.

By 199 strain T ₁carry out Molecular Identification for individual plant, method, with step 6, detects positive transgenic plant 47 strain altogether, and wherein, turn agriculture 199 plant 20 strain of pAHC25-RCR1 section, turn pAHC25-RCR1 and raise the strain of wheat 16 plant 27, part individual plant PCR detected result is shown in Fig. 2.With recombinant expression plasmid pAHC25-RCR1 for positive control, raise wheat 16, the genomic dna of section's agriculture 199 is negative control, expection amplified production fragment is about 291bp.

Three, the acquisition of empty carrier plant is turned

Replace recombinant plasmid pAHC25-RCR1 with carrier pAHC25, other same step 2, obtain turning empty carrier section agriculture 199 plant and turning empty carrier raising wheat 16 plant, as the contrast of transfer-gen plant.

Four, the transcriptional level of the qualification of transgenic plant sharp eyespot resistance and RCR1 gene

1, wheat hypochnus mycelia is cultivated

Toothpick section is erect and fills up small beaker, preparation MS liquid nutrient medium, pour in the small beaker of dress toothpick section, after sterilizing by Rhizoctonia cerealis (Rhizoctonia cerealis) the R0301 inoculated by hypha block of preserving in beaker, 25 DEG C of constant temperature culture are covered with toothpick thick and fast to mycelia.

Prepare wheat vermiculite substratum (ripe wheat: sand=1:1, adds suitable quantity of water, mixing), after sterilizing, inoculation Rhizoctonia cerealis (Rhizoctonia cerealis) R0301,25 DEG C of constant temperature culture are covered with wheat thick and fast to mycelia.

2, sharp eyespot resistance qualification

For the identification of experiment material be 47 strain T of step 2 ₁turn empty carrier raise wheat 16 plant and 40 strain WT lines (raising wheat 16 and each 20 strains of Ke Nong 199) for positive transgenic plant (turn agriculture 199 plant 20 strain of pAHC25-RCR1 section, turn pAHC25-RCR1 and raise the strain of wheat 16 plant 27), 10 Zhu Zhuan empty carrier section agriculture 199 plant of step 3,10 strains.

At wheat during jointing stage, the toothpick two being covered with Singularity SCAR mark of wheat rhizoctonia R0301 is embedded between wheat base portion 1-2 leaf sheath, and what keep leaf sheath during inoculation embraces bulbous state as far as possible naturally, water spray moisturizing 5-7 days after inoculation; The investigation banded sclerotial blight state of an illness when wheat dough stage, results.

Banded sclerotial blight severity Scaling standard, according to the methods such as Li Sishen carry out (.1997 such as Li Sishen, Li Anfei, Li Xianbin, wheat germplasm identifies preliminary study to sharp eyespot resistance. Crop Germplasm Resources. (4): 31-33):

0 grade (IT 0): complete stool is anosis;

1 grade (IT 1): 1st, 2 leaf sheath morbidities, but stem stalk is anosis;

2 grades (IT 2): 1st, 2 leaf sheath morbidities, but scab is less than 1/3 around stem stalk;

3 grades (IT 3): 3rd, 4 leaf sheath morbidities, or scab is around stem stalk 1/3-2/3;

4 grades (IT 4): 5th, 6 leaf sheath morbidities, or scab is around stem stalk 2/3-1 week;

5 grades (IT 5): occur withered, dead ears or whole strain withered.

Turn pAHC25-RCR1 plant to significantly improve sharp eyespot resistance, the results are shown in Table 2.It is 1.11 that 27 strains turn the average sick level of banded sclerotial blight that pAHC25-RCR1 raises wheat 16 plant, and disease index is the average sick level of banded sclerotial blight of 22.2%, 20 Zhu Zhuan pAHC25-RCR1 section agriculture 199 plant is 1.2, and disease index is 24%; And wild-type raises wheat 16, the average sick level of banded sclerotial blight of section's agriculture 199 plant is respectively 2.7,2.9, disease index is respectively 54%, 58%; Turn empty carrier section agriculture 199 plant and turn the average sick level of banded sclerotial blight that empty carrier raises wheat 16 plant and be respectively 2.6,2.7, disease index is respectively 52%, 54%, illustrates that turning RCR1 gene can strengthen plant to sharp eyespot resistance.In table 2,27 plant that strain is numbered Y1, Y2, Y4, Y12, Y13, Y24, Y27 and Y37 raise wheat 16 plant for turning pAHC25-RCR1; Strain is numbered 20 plant of K7, K9, K15, K16, K19, K39, K56, K57 and K58 for turning pAHC25-RCR1 section agriculture 199 plant; Strain be numbered raise wheat 16 20 plant for raising wheat 16 WT lines (recipient plant), 20 plant that strain is numbered section's agriculture 199 are section's agriculture 199 WT lines (recipient plant), strain is numbered 10 plant turning empty carrier-Yang Mai 16 and raises wheat 16 plant for turning empty carrier, strain be numbered turn empty carrier-Ke Nong 199 10 plant for turning empty carrier section agriculture 199 plant.

Table 2, transgenosis and recipient plant sharp eyespot resistance investigation result

3, the transcriptional expression analysis of RCR1 gene and line withered bacterium actin gene in transgenic plant

Transfer-gen plant and acceptor section agriculture 199 and the withered bacterium of Yang Mai 16 plant inoculation line, after 12 days, extract its inoculation line withered bacterium position leaf sheath RNA, reverse transcription synthesis cDNA, and utilize primer RCR1T-F/RCR1T-R, fluorescence quantitative RT-RCR analyzes the transcriptional level of RCR1 gene; Utilize the analysis of primer RCactin-F/RCactin-R fluorescence quantitative RT-RCR to invade the relative expression quantity of line withered bacterium actin gene in leaf sheath, reflect with this biomass invading the withered bacterium of line in wheat plant.As shown in Figure 3 A and Figure 3 B, in transfer-gen plant, the transcriptional level of RCR1 gene is higher than non-transgene receptor section agriculture 199 and Yang Mai 16 for result; In transfer-gen plant, the relative expression quantity of line withered bacterium actin gene is significantly lower than non-transgene receptor section agriculture 199 and Yang Mai 16, and the quantity namely invading the withered bacterium of line in transfer-gen plant is less than the withered bacterium quantity of line invaded in non-transgene receptor section agriculture 199 and Yang Mai 16.This illustrates, the high expression level of RCR1 gene significantly improves the resistance of wheat plant to the withered bacterium of line.In Fig. 3 A and Fig. 3 B, Yangmai16 raises wheat 16 for non-transgene receptor, and Kenong199 is non-transgene receptor section agriculture 199, and the numbering of other transfer-gen plant is with table 2.

RCR1 Gene Transcript Analysis primer pair:

RCR1T-F：5’-ACTAAGGAGACAATGAAAGCAC-3’

RCR1T-R：5’-CCCTCGTCGTTGAGCACC-3’

Line withered bacterium actin Gene Transcript Analysis primer pair:

RCactin-F:5’-gcatccacgagaccacttac-3’

RCactin-R:5’-gcgtcccgctgctcaagat-3’

The transgenic wheat that embodiment 3, cultivation banded sclerotial blight reduce

One, the RCR1 gene in the reticent wheat CI12633 of virus-mediated gene silent technology is adopted

1, by two ends of the DNA fragmentation shown in the 2716-3030 position Nucleotide (in sequence table sequence 3) of sequence in sequence table 1 respectively with NheI recognition sequence.After NheI enzyme is cut, DNA fragmentation (315bp) shown in the 2716-3030 position Nucleotide of sequence in sequence table 1 is inserted on the BSMV-γ after the linearizing of NheI enzyme (the γ carrier of BMSV virus) with reverse interpolation, to make with the DNA molecular (antiRCR1) of the DNA fragmentation reverse complemental shown in the 2716-3030 position Nucleotide of sequence in sequence table 1, by the T7 promoters driven of γ carrier, to obtain recombinant vectors BSMV-γ: antiRCR1.

2, in two leaf one heart stages, with the anti-wheat hypochnus material of recombinant vectors BSMV-γ: antiRCR1 transfection---second leaf of wheat CI12633, concrete steps are as follows:

(1) rubbing manipulation inoculation BSMV-γ: antiRCR1(or BSMV-γ is adopted) on second leaf of the disease-resistant material C I12633 of second leaf full extension.During inoculation, with not inoculating the base portion that hand fixes wheat seedling, thumb and the forefinger of inoculation hand push down blade, along the direction of vane extension, rub to blade tip continuously from bottom leaf, one, two two panels leaf Simultaneous vaccination.

(2) after having inoculated, spray DEPC water to wheat seedling, preservative film covers moisturizing 24h, removes preservative film afterwards, sprays a DEPC water every 1-2h.

(3) the 4th blade is got in inoculation on the 14th day, extracts RNA, employing Q-RT-PCR detection gene silencing situation (YZ-F:5 '-GCACCTCAGAGTCAATAGCAC-3 ', YZ-R:5 '-CGCAGCAGTGTCCTGTTTTT-3 ').

Result is as shown in Figure 4: import RCR1 gene in the CI12633 plant of BSMV-γ: antiRCR1 and be silenced, obtain CI12633(called after BSMV-γ: antiRCR1-CI12633 of RCR1 gene silencing); And import the CI12633(called after BMSV-γ-CI12633 of BSMV-γ, in contrast) there is no noticeable change with the expression amount of RCR1 gene in wild-type wheat CI12633.

Two, the Disease Resistance Identification of plant is silenced

The wheat transfection of step one is after BSMV20 days, toothpick inoculation is adopted to inoculate Rhizoctonia cerealis (Rhizoctonia cerealis) R0301 to it, the toothpick covering with Rhizoctonia cerealis (Rhizoctonia cerealis) R0301 mycelia is embedded between base portion 1-2 leaf sheath, with moistening absorbent cotton, it is surrounded gently, moisturizing 3 days.After wheat jointing, adopt bacterium liquid injection inoculation Rhizoctonia cerealis (Rhizoctonia cerealis) R0301: cultivate Rhizoctonia cerealis (Rhizoctonia cerealis) about R030115 days with PDA liquid nutrient medium; Then vibrate 30s, draws 10 μ l cultured bacterium liquid and be injected between leaf sheath third from the bottom and stem stalk, spraying and moisturizing 10 days.

Inoculation Rhizoctonia cerealis (Rhizoctonia cerealis), after R030120 days, is extracted the RNA of site of pathological change stem stalk, is carried out quantitative and semi-quantitative RT-PCR analysis, detects the relative expression quantity of line withered bacterium Actin gene.Q-RTPCR primer used is: RCactin-F:5 '-gcatccacgagaccacttac-3 '; RCactin-R:5 '-gcgtcccgctgctcaagat-3 '.Result shows, in BSMV-γ: antiRCR1-CI12633 plant, the relative expression quantity of Rhizoctonia cerealis (Rhizoctonia cerealis) R0301 Actin gene is significantly higher than the expression amount (Fig. 5) in BMSV-γ-CI12633, namely in BSMV-γ: the antiRCR1-CI12633 plant after RCR1 silenced gene expression, the accumulation volume of Rhizoctonia cerealis (Rhizoctonia cerealis) R0301 is significantly higher than the accumulation volume of Rhizoctonia cerealis (Rhizoctoniacerealis) R0301 in contrast (BMSV-γ-CI12633) plant of RCR1 gene normal expression and wild-type wheat CI12633 plant.

Inoculation Rhizoctonia cerealis (Rhizoctonia cerealis), after R030145 days, carries out Disease Resistance Identification.Result as shown in Figure 6, BSMV-γ: antiRCR1-CI12633 plant base of leaf position third from the bottom banded sclerotial blight scab after RCR1 silenced gene expression is obviously greater than the scab of contrast (BMSV-γ-CI12633) plant and wild-type wheat CI12633 plant, the above results illustrates that RCR1 gene is the gene needed for the anti-banded sclerotial blight reaction of CI12633, is also the anti-banded sclerotial blight important gene of wheat.

Claims

1. protein, the protein be made up of the aminoacid sequence shown in sequence in sequence table 2.

2. the biomaterial relevant to protein described in claim 1 is following B1) to B7) in any one:

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

B2) containing B1) expression cassette of described nucleic acid molecule;

B3) containing B1) recombinant vectors of described nucleic acid molecule or containing B2) recombinant vectors of described expression cassette;

B4) containing B1) recombinant microorganism of described nucleic acid molecule or containing B2) recombinant microorganism of described expression cassette or containing B3) recombinant microorganism of described recombinant vectors;

B5) containing B1) the transgenic plant cells system of described nucleic acid molecule or containing B2) the transgenic plant cells system of described expression cassette or containing B3) the transgenic plant cells system B6 of described recombinant vectors) and with the DNA molecular of arbitrary fragment reverse complemental in the DNA molecular shown in the Nucleotide of 270-3107 position of sequence 1 in sequence table;

B7) containing B6) expression cassette of described DNA molecular, recombinant vectors, recombinant microorganism or transgenic plant cells system;

Described transgenic plant cells system does not comprise the reproductive material of plant.

3. relevant biological material according to claim 2, is characterized in that: B1) described nucleic acid molecule is following 1) or 2) shown in gene:

2) nucleotide sequence is the DNA molecular of sequence 1 in sequence table.

4. the application of relevant biological material in regulating plant disease resistance described in protein described in claim 1 or Claims 2 or 3; Described disease resistance is anti-banded sclerotial blight.

5. cultivate a method for disease resistant transgenic plants, comprise and in recipient plant, import the encoding gene of protein described in claim 1 obtain the step of disease resistance higher than the disease resistant transgenic plants of described recipient plant; Described disease resistance is anti-banded sclerotial blight.

6. method according to claim 5, is characterized in that: described recipient plant is wheat, and the encoding sequence of the encoding gene of protein described in claim 1 is the DNA molecular of the 270-3107 Nucleotide of sequence 1 in sequence table.

7. cultivate the method for the transgenic plant that disease resistance reduces, comprise the expression of the encoding gene being protein described in claim 1 in reduction object plant, obtain the transgenic plant of disease resistance lower than described object plant; Described object plant is wheat, and the expression reducing the encoding gene of protein described in claim 1 in object plant realizes by the DNA molecular with the DNA fragmentation reverse complemental shown in the 2716-3030 position Nucleotide of sequence in sequence table 1 is imported described object plant; Described disease resistance is anti-banded sclerotial blight.