CN111763249B

CN111763249B - Plant powdery mildew resistance-related protein Pm5e and its encoding gene and application

Info

Publication number: CN111763249B
Application number: CN201910174567.3A
Authority: CN
Inventors: 刘志勇; 郭广昊; 谢菁忠; 王勇; 吴秋红; 陆平; 陈永兴; 董玲丽
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2019-03-08
Filing date: 2019-03-08
Publication date: 2021-11-16
Anticipated expiration: 2039-03-08
Also published as: CN111763249A

Abstract

The invention discloses a plant powdery mildew resistance related protein Pm5e, and a coding gene and application thereof. The protein provided by the invention is obtained from wheat, is named as Pm5e protein and is a protein shown in a sequence 1 in a sequence table. The nucleic acid molecule encoding the Pm5e protein also belongs to the protection scope of the invention. The invention also protects the application of the Pm5e protein, and aims to improve the disease resistance of plants to powdery mildew. The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the nucleic acid molecule is introduced into a receptor plant to obtain a transgenic plant with enhanced powdery mildew disease resistance. The method has great application and popularization values for breeding the powdery mildew of the plants.

Description

Plant powdery mildew resistance related protein Pm5e, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant powdery mildew resistance related protein Pm5e, and a coding gene and application thereof.

Background

Wheat is one of the world's most prominent food crops, providing approximately 20% of the heat required globally. Wheat powdery mildew is a main fungal disease caused by Blumeria graminis f.sp.tritici, Bgt, and can obviously affect the tiller number, ear number and thousand kernel weight of wheat and seriously reduce the yield and quality of wheat. Therefore, controlling the powdery mildew hazard has important significance for improving the wheat yield, reducing the pesticide and labor cost and protecting the environment. Chemical prevention and control not only harms the environment and damages human health, but also can cause pathogenic bacteria to generate drug resistance, thereby causing more serious loss. The most economical, environment-friendly and effective measures for preventing and treating the wheat powdery mildew are to excavate the disease-resistant genes and cultivate the disease-resistant varieties.

At present, the disease-resistant breeding of wheat powdery mildew in China faces the problems that the variation speed of powdery mildew is too high, the epidemic range of new highly toxic microspecies is continuously expanded and available resistance genes are limited, and the problem of narrowing of the genetic basis of bred varieties is increasingly serious, so that new disease-resistant genes need to be continuously excavated, the sources of the disease-resistant genes are expanded, and the existing disease-resistant genes are reasonably distributed, so that the multiple genes are effectively polymerized to breed broad-spectrum and durable-resistance varieties, pathogenic bacteria are difficult to break out and spread in a large scale, the harm of the wheat powdery mildew is effectively prevented and controlled, the investment is reduced, and the benefit is improved.

Under the influence of long-term wheat cultivation process, different geographical environments and complex natural conditions, valuable local wheat variety germplasm resources are enriched, and excellent genes which are lacked by the bred varieties are hidden. Wheat in China has various local varieties and is a huge treasury of excellent genetic resources.

Disclosure of Invention

The invention aims to provide a plant powdery mildew resistance related protein Pm5e, and a coding gene and application thereof.

The protein provided by the invention is obtained from wheat (Triticum aestivum L.), is named as Pm5e protein, and is (a1) or (a2) or (a3) or (a4) as follows:

(a1) protein shown as a sequence 1 in a sequence table;

(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and is related to the disease resistance of the powdery mildew of plants;

(a4) a protein derived from wheat, having 98% or more identity to (a1) and involved in plant powdery mildew resistance.

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

The protein can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The nucleic acid molecule encoding the Pm5e protein also belongs to the protection scope of the invention.

The nucleic acid molecule is (b1) or (b2) or (b3) or (b4) as follows:

(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) DNA molecule shown in sequence 3 in the sequence table;

(b3) a DNA molecule derived from wheat and having 95% or more identity to (b1) or (b2) and encoding said protein;

(b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encodes the protein.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

Expression cassettes, recombinant vectors or recombinant microorganisms containing the nucleic acid molecules are within the scope of the invention.

The recombinant expression vector containing the nucleic acid molecule can be constructed using existing expression vectors. When the nucleic acid molecule is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when recombinant expression vectors are constructed using the nucleic acid molecules, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or adjacent regions initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of the transgenic plant or the transgenic microorganism, an expression vector to be used may be processed, for example, a gene for expressing an enzyme or a luminescent compound which produces a color change in the plant or the microorganism, a gene for an antibiotic marker having resistance or a chemical-resistant agent marker, etc. From the viewpoint of safety of transgenes, the transformed plants or microorganisms can be directly screened phenotypically without adding any selectable marker gene.

The recombinant vector may specifically be a recombinant expression vector. The recombinant expression vector can be specifically a recombinant plasmid obtained by inserting the nucleic acid molecule into an existing plant expression vector. The existing plant expression vector can be a pCAMBIA1300 vector or a pTCK303 vector.

The invention also protects the application of the Pm5e protein, which is (c1) or (c 2):

(c1) regulating and controlling powdery mildew disease resistance of plants;

(c2) improve the powdery mildew resistance of the plants.

The invention also protects the application of the nucleic acid molecule, which is (d1) or (d 2):

(d1) cultivating transgenic plants with changed powdery mildew resistance;

(d2) cultivating transgenic plants with enhanced powdery mildew resistance.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the nucleic acid molecule is introduced into a receptor plant to obtain a transgenic plant with enhanced powdery mildew disease resistance.

The invention also protects the application of the method in plant breeding. The purpose of the plant breeding is to obtain plants with enhanced powdery mildew disease resistance.

The invention also provides a plant breeding method, which comprises the following steps: the content and/or the activity of the Pm5e protein in the target plant are/is increased, so that the powdery mildew resistance of the target plant is improved.

Any of the above plants may be a monocot. Any of the above plants may be a graminaceous plant. Any of the above plants may be a plant of the genus Triticum. Any of the above plants may be wheat Fielder.

Any one of the above powdery mildews can be powdery mildews caused by powdery mildew pathogenic bacteria. The powdery mildew pathogenic bacteria can be Blumeria brucei wheat specialization type. The powdery mildew pathogenic bacteria can be E20 physiological race.

The inventor obtains the powdery mildew resistance gene Pm5e contained in local wheat varieties in China, and provides richer powdery mildew resistance gene resources for wheat breeding work. The Pm5e protein coding gene is introduced into powdery mildew disease resistant wheat, so that the disease resistance of a transgenic plant is remarkably improved, and the powdery mildew resistance is realized. The method has great application and popularization values for breeding the powdery mildew of the plants.

Drawings

FIG. 1 shows the results of expression analysis of Pm5e gene at different inoculation times.

FIG. 2 shows the results of the identification of powdery mildew resistance in step one of example 3.

FIG. 3 shows the results of the identification of powdery mildew resistance in step two of example 3.

FIG. 4 is a photograph of a phenotype of disease resistance identification in example 4.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The wheat variety rejuvenation 30, referred to as wheat rejuvenation 30 for short, is a local variety of Chinese wheat with high powdery mildew resistance. The wheat variety Nongda 015, called Nongda wheat 015 for short, is a variety with high susceptibility to powdery mildew. Wheat variety Fielder, abbreviated as wheat Fielder, is a variety of powdery mildew disease. The wheat variety Xue Zao, short for Xue Zao, is a variety of powdery mildew.

The powdery mildew pathogenic bacteria adopted in the examples are powdery mildew (Blumeragramis, sp.tritici, Bgt) E20 physiological races.

According to the phenotype, the powdery mildew disease resistance of the plant adopts a grading standard of 0-4 grade. 0 is immunization. 0; it is near immunity. 1 is highly disease resistant. 2 moderate disease resistance. 3, moderate susceptibility. 4 is highly susceptible. According to the broad classification, 0-2 is disease-resistant, and 3-4 is susceptible. The corresponding phenotypes for each of the grading criteria are shown in Table 2.

TABLE 2

Rank of	Phenotype
		0	The plant has no related symptoms of powdery mildew
0；	Allergic necrosis and chlorosis
		1	Thin mycelium layer, less lesion, and green penetration
2	The mycelium layer is thick and impermeable to green, and can produce a certain amount of spores
		3	The hypha layer is thicker, the disease spots are more, the spore yield is large, but the hypha is not connected into pieces
4	Thick hypha layer, dense hypha, many scabs, large spore yield, and continuous hypha into tablets

Example 1 discovery of proteins and genes encoding the same

Developing molecular markers by using a mixed pool transcriptome sequencing (BSR-Seq) method, and using filial generations of wheat rejuvenation 30 and wheat Nongda 0153705F_2：3The family is a mapping population, and fine positioning is carried out, and finally the Pm5e is positioned between chromosome 7BL molecular markers WGGB2 and WGGB3, and the physical interval between the markers is about 13.5 kb. The physical sequences of the wheat rejuvenation 30 and wheat agriculture 015 positioning intervals are amplified by PCR by utilizing Novozam high-fidelity DNA polymerase.

Finally, a new protein is found from the wheat rejuvenation 30, and is named as Pm5e protein as shown in a sequence 1 of a sequence table. The open reading frame of the Pm5e protein encoded in the cDNA of rejuvenation 30 is shown as a sequence 2 in the sequence table. In the genome DNA of rejuvenation 30, the gene coding the protein Pm5e is shown as a sequence 3 (with 4 exons) in the sequence table.

Example 2 analysis of Pm5e Gene expression at different vaccination times

The test plants were wheat rejuvenated 30 plants.

And (3) culturing the tested plant to one leaf and one heart stage, then inoculating powdery mildew pathogenic bacteria, and taking the first leaf of the plant after inoculating for 0h, 4h, 6h, 12h, 24h, 48h and 72h respectively, and detecting the relative expression level of the Pm5e gene. An inoculation mode comprises the following steps: the wheat Xuehao plant infected with powdery mildew pathogenic bacteria and fully diseased (fully diseased phenotype: thick hypha, dense hypha, more lesion, large spore yield and continuous hypha) is manually brushed and dusted above a tested plant. The method for detecting the relative expression level of the Pm5e gene comprises the following steps: extracting total RNA of the leaf, taking an Actin gene as an internal reference gene, and detecting the relative expression level of the Pm5e gene by qRT-PCR.

The primers for identifying the Pm5e gene expression level are as follows:

NLR-QF：5’-GAAGGCAAGGAAAGCAGGTC-3’；

NLR-QR：5’-GTACTGCTGCCCTTGACTTG-3’。

the results are shown in FIG. 1. The expression of the Pm5e gene is induced by powdery mildew pathogenic bacteria, the expression level reaches the highest after inoculating the powdery mildew pathogenic bacteria for 6 hours, and then the expression level is restored to a normal level.

Example 3 transgene validation of protein function

Verification of target gene in transgenic group

1. Construction of recombinant plasmids

(1) Taking 30 rejuvenated wheat leaves, and extracting genome DNA.

(2) And (2) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and adopting a primer pair consisting of NLR-com-VF and NLR-com-VR, and recovering a PCR amplification product.

NLR-com-VF：5’-AATTCGAGCTCGGTACCCGGG ACATTCTGAGCTTCCACTTC-3’；

NLR-com-VR：5’-TGTAAAACGACGGCCAGTGCCA GACCTGAAACCTGAAGTGTT-3’。

(3) Taking pCAMBIA1300 vector, carrying out double enzyme digestion by using restriction enzymes BamHI and HindIII, and recovering the vector skeleton.

(4) And (3) carrying out homologous recombination on the PCR amplification product obtained in the step (2) and the vector skeleton obtained in the step (3) to obtain the recombinant plasmid pCAMBIA 1300-Bar-NLR. According to the sequencing result, the recombinant plasmid pCAMBIA1300-Bar-NLR is structurally described as follows: the DNA molecule shown in the sequence 3 of the sequence table is inserted into the pCAMBIA1300 vector.

2. Preparation of transgenic plants

(1) And (3) introducing the recombinant plasmid obtained in the step (1) into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium tumefaciens.

(2) And (2) taking the recombinant agrobacterium obtained in the step (1), carrying out genetic transformation on the embryonic callus of the wheat Fielder, and then culturing to obtain a plant, namely a T0 generation plant.

(3) Selfing the T0 generation plants to obtain grains, and culturing the grains into plants, namely T1 generation plants.

(4) Transgenic plants were selected from the T1 generation plants.

The method for screening transgenic plants comprises the following steps: extracting genome DNA of plant leaves, performing PCR amplification by using the genome DNA as a template and a primer pair consisting of NLR-MD1F and NLR-MD1R, and if a 164bp amplification product is obtained, the plant is a transgenic plant.

NLR-MD1F：5’-TCGTCCAAAGTGCTCGAGAT-3’；

NLR-MD1R：5’-GTTGAAGGCTTCCCACGTG-3’。

3. Preparation of transgenic empty vector plants

And (3) replacing the recombinant plasmid with the pCAMBIA1300 vector, and performing operation according to the step 2 to obtain a transgenic empty vector plant.

4. Identification of powdery mildew disease resistance

Test plants: 20 strains of T₁L NLR _ com1 plant, 20T plants₁L NLR _ com2 plant, 20T plants₁A _NLR _ com3 plant, 20T 1 transgenic empty vector plants, 20 wheat rejuvenated 30 plants (denoted FZ 30) and 20 wheat Fielder plants. T is₁The _NLR _ com1 plant is a transgenic plant screened from the selfed progeny of the same T0 generation plant. T is₁The _NLR _ com2 plant is a transgenic plant screened from the selfed progeny of the same T0 generation plant. T is₁The _NLR _ com3 plant is a transgenic plant screened from the selfed progeny of the same T0 generation plant.

The test plant is cultured to one-leaf one-heart stage, then powdery mildew pathogenic bacteria are inoculated, and 10 days after inoculation, the phenotype of the first leaf of the plant is observed and photographed. An inoculation mode comprises the following steps: the wheat Xuehao plant infected with powdery mildew pathogenic bacteria and fully diseased (fully diseased phenotype: thick hypha, dense hypha, more lesion, large spore yield and continuous hypha) is manually brushed and dusted above a tested plant.

The photographs are shown in FIG. 2 (each test plant provides an exemplary photograph of 3 plants). The phenotype of the transgenic plant is consistent with that of a rejuvenated 30-plant wheat, and the transgenic plant shows remarkable powdery mildew resistance. The phenotype of the empty vector transferred plant is consistent with that of the wheat Fielder plant, and the phenotype shows obvious powdery mildew susceptibility. T is₁L NLR _ com1 plant, T₁L NLR _ com2 plant and T₁The disease-resistant grades of the _NLR _ com3 plants are all 1 grade. The disease-resistant grade of the wheat rejuvenation 30 plants is 0; and (4) stages. The disease-resistant grades of the empty vector-transferred plant and the wheat Fielder plant are both 4 grades.

Secondly, verifying the sequence of the transgenic coding region to the target gene

1. Construction of recombinant plasmids

(1) Taking rejuvenated 30 leaves of wheat, extracting total RNA and carrying out reverse transcription to obtain cDNA.

(2) And (2) taking the cDNA obtained in the step (1) as a template, carrying out PCR amplification by adopting a primer pair consisting of NLR-OE-VF and NLR-OE-VR, and recovering a PCR amplification product.

NLR-OE-VF：5’-TCTAGAGGATCCCCGGGTACC ATGGCGCAGATGGAGGATGTAG-3’；

NLR-OE-VR：5’-TTCGAGCTCTCTAGAACTAGT TTAGCGCTTGGGTTGGGGCGGG-3’。

(3) The pTCK303 vector was digested with restriction enzymes KpnI and SpeI, and the vector backbone was recovered.

(4) And (3) carrying out homologous recombination on the PCR amplification product obtained in the step (2) and the vector skeleton obtained in the step (3) to obtain the recombinant plasmid pTCK 303-Hyg-NLR. According to the sequencing result, the structure of the recombinant plasmid pTCK303-Hyg-NLR is described as follows: the DNA molecule shown in the sequence 2 of the sequence table is inserted into the pTCK303 vector.

2. Preparation of transgenic plants

(4) Transgenic plants were selected from the T1 generation plants.

The method for screening transgenic plants comprises the following steps: extracting genome DNA of plant leaves, performing PCR amplification by using the genome DNA as a template and a primer pair consisting of NLR-MD2F and NLR-MD2R, and if an amplification product is obtained, the plant is a transgenic plant.

NLR-MD2F：5’-TTAGCCCTGCCTTCATACGC-3’；

NLR-MD2R：5’-CACTTTGGACGAAGTTGAGG-3’。

3. Preparation of transgenic empty vector plants

The recombinant plasmid was replaced with pTCK303 vector, and the procedure was performed in reference to step 2 to obtain a transgenic empty vector plant.

4. Identification of powdery mildew disease resistance

Test plants: 20 strains of T₁L NLR-OE 1 plant, 20T₁L NLR-OE 2 plant, 20T₁L NLR-OE 3 plant, 20T 1 generation empty carrierPlants, 20 wheat rejuvenated 30 plants (denoted FZ 30) and 20 wheat Fielder plants. T is₁A _nlr _ OE1 plant is a transgenic plant screened from the selfed progeny of the same T0 generation plant. T is₁A _nlr _ OE2 plant is a transgenic plant screened from the selfed progeny of the same T0 generation plant. T is₁A _nlr _ OE3 plant is a transgenic plant screened from the selfed progeny of the same T0 generation plant.

The photographs are shown in FIG. 3 (each test plant provides an exemplary photograph of 3 plants). The phenotype of the transgenic plant is consistent with that of a rejuvenated 30-plant wheat, and the transgenic plant shows remarkable powdery mildew resistance. The phenotype of the empty vector transferred plant is consistent with that of the wheat Fielder plant, and the phenotype shows obvious powdery mildew susceptibility. T is₁L NLR _ OE1 plant, T₁L NLR _ OE2 plants and T₁The disease resistance grades of the _NLR _ OE3 plants are all 0 grade. The disease-resistant grade of the wheat rejuvenation 30 plants is 0; and (4) stages. The disease-resistant grades of the empty vector-transferred plant and the wheat Fielder plant are both 4 grades.

Examples 4,

EMS reagent with the concentration of 0.4 percent is used for treating 30 rejuvenated wheat seeds, and 3356M are obtained by collecting the wheat seeds in a single plant mode₂Seeds of the family. Powdery mildew resistance identification was performed for each pedigree (3 of step one of example 3) to obtain 3 independent susceptible mutant pedigrees (M2384 pedigree, M2586 pedigree, M3208 pedigree).

The phenotype photographs of the identification of powdery mildew resistance of 30 plants of wheat rejuvenation, of plants of wheat ficus pumila and of 3 independent susceptible mutant families are shown in fig. 4.

Amplifying target genes in the genome DNA and cDNA of the disease-sensitive mutant family. Mutant Mut2384 is mutated at the exon-intron boundary resulting in an altered splicing pattern. Mutant Mut2586 causes premature translation termination. Mutant Mut3208 has a 15bp deletion.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

<120> plant powdery mildew resistance related protein Pm5e, and coding gene and application thereof

<130> GNCYX190094

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1067

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<213> Triticum aestivum L.

<400> 1

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Ala Leu Asp Pro Asp Leu His Ala Ala Ile Arg Ala Pro Val Thr Val

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Met Leu Gly Pro Ser Ala Leu Leu Leu Arg Glu Leu Asp Ser Ser Gly

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Gln Ser Leu Leu Gly Ala Glu Glu Leu Arg Leu Leu Arg Asp Ala Leu

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Glu Phe Ser Glu Leu Pro Asp Arg Ala Lys Ala Val Tyr Ser Gly Leu

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Leu Ala Arg Ala Ile Asp Ala Arg Glu Arg Arg Arg Gly Phe Lys Trp

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Ser Pro Lys Thr Thr Arg Ser Asp Thr Trp Glu Ala Phe Asn Arg Arg

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Asp Asp Leu Trp Thr Ala Ser Val Trp His Ile Val Ser Arg Ala Phe

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Leu Leu Lys Tyr Leu Thr Val Glu Cys Asn Ile Thr Val Lys Leu Pro

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Ile Glu Ile Leu Glu Arg Met Pro Ala Leu Ala Ala Leu Ala Leu Tyr

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Tyr Arg Leu Thr Tyr Phe Lys Phe Leu Cys Ala Ala Pro Cys Leu Ser

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Phe Glu Gln Gly Ala Met Pro Lys Leu Gln Asn Leu Asn Leu Gly Phe

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<213> Triticum aestivum L.

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atggcgcaga tggaggatgt agatgccgcg tcccctctgc cagcgatggc gttggaccct 60

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ctccgggaac tcgattcttc cggacagagt cttctcggtg cggaggagct tcgtcttctt 180

agagatgctc tcagggaagt atgcatcccc ctgaagagta tgtccgaaga cgacggtgct 240

agctttatgg cccggtggtg gatgaagata gttcgggagc tttgttatga tacgcaggat 300

tacctcaact tcgtccaaag tgctcgagat cgtcctgaat tttcagagtt acctgatcgt 360

gccaaggctg tttattcagg cttacttgct cgcgccattg atgcgaggga aagacgcaga 420

ggtttcaagt ggtctcccaa gaccacccgg tctgacacgt gggaagcctt caaccgccgc 480

ttctccaaaa aacttgtcca gctgctgggt tccttcggtg tccataccac acccggtgcc 540

gtggtcgtgg aggccccaaa caagcttgtc cagctgctag ctttagatga tgatgtcaac 600

gacaagacac tcaaggtgat acctataatt ggatgtgcag gtgttggaaa gacaacagct 660

gccagaacct tgtatcacaa gcatggaggg aaatttcagt gccgggcttt tgtaagtgtg 720

tctcagaatc cagatatgag gggaatcctc accagcatgc tagcacaact taaggcacca 780

cggccccctg gctttcctga tgtgctggac cttattggcg ctatcagcag gcatctccaa 840

ggcaaagggt acttgatcgt acttgatgat ttatggactg catcggtatg gcatattgtt 900

agccgcgctt ttcctcgtgg tgatcaccgc agcagaataa taacaactac acaagtgcat 960

gacgtagcat tggcatgctc tggttatcac ccggtccgta tatataagat ggaacttctt 1020

gatgaatatg aatctcgaaa gttattcttc cgtagggtgt ttagctctgc ccctggagat 1080

ggttgttctc cagctaccaa agaagtctca tacgagatta tcagaaaatg tgaaggtttg 1140

ccgttagcaa ttgtaagtat agcaggtctg ttagcaagcg aattaagcat cgtcatggaa 1200

gattggaggc acatacaaaa ttctttttcc tccacttccg aagggatgaa agatatttta 1260

aaccttatct acaatagtct tccacctggt ttgaggacat gcttgctata tctgagtatg 1320

tatccacagg gctacgtgat gaagaaggct gagttggtga agcactgggt agccgaaggt 1380

tttatcggtg ttgtggaagg gacagtcgca atgaaaattg ctgagcgtta ttttgatgag 1440

cttgtcagca gagcaatggt ccaggccgtg gacaccgatt atactggcaa ggtgttgtca 1500

tgtacggttc accatttggt actggatttt attaggtcca aatctttgga tgagaatttt 1560

gtcaccactg tggactattc tgaatcaact ctagcacacc ctgacaaagt tcgtcggtta 1620

tccatccagt ttggaggggt aaaaagcgca tacattccag aaaccatcgt aacatcgaaa 1680

gttcggtcac ttgtattttg gggtttcttc aagtgtgcgc ctccttccat tatggattat 1740

ggatttcttc gtattctgaa tcttcatatt tgggctgatg aagacaatga gatttttgac 1800

ctcattggaa ttgggaactt atttcttctg aagtatctca cggttgaatg taatatcacc 1860

gtcaaacttc cagagaagat tggaatgctc cgatacttgg agacactgga agtagatgca 1920

agattgtttg ctgttccatc agatatggat aatctggaga ggttactgca cctccgtctt 1980

ccgagcgaat ctatcctgcc tcaaggagta gcccacatga catctcttcg cactttgggg 2040

aattttgatc tcagccgccg ttactcaata gagaatgtat tacaacttgg agggctgtct 2100

aatctccagg atctccagct cacctgtgct atggcacagc aagcagaaaa cctggaaaag 2160

aatgtgctac tccttggctg gattgttgag aggcttagtt tcttgcagac cataactttg 2220

gtacctgctt cagtgtcctc tcatcaggat gatggtcagg ctgcagcacc tacaagcata 2280

attattcccc ctgatggatt taacatggag cctcctccag acctgcttct acagaggatc 2340

gaaatgtcgc ggcactgctg catcttcttc tgcataccta agtgctttgg agagctaagg 2400

aaactctgca tcctaaagat tgcaattagg agtctgtcga ggagtgatat tgaaatcctg 2460

gaaagaatgc cggccctcgc tgctctcgcc ctgtataatc agacaactcc cacagaaaag 2520

atgatcatga ctgacggggg attctataga ctcacgtact tcaagttctt gtgtgctgca 2580

ccatgcctgt cctttgaaca aggagcgatg cccaaactcc aaaacctcaa cctaggattc 2640

aactcagacc aatggagatc ggataccttt gaaactcttg gcttgagtca cttgagaggc 2700

cttacggatg tatgtgtaag acttggtact ggggctgctg acaacttcaa cgtaaaagtt 2760

gccgagtcgg cattggaggc tgtcgtcagg aatcacccca actctcctag gatcagaata 2820

aaatttgtgg atctgatttt tgatggtaag gaggatgata gcaccgcaac tcaccagtat 2880

caagaaggca aggaaagcag gtctgccaga ggccaagatg gcaaacaaga tgcaacaagg 2940

ggcgaacgtc aacaacaggg gccaccaatg agcaaacaag atgcacggag gagcaaagca 3000

gtgggagcgt catcaaaggc agtaccaccg ataagcaaag ctgtaaatgc tagcaagtca 3060

agggcagcag tacctcctcc caccagctct agcccatcaa agccgatgcc atcttcctcc 3120

cttaccagtc caccaaaacc gaggcgacgt ggctcccgca ccagccctcc ccgacccacg 3180

agcccgcccc aacccaagcg ctaa 3204

<210> 3

<211> 7747

<212> DNA

<213> Triticum aestivum L.

<400> 3

acattctgag cttccacttc cactcaggtc gagtgtctct agctgattca gcttccagat 60

ttcccttgga agcctatcaa tgatgcccga tttgatgctc aggtatttca tcagcaccag 120

gctgtagata gcctgaatat ggcccgcact aaggtgagca cactctttca ggtccaacac 180

tcgcagcagt ttatacttgg taaaatctaa aacagcttca taactatcta gagttgccgc 240

cgcgccagtg gagaatactg ccagggtccg gagaagagat aaactctgag gcaaattcag 300

atgggcgttt gcacaagcat gcacagatag cctgcggagg tgttcgttct gcaattcggc 360

cacatcgtga gataaccata tgaaattctc cgacatggac cgttgggtga tgtactgccg 420

aatcgcatcg ccggtaggct ggcatctctt caccttccca ttgctgcttc tctgggtgga 480

cctgatgata ctggaaccaa caagcatgcc caaattgctg gctgcgcctt gttctcctgg 540

tacaagtcct tcagccgtcc atctcctttc taggggctta gtcctgacat gatgttcatg 600

agggaacaag cgcagatata gcaagcagtt gtggtaatca ccattaagat tacctcccct 660

catcaggaaa tgaaaattct gaatctccgc ggggacagca gcagccgccg cttgtgctgc 720

agccccttgt acaggaaagc caaacctctt tatccgttca gtcgttgcag ttgatctctc 780

cttgagttgg gcaattttgt cggcaaattc atcatcggtc gtcatcttgg cgttgaagct 840

gtctatacag tcttggatgt cataagccaa ggccctcagt tgtgcaaaat attctgcctg 900

caagaaactc atcggctcga cactggaagc atgatccttc atggcagctt cgatccactt 960

gagttcttca ctgatgatgg tagcatcagt atttagcttc cccttcacgc tgcagagctt 1020

cgacaaggcg acgccagcta ccgtgctaaa tgccgctaca gcaaactcca tcggtctctc 1080

tttccttgtc tccacagatc actcctcctc cctagatctg catgcacggc aaagagcggt 1140

tagtgttgct tcaaatggaa gagagaggga gagggaaggg gccgggagac agagcttctc 1200

tacctcggtg gagggagctt cagagttccc ctcctcggct gcagtgactt gatagagtca 1260

caagtcacaa gtcacaattc acaagtggca aggaggactg ggctaacata tactccctcc 1320

gttccaaaat agatgactca actttgtact aactttgtac taaagttagt acaaagttga 1380

gtcatctatt ttggaacgga gggagtactc tgctagtgtt tactatgaac ttgtttggga 1440

aactttttct ccagaacggt tttggatcta acgggactca gttttttttt catacatatc 1500

taattagagg tttgatttaa atttcagata attttgcaat agttggaatc atcttgtacg 1560

atcgatgatc agagaactaa ttaaatttgt atcatctcag acactcagag gtgctcgtag 1620

ctagtgataa gatatgtgtg cgtgtgttaa taggggtata tgtgatgatt gtcttcattt 1680

ataccgtgtt tacaaaaagg tctattctat atatattttt cattatttaa atgtgttaag 1740

atgcttcctc ttgctccctc tgtaaagaaa tataagagtg ttcaaatgct cttatagttt 1800

ttttacggag ggagcatctc ccaaggagta ttggacgtgt tatgtactta tattagcttc 1860

ctatataggt aagagtaatg gtagccgggt caatcaaagg ttcggcttgc ttctccccca 1920

tcctctctta tgaaatggat aacccaatgg atggttcacg ttacttttca cactgtctct 1980

ttttctaacc tgtaaccgtc actactataa aatcgttttc caagtaagaa aaatggacta 2040

agaggatcca tttgttttct atgttgatgt caattaatct aacccagatg gcagattcgt 2100

tccagatgtt aaccaccagt ggttgttgat agaataggca gcaatcacag tttttctttg 2160

gaggaggggc ggttccgtgg ggcggaggct tggccggttc ttccagtcac aggtcaggtc 2220

ttctaagcac cctccccttc cccatgttcc atgtacgtat agggggctgt tgagttgtgg 2280

tgttccactt tgtccacgca ctactttttc gcccatctga tagcttcgtc caaatcgaga 2340

tttccctggt agtgtctccc ctcccgagct ttctttgtcg ttcattcata caattacaaa 2400

tcatctggtt gtctatgagg gatcaatagt aatcatctgt ttcttgcttc aggtgaagat 2460

ggcgcagatg gaggatgtag atgccgcgtc ccctctgcca gcgatggcgt tggaccctga 2520

tctccacgca gcgattcgag ctcctgttac tgttatgctg ggtccgtcgg ccctcctcct 2580

ccgggaactc gattcttccg gacagagtct tctcggtgcg gaggagcttc gtcttcttag 2640

agatgctctc agggaagtat gcatccccct gaagagtatg tccgaagacg acggtgctag 2700

ctttatggcc cggtggtgga tgaagatagt tcgggagctt tgttatgata cgcaggatta 2760

cctcaacttc gtccaaagtg ctcgagatcg tcctgaattt tcagagttac ctgatcgtgc 2820

caaggctgtt tattcaggct tacttgctcg cgccattgat gcgagggaaa gacgcagagg 2880

tttcaagtgg tctcccaaga ccacccggtc tgacacgtgg gaagccttca accgccgctt 2940

ctccaaaaaa cttgtccagc tgctgggttc cttcggtgtc cataccacac ccggtgccgt 3000

ggtcgtggag gccccaaaca agcttgtcca gctgctagct ttagatgatg atgtcaacga 3060

caagacactc aaggtgatac ctataattgg atgtgcaggt acagtaataa tctatcctca 3120

acttgaatat ctctagtctt tttctgcccg gcccattaat gctagctgga acatatgcca 3180

agtttctttc atcttctttc aggtgttgga aagacaacag ctgccagaac cttgtatcac 3240

aagcatggag ggaaatttca gtgccgggct tttgtaagtg tgtctcagaa tccagatatg 3300

aggggaatcc tcaccagcat gctagcacaa cttaaggcac cacggccccc tggctttcct 3360

gatgtgctgg accttattgg cgctatcagc aggcatctcc aaggcaaagg gtgattgatc 3420

tcaactatac tgtctaaata agcaaattcc gtgttctctt ctcataaata aatcataggc 3480

ataattttat tttatgaggt tccagttatg gcgtgtcagg cattgccata gcattttgag 3540

aaattatgaa tattattaat gggtgttgac cctcaaaaaa aaattatagc acatgctcag 3600

gtgaacctag gcaaattaat atatatgctg ccgcttgagc aaggtatata ttaattttta 3660

ttttttctga cctaacatac aaaattatgc aggtacttga tcgtacttga tgatttatgg 3720

actgcatcgg tatggcatat tgttagccgc gcttttcctc gtggtgatca ccgcagcaga 3780

ataataacaa ctacacaagt gcatgacgta gcattggcat gctctggtta tcacccggtc 3840

cgtatatata agatggaact tcttgatgaa tatgaatctc gaaagttatt cttccgtagg 3900

gtgtttagct ctgcccctgg agatggttgt tctccagcta ccaaagaagt ctcatacgag 3960

attatcagaa aatgtgaagg tttgccgtta gcaattgtaa gtatagcagg tctgttagca 4020

agcgaattaa gcatcgtcat ggaagattgg aggcacatac aaaattcttt ttcctccact 4080

tccgaaggga tgaaagatat tttaaacctt atctacaata gtcttccacc tggtttgagg 4140

acatgcttgc tatatctgag tatgtatcca cagggctacg tgatgaagaa ggctgagttg 4200

gtgaagcact gggtagccga aggttttatc ggtgttgtgg aagggacagt cgcaatgaaa 4260

attgctgagc gttattttga tgagcttgtc agcagagcaa tggtccaggc cgtggacacc 4320

gattatactg gcaaggtgtt gtcatgtacg gttcaccatt tggtactgga ttttattagg 4380

tccaaatctt tggatgagaa ttttgtcacc actgtggact attctgaatc aactctagca 4440

caccctgaca aagttcgtcg gttatccatc cagtttggag gggtaaaaag cgcatacatt 4500

ccagaaacca tcgtaacatc gaaagttcgg tcacttgtat tttggggttt cttcaagtgt 4560

gcgcctcctt ccattatgga ttatggattt cttcgtattc tgaatcttca tatttgggct 4620

gatgaagaca atgagatttt tgacctcatt ggaattggga acttatttct tctgaagtat 4680

ctcacggttg aatgtaatat caccgtcaaa cttccagaga agattggaat gctccgatac 4740

ttggagacac tggaagtaga tgcaagattg tttgctgttc catcagatat ggataatctg 4800

gagaggttac tgcacctccg tcttccgagc gaatctatcc tgcctcaagg agtagcccac 4860

atgacatctc ttcgcacttt ggggaatttt gatctcagcc gccgttactc aatagagaat 4920

gtattacaac ttggagggct gtctaatctc caggatctcc agctcacctg tgctatggca 4980

cagcaagcag aaaacctgga aaagaatgtg ctactccttg gctggattgt tgagaggctt 5040

agtttcttgc agaccataac tttggtacct gcttcagtgt cctctcatca ggatgatggt 5100

caggctgcag cacctacaag cataattatt ccccctgatg gatttaacat ggagcctcct 5160

ccagacctgc ttctacagag gatcgaaatg tcgcggcact gctgcatctt cttctgcata 5220

cctaagtgct ttggagagct aaggaaactc tgcatcctaa agattgcaat taggagtctg 5280

tcgaggagtg atattgaaat cctggaaaga atgccggccc tcgctgctct cgccctgtat 5340

aatcagacaa ctcccacaga aaagatgatc atgactgacg ggggattcta tagactcacg 5400

tacttcaagt tcttgtgtgc tgcaccatgc ctgtcctttg aacaaggagc gatgcccaaa 5460

ctccaaaacc tcaacctagg attcaactca gaccaatgga gatcggatac ctttgaaact 5520

cttggcttga gtcacttgag aggccttacg gatgtatgtg taagacttgg tactggggct 5580

gctgacaact tcaacgtaaa agttgccgag tcggcattgg aggctgtcgt caggaatcac 5640

cccaactctc ctaggatcag aataaaattt gtggatctga tttttgatgg taaggaggat 5700

gatagcaccg caactcacca gtatcaagaa ggcaaggaaa gcaggtacgg ctttttcttc 5760

cgtttacaag aattttgtag ggggtttaaa ggataggaat tttataaaag gatttctttg 5820

gaacccagtg atttgtagga atagaatctc attcctgtcc agcatagaaa ccaatccttt 5880

ctatttcaaa ggggggaaaa agcattagcc tagacccaaa cccaatgaaa aaaacaatca 5940

tatcctgtga atcagatgaa atgacatact tgtcacctca cttcatatga ttttccaatt 6000

ttgatgatat gcctattcaa tgaaccaaaa gagaccttaa cagggcattc gtccaatctt 6060

gcactgctaa tttgttcgtc atttttcctt caatcgcgac aggtctgcca gaggccaaga 6120

tggcaaacaa gatgcaacaa ggggcgaacg tcaacaacag gggccaccaa tgagcaaaca 6180

agatgcacgg aggagcaaag cagtgggagc gtcatcaaag gcagtaccac cgataagcaa 6240

agctgtaaat gctagcaagt caagggcagc agtacctcct cccaccagct ctagcccatc 6300

aaagccgatg ccatcttcct cccttaccag tccaccaaaa ccgaggcgac gtggctcccg 6360

caccagccct ccccgaccca cgagcccgcc ccaacccaag cgctaagctt ttggtacgat 6420

tatttcttct tcgtgcacca aaaatagaaa acgcttctaa tgtaccattt acagctttgc 6480

tcaagttttc caagctcttg gtatgatttt cccaaggaca taacagagtg tcgacctttt 6540

ctttcatgtc atgaagtcca tctgctacct actttcttcc ctctagaaac tctcttgtgc 6600

tggaaagtaa cttattcaaa gccagaactg ggctaggtgc ttattttcat tcatccctga 6660

cccaggccag gggttgagcg catcatcccg cttattagcg ccagggacat ccgacagaca 6720

gacggacgat gcaccaagcg gtgaaccatg cggcgggaag aacgagcatc cgaggagcag 6780

tccccaccct gatgccatcc aaactgatca gctctctaaa tattacagca tctattctac 6840

atccgtatgt acacatgtct gcttttcttg tgcactaagt tctgtactgt gttttcttat 6900

aggctggcac ttttcaaaaa aaattcaaaa tgaggatgta cccctcagcc tctgagtgat 6960

gcacacagcc ttataggctg gcattatacc atttgatact tctgtgacca tgccgtgagc 7020

tgcgggatcg acatgttctc gtttgaaatg gggaagacat aaacatatat gtcatacgaa 7080

gaaagcaaaa aaaatgtttc tgttgtaaat aatgagagaa ttcaagtgct atgagtgact 7140

tacttaggtt ttaaaacaaa acaaataggg cttgtgtagc ataggagtca tgagcacagc 7200

accagaacag cacccaagca cagcctccaa accccaaaac aatatcaacg aaaatttgaa 7260

attcaacacg tgaattctac caacatcacc agagcagaac tcaggaatca gtattaaaaa 7320

tctcgggatt gacaaatccc acaaacctaa atttaccact catcatgagt agtacatcaa 7380

acaatgcaaa tttattgaac actcacaaaa cactaccagg ttcaaacagg tgacactata 7440

attaggctta catctatgtc aataactatt tcgaaacaga aaaaaggagt ctcgccaaga 7500

gacatcaagg gtatatccaa acaaagagca tcacaaaaca cttccagctt attgaggcaa 7560

atttattgag ccacaaatct cattcgaaat cacattctac tcaccacaca ccaggcgtat 7620

atcaaacaag tcaaatttat tgggccacaa atctcattcg gaatcacata ctattcacca 7680

cataccatgg cgtatatcaa acaaggcaaa tttattgagc atcacaaaac acttcaggtt 7740

tcaggtc 7747

Claims

1. The application of Pm5e protein is as follows (c1) or (c2):

(c1) Regulate powdery mildew resistance of plants;

(c2) improving the powdery mildew resistance of plants;

The Pm5e protein is shown in sequence 1 in the sequence listing;

The plant is a Triticum genus.

2. The use of nucleic acid molecules encoding Pm5e proteins, as follows (d1) or (d2):

(d1) Breeding transgenic plants with altered resistance to powdery mildew;

(d2) Breeding transgenic plants with enhanced resistance to powdery mildew;

The Pm5e protein is shown in sequence 1 in the sequence listing;

The plant is a Triticum genus.

3. application as claimed in claim 2, is characterized in that:

The nucleic acid molecule encoding Pm5e protein is as follows (b1) or (b2):

(b1) A DNA molecule whose coding region is shown in SEQ ID NO: 2 in the sequence listing;

(b2) The DNA molecule shown in SEQ ID NO: 3 in the sequence listing.

4. A method for preparing a transgenic plant, comprising the steps of: introducing a nucleic acid molecule encoding a Pm5e protein into a recipient plant to obtain a transgenic plant with enhanced powdery mildew resistance; the plant is a Triticum genus plant; the Pm5e protein As shown in sequence 1 in the sequence listing.

5. The method of claim 4, wherein:

The nucleic acid molecule encoding Pm5e protein is as follows (b1) or (b2):

(b2) The DNA molecule shown in SEQ ID NO: 3 in the sequence listing.

6. A plant breeding method, comprising the steps: increase the content of Pm5e protein in the target plant, thereby improving the powdery mildew resistance of the target plant; the plant is a Triticum genus; the Pm5e protein is as sequence 1 in the sequence table shown.