CN108484745B

CN108484745B - A Wheat Powdery Mildew Resistance-Related Protein TaSARD1 and Its Encoding Gene and Application

Info

Publication number: CN108484745B
Application number: CN201810472228.9A
Authority: CN
Inventors: 常诚; 智朋飞; 岳同卿; 樊庆鑫
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2018-05-17
Filing date: 2018-05-17
Publication date: 2021-06-25
Anticipated expiration: 2038-05-17
Also published as: CN108484745A

Abstract

The invention provides a wheat powdery mildew resistance-related protein TaSARD1 and its encoding gene and application. The protein is a protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID No. 2; (b) the amino acid sequence shown in SEQ ID No. 2 passing through one or several amino acids A protein derived from (a) that has substitution, deletion or addition of residues and is associated with disease resistance or has transcriptional activating activity. The present invention finds that the wheat protein TaSARD1 is transiently overexpressed, and it is found that it can resist wheat powdery mildew caused by the physiological race E09 of wheat powdery mildew. The protein provided by the invention can lay a foundation for the research on cultivating transgenic wheat with resistance to wheat powdery mildew.

Description

Wheat powdery mildew resistance-related protein TaSARD1, and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a wheat powdery mildew resistance-related protein TaSARD1, and a coding gene and application thereof.

Background

Wheat powdery mildew is a fungal disease caused by the obligate parasitic fungus erysiphe Blumeria graminis f sp. In recent years, the damage caused by wheat powdery mildew is increasingly serious due to the increase of the planting density of crops, the increase of the application amount of nitrogen fertilizer and the single planting of the crops. The disease can affect various organs of the overground part of a wheat plant, mainly leaves and leaf sheaths, chaffs and awns can be damaged when the disease is serious, the yield can be reduced by 10 to 40 percent generally, and the yield in a seriously ill field can be reduced by more than 50 percent. The powdery mildew has large population, wide application range, numerous physiological species and high variation speed, so that a plurality of effective disease-resistant genes lose resistance. At present, breeding workers always select and popularize disease-resistant varieties as main prevention and treatment means for diseases, the effects are achieved for years, but the problem of resistance loss is unsolved all the time, and the diversity of resistance sources is an effective way for realizing the lasting disease resistance. Therefore, cloning a new disease-resistant gene by a biological method, and improving the resistance of wheat to powdery mildew by using a transgenic method is one of effective strategies for preventing and treating powdery mildew in the future. However, the existing wheat powdery mildew resistance gene resources are relatively deficient, and the development of wheat disease resistance gene resources is urgently needed.

Disclosure of Invention

The invention aims to provide a wheat powdery mildew resistance related protein TaSARD1, and a coding gene and application thereof.

The invention provides a wheat powdery mildew resistance related protein, which is a protein of the following (a) or (b):

(a) protein composed of amino acid sequence shown in SEQ ID No. 2;

(b) and (b) protein which is derived from (a) and related to disease resistance or has transcription activation activity, wherein the amino acid sequence shown in SEQ ID No.2 is subjected to substitution, deletion or addition of one or more amino acid residues.

The protein of (a) or (b) may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression. The gene encoding the protein of (b) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in SEQ ID No.1 of the sequence Listing, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 2 above at the 5 'end and/or 3' end thereof.

Substitution, substitution and/or addition of one or several amino acid residues in the amino acid sequence of the above-mentioned protein may be caused by naturally occurring variation or by artificial mutagenesis.

The present invention also provides a DNA molecule according to any one of the following 1) to 3):

1) DNA molecule shown in SEQ ID No. 1;

2) a DNA molecule which hybridizes with the DNA sequence defined in 1) under strict conditions and codes a protein which is related to disease resistance or has transcription activation activity;

3) a DNA molecule which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in 1) and which encodes a protein which is associated with disease resistance or has transcriptional activation activity.

The stringent conditions are as follows: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The invention also provides the gene recombinant vector, the expression cassette, the transgenic cell line or the recombinant strain. Wherein, the recombinant vector can be a vector obtained by inserting SEQ ID No.1 into pUbi-Adaptor-NOS vector; the vector may be a vector obtained by inserting SEQ ID No.1 between the BamHI and speI cleavage sites of the 35S-BD vector.

The invention also provides application of the protein in culturing transgenic plants with wheat powdery mildew disease resistance.

Further introducing the coding gene into plant wheat to obtain the wheat powdery mildew disease-resistant transgenic wheat.

The invention also provides application of the protein as a transcription activator. The protein is used as a transcription activator to activate gene expression.

The invention discovers a wheat protein TaSARD1, and the coding gene thereof is transiently overexpressed, so that the wheat protein TaSARD1 can resist wheat powdery mildew caused by wheat powdery mildew physiological race E09, and further research shows that the protein also has transcription activation activity and can be used as a transcription activation factor. The protein provided by the invention can lay a foundation for the research of culturing transgenic wheat with wheat powdery mildew resistance.

Drawings

FIG. 1 is a graph showing the effect of transient overexpression of TaSARD1 on wheat powdery mildew formation index in example 2.

FIG. 2 is a graph showing the results of transcriptional activation reporter gene expression of TaSARD1 in example 2.

FIG. 3 is a graph showing the results of example 3 in which TaSARD1 is localized in the wheat cell nucleus.

FIG. 4 is a graph showing the expression of TaSARD1 induced by Ubberella graminis in example 3.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials used in the examples described below are all available from Qingdao university.

Some of the materials in the following examples are as follows:

the wheat variety Shannong 20 is described in research paper on molecular detection of disease-resistant gene of new wheat variety "Shannong 20". Crop academic newspaper, 2014, 40: 611-621. Publicly available from Qingdao university;

the wheat variety Jing 411 is recorded in a research paper, and the Jing 411 is used as a backbone parent to culture a new high-yield wheat variety. Crop academic newspaper, 2009, 4: 1-5. Publicly available from Qingdao university;

wheat cultivar Kenong 199 is described in research paper high-yield widely-applicable wheat cultivar Kenong 199. Wheat crop press, 2007, 27 (2): 368-370. Publicly available from Qingdao university;

wheat variety Yumai 66 is described in research paper about the identification and molecular marker of powdery mildew resistance gene of common wheat variety Yumai 66. Crop literature, 2008, 34 (4): 545-550. Publicly available from Qingdao university;

wheat powdery mildew physiological race E09 is described in research paper, and the wheat head powdery mildew resistant gene resource GB4 is cultivated by utilizing artificially synthesized wheat. Plant genetic resources journal, 2007, 8: 378. publicly available from Qingdao university;

columbia ecotype Arabidopsis thaliana (col-0) is described in the research paper Molecular characterization of the subergence response of the Arabidopsis thaliana ecotype Columbia.New Photobiolist.2011, 190: 457-;

the vector pUbi-GUS is described in research paper A transfer assay system for the functional assays in leather. molecular μ lar Plant-Microbe Interactions,1999,12:647-654, publicly available from the university of Qingdao;

the vector pUbi-Adaptor-NOS is described in the research paper Recognition specificity and RAR1/SGT1 dependency in barrel MLA disease resistance genes to the powder mutationPlant Cell,2003,15: 732-;

vector 35S-BD is described in the research paper Soybean GmPHD-type transformation regulation restriction complete in transgenic Arabidopsis plants PLoS One,2009,4(9) e7209. publicly available from Qingdao university;

vector 35S-BD-VP16 is described in the research paper Soybean GmPHD-type transcription regulation expression complete in transgenic Arabidopsis plants PLoS One,2009,4(9): e7209. publicly available from the university of Qingdao;

vector 5 XGAL 4-LUC records a research paper in Soybean GmPHD-type transcription regulation expression level in transgenic Arabidopsis plants PLoS One,2009,4(9): e7209. publicly available from the university of Qingdao;

the vector Pptrl is described in the research paper Soybean GmPHD-type transformation regulation stress tolerance in transgenic Arabidopsis plants, plos One,2009,4(9) e7209. publicly available from the university of Qingdao;

the vector pUbi-Gateway-mYFP is described in The research paper The CC-NB-LRR-type Rdg2a resistance gene transfer immunity to The seed-borne base belt strain stress vector in The present invention of a highly reactive cell de.

Example 1 obtaining of TaSARD1 Gene

1. Obtaining RNA

Inoculating 7-day seedling of Shannong 20 wheat with wheat white powder physiological strain E09, collecting materials in 48 hr, and extracting RNA.

2. Reverse transcription to obtain cDNA

Reverse transcription system:

RNA 2.5. mu.L, Oligo-dT primer 1. mu.L, DEPC water 6.5. mu.L, mix the above solution in a centrifuge tube, incubate at 65 ℃ for 5 minutes, and keep on ice for 5 minutes.

5. mu.L of 5 Xreverse transcription buffer solution, 1.25. mu.L of dNTP mix, 0.625. mu.L of inhibitor, 1. mu.L of M-MLV reverse transcriptase, 7.125. mu.L of DEPC water, and carrying out warm bath at 42 ℃ for 1 hour and 5 minutes at 95 ℃ to obtain cDNA.

3. Clone TaSARD1

Adding 25 μ L of double distilled water into the cDNA, taking 1 μ L of the cDNA to perform subsequent PCR reaction as a template, performing PCR amplification by using the following primers,

a forward primer: CTTCTTCCTCCTCCTTCCAG (SEQ ID No.3)

Reverse primer: GAAATGCTAGAAAAAGGTGAAG (SEQ ID No.4)

The PCR system was as follows: KOD buffer 5. mu.L, MgSO₄mu.L, dNTP 5. mu.L, template 100ng, forward primer 2. mu.L, reverse primer 2. mu.L, KOD plus 1. mu.L, water 32. mu.L.

The PCR procedure was as follows: 94 ℃ for 5min, then 94 ℃ for 30s, 60 ℃ for 30s, 68 ℃ for 90s, for 29 cycles, and finally 68 ℃ for 10min and 16 ℃ for 10 min.

Sending the obtained PCR product to sequencing, wherein the result shows that the PCR product has the nucleotide shown by SEQ ID No.1 in the sequence table, the gene shown by the sequence is named TaSARD1, and the coding region is the 1 st-1143 rd nucleotide from the 5' end of the SEQ ID No.1 in the sequence table; the protein coded by the gene is named TaSARD1, and the amino acid sequence of the protein is SEQ ID No.2 in the sequence table.

Example 2 application of TaSARD1 Gene in powdery mildew resistance

First, the gene gun transient overexpression technology identifies the regulation and control effect of TaSARD1 on wheat powdery mildew resistance

1. Vector construction

1) Using the PCR product (or the artificially synthesized sequence 1) obtained in example 1 as a template, PCR amplification was carried out using the following primers to obtain a PCR product of about 1.2 Kb.

A forward primer: GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGGCGGCTCACAAGCGGCT (SEQ ID No.5)

Reverse primer: GGGGACCACTTTGTACAAGAAAGCTGGGTC TTAGCTGAAGTTGCTCTGGAA (SEQ ID No.6)

2) BP reaction is carried out on the PCR product obtained in the step 1) and a pDNOR201 vector (purchased from invitrogen) to obtain an ENTRY vector.

The BP reaction system described above: PCR product 75ng, pDNOR 20175 ng, BP enzyme 0.5. mu.L, 25 ℃ overnight.

3) The ENTRY vector and pUbi-Adaptor-NOS vector are subjected to LR reaction to generate pUbi-TaMYB1 vector.

LR reaction system: ENTRY vector 75ng, pUbi-Adaptor-NOS vector 75ng, LR enzyme 0.5. mu.L, 25 ℃ overnight.

The vector pUbi-TaSARD1 is identified by sequencing, and is obtained by inserting SEQ ID No.1 in a sequence table into pUbi-Adaptor-NOS vector.

2. Gene gun transient overexpression test

1) Preparation of gold powder

(1) Weighing 9mg (w) of gold powder, placing the gold powder in a centrifuge tube, and placing the centrifuge tube for more than 4 hours at 65 ℃;

(2) adding 70% ethanol, vortex oscillating for 8 min, and standing for 15 min;

(3) centrifuging at 2000r/min for 2s, and removing supernatant;

(4) adding 1mL of sterilized distilled water, carrying out vortex oscillation for 2 minutes, standing for 1 minute, centrifuging at 2000rpm/min for 2s, and removing supernatant;

(5) repeating the step (4) for three times;

(6) adding 50% glycerol into the precipitated gold powder, and carrying out vortex oscillation.

2) Preparation of DNA bullets

(1) Shaking gold powder in 50% glycerol for 5min to obtain suspension;

(2) taking 50 mu l of suspension liquid in a centrifuge tube;

(3) mu.l of plasmid DNA (2. mu.g) was added and 50. mu.l of CaCl was added with shaking₂Adding 20 μ l spermidine (0.1M) into the aqueous solution (2.5M) during shaking, shaking for 3 min, standing for 1min, centrifuging at 2000rpm/min for 2s, and removing supernatant;

(4) adding 140 μ l 70% ethanol, vortex vibrating to disperse the precipitate uniformly, centrifuging (2000rpm/min 2s), and removing supernatant;

(5) adding 140 μ l 100% ethanol, vortex vibrating to disperse the precipitate uniformly, centrifuging (2000rpm/min 2s), and discarding the supernatant;

(6) add 12. mu.l of 100% ethanol and vortex to disperse the precipitate evenly.

3) Gene gun bombardment transformation method

Gene gun-mediated methods: respectively carrying out moisturizing and bud blowing on seeds of 20 wheat shannon, 411 wheat koong 199 and 66 wheat yunnan, growing for about one week, cutting off a first leaf with the leaf surface upward, placing on a culture dish containing a culture medium (1% agar and 100mg/L benzimidazole), recovering for 4 hours to obtain target materials of 20 wheat shannon, 411 wheat koong 199 and 66 wheat yunnan, and carrying out bombardment by using a gene gun;

the objective plasmid pUbi-TaSARD1 and the plasmid pUbi-GUS were expressed as 1: 1 volume, wrapping on gold powder particles with the diameter of 1 μm, bombarding the target materials of wheat Shannon 20, Jing 411, Kenong 199 and Yumai 66 by PDS-1000/He (American Bio-Rad) according to the method, wherein the bombardment parameters of a gene gun are as follows: the distance between the barrier net and the bombarding material was 5.5cm and the pressure of the splittable film was 1100 Pa).

After the gene gun bombardment is finished, respectively putting the bombarded target material leaves of the wheat Shannon 20, the Jing 411, the Kenong 199 and the Yumai 66 into an incubator to recover and culture for 4 hours (the culture condition is 22 ℃, 16h illumination/18 ℃ and 8h darkness) to obtain the wheat Shannon 20, the Jing 411, the Kenong 199 and the Yumai 66 leaves which are transferred into pUbi-TaSARD1 and pUbi-GUS; standing for 15 hours, and then inoculating spores of wheat powdery mildew physiological race E09;

the inoculation methods are as follows: shaking spores of corresponding microspecies on corresponding wheat leaves to ensure 2 spores/mm²。

Standing and culturing the inoculated leaves of 20 Shannong, 411 Jing, 199 Konong and 66 Yumai for 48 hours (under the culture condition of 22 ℃, 16h of illumination and 8h of darkness at 18 ℃), performing GUS staining, standing overnight at 37 ℃, decoloring after the staining is finished, and observing the cells by using a microscope after two days.

The ratio of the susceptible cells in the GUS-expressing cells was counted to calculate the haustorium index, and the function of the gene TaSARD1 was judged from the haustorium index. The lower the haustorium index, the higher the resistance.

The judgment standard of the disease-resistant cells is as follows: GUS is expressed in cells, the cells do not contain haustoria, and the cells with conidium attached to the surfaces of the cells are disease-resistant cells.

Judgment standard of the infected cells: GUS is expressed in the cells, and the cells containing haustorium in the cells are susceptible cells.

The calculation formula of the sucker index is as follows: the haustorium index is the number of susceptible cells/(number of resistant cells + number of susceptible cells) × 100%.

Wheat Shannon 20 leaf cells (OE-EV) transferred with pUbi-Adaptor-NOS and pUbi-GUS, wheat Jing 411 leaf cells (OE-EV) transferred with pUbi-Adaptor-NOS and pUbi-GUS, wheat Kenong 199(KN199) leaf cells (OE-EV) transferred with pUbi-Adaptor-NOS and pUbi-GUS, and wheat Yumai 66 leaf cells (OE-EV) transferred with pUbi-Adaptor-NOS and pUbi-GUS were used as air controls. The experiment was repeated 3 times and the results averaged.

The results are shown in FIG. 1:

the haustorium index of the wheat shannon 20 leaf cells transferred into pUbi-Adaptor-NOS and pUbi-GUS is 49.61%, and the haustorium index of the wheat shannon 20 leaf cells transferred into pUbi-TaSARD1 and pUbi-GUS is 31.32%;

the haustorium index of the wheat Jing 411 leaf cell transferred with pUbi-Adaptor-NOS and pUbi-GUS is 52.32%, and the haustorium index of the wheat Jing 411 leaf cell transferred with pUbi-TaSARD1 and pUbi-GUS is 36.74%;

the haustorium index of the wheat family agro 199 leaf cells transferred with pUbi-Adaptor-NOS and pUbi-GUS was 51.49%, and the haustorium index of the wheat family agro 199 leaf cells transferred with pUbi-TaSARD1 and pUbi-GUS was 37.93%;

the haustorium index of the wheat Yumai 66 leaf cells transferred into pUbi-Adaptor-NOS and pUbi-GUS is 46.78%, and the haustorium index of the wheat Yumai 66 leaf cells transferred into pUbi-TaSARD1 and pUbi-GUS is 25.89%.

The results show that after TaSARD1 is over-expressed in wheat leaves, the haustorium index is obviously reduced, which indicates that TaSARD1 positively regulates the powdery mildew resistance of wheat.

Second, analysis of transcriptional activation Activity Using TaSARD1 as a transcriptional activator

1. 35S-BD-TaSARD1 vector construction

A forward primer: AAATTTGGATCCATGGCGGCTCACAAGCGGCTC (SEQ ID No.7)

Reverse primer: GGGCCTACTAGT TTAGCTGAAGTTGCTCTGGAAC (SEQ ID No.8)

And carrying out double enzyme digestion on the prepared PCR product by BamHI and XhoI, and then connecting the product with a 35S-BD vector subjected to the same enzyme digestion to obtain a vector 35S-BD-TaSARD 1.

The 35S-BD-TaSARD1 vector is identified by sequencing, and the vector is obtained by inserting the sequence 1 in the sequence table into the enzyme cutting sites of BamHI and XhoI of the 35S-BD vector.

2. Protoplast preparation

Three weeks Columbia type Arabidopsis thaliana young and tender leaves are cut into 1 mm strips by a blade, enzymolysis is carried out for 4 hours at 25 ℃ in the dark, counting is carried out under a microscope by a cell counting plate after a series of operations such as filtration, centrifugation and the like, and generally 2 multiplied by 10 are contained in each 100 mu L⁵And (4) protoplasts.

3. Plasmid transformation and fluorescence value determination

100 μ L of protoplasts were dispensed into 2mL EP tubes and the following plasmids were added: effects 35S-BD-TaSARD 1; CK-: 35S-BD; CK +:35S-BD-VP 16; reporter, 5 XGAL 4-LUC; internal control: Pptrl. The expression of the reporter gene LUC was examined after 16 hours of dark culture at 25 ℃.

Detecting the fluorescence values of firefly luciferase and Renilla luciferase,the ratio of the two (firefly luciferase fluorescence value/Renilla luciferase fluorescence value) is obtained, which is the relative activity of the reporter gene. (details of the detection method are described in the Promega kit Dual-

Reporter Assay System goods number E1910)

As shown in FIG. 2, the relative activity of LUC in protoplasts transformed with 35S-BD-TaSARD1 and 5 XGAL 4-LUC was 2.35;

the relative activity of LUC in protoplasts transformed with 35S-BD and 5 XGAL 4-LUC was 1;

the relative activity of LUC in protoplasts transformed with 35S-BD-VP16 and 5 XGAL 4-LUC was 12.98;

the above results indicate that TaSARD1 can activate the expression of reporter gene, and the fluorescence value is 2.35 times of that of control plasmid 35S-BD, so that TaSARD1 can be determined to have transcription activation activity and be transcription activator.

Example 3 subcellular localization and Erysiphe-induced expression Pattern analysis of TaSARD1

Subcellular localization of TaSARD1

1. Vector construction

A forward primer: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGCTCACAAGCGGCTC (SEQ ID No.9)

Reverse primer: GGGGACCACTTTGTACAAGAAAGCTGGGTCGCTGAAGTTGCTCTGGAAC (SEQ ID No.10)

2) BP reaction is carried out on the PCR product prepared in the step 1) and a pDNOR201 vector (purchased from invitrogen company) to obtain an ENTRY-TaSARD1 vector.

3) The ENTRY-TaSARD1 vector is reacted with pUbi-Gateway-mYFP vector LR to generate pUbi-TaSARD1-mYFP vector.

LR reaction system:

75ng of intermediate vector, 75n of pUbi-Gateway-mYFP vector, 0.5 mu L of LR enzyme and overnight at 25 ℃.

The pUbi-TaSARD1-mYFP vector is identified by sequencing, and the vector is obtained by inserting the sequence 1 in the sequence table into the pUbi-Gateway-mYFP vector.

2. Gene gun-mediated single cell transient transformation technology for identifying subcellular localization condition of TaSARD1

After the 20 wheat shannong seeds are subjected to moisturizing and bud blowing planting, after the seeds grow for about one week, shearing off a first leaf with the leaf surface facing upwards, placing the first leaf on a culture dish containing a culture medium (1% agar and 100mg/L benzimidazole), recovering for 4 hours, and performing gene gun bombardment; wrapping a target plasmid pUbi-TaSARD1-mYFP on gold powder particles with the diameter of 1 μm, bombarding the target material by adopting PDS-1000/He (American Bio-Rad), wherein the bombardment parameters of a gene gun are as follows: the distance between the blocking net and the bombarding material is 5.5cm, and the pressure of the splittable film is 1100 Pa.

After the leaves are bombarded by a gene gun, the leaves are placed in a plant culture chamber to normally grow for 36 hours, DAPI staining is carried out to mark cell nuclei, and the subcellular localization condition of TaSARD1 is observed through different fluorescence channels of a confocal microscope.

As a result, as shown in FIG. 3, the fluorescence of TaSARD1-mYFP completely overlapped the fluorescence of DAPI, so that TaSARD1 was localized in the nucleus.

Second, analysis of powdery mildew-induced expression pattern of TaSARD1

After 20 seeds of wheat shannong are planted through moisturizing and bud blowing, after the seeds grow for about one week, a first leaf is cut off, the leaf surface faces upwards, the seeds are placed on a culture dish containing a culture medium (1% agar, 100mg/L benzimidazole) and recovered for 24 hours, physiological microspecies E09 of erysiphe graminis are inoculated, the materials are obtained at different time points, RNA is extracted, reverse transcription is carried out to obtain cDNA, the cDNA is diluted by 10 times, and 2 mu L is taken for Real time PCR. The Real time PCR system and the program were described in the Promega kit GoTaq-qPCR Master Mix (catalog No. A6001). The control was a non-inoculated treatment.

Primers for Real time PCR were as follows:

a forward primer: CGTGGAGACCGTGCAGGAG (SEQ ID No.11)

Reverse primer: CCGGACCAGCTGGCAGAG (SEQ ID No.12)

The Real time PCR system is as follows:

qPCR Master Mix 5. mu.L, template 2. mu.L, forward primer 0.2. mu.L, reverse primer 0.2. mu.L, CXR 1. mu.L, water 1.6. mu.L.

The Real time PCR program is as follows: the first stage is at 96 deg.C for 2min, the second stage is at 95 deg.C for 15s, and the second stage is at 61 deg.C for 1min, and the dissolution curve is prepared in the third stage after 45 cycles.

The results are shown in fig. 4, the expression of TaSARD1 can be strongly induced by the inoculation treatment of Erysiphe cichoracearum physiological race E09, the expression level is improved by about 1.9 times after 3 hours of inoculation, the expression of TaSARD1 is at a peak after 24 hours of inoculation and reaches 7.8 times when the inoculation is not carried out, the expression level is reduced, and the expression level of TaSARD1 after 36 hours of inoculation is 4.9 times when the inoculation is not carried out.

Sequence listing

<110> Qingdao university

<120> wheat powdery mildew resistance related protein TaSARD1, and coding gene and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

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<213> wheat (Triticum aestivum)

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caggtccaga gtgtgttcct ggtgctggag cctctcctgc gccgagtggt gcaggaggag 180

atccaggcgg ggctggtgcg cagcccgcgg tacatcgaga ggtcgtcgcc ggagacatcg 240

ccggcggcgg agccgcccgc gttgaggctg gcgttcctgt tccagccggc gctgccgatc 300

ttcaccggca gcaagatcga ggacgtgcac ggcgagccgc tccaggtcat cctcgtcgac 360

gcggtcaccg ggtcgccctg cggcgcgctc ccgcagttca tgcgcgtcga gctggtgccg 420

ctcttcgggg acttcccgcc ggacggccgc gaggactgga cgactggcga gttcgcccgc 480

ggcgtcgtca aggagcgcgc ggggaagcgc ccgctcctca ccggcgacgt cggcctcacc 540

atgcgggacg ggcgcgccgt cgtgaacgac ctccagttca ccgacaactc ctcctgggtc 600

cgctgccgca agttccgcat cggcgcgcgc gtgatgccgg gcagctacga gggcggcagg 660

gtcgccgagg ccatgaccga cgccttcaac gtccgtgatc accgcggcga actgtaccgg 720

aagcactacc cgccggcgct caccgacgac gtgtggcggc tggagaagat cggcaaggag 780

ggggccttcc accggaagct gcggcagaac ggcgtggaga ccgtgcagga gttcgtgcgg 840

atgctcaccg tgaggccgga aatactgcgc gcgataatgg gcgacggcat gacggaccgc 900

atgtgggagg tgaccacgag ccacgccaag acgtgcgacg ccggcgacaa ggtgtacgcg 960

tacgccgggc acggcgccac cgtctacgtc aactccctct gccagctggt ccggctcgag 1020

ttcgccggcg tcgagtgcgc ggcgcagcag ctgagcaggg accagaaggc gtacgtgcac 1080

cggctgtacg tggaggcgtt cgagcagcgg cacagcctcc aggaggccga gcccctgccc 1140

gccgccatgc tcctccacgc cagcagcagc agcaacagcc tcccaatgct gcagaacgct 1200

gcgccggtcg cgccgccgcc gcttccggcg acgccgctct ggttccaggg caaccaggag 1260

ctggacctcc agatcgtcga cgagctctcc ggcggccagg gcaactttgg cttccagatg 1320

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Met Ala Ala His Lys Arg Leu His Asp Gly Phe Glu Gln Asp Pro Asp

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Gln Pro Glu Lys Lys Arg Met Glu Arg Ser Val Ser Phe Ser Thr Val

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Ile Arg Glu Ala Met Val Met Lys Gln Val Gln Ser Val Phe Leu Val

35 40 45

Leu Glu Pro Leu Leu Arg Arg Val Val Gln Glu Glu Ile Gln Ala Gly

50 55 60

Leu Val Arg Ser Pro Arg Tyr Ile Glu Arg Ser Ser Pro Glu Thr Ser

65 70 75 80

Pro Ala Ala Glu Pro Pro Ala Leu Arg Leu Ala Phe Leu Phe Gln Pro

85 90 95

Ala Leu Pro Ile Phe Thr Gly Ser Lys Ile Glu Asp Val His Gly Glu

100 105 110

Pro Leu Gln Val Ile Leu Val Asp Ala Val Thr Gly Ser Pro Cys Gly

115 120 125

Ala Leu Pro Gln Phe Met Arg Val Glu Leu Val Pro Leu Phe Gly Asp

130 135 140

Phe Pro Pro Asp Gly Arg Glu Asp Trp Thr Thr Gly Glu Phe Ala Arg

145 150 155 160

Gly Val Val Lys Glu Arg Ala Gly Lys Arg Pro Leu Leu Thr Gly Asp

165 170 175

Val Gly Leu Thr Met Arg Asp Gly Arg Ala Val Val Asn Asp Leu Gln

180 185 190

Phe Thr Asp Asn Ser Ser Trp Val Arg Cys Arg Lys Phe Arg Ile Gly

195 200 205

Ala Arg Val Met Pro Gly Ser Tyr Glu Gly Gly Arg Val Ala Glu Ala

210 215 220

Met Thr Asp Ala Phe Asn Val Arg Asp His Arg Gly Glu Leu Tyr Arg

225 230 235 240

Lys His Tyr Pro Pro Ala Leu Thr Asp Asp Val Trp Arg Leu Glu Lys

245 250 255

Ile Gly Lys Glu Gly Ala Phe His Arg Lys Leu Arg Gln Asn Gly Val

260 265 270

Glu Thr Val Gln Glu Phe Val Arg Met Leu Thr Val Arg Pro Glu Ile

275 280 285

Leu Arg Ala Ile Met Gly Asp Gly Met Thr Asp Arg Met Trp Glu Val

290 295 300

Thr Thr Ser His Ala Lys Thr Cys Asp Ala Gly Asp Lys Val Tyr Ala

305 310 315 320

Tyr Ala Gly His Gly Ala Thr Val Tyr Val Asn Ser Leu Cys Gln Leu

325 330 335

Val Arg Leu Glu Phe Ala Gly Val Glu Cys Ala Ala Gln Gln Leu Ser

340 345 350

Arg Asp Gln Lys Ala Tyr Val His Arg Leu Tyr Val Glu Ala Phe Glu

355 360 365

Gln Arg His Ser Leu Gln Glu Ala Glu Pro Leu Pro Ala Ala Met Leu

370 375 380

Leu His Ala Ser Ser Ser Ser Asn Ser Leu Pro Met Leu Gln Asn Ala

385 390 395 400

Ala Pro Val Ala Pro Pro Pro Leu Pro Ala Thr Pro Leu Trp Phe Gln

405 410 415

Gly Asn Gln Glu Leu Asp Leu Gln Ile Val Asp Glu Leu Ser Gly Gly

420 425 430

Gln Gly Asn Phe Gly Phe Gln Met Phe Gln Ser Asn Phe Ser

435 440 445

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cttcttcctc ctccttccag 20

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gaaatgctag aaaaaggtga ag 22

<210> 5

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggggacaagt ttgtacaaaa aagcaggctt catggcggct cacaagcggc t 51

<210> 6

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggggaccact ttgtacaaga aagctgggtc ttagctgaag ttgctctgga a 51

<210> 7

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaatttggat ccatggcggc tcacaagcgg ctc 33

<210> 8

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gggcctacta gtttagctga agttgctctg gaac 34

<210> 9

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggggacaagt ttgtacaaaa aagcaggctt catggcggct cacaagcggc tc 52

<210> 10

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggggaccact ttgtacaaga aagctgggtc gctgaagttg ctctggaac 49

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgtggagacc gtgcaggag 19

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccggaccagc tggcagag 18

Claims

1. The application of the wheat powdery mildew resistance related protein in cultivating transgenic wheat with wheat powdery mildew disease resistance is characterized in that the amino acid sequence of the wheat powdery mildew resistance related protein is shown as SEQ ID No.2, and the coding gene for expressing the wheat powdery mildew resistance related protein in the wheat is shown as SEQ ID No. 1.

2. The use of claim 1, wherein the gene encoding the powdery mildew resistance-associated protein is introduced into wheat, which is a plant, to obtain powdery mildew-resistant transgenic wheat.

3. The use according to claim 2, wherein the gene encoding the powdery mildew resistance-associated protein is introduced via a recombinant expression vector inserted into a pUbi-Adaptor-NOS vector.

4. An application of a wheat powdery mildew resistance related protein as a transcription activating factor is disclosed, wherein the amino acid sequence of the wheat powdery mildew resistance related protein is shown as SEQ ID No.2, and a coding gene for expressing the wheat powdery mildew resistance related protein is shown as SEQ ID No. 1.

5. The use of claim 4, wherein the gene encoding the protein associated with powdery mildew resistance is inserted into the 35S-BD vectorBamHI andSpeand (I) obtaining a recombinant vector between enzyme cutting sites.