Wheat Urala NDR1 / HIN1 Like gene TuNHL and application thereof

Abstract

The invention discloses an NDR1 / HIN1 Like gene TuNHL of Wuraili wheat and an application of the NDR1 / HIN1 Like gene TuNHL. The nucleotide sequence of the Wurata wheat NDR1 / HIN1 Like gene TuNHL is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein coded by the Wurata wheat NDR1 / HIN1 Like gene TuNHL is as shown in SEQ ID NO. 2. According to the present invention, the Wurata wheat NDR1 / HIN1 Like gene TuNHL is indispensable for the normal resistance function of the wheat stripe rust resistance function gene YrU1, and the powdery mildew resistance of wheat can be significantly improved by the single-cell instantaneous overexpression of the TuNHL on the common wheat powdery mildew susceptible leaf; overexpression of TuNHL in the arabidopsis thaliana clo ecotype can also significantly improve the powdery mildew resistance of arabidopsis thaliana. Therefore, the Wurata wheat NDR1 / HIN1 Like gene TuNHL has relatively high application value and potential in plant resistance breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and specifically relates to a kind of Urartu wheat NDR1 / HIN1 Like Gene TuNHL And the application of this gene in plant resistance breeding. Background Art

[0002] Common wheat ( Triticum aestivum ) is one of the most important staple crops in the world. Nearly half of the world's population relies on wheat for food. However, wheat faces serious threats from diseases such as stripe rust, powdery mildew and fusarium head blight during its growth, which greatly threatens the yield and quality of wheat. Wheat stripe rust is caused by Puccinia striatum ( Puccinia striiformis f. sp. tritici , Pst ) is a parasitic fungal disease that infects wheat leaves and other parts, and wheat powdery mildew is caused by Blumeria graminis wheat-specific type ( Blumeria graminis formaspecialis tritici ) are both parasitic pathogenic fungi. Both wheat stripe rust and powdery mildew can harm wheat and its closely related species. After the pathogens infect and spread, the hyphae parasitic in the plant will compete with the host for nutrients, and then the large number of conidia produced by the pathogen will cover the leaf surface, hindering the photosynthesis of the host plant. The use of chemical spraying to control stripe rust and powdery mildew not only greatly increases the cost of grain production, but also causes excessive pesticide residues in food and serious deterioration of the ecological environment. Relatively speaking, cultivating and promoting the planting of wheat varieties that have excellent pathogen resistance is obviously an effective, economical and environmentally friendly response strategy. The top priority for cultivating resistant wheat varieties is to step up the search and excavation of excellent resistance genes in local wheat varieties and their closely related species and study their resistance mechanisms, which is also the key to the success of resistance breeding.

[0003] Urartu wheat ( Triticum urartu ) is the original ancestor of the A genome of common wheat and belongs to the diploid wild einkorn wheat. Compared with Aegilops pseudospeltii and Aegilops tauschii (the two are the donor ancestor species of the B and D genomes of common wheat, respectively), Triticum urartuThe characteristics of plant and ear morphological development are closer to hexaploid common wheat. A representative material of Urartu wheat, G1812, contains 7 pairs of chromosomes, and its whole genome sequence has been sequenced and published. The genome size of G1812 is about 5.6 G, and the functional genes have been basically annotated. PI428309 is another Urartu wheat material. Although its genome sequence information is unknown, it has been found through resistance screening that it has excellent resistance to wheat powdery mildew and wheat stripe rust. By constructing a genetic positioning population between PI428309 and the susceptible Urartu wheat material G1812, and using experimental methods such as BSR, BSE and other population segregation analysis, complementary resistance verification, etc., several major and dominant resistance functional genes have been cloned and reported in the resistant material PI428309. Among them is the wheat powdery mildew resistance functional gene Pm60 , which encodes a typical CC-NBS-LRR (CNL) protein; the other resistance gene is YrU1 , which encodes a special CNL protein with an ankyrin domain at the N-terminus and a WRKY structure at the C-terminus. This protein confers resistance to wheat stripe rust in Urartu wheat PI428309. The discovery of these genes provides an excellent source of resistance in wheat resistance breeding.

[0004] At present, the number of resistance genes that can be effectively applied to wheat resistance breeding is still very limited, and the mechanism of these resistance genes regulating immune response is not yet deeply understood. Current research results generally believe that the innate immune system of plants against pathogens includes two levels, namely, pattern-triggered immunity (PTI) triggered by pathogen- or damage-related molecular patterns and effector-triggered immunity (ETI) activated by effector factors secreted by pathogens. The two also have a mutually synergistic and promoting relationship. Therefore, the plant immune system involves a large number of resistance-related genes and complex immune pathways. In order to cultivate excellent disease-resistant wheat varieties, we urgently need to step up the exploration and cloning of more wheat resistance-related genes and conduct in-depth research on the mechanism of their participation in immune response.

[0005] Based on the above background, we conducted transcriptome sequencing analysis on samples of Urartu wheat material PI428309 before and after inoculation with wheat powdery mildew and found a NDR1 / HIN1 Like Gene TuNHL The transcription level increased significantly after inoculation with powdery mildew. Comparison with the genome sequence of Urartu wheat material revealed that important disease resistance-related genes in Arabidopsis NDR1 (Non-race-specific disease resistance 1) and TuNHLThe similarity is the highest, and it may be a homologous gene. Through single-cell transient overexpression and stable transformation of Arabidopsis, it was found that TuNHL It plays an important role in wheat powdery mildew resistance and Arabidopsis powdery mildew resistance. In addition, after inoculation with wheat stripe rust, the Urartu wheat material PI428309 TuNHL The transcription level of did not change significantly. However, virus-induced gene silencing experiments confirmed that TuNHL Wheat stripe rust resistance genes of Urartu wheat material PI428309 YrU1 It is essential to exert resistance function. TuNHL Participated YrU1 Mediated resistance to stripe rust. TuNHL The present invention provides a new and efficient resistance gene for wheat resistance breeding, and also provides a new opportunity for further exploration of wheat stripe rust resistance genes. YrU1 It lays the foundation for the mechanism of regulating immune response. Summary of the invention

[0006] The object of the present invention is to provide a kind of Urartu wheat NDR1 / HIN1 Like Gene TuNHL and its application, to advance the understanding of the mechanism of plant disease resistance genes regulating immune response, thereby providing novel and efficient resistance source genes for crop resistance breeding, and achieving the goal of cultivating excellent resistant varieties.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: The present invention first provides a kind of Urartu wheat E NDR1 / HIN1 Like Gene TuNHL , the Urartu wheat NDR1 / HIN1 Like Gene TuNHL The nucleotide sequence of the coding region is shown in SEQ ID NO.1.

[0008] The present invention also provides a kind of Urartu wheat NDR1 / HIN1 Like Gene TuNHL The encoded protein has an amino acid sequence as shown in SEQ ID NO.2.

[0009] The present invention also provides a kind of Urartu wheat containing the above NDR1 / HIN1 Like Gene TuNHL of the recombinant vector.

[0010] The present invention also provides the above-mentioned Urartu wheat NDR1 / HIN1 Like Gene TuNHL Application in constructing powdery mildew-resistant wheat varieties.

[0011] The present invention also provides the above-mentioned Urartu wheat NDR1 / HIN1 Like Gene TuNHL Application in constructing powdery mildew-resistant Arabidopsis materials.

[0012] The present invention also provides the above-mentioned Urartu wheat NDR1 / HIN1 Like Gene TuNHL Weakening disease resistance genes YrU1 Application of mediated resistance to wheat stripe rust.

[0013] Furthermore, the wheat varieties include Urartu wheat PI428309 and common wheat.

[0014] The beneficial effects of the present invention are: The present invention cloned in Urartu wheat PI428309 NDR1 / HIN1 Like Gene TuNHL , which is for the stripe rust resistance gene in Urartu wheat PI428309 YrU1 In addition, Urartu wheat NDR1 / HIN1 Like Gene TuNHL It can also exert powdery mildew resistance function in hexaploid wheat and Arabidopsis, indicating that this gene can play a resistance role not only in wheat plants, but also in dicotyledonous model plants. NDR1 / HIN1 Like Gene TuNHL It has resistance to wheat stripe rust and powdery mildew and can exert resistance in the heterologous plant Arabidopsis thaliana, and has important application value in the field of plant disease resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Urartu wheat NDR1 / HIN1 Like Gene TuNHL Phylogenetic tree analysis (a) and sequence comparison (b) of the protein sequence with Arabidopsis NDR1 and NDR1 homologous proteins in other plants.

[0016] Figure 2 : Silence of Urartu Wheat NDR1 / HIN1 Like Gene TuNHL Puccinia striiformis CYR33 The resistance of barley streak virus was significantly weakened; (a) Barley streak virus induced silencing of Urartu wheat PI428309 TuNHL Puccinia striiformis CYR33 Observe the phenotype after 14 days; (b) Detect the phenotype of each sample before inoculation with stripe rust TuNHL Expression level (** indicates extremely significant difference, P < 0.01, using t test).

[0017] Figure 3:BSMV: GFP and BSMV: TuNHL Inoculated with wheat stripe rust after infection with Urartu wheat PI428309 CYR33 Histological observation of fungal growth; (a) Urartu wheat PI428309 was infected with BSMV: GFP and BSMV: TuNHL Infection, inoculation with wheat stripe rust CYR33 , Wheat germ agglutinin (WGA) staining was used to observe the structural morphological changes of substomatal vesicles (SSV), infected hyphae (IH), haustorium mother cells (HMC), and haustorium (H) after the stripe rust grew on wheat leaves at 2 dpi, 3 dpi, and 5 dpi; (b) BSMV was counted 2 dpi after the stripe rust was inoculated: GFP and BSMV: TuNHL After treatment, the numbers of hyphae branches (HB), haustoria mother cells (HMC) and haustoria (H) of stripe rust were counted; (c) 3 dpi after inoculation with stripe rust, the length of mycelium infected by stripe rust was counted (P<0.001); (d) 5 dpi after inoculation with stripe rust, the area of ​​mycelium infection was counted (P<0.05).

[0018] Figure 4 : Overexpression TuNHL Can enhance wheat's resistance to powdery mildew; (a) Urartu wheat PI428309 inoculated with powdery mildew Bgt After E09 TuNHL Transcription levels; (b) Single-cell transient expression in leaves of common wheat Fielder TuNHL Can significantly reduce its vaccination Bgt The haustorium index after E09 improves the resistance to powdery mildew.

[0019] Figure 5 : Overexpression TuNHL It can enhance the powdery mildew resistance of Arabidopsis thaliana; (a) The phenotype of Arabidopsis thaliana grown for four weeks after inoculation with powdery mildew for 7 days, WT is Arabidopsis thaliana Columbia ( Col ) ecotype, among which TuNHL-1 # and TuNHL-4 # For overexpression TuNHL Arabidopsis transgenic T 2 (a) Trypan blue staining results of Arabidopsis leaves inoculated with powdery mildew for 7 days, Bars = 100 µm; (c) Statistical results of conidiophores of Arabidopsis materials grown for four weeks after quantitative inoculation with powdery mildew for 5 days. ndr1 Arabidopsis thaliana NDR1 Gene mutants; (d) Trypan blue staining results of Arabidopsis leaves inoculated with bacteria for 5 days, Bars = 100µm. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate and explain the present invention, but are not used to limit the scope of the present invention.

[0021] The present invention discovered that Urartu wheat NDR1 / HIN1 Like Gene TuNHL After inoculation with wheat stripe rust, the expression level did not change significantly, but it was silent. TuNHL Can significantly weaken YrU1 Mediated resistance to stripe rust. TuNHL After inoculation with wheat powdery mildew, the expression level increased significantly, and the gene was transiently overexpressed on wheat leaves. TuNHL It can also significantly enhance the powdery mildew resistance of wheat; and it can be stably overexpressed in Arabidopsis TuNHL , and can also significantly improve the powdery mildew resistance of Arabidopsis thaliana. The present invention proves TuNHL It plays an important role in wheat stripe rust, powdery mildew resistance and Arabidopsis powdery mildew resistance. In order to facilitate the reader to deeply understand the content and purpose of the present invention, the specific technical implementation steps of the present invention will be described in detail through the following examples. The technical means described in the examples are conventional means familiar to people in the field. The disease resistance or susceptibility described in the following examples refers to wheat stripe rust, wheat powdery mildew or Arabidopsis powdery mildew; stripe rust refers to wheat stripe rust, caused by wheat stripe rust ( Puccinia striiformis f. sp. tritici , Pst ), unless otherwise specified, wheat stripe rust refers to the wheat stripe rust strain CY33 prevalent in northwest China ( Pst CYR33). The powdery mildew refers to wheat powdery mildew and Arabidopsis powdery mildew, which are caused by Blumeria graminis ( Blumeria graminis f.sp. tritici , Bgt ) and Arabidopsis thaliana powdery mildew ( Golovinomyces cichoracearum ). Unless otherwise specified, wheat powdery mildew refers to the wheat powdery mildew strain E09 prevalent in northern China ( Bgt E09). The Arabidopsis thaliana mentioned in this article is Arabidopsis thaliana Columbia ( Col ) Ecological type.

[0022] Example 1 Urartu wheat material PI428309 NDR1 / HIN1 Like Gene TuNHL Clone Using the published genome sequence of common wheat Chinese Spring as a reference, we discovered proteins that play a key role in the immune response of Arabidopsis thaliana NDR1(Non-race-specific disease resistance 1) Similar genes in common wheat Chinese spring are TraesCS2A02G218400.1, TraesCS2B02G243800.1 and TraesCS2D02G225000.1. By comparing the transcriptome database of Urartu wheat PI428309, the homologous genes of TraesCS2A02G218400.1 in Urartu wheat PI428309 were preliminarily obtained. TuNHL To obtain the mRNA sequence of Urartu wheat PI428309 TuNHL Full-length gene, design specific primers ( TuNHL -F and TuNHL -R), obtained by reverse transcription with Promega kit and high-fidelity amplification with KOD-Fx DNA polymerase (TOYOBO) TuNHL Using online coding region prediction software, we can obtain TuNHL The coding region sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. TuNHL -F and TuNHL The nucleotide sequence of -R is as follows: TuNHL -F:5'-ATCGACCAGCCATGCCGCGGAACTA-3', TuNHL -R:5'-TCAGCGCCCTTTGATGCTCACGT-3'.

[0023] After the Urartu wheat PI428309 grew to the one-leaf-one-heart stage, it was inoculated with wheat powdery mildew E09. The leaves of the Urartu wheat PI428309 were sampled at 0 h and 36 h after inoculation, and transcriptome sequencing analysis was performed. The results showed that after inoculation with wheat powdery mildew, TuNHL The transcription level of TuNHL The transcription levels at 24 h, 48 h, 72 h, and 96 h after inoculation with wheat powdery mildew were found to be the highest at 48 h, while the transcription levels at each time point after inoculation with wheat stripe rust CY33 were TuNHL There was no significant difference in the transcription levels.

[0024] TuNHL has very similar amino acid sequences to NDR1 analogs in the wheat tribe, such as TraesCS2A02G218400.1, but they have very low sequence similarity to Arabidopsis NDR1. Figure 1The amino acid sequence comparison results of Arabidopsis NDR1, TuNHL and other Poaceae NDR1-like proteins are shown, and their phylogenetic relationships are analyzed.

[0025] Example 2 TuNHL Regulation of Urartu wheat PI428309 YrU1 Wheat stripe rust resistance RNA from leaves of Urartu wheat PI428309 was reverse transcribed to obtain cDNA, which was used as a template and the primers γ- TuNHL -F and γ- TuNHL -R was amplified to obtain the PCR product ( TuNHL The Barley stripe mosaic virus RNAγ (BSMV) vector was digested with restriction endonuclease Nhe Ι to obtain a linearized plasmid carrying restriction sites. The Vazyme ClonExpress® II One Step Cloning Kit C112 homologous recombination kit was used to TuNHL The sequence fragment was connected with the linearized plasmid by homologous recombination to construct the silencing vector BSMV: TuNHL In the same way, a BSMV vector with a GFP fragment was prepared: GFP As a control group. Referring to the experimental method described in the literature [Holzberg S, Brosio P, Gross C, Pogue GP. 2002. Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J 30: 315-327], the constructed silencing vector BSMV: TuNHL Infect the leaves of the plants and then detect the fourth leaf of the infected plants. TuNHL The expression level of γ-1 was measured and the stripe rust resistance phenotype was detected by inoculation with wheat stripe rust fungus CY33. The Urartu wheat material G1812 susceptible to wheat stripe rust fungus CY33 was used as a negative control, and the Urartu wheat material PI428309 (CK) not treated with the virus was used as a positive control. TuNHL -F and γ- TuNHL The sequence of -R is as follows: γ- TuNHL -F:5'-TTTTTTTTTTTTTAGCTAGCTGGCAGCGCTTCGCCAGCGA-3', γ- TuNHL-R: 5'-GATTCTTCTTCCGTTGCTAGCCTAGAAGTAGAGCGTGCACTGC-3'.

[0026] The experimental results are as follows Figure 2 As shown, relative to the control group BSMV: GFP , with BSMV: TuNHL Infection of Urartu wheat PI428309 can interfere with TuNHL After inoculation with wheat stripe rust CY33, TuNHL It can significantly reduce the stripe rust resistance of Urartu wheat PI428309. This experiment proves TuNHL Adjustable Urartu wheat PI428309 YrU1 Mediated resistance to stripe rust.

[0027] After inoculation with wheat stripe rust, samples were taken at 2 dpi, 3 dpi, and 5 dpi, and the fungal structure in the leaves was stained with wheat germ agglutinin (WGA). The germination of stripe rust spores, hyphae length, and hyphae infection area were then counted. Figure 3 As shown, 2 days after inoculation, BSMV: TuNHL BSMV group and control group: GFP Compared with the control group, the number of HB (hyphae branches), HMC (haustoria mother cells), and H (haustoria) all showed significant changes; 3 days after inoculation, BSMV: TuNHL BSMV group and control group: GFP Compared with the control group, the length of mycelium germination increased significantly; 5 days after inoculation, BSMV: TuNHL BSMV group and control group: GFP The mycelial infection area was significantly larger than that of TuNHL Down-regulation of transcription levels is beneficial to the development of stripe rust. TuNHL Stripe rust resistance gene in Urartu wheat PI428309 YrU1 Plays an important role in mediating immune responses.

[0028] Example 3 Overexpression in common wheat TuNHL Enhances resistance to powdery mildew Transcriptome sequencing analysis revealed that the Urartu wheat material PI428309 had a significant difference in the number of samples before and after inoculation with wheat powdery mildew. TuNHL The transcription level of α was significantly increased after inoculation with powdery mildew. TuNHL The same conclusion can be drawn at the transcription level ( Figure 4a). Referring to the experimental method described in [Shen QH, Saijo Y, Mauch S, Biskup C,Bieri S, Keller B, Seki H, Ülker B, Somssich IE,Schulze-Lefert P. 2007. Nuclear Activity of MLA Immune Receptors Links Isolate-Specific and BasalDisease-Resistance Responses. Science 315: 1098-1103.], single cell transient overexpression experiment was performed to TuNHL The powdery mildew resistance function of the gene was verified by single-cell transient overexpression mediated by gene gun on leaves of common wheat Fielder susceptible to powdery mildew. TuNHL (Connect the system with gateway TuNHL The coding region was connected to the intermediate vector pDONR207 and then transferred to the final vector pUBI-GW for amplification. TuNHL The primers used for the coding region of TuNHL -attB-F and TuNHL -attB-R), inoculated with Blumeria graminis E09 4 h after expression, and stained with GUS 48 h later, and the haustoria index was statistically analyzed. TuNHL -attB-F and TuNHL The nucleotide sequence of -attB-R is as follows: TuNHL -attB-F: 5'-GGGGACAAGTTGTACAAAAAAGCAGGCTTCATGCCGCGGAACTACCGC-3', TuNHL -attB-R: 5'-GGGGACCACTTGTTGTACAAGAAAGCTGGGTCCTAGAAGTAGAGCGTGCA-3'.

[0029] From the experimental results, we can know that ( Figure 4 b) Overexpression TuNHL Compared with the control treatment (overexpression of PGY protein), the powdery mildew haustoria index on the leaves of wheat Fielder susceptible to powdery mildew was significantly reduced, thereby improving the powdery mildew resistance of wheat.

[0030] Example 4 Heterologous Overexpression TuNHL Enhancing powdery mildew resistance in Arabidopsis Construct driven by the constitutive CaMV 35S promoter NDR1 / HIN1 Like Gene TuNHLThe plant expression vector pYBA 1143 was used to introduce the Agrobacterium GV3101 strain by freeze-thaw method, and Arabidopsis thaliana was genetically transformed by inflorescence infiltration method. The positive plants were screened with the herbicide Basta, and the positive plants were tested for TuNHL The expression level of Arabidopsis mutant ndr1 , Col For control.

[0031] To observe heterologous overexpression TuNHL Effect on the powdery mildew resistance of Arabidopsis thaliana. The transgenic Arabidopsis thaliana at 4 weeks old was inoculated with powdery mildew. The results showed that ( Figure 5 ), 7 days after inoculation, wild-type Arabidopsis Col The leaf surface of WT was densely covered with conidia and no cell death was found. TuNHL There was obvious cell death at the locations infected by powdery mildew in the transgenic lines ( Figure 5 a); Trypan blue staining revealed ( Figure 5 b), TuNHL The number of powdery mildew conidiophores and hyphae on the leaves of transgenic plants was significantly reduced, and multiple cell deaths were observed. Quantitative inoculation of Arabidopsis powdery mildew and statistical analysis also found that at 4 weeks of age, 2 The number of conidiophores produced by the germination of a single powdery mildew spore on the leaves of the positive transgenic plants (50 single spore colonies were measured for each material) was significantly less than that of the wild-type Arabidopsis. Col and ndr1 Mutants ( Figure 5 cd). The above experimental data show that heterologous expression in Arabidopsis TuNHL It can induce cell death in Arabidopsis leaves, inhibit the invasion and growth of pathogens, and enhance Arabidopsis's resistance to powdery mildew.

[0032] The above content has been described in detail through general description and specific implementation schemes. For those in the industry in this field, it is easy to make some changes and derivatives to the present invention, so any changes made on the basis of not deviating from the basic content of the present invention belong to the scope of protection claimed by the present invention.

Claims

1. A kind of Urartu wheat NDR1 / HIN1 Like Gene TuNHL , characterized in that: The Urartu wheat NDR1 / HIN1 Like Gene TuNHL The nucleotide sequence is shown in SEQ ID NO.

1.

2. The Urartu wheat of claim 1 NDR1 / HIN1 Like Gene TuNHL The encoded protein is characterized by: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A kind of wheat containing the Urartu wheat according to claim 1 NDR1 / HIN1 Like Gene TuNHL of the recombinant vector.

4. Overexpression of the Urartu wheat of claim 1 NDR1 / HIN1 Like Gene TuNHL Application in constructing powdery mildew-resistant wheat varieties.

5. Overexpression of the Urartu wheat of claim 1 NDR1 / HIN1 Like Gene TuNHL Application in constructing powdery mildew-resistant Arabidopsis materials.

6. The Urartu wheat of claim 1 NDR1 / HIN1 Like Gene TuNHL Weakening disease resistance genes Yj Y Application of mediated resistance to wheat stripe rust.

7. The use according to claim 4, characterized in that: The wheat varieties include Urartu wheat PI428309 and common wheat.

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