Application of wheat negative regulation anti-disease gene TaZF10 in gibberellic disease resistance
By targeting the wheat negative regulatory disease resistance gene TaZF10 using CRISPR-Cas9 gene editing technology, the problem of scarce wheat scab resistance resources was solved, and a loss-of-function mutant that significantly improves scab resistance was created, achieving durable resistance to scab in wheat.
Patent Information
- Application Number
- CN202511092248.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-05
- Publication Date
- 2025-12-19
AI Technical Summary
There is a lack of germplasm resources resistant to wheat scab, traditional breeding has progressed slowly, chemical fungicides have led to resistance, and the resistance of existing varieties is unstable, making it difficult to meet production needs.
By revealing the functional mechanism of the wheat negative regulation of the disease resistance gene TaZF10, we used CRISPR-Cas9 gene editing technology to construct a gene editing vector that targets TaZF10, causing a frameshift mutation that causes the protein TaZF10 to lose its function, thus creating a loss-of-function mutant.
It significantly improves wheat's resistance to Fusarium head blight, reduces the severity of the disease by about 50%, breaks the pathogen's interference with the host's defense network, and provides durable disease-resistant material.
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Figure CN121160792A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology and relates to wheat negative regulatory disease resistance genes. TaZF10 in Application in the treatment of Fusarium head blight. Background Technology
[0002] Wheat scab (Fusarium head blight, FHB) is caused by Fusarium graminearum (… Fusarium graminearum Fusarium head blight (FHB) and its complexes cause fungal diseases that seriously threaten wheat production. This pathogen is saprophytic and parasitic, surviving on plant debris or living hosts. Its infection not only directly leads to wheat yield loss and quality decline, but also poses a significant risk to human health and livestock due to the contamination of grains with trichothecene toxins such as deoxynivalenol (DON). The life cycle of Fusarium head blight involves both asexual and sexual reproduction stages. Ascospores produced on diseased plant debris in the field are the main primary source of infection for wheat ears during the spring flowering stage, a characteristic that exacerbates the complexity of disease transmission. Currently, Fusarium head blight control mainly relies on chemical fungicides, but long-term use has led to widespread resistance of Fusarium head blight to benzimidazole fungicides (such as carbendazim) and QoI fungicides, significantly reducing control effectiveness. Traditional breeding methods have progressed slowly due to the quantitative genetic nature of wheat resistance to Fusarium head blight, which is controlled by multiple genes, and the scarcity of resistance gene resources makes the selection of highly resistant varieties difficult. Existing disease-resistant varieties generally suffer from problems such as unstable resistance or unfavorable linkage with agronomic traits, making it difficult to meet production needs.
[0003] Breeding disease-resistant varieties is the most economical, sustainable, and effective method for controlling wheat scab. However, the scarcity of scab-resistant germplasm resources severely hinders the breeding and application of resistant varieties. Traditional disease-resistant breeding mainly relies on the discovery and utilization of disease-resistant genes. In recent years, the functions of negatively regulating disease-resistant genes and susceptible genes have gradually gained attention. Negatively regulating disease-resistant genes usually play a role in the trade-off between disease resistance and quality. Some negatively regulating disease-resistant genes are directly targeted and manipulated by pathogen effector proteins, becoming susceptible genes. Others are not directly utilized by pathogens, but other genes in their interaction network can be targeted by pathogens. Regardless of how pathogens establish contact with negatively regulating disease-resistant genes, interfering with pathogen utilization of these genes through gene editing and other means is an effective way to improve wheat scab resistance. Discovering genes that negatively regulate scab resistance and modifying them using gene editing and other means can provide important molecular targets for creating broad-spectrum, durable scab-resistant materials, and is a key step in promoting molecular design breeding for scab resistance. Summary of the Invention
[0004] The scarcity of genetic resources for resistance to Fusarium head blight in wheat severely restricts the effectiveness of integrated disease control. This invention breaks away from the traditional model of relying on the discovery of resistance sources for Fusarium head blight, focusing instead on the discovery and targeted modification of genes that negatively regulate resistance. By elucidating the function of key negatively regulating resistance genes in Fusarium head blight resistance and applying CRISPR-Cas9 gene editing technology to precisely create loss-of-function mutants, wheat materials with significantly enhanced resistance to Fusarium head blight are obtained. This invention provides more strategies and ideas for enriching resistance and susceptibility gene resources and creating durable Fusarium head blight-resistant materials.
[0005] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the protein TaZF10 described in this invention is represented by "TaZF10" in non-italicized font, and the gene... TaZF10 Use italic font TaZF10 This indicates that, of course, those skilled in the art can clearly and completely understand the meaning and description of the relevant genes and their encoded proteins based on the description in this invention.
[0006] On one hand, the present invention relates to a method for obtaining plants resistant to Fusarium head blight, comprising: making a gene... TaZF10 Frameshift mutations may cause the protein TaZF10 to lose its function; The gene TaZF10 The protein TaZF10 is encoded by this protein; The amino acid sequence of the protein TaZF10 is shown in SEQ ID NO: 1.
[0007] SEQ ID NO: 1 is as follows: MSGNDSQAAADRIKAQALSNAKGLSRAQAERAAEAAARNVNAYGQKEEGPSRWQERKEAKRQMYLMSTEKAVRLGVRPNAAPASATAGGQCQKCFQPGHWTYECKNERVYISRPSRTDQLKDPRLKKAWLPAPS QFVNPDLEKEMEEERKLMREKLKKEKSEKRKARSKSKSKRKHRALVSDSDSESSGTGSEYSSESGSSSYSSSDSEDKKKRRRKTMQKKRRHRRDTSTSSSSSRSESESESDSDSDGKGSRKKSKKRGDKRRS.
[0008] Furthermore, in the method for obtaining plants resistant to Fusarium head blight provided by the present invention, the gene... TaZF10 The nucleotide sequence is shown in SEQ ID NO: 2.
[0009] SEQ ID NO: 2 is as follows: .
[0010] Furthermore, in the method for obtaining plants resistant to Fusarium head blight provided by the present invention, the plant is wheat.
[0011] Furthermore, in the method for obtaining Fusarium head blight-resistant plants provided by the present invention, the pathogen of Fusarium head blight is Fusarium graminearum. Fusarium graminearum .
[0012] Furthermore, in the method for obtaining plants resistant to Fusarium head blight provided by the present invention, the aforementioned... TaZF10 A gene editing vector is used to introduce the gene editing vector into the plant.
[0013] Furthermore, in the method for obtaining plants resistant to Fusarium head blight provided by the present invention, the gene editing vector targets the gene. TaZF10 gRNA1 and gRNA2 in the cell; The nucleotide sequence of the gRNA1 is shown in SEQ ID NO: 3; The nucleotide sequence of the gRNA2 is shown in SEQ ID NO: 4.
[0014] SEQ ID NO: 3 is as follows: CAGGCCTTATCAAATGCAAA.
[0015] SEQ ID NO: 4 is as follows: CTCGCAATGTCAATGCCTAT.
[0016] On the other hand, the present invention relates to a plant resistant to Fusarium head blight, which is obtained by the method described above for obtaining a plant resistant to Fusarium head blight.
[0017] On the other hand, this invention relates to genes. TaZF10 Application in the prevention and control of Fusarium head blight, gene TaZF10 Negative regulation of wheat resistance to Fusarium head blight; Make genes TaZF10 Frameshift mutations may cause the protein TaZF10 to lose its function; The gene TaZF10 The protein TaZF10 is encoded by this protein; The amino acid sequence of the protein TaZF10 is shown in SEQ ID NO: 1.
[0018] Furthermore, in the gene provided by this invention TaZF10 In the application of the gene in the prevention and control of Fusarium head blight TaZF10 The nucleotide sequence is shown in SEQ ID NO: 2.
[0019] Furthermore, in the gene provided by this invention TaZF10 In the application of Fusarium head blight control, the plant in question is wheat, and the pathogen causing Fusarium head blight is *Fusarium graminearum*. Fusarium graminearum .
[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention reveals the negative regulatory genes for disease resistance in wheat. TaZF10This invention utilizes CRISPR-Cas9 gene editing technology to explore the functional mechanism of wheat and provides a technical solution for enhancing wheat resistance to Fusarium head blight. The invention verifies the gene's... TaZF10 The negative regulation of wheat resistance to Fusarium head blight involves the encoding protein TaZF10, which interacts with the Fusarium head blight resistance protein TaSnRK1α. Furthermore, TaSnRK1α promotes the degradation of TaZF10 by interfering with its stability. This invention utilizes a specific gRNA targeting gene. TaZF10 Constructing a CRISPR-Cas9 editing vector to create genes TaZF10 Frameshift mutations cause the TaZF10 protein to lose function, creating a loss-of-function mutant. Field disease resistance assessments show that... TaZF10 Editing the plant significantly reduced the incidence of Fusarium head blight by about 50%, demonstrating that targeted modification of this negative regulatory gene can effectively break the pathogen's interference with the host's defense network. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 It's a gene. TaZF10 Electrophoresis results. The left side shows the 2K marker bands, and the right side shows... TaZF10 The band size is 804 bp.
[0023] Figure 2 This is a Western blot result of the protein immunoprecipitation technique based on tobacco in vivo to verify the interaction between TaSnRK1α and TaZF10.
[0024] Figure 3 This figure shows the protein degradation validation results based on the tobacco transient expression system.
[0025] Figure 4 yes TaZF10 A schematic diagram of a gene-editing vector structure. (The diagram shows...) TaZF10 The gene-editing vector contains the following core elements linked in sequence: the maize ubiquitin promoter (ZmUbi), the N-terminal nuclear localization signal (NLS), and Streptococcus pyogenes. Cas9 Gene( spCas9The sequence includes the C-terminal nuclear localization signal (NLS), the caustic acid synthase terminator (NOS), the wheat U6.1 promoter (TaU6.1), the first guide RNA sequence (gRNA1), the PolyT termination sequence, the wheat U6.3 promoter (TaU6.3), the second guide RNA sequence (gRNA2), and the PolyT termination sequence.
[0026] Figure 5 for TaZF10 Electrophoretic detection results of PCR amplification products of gRNA1 / gRNA2, the target sites in gene-edited plants. Lane labeling: "M" indicates 2K Marker; "L1-L8" are 8 independent lanes. TaZF10 Genomic DNA of the gene-edited strain; "Fielder" is wild-type non-transgenic wheat; "H2O" is a template-free negative control.
[0027] Figure 6 for TaZF10 Phenotypic and disease incidence statistics of Fusarium head blight inoculation in the field of gene-edited lines. "Fielder" represents the wild-type control; TaZF10 -KO" for TaZF10 Gene-edited mutant lines; Figure 6 The 'a' in the image represents a photograph of wheat. Figure 6 In this text, 'b' represents the statistical result of the number of diseased spikelets, and **** indicates... p <0.0001. Detailed Implementation
[0028] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.
[0029] Example 1 This embodiment provides genes TaZF10 Molecular cloning and verification of its interaction with the Fusarium head blight resistance protein TaSnRK1α.
[0030] Fielder wheat leaves grown in soil in a greenhouse were used as the experimental material. Total RNA was extracted and cDNA was reverse transcribed to prepare PCR templates. Specific primers TaZF10-F (5'-ATGTCTGGAAATGATTCTCAG-3') and TaZF10-R (5'-TCAGGATCGGCGCTTGTCGCC-3') were used for PCR amplification. TransStart® FastPfuDNA Polymerase kit (Beijing TransGen Biotech Co., Ltd.), amplifying genes TaZF10 The full-length encoded sequence.
[0031] Table 1. TaZF10 Gene PCR reaction system
[0032] The PCR reaction program was as follows: pre-denaturation at 95℃ for 2 min; 35 cycles of 95℃ for 20 sec, 58.5℃ for 20 sec, and 72℃ for 30 sec; final extension at 72℃ for 10 min; and storage at 4℃. The gel electrophoresis results are as follows: Figure 1 As shown, the band size is as expected.
[0033] The PCR amplification products were sequenced using Sanger sequencing, confirming that the obtained cDNA sequence is as shown in SEQ ID NO: 2, and that its encoded amino acid sequence is consistent with SEQ ID NO: 1.
[0034] An immunoprecipitation (Co-IP) experiment was performed in *Nicotiana benthamiana* using an Agrobacterium transient expression system to verify the interaction between protein TaZF10 and Fusarium head blight resistance protein TaSnRK1α. The sequence of Fusarium head blight resistance protein TaSnRK1α is shown in SEQ ID NO: 5.
[0035] SEQ ID NO: 5 is as follows: MSTMDAAGRDANPLAGYRIGKTLGIGSFGKVKIAEHIITGHKVAIKILNRRKIKSMEMEEKVKREIKILRLFMHPHIIRLYEVIDTPADIYVVMEYVKSGELFDYIVEKGRLQEEEARRFFQQIIS GVEYCHRNMVVHRDLKPENLLLDSKCNVKIADFGLSNVMRDGHFLKTSCGSPNYAAPEVISGKLYAGPEVDVWSCGVILYALLCGTLPFDDENIPNLFKKIKGGIYTLPSHLSPLARDLIPRMLVV DPMKRITIREIREHSWFKARLPRYLAVPPPDTAQQVKKLDDETLNDVIKMGFDKNQLTESLQKRLQNEATVAYYLLLDNKLRTTSGYLGAEYQESMDSSFSQVSPETPSSASEARQYGSPGFGLRH HFAAERKWALGLQSRAHPREIITEVLKALQELNVCWKKIGHYNMKCRWSPGFVETMMHNNNGFGVESAIIEADGLSDKSTHIVKFEIQLYKTRDEKYLLDLQRVSGPQLLFLDLCSAFLTQLRVL.
[0036] like Figure 2 As shown, the experimental group co-expressed the GFP-TaZF10 and TaSnRK1α-mCherry fusion protein in tobacco cells, while the control group co-expressed GFP-TaZF10 and an empty mCherry tag protein. After enriching the GFP-TaZF10 fusion protein using Anti-GFP Affinity Beads4FF (purchased from Tiandi Renhe Biotechnology Co., Ltd.), TaSnRK1α-mCherry was specifically detected, while the empty mCherry tag protein was not enriched. These results indicate that TaZF10 and TaSnRK1α have a specific interaction in tobacco cells.
[0037] Example 2 This embodiment describes how the Fusarium graminearum resistance protein TaSnRK1α promotes the degradation of the protein TaZF10.
[0038] To verify the promoting effect of the Fusarium head blight resistance protein TaSnRK1α on the degradation of protein TaZF10, this study used an Agrobacterium transient expression system to conduct in vivo protein degradation experiments in *Nickelback*. Figure 3As shown, the experimental group co-expressed the GFP-TaZF10 and TaSnRK1α-mCherry fusion protein in tobacco cells, while the control group co-expressed GFP-TaZF10 and an empty mCherry tag protein. The protein expression levels of TaZF10 and TaSnRK1α (or empty mCherry) were detected using GFP-specific antibodies and mCherry-specific antibodies, respectively. The results showed that the expression level of TaZF10 protein co-expressed with TaSnRK1α was significantly lower at 36 hours (h) and 48 hours (h) compared to the control group co-expressed with the empty mCherry tag protein, indicating that TaZF10 protein was degraded.
[0039] Example 3 This embodiment describes genes. TaZF10 Construction of gene-editing vectors and acquisition of gene-edited plants.
[0040] Genes targeting wheat chromosomes 7A, 7B, and 7D were designed using sequence analysis. TaZF10 Two conserved gRNAs (gRNA1 5' CAGGCCTTATCAAATGCAAA 3', gRNA2 5' CTCGCAATGTCAATGCCTAT 3') were synthesized at Shaanxi Zhongke Yutong Biotechnology Co., Ltd. The primer sequences for gRNA1, gRNA1-F (5' CAGGCCTTATCAAATGCAAA 3') and gRNA1-R (5' TTTGCATTTGATAAGGCCTG 3'), and the primer sequences for gRNA2, gRNA2-F (5' CTCGCAATGTCAATGCCTAT 3') and gRNA2-R (5' ATAGGCATTGACATTGCGAG 3'), were also synthesized. gRNA1 was replaced at the gRNA1 replacement site using BtgZI restriction endonuclease, and gRNA2 was replaced at the RNA2 replacement site using BsaI restriction endonuclease. After diluting gRNA1 and gRNA2 primers F and R to 100mM, the dimerization reaction systems of gRNA primers were prepared according to Table 2. The reaction conditions were boiling water bath for 10 min and cooling at room temperature for 1-2 h.
[0041] Table 2. gRNA dimerization reaction system
[0042] The linear pENTR4 intermediate vector was digested with BtgZI restriction endonuclease. Then, following the ligation system shown in Table 3, the gRNA1 dimer fragment was directionally inserted into the multiple cloning site of the vector using T4 DNA Ligase (Baori Biotechnology Co., Ltd.). The ligation reaction was performed at 16°C for 12–16 hours. The recombinant plasmid gRNA1-pENTR4 was successfully constructed after transformation and colony PCR verification.
[0043] Table 3. T4 Connection Reaction System
[0044] Repeat the above steps to construct gRNA2 into the linearized pENTR4-gRNA1 vector, obtaining the gRNA1-gRNA2-pENTR4 intermediate vector. Using the Gateway® LR Clonase™ II enzyme mixture (Invitrogen, USA), the gRNA1-gRNA2-pENTR4 intermediate vector was recombined into the gene editing vector Cas9 via Gateway LR recombination reaction, constructing the gRNA1-gRNA2-Cas9 recombinant expression vector. Its structure is illustrated in the figure. Figure 5 .
[0045] The recombinant expression vector gRNA1-gRNA2-Cas9 was introduced into Agrobacterium EHA105 competent cells, and the cells were transformed using an Agrobacterium-mediated genetic transformation system with Fielder as the recipient. TaZF10 Gene-edited immature wheat embryos. Transplanted into T0 generation edited plants using a callus induction-regeneration system. TaZF10 Sequencing primers gRNA-check-F (5' GGAGTGAGTACGGTGTGCCAGGCTGCAGCTGACAGAATAA 3') and gRNA-check-R (5' GAGTTGGATGCTGGATGGAGCAGTTGCAGAAGCTGGTG 3') were designed near the editing target sites gRNA1 and gRNA2. Genomic DNA was extracted from leaves of T0 generation plants using the CTAB method. The expected 279 bp target region was amplified by PCR, and gel electrophoresis verification was performed (see [link to gel electrophoresis documentation]). Figure 6 The amplified products were analyzed for high-throughput editing efficiency using the Hi-TOM platform at the Rice Research Institute of the Chinese Academy of Agricultural Sciences. Genes from wheat chromosomes 7A, 7B, and 7D were selected. TaZF10 T2 generation plants that underwent editing were analyzed for resistance to Fusarium head blight.
[0046] Example 4 This embodiment describes genes. TaZF10 Field identification of Fusarium head blight resistance in gene-edited wheat.
[0047] After approval, T2 generation homozygous strains were sown in the transgenic isolation test site. TaZF10 Gene-edited plants ( TaZF10 -KO) and wild-type Fielder. The test plots were 1.5 m × 1.5 m rectangular units (area 2.25 m²). 2 The plot contains six parallel planting strips, with a row spacing of 25 cm. 50 wheat seeds are manually sown per row, for a total of 300 seeds per plot. Sufficient antifreeze water is applied before winter. At the wheat flowering stage, a spore suspension of the wild-type PH-1 strain of *Fusarium graminearum* (concentration 2×10⁻⁶) is used. 5 Single-flower drip inoculation was performed using conidia (10 μL / flower, one floret in the middle of each spike). After inoculation, the plants were sprayed to maintain humidity and covered with a film for 3 days to maintain moisture. Disease incidence was assessed 14 days after inoculation, and the number of diseased grains per spike was counted. A t-test was used to analyze the significant differences between the transgenic lines and the control. Results are as follows: Figure 6 As shown in a, compared to wild-type Fielder wheat plants, TaZF10 Genetically edited wheat plants showed a significant reduction in the number of diseased grains per ear after inoculation with the wild-type strain of Fusarium graminearum PH-1. The number of diseased grains was counted, as shown below. Figure 6 The b in the text shows that, compared to Fielder, TaZF10 Gene-edited wheat plants showed a reduction of approximately 50% in the number of diseased grains. This indicates that gene editing... TaZF10 The absence of this component can significantly improve wheat's resistance to Fusarium head blight.
[0048] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate preferred embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.
Claims
1. A method for obtaining plants resistant to Fusarium head blight, characterized in that, Make genes TaZF10 Frameshift mutations lead to the loss of function of the protein TaZF10; The gene TaZF10 The protein TaZF10 is encoded by this protein; The amino acid sequence of the protein TaZF10 is shown in SEQ ID NO:
1.
2. The method for obtaining plants resistant to Fusarium head blight according to claim 1, characterized in that, The gene TaZF10 The nucleotide sequence is shown in SEQ ID NO:
2.
3. The method for obtaining plants resistant to Fusarium head blight according to claim 1, characterized in that, The plant in question is wheat.
4. The method for obtaining plants resistant to Fusarium head blight according to claim 1, characterized in that, The pathogen causing Fusarium head blight is Fusarium graminearum. Fusarium graminearum .
5. The method for obtaining plants resistant to Fusarium head blight according to claim 1, characterized in that, Construct the TaZF10 A gene editing vector is used to introduce the gene editing vector into the plant.
6. The method for obtaining plants resistant to Fusarium head blight according to claim 5, characterized in that, The gene-editing vector targets the gene. TaZF10 gRNA1 and gRNA2 in the cell; The nucleotide sequence of the gRNA1 is shown in SEQ ID NO: 3; The nucleotide sequence of the gRNA2 is shown in SEQ ID NO:
4.
7. A plant resistant to Fusarium head blight, characterized in that, The method for obtaining plants resistant to Fusarium head blight as described in any one of claims 1 to 6 is used.
8. Genes TaZF10 Its application in the prevention and control of Fusarium head blight is characterized by... Gene TaZF10 Negative regulation of wheat resistance to Fusarium head blight; This can cause frameshift mutations in the TaZF10 gene or cause the TaZF10 protein to lose its function. The gene TaZF10 The protein TaZF10 is encoded by this protein; The amino acid sequence of the protein TaZF10 is shown in SEQ ID NO:
1.
9. The gene according to claim 8 TaZF10 Its application in the prevention and control of Fusarium head blight is characterized by... The gene TaZF10 The nucleotide sequence is shown in SEQ ID NO:
2.
10. The gene according to claim 8 TaZF10 Its application in the prevention and control of Fusarium head blight is characterized by... The plant in question is wheat, and the pathogen causing the Fusarium head blight is Fusarium graminearum. Fusarium graminearum .