Application of wheat nucleoside diphosphate kinase TaNDK in resistance to scab

Abstract

The application belongs to the technical field of biology, and discloses application of a wheat nucleoside diphosphate kinase TaNDK in resistance to scab. TaNDK The gene can reduce the infection degree of wheat to scab and significantly improve the resistance of wheat to scab after overexpression in wheat. The application provides a gene resource and a technical idea for breeding and improvement of wheat resistance to scab.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the wheat nucleoside diphosphate kinase (NDK) gene in wheat scab resistance. Background Technology

[0002] Wheat (Triticum aestivum L.) is an important food crop, providing starch, protein, and dietary fiber for more than 40% of the world's population. However, global wheat yields are constantly threatened by various diseases. Fusarium head blight is one of the most destructive wheat diseases, a fungal disease caused by Fusarium graminearum. This disease can occur throughout the entire wheat growth cycle, causing root rot, stem rot, and ear rot, and is found in most wheat-growing areas globally. With changes in temperature and farming practices, Fusarium head blight has occurred in 20% of wheat-growing areas in recent years, severely impacting wheat quality and yield. The accumulation of large amounts of toxins in the grains not only affects seed germination but can also lead to poisoning in humans and animals.

[0003] Developing wheat varieties resistant to Fusarium head blight using resistance breeding techniques is one of the effective ways to control this disease. Hundreds of resistance QTLs have been identified, but only a few, such as Fhb1, have shown stable effects. Therefore, it is necessary to find and utilize more effective methods to control Fusarium head blight in wheat. Although utilizing the plant's innate defense mechanisms is a potentially effective strategy to enhance crop resistance to diseases, our understanding of how to apply this mechanism to enhance wheat resistance to Fusarium head blight remains incomplete. Since DON toxin accumulation is closely related to the spread of Fusarium head blight, controlling the accumulation of DON toxin in wheat can effectively reduce the damage caused by Fusarium head blight. However, current research on the interaction between DON toxin and crops is still insufficient and requires further exploration.

[0004] Nucleoside diphosphate kinases (NDKs) are a class of highly conserved genes whose encoded proteins are ubiquitous in organisms. As kinases, NDKs catalyze substrate phosphorylation and autophosphorylation, and are closely related to energy metabolism and biosynthesis in organisms. Cytoplasmic type I NDKs participate in metabolism, growth, and stress responses (Dorion S, Rivoal J. Clues to the functions of plant NDK isoforms. Naunyn Schmiedebergs Arch Pharmacol. 2015; 388(2):119-132.). Immunolocalization experiments on potato root tips and apical buds showed that type I NDK was mainly expressed in the meristematic and vascular tissues of the apical region (Dorion S, Matton DP, Rivoal J (2006b) Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato. Planta 224:108–124). OsNDK1 expression was significantly upregulated after rice infection with bacterial blight or Burkholderia (Cho SM, Shin SH, Kim KS, et al. Enhanced expression of a gene encoding anucleoside diphosphate kinase 1 (OsNDPK1) in rice plants upon infection with bacterial pathogens[J]. Mol Cells, 2004, 18(3):390–395.). Summary of the Invention

[0005] This invention utilizes genetic engineering techniques and a pathogen-crop interaction system to screen for the wheat disease resistance-related gene TaNDK. Functional analysis of the protein and gene reveals its disease resistance mechanism in the interaction between wheat and Fusarium head blight, providing a solution for breeding Fusarium head blight-resistant materials. The technical solution adopted in this invention is as follows.

[0006] The present invention first provides a TaNDK protein, the amino acid sequence of which is shown in SEQ ID No.2.

[0007] The present invention provides a gene encoding the TaNDK, the specific nucleotide sequence of which is shown in SEQ ID No. 1.

[0008] This invention provides an expression element containing the aforementioned gene, a recombinant vector, and a host cell.

[0009] The present invention further provides the application of the TaNDK protein or its encoding gene in improving plant disease resistance.

[0010] Specifically, the plant is a monocotyledonous plant, preferably wheat; more preferably, the disease resistance refers to wheat scab caused by Fusarium.

[0011] This invention also provides the application of the TaNDK protein or its encoding gene, the expression element, the recombinant vector, and the host cell in the preparation of transgenic plants with enhanced disease resistance.

[0012] Specifically, the plant is a monocotyledonous plant, preferably wheat; more preferably, the disease resistance refers to wheat scab caused by Fusarium.

[0013] The present invention provides a method for preparing transgenic plants with enhanced disease resistance, comprising the steps of overexpressing the gene encoding TaNDK in transgenic plants by transgenic methods, and screening to obtain transgenic plants that can enhance resistance to wheat scab.

[0014] Specifically, the plant is a monocotyledonous plant, preferably wheat; more preferably, the disease resistance refers to wheat scab caused by Fusarium.

[0015] This invention relates to the application of TaNDK in disease resistance. Transgenic plants overexpressing the TaNDK gene exhibit significant resistance to wheat scab, effectively inhibiting its spread within host cells. The transgenic plants involved in this invention are not affected during normal growth and fruiting. This invention provides a method for applying the wheat disease-resistant TaNDK protein in the breeding and improvement of disease-resistant wheat varieties. Specifically, this invention enables the overexpression of the TaNDK gene and successfully cultivates corresponding transgenic plants, laying a solid foundation for further research on disease resistance and variety improvement. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly introduced below.

[0017] Figure 1 A schematic diagram of the pUbiGW vector with the Ubiquitin promoter;

[0018] Figure 2 The relative expression level of TaNDK in overexpressing wheat lines;

[0019] Figure 3 Phenotypic representation of wheat resistance to Fusarium head blight in wheat with TaNDK overexpression;

[0020] Figure 4 Statistical graph showing the identification of wheat resistance to Fusarium head blight by overexpression of TaNDK in wheat. Detailed Implementation

[0021] The present invention will be described below through specific embodiments to provide a better understanding of it, but these embodiments do not constitute a limitation thereof. The specific details are as follows:

[0022] Example 1: Construction of transgenic wheat overexpressing the TaNDK gene

[0023] This invention identifies the key target protein TaNDK in wheat's response to Fusarium head blight by using genetic engineering techniques and a biochemical system of interaction between Fusarium head blight pathogens and wheat. Through functional analysis of the protein and gene, construction of transgenic materials, and verification of their resistance to wheat Fusarium head blight, the invention reveals the disease resistance mechanism in the interaction between wheat and Fusarium head blight.

[0024] In this invention, the CDS nucleotide sequence of the TaNDK gene is shown in SEQ ID No. 1, and is as follows:

[0025] TGGCGGAGCAGACCTTCATGATCAAGCCCGACGGCGTCCAGAGGGGCCTCATCGGCGAGGTCATCAGCCGCTTCGAGAAGAAGGGCTTCTACCTCAAAGGTTTGAAGCTCCAGAACGTGGAAGTCGTTCGCCGAGCAGCACTACGCCGATCTGTCCTCCAAGCCCTTCTTCGCTGGGCTCGTGGAGTACATCGTCTCCGGCCCGGTCGTCGCTATGGTCTG GGAGGGCAAGAGCGTCGTCTCCACTGGACGCAAGATCATTGGCGCCACCAACCCCCTGGCCTCTGAGCCCGGCACCATCCGTGGTGACTTCGCCGTCGACATCGGCAGGAACGTCATCCATGGAAGCGACTCAGTTGAGAGTGCCAGGAAGGAGATCGCCCTGTGGTTCCCTGAGGGCATTGCCGAGTGGAGGAGCAGCCAGCACAACTGGATCTACGAGGCCTAA.

[0026] In this invention, the amino acid sequence of the protein encoded by the TaNDK gene is shown in SEQ ID No. 2, and is as follows:

[0027] MAEQTFIMIKPDGVQRGLIGEVISRFEKKGFYLKGLKLQNVEKSFAEQHYADLSSKP FFAGLVEYIVSGPVVAMVWEGKSVVSTGRKIIGATNPLASEPGTIRGDFAVDIGRNVIHGS DSVESARKEIALWFPEGIAEWRSSQHNWIYEA.

[0028] In this invention, the application preferably includes the following steps:

[0029] (1) The full-length fragment of TaNDKCDS was inserted into the pUbiGW vector with the Ubiquitin promoter via the BamH I site using In-Fusion cloning technology (Clontech, catalog number 638910);

[0030] The nucleotide sequence of the full-length TaNDK CDS fragment is shown in SEQ ID No. 1;

[0031] (2) The ligation vector obtained in step (1) is transferred into Escherichia coli DH5α. After screening and sequencing, the corresponding recombinant plasmid is extracted after confirming that the NDK gene has been successfully ligated.

[0032] (3) The plasmid obtained in step (2) is transformed with Agrobacterium EHA105 to obtain Agrobacterium carrying the NDK plasmid;

[0033] (4) The Agrobacterium obtained in step (3) is transformed into the Fielder wheat line to obtain multiple independent transformed offspring. After gene identification, wheat overexpressing the TaNDK gene is obtained.

[0034] The wheat conversion was carried out according to the previously described method (Goetz H., Cornelia M., and Jochen K. (2021). Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos. Rom. Agric. Res. 38, 99-107 (2021)).

[0035] The gene identification was further confirmed by qRT-PCR to determine whether the TaNDK gene was overexpressed in T0 generation wheat. Figure 2 As shown, the overexpression lines #1, #2, and #3 expressed 36.9, 22.8, and 27.7 times the wild type, respectively. Therefore, the overexpression lines #1, #2, and #3 were selected for subsequent Fusarium head blight phenotypic identification in this invention.

[0036] Example 2: Identification of Fusarium head blight resistance phenotype in transgenic wheat overexpressing the TaNDK gene

[0037] The transgenic wheat obtained in Example 1 was subjected to Fusarium head blight resistance phenotype identification, and the specific steps are as follows:

[0038] (1) Inoculate Fusarium graminearum PH1 into wheat florets using the single-floret drip method during the mid-flowering stage. Specifically, inject 10 μl (approximately 1 × 10⁻⁶) of the prepared spore suspension into a small floret on a small spikelet slightly above the middle of the wheat ear using a micropipette. 5 Each strain should be inoculated with at least 30 spores and labeled with the inoculation date and number.

[0039] (2) Cover the inoculated wheat ears in step (1) with a bag to keep them moist for 2 days. After removing the plastic bag, spray water on the inoculated wheat ears regularly every day to keep them moist.

[0040] (3) The number of diseased spikelets in each inoculated wheat ear was investigated on the 7th, 15th and 21st day after inoculation of the wheat ears in step (1).

[0041] The phenotypic identification was performed using the Enviologix QuickStix Kit (Envirologix, catalog number AS013) to evaluate the genotype of the transgenic plants.

[0042] (4) Figure 3 The disease symptoms in both spikelets are clearly shown, and these symptoms are consistent with typical characteristics of wheat scab. Specifically, on day 13 after inoculation, the transgenic TaNDK-OE had 6 diseased spikelets, while the Fielder had 9, indicating that the disease incidence in the transgenic type was significantly lower than in the wild type; Figure 4 On days 7, 15, and 21, the average number of diseased spikelets in the transgenic TaNDK-OE strain was 1.7, 10.6, and 16.6, respectively, while the average number of diseased spikelets in the Fielder strain was 3.4, 12.8, and 19.1. The rate of diseased spikelets in the transgenic TaNDK-OE strain was reduced by 8.93%, 10.93%, and 12.36% compared to the wild-type strain on days 7, 15, and 21, respectively. These data strongly demonstrate that the TaNDK-OE transgenic line can significantly enhance wheat resistance to Fusarium head blight. Identification showed that under greenhouse conditions, the TaNDK-OE transgenic wheat exhibited a significant difference in resistance compared to the wild-type Fielder. After the onset of the disease, the severity of disease in the spikelets of the TaNDK-OE transgenic wheat was significantly lower than that of the wild-type Fielder. This result not only confirms the potential of the TaNDK-OE transgenic wheat in resistance to Fusarium head blight but also provides important genetic improvement material for future wheat disease resistance breeding.

Claims

1. A kind TaNDK The application of genes or their encoded proteins in enhancing plant disease resistance; among them, The plant is wheat, the disease resistance refers to the wheat scab caused by Fusarium; the TaNDK The amino acid sequence of the protein encoded by the gene is shown as SEQ ID No.

2.

2. Use according to claim 1, wherein The TaNDK The nucleotide sequence of the gene is shown as SEQ ID No.

1.

3. A kind TaNDK Gene or its encoded protein, containing the TaNDK Gene expression elements, containing the TaNDK Gene recombination vectors or containing the above TaNDK The application of host cells for genes in the preparation of transgenic plants with enhanced disease resistance; wherein The plant is wheat, the disease resistance refers to the wheat scab caused by Fusarium; the TaNDK The amino acid sequence of the protein encoded by the gene is shown as SEQ ID No.

2.

4. The use according to claim 3, wherein the compound is ###0002### The TaNDK The nucleotide sequence of the gene is shown in SEQ ID No.

1.

5. A method of making a transgenic plant with enhanced disease resistance, comprising, comprising overexpressing in a transgenic plant by a transgenic method a gene encoding TaNDK and screening for transgenic plants having enhanced resistance to wheat scab; The plant is wheat, the disease resistance refers to the wheat scab caused by Fusarium; the TaNDK The amino acid sequence of the protein encoded by the gene is shown as SEQ ID No.

2.

6. The use according to claim 5, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The TaNDK TaNDK The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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