Application of wheat nucleoside diphosphate kinase TaNDK in gibberellic disease resistance

Abstract

The invention belongs to the technical field of biology, and discloses application of wheat nucleoside diphosphate kinase TaNDK in resisting gibberellic disease. After the TaNDK gene provided by the invention is over-expressed in wheat, the infection degree of the wheat to the gibberellic disease can be reduced, and the resistance of the wheat to the gibberellic disease is remarkably improved. The invention provides a gene resource and a technical thought for gibberellic disease-resistant breeding improvement of wheat.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of wheat nucleoside diphosphate kinase NDK gene in wheat fusarium head blight resistance. Background Art

[0002] Wheat (Triticum aestivum L.) is an important food crop that provides starch, protein and dietary fiber for more than 40% of the world's population. However, global wheat production is constantly threatened by various diseases. Wheat head blight (Fusarium head blight) is one of the most destructive wheat diseases. It is a fungal disease caused by wheat Fusarium. The disease can occur throughout the entire growth period of wheat and can cause root rot, stem base rot and ear rot. It occurs in most wheat-growing areas around the world. With changes in temperature and farming methods, 20% of wheat-growing areas have suffered from head blight in recent years, seriously affecting wheat quality and yield. A large amount of toxins accumulate in wheat grains, which not only affects seed germination, but also causes poisoning in humans and animals.

[0003] Using resistance breeding technology to cultivate wheat varieties that are resistant to Fusarium head blight is one of the effective ways to prevent and control wheat Fusarium head blight. Hundreds of resistance QTLs have been identified, but only a few resistance QTLs such as Fhb1 show stable effects. Therefore, it is necessary to find and utilize more effective methods to control wheat Fusarium head blight. Although utilizing the innate defense mechanism of plants is a potential and effective strategy to improve crop resistance to diseases, we still lack a deep understanding of how to use this mechanism to enhance wheat resistance to Fusarium head blight. Since the accumulation of DON toxins is closely related to the expansion of Fusarium head blight, controlling the accumulation of DON toxins in wheat can effectively reduce the damage caused by Fusarium head blight to wheat. However, the current research on the interaction between DON toxins and crops is still insufficient and needs further exploration.

[0004] Nucleoside diphosphate kinase (NDK) is a highly conserved gene whose encoded protein is ubiquitous in organisms. As a kinase, NDK has the function of catalyzing substrate phosphorylation and self-phosphorylation, and is closely related to the energy metabolism and biosynthesis metabolism of organisms. Cytoplasmic type I NDK is involved in metabolism, growth and stress response (Dorion S, Rivoal J. Clues to the functions of plant NDKisoforms. NaunynSchmiedebergs 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 meristem and vascular tissue 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). After rice was infected with Xanthomonas solani or Burkholderia ovale, the expression of OsNDK1 was significantly upregulated (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] The present invention screens out the wheat disease resistance-related gene TaNDK through genetic engineering means and pathogen-crop interaction system, and reveals its disease resistance mechanism in the interaction process between wheat and fusarium head blight by functional analysis of the protein and gene, providing a solution for cultivating fusarium head blight-resistant materials. The present invention adopts the following technical scheme.

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

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

[0008] The invention provides expression elements, recombinant vectors and host cells containing the gene.

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

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

[0011] The present invention also provides the use of the TaNDK protein or its encoding gene, the expression element, the recombinant vector, and the host cell in preparing transgenic plants with improved disease resistance.

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

[0013] The present invention provides a method for preparing a transgenic plant with enhanced disease resistance, which comprises the steps of overexpressing a gene encoding the TaNDK in a transgenic plant by a transgenic method, and screening and obtaining a transgenic plant with enhanced resistance to wheat fusarium head blight.

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

[0015] The present invention relates to the application of TaNDK in the disease resistance process. Transgenic plants that overexpress the TaNDK gene have significant resistance to wheat scab and can effectively inhibit the spread of wheat scab in host cells. The transgenic plants involved in the present invention are not affected during normal growth and fruiting. The present invention provides an application method of the TaNDK protein related to wheat disease resistance in cultivating wheat disease-resistant variety improvement. Specifically, the present invention can achieve overexpression of the TaNDK gene and successfully cultivate corresponding transgenic plants, which lays a solid foundation for further disease resistance research and variety improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 Schematic diagram of pUbiGW vector with Ubiquitin promoter;

[0018] Figure 2 is the relative expression level of TaNDK in the overexpression wheat lines;

[0019] Figure 3 This is the phenotype of wheat resistance to wheat fusarium head blight by overexpressing TaNDK in wheat;

[0020] Figure 4 This is a statistical chart for identifying resistance to wheat fusarium rust by overexpressing TaNDK in wheat. DETAILED DESCRIPTION

[0021] The present invention is described below through specific embodiments to facilitate a better understanding of the present invention, but does not constitute a limitation of the present invention. The specific examples are as follows:

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

[0023] The present invention uses genetic engineering methods and the biochemical system of the interaction between the pathogenic factors of Fusarium fusarium and wheat to identify the key target protein TaNDK of wheat in response to Fusarium fusarium. By performing functional analysis on the protein and gene, constructing transgenic materials and verifying their resistance to wheat Fusarium fusarium, the disease resistance mechanism in the interaction between wheat and Fusarium fusarium is revealed.

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

[0025] TGGCGGAGCAGACCTTCATGATCAAGCCCGACGGCGTCCAGAGGGGCCTCATCGGCGAGGTCATCAGCCGCTTCGAGAAGAAGGGCTTCTACCTCAAAGGTTTGAAGCTCCAGAACGTGGAAGTCGTTCGCCGAGCAGCACTACGCCGATCTGTCCTCCAAGCCCTTCTTCGCTGGGCTCGTGGAGTACATCGTCTCCGGCCCGGTCGTCGCTATGGTCTG GGAGGGCAAGAGCGTCGTCTCCACTGGACGCAAGATCATTGGCGCCACCAACCCCCTGGCCTCTGAGCCCGGCACCATCCGTGGTGACTTCGCCGTCGACATCGGCAGGAACGTCATCCATGGAAGCGACTCAGTTGAGAGTGCCAGGAAGGAGATCGCCCTGTGGTTCCCTGAGGGCATTGCCGAGTGGAGGAGCAGCCAGCACAACTGGATCTACGAGGCCTAA.

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

[0027] MAEQTFIMIKPDGVQRGLIGEVISRFEKKGFYLKGLKLQNVEKSFAEQHYADLSSKP FFAGLVEYIVSGPVVAMVWEGKSVVSTGRKIIGATNPLASEPGTIRGDFAVDIGRNVIHGS DSVESARKEIALWFPEGIAEWRSSQHNWIYEA.

[0028] In the present invention, the application preferably comprises the following steps:

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

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

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

[0032] (3) transforming the plasmid obtained in step (2) into Agrobacterium EHA105 to obtain Agrobacterium carrying the NDK plasmid;

[0033] (4) transforming the Agrobacterium obtained in step (3) into the wheat Fielder strain to obtain multiple independent transformed offspring, and obtaining wheat overexpressing the TaNDK gene after gene identification;

[0034] The wheat transformation 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 whether the TaNDK gene was overexpressed in T0 generation wheat. Figure 2 As shown, the expression levels of overexpression strains #1, #2, and #3 were 36.9, 22.8, and 27.7 times that of the wild type, respectively. Therefore, the present invention selected overexpression strains #1, #2, and #3 for subsequent phenotypic identification of scab.

[0036] Example 2: Phenotypic identification of resistance to Fusarium head blight in transgenic wheat overexpressing the TaNDK gene

[0037] The transgenic wheat obtained in Example 1 was subjected to phenotypic identification of resistance to fusarium head blight, and the specific steps were as follows:

[0038] (1) In the middle of the wheat flowering period, the single flower drip method was used to inoculate Fusarium graminearum PH1, that is, 10 μl (about 1×10 5 Each strain should be inoculated with more than 30 ears, and the inoculation date and number should be marked on a sign;

[0039] (2) bagging the wheat ears inoculated in step (1) 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) Investigate the number of diseased spikelets in each inoculated wheat ear on the 7th, 15th and 21st day after the wheat ears are inoculated in step (1).

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

[0042] (4) Figure 3 The disease symptoms of the two ears were clearly shown, and these symptoms were consistent with the typical characteristics of wheat fusarium head blight. Specifically, on the 13th day after inoculation, the number of diseased spikelets of transgenic TaNDK-OE was 6, and the number of diseased spikelets of Fielder was 9, which showed that the disease of transgenic was significantly lower than that of wild type; Figure 4 The average number of diseased spikelets of transgenic TaNDK-OE on the 7th, 15th and 21st days was 1.7, 10.6 and 16.6, and the average number of diseased spikelets of Fielder was 3.4, 12.8 and 19.1. The diseased spikelet rate of transgenic TaNDK-OE was reduced by 8.93%, 10.93% and 12.36% compared with the wild type on the 7th, 15th and 21st days, respectively. The above data strongly prove that the TaNDK-OE transgenic line can significantly enhance the resistance of wheat to Fusarium head blight. It was identified that under greenhouse conditions, TaNDK-OE transgenic wheat showed significant resistance differences compared with wild-type Fielder. After the disease occurred, the severity of the disease in the ear of TaNDK-OE transgenic wheat was significantly lower than that of wild-type Fielder. This result not only confirms the potential of TaNDK-OE transgenic wheat in resistance to Fusarium head blight, but also provides important genetic improvement materials for future wheat disease resistance breeding.

Claims

1. A TaNDK protein, characterized in that Its amino acid sequence is shown in SEQ ID No.

2.

2. Encoding as claimed in claim 1 TKM The gene, preferably, its specific nucleotide sequence is shown as SEQ ID No.

1.

3. An expression element, a recombinant vector, and a host cell containing the gene according to claim 2.

4. The method according to claim 1 TKM Application of the gene or its encoded protein in improving the disease resistance of plants.

5. The use according to claim 4, characterized in that The plant is a monocotyledonous plant, preferably the plant is wheat; more preferably, the disease resistance refers to wheat fusarium rust caused by Fusarium.

6. The method according to claim 1 TKM Use of the gene or its encoded protein, the expression element, the recombinant vector and the host cell as claimed in claim 3 in preparing transgenic plants with improved disease resistance.

7. The use according to claim 6, characterized in that The plant is a monocotyledonous plant, preferably the plant is wheat; more preferably, the disease resistance refers to wheat fusarium rust caused by Fusarium.

8. A method for preparing a transgenic plant with enhanced disease resistance, characterized in that: comprising overexpressing the gene encoding the gene according to claim 2 in a transgenic plant by a transgenic method TKM Genes, and steps for screening transgenic plants with enhanced resistance to wheat fusarium rust.

9. The use according to claim 8, characterized in that The plant is a monocotyledonous plant, preferably the plant is wheat; more preferably, the disease resistance refers to wheat fusarium rust caused by Fusarium.

Images