Application of wheat TaALIX protein in reduction of vomitoxin accumulation and wheat gibberellic disease resistance

Abstract

The invention relates to the field of biotechnology and plant protection, and discloses application of ESCRT related protein in prevention and treatment of wheat scab. It is found for the first time that by improving the expression quantity of the wheat ESCRT III protein TaALIX, the resistance of wheat to gibberellic disease is enhanced, the content of deoxynivalenol (DON) is reduced, and the application has practical significance in plant disease control.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of a protein TaALIX related to the ESCRT system in reducing the accumulation of vomitoxin in wheat grains and enhancing the resistance of wheat to Fusarium head blight. Background Art

[0002] Fusarium head blight (FHB) is one of the most destructive fungal diseases in global wheat production areas, causing large-scale food production reduction and economic losses. There are more than 20 species of Fusarium pathogens that cause FHB in wheat, and in China, Fusarium graminearum Fusarium graminearum is the main dominant species. During the infection process, Fusarium graminearum produces a variety of mycotoxins, among which deoxynivalenol (DON) is one of the most important mycotoxins. It can cause neurotoxicity, reproductive toxicity, and immunotoxicity. After humans and animals ingest contaminated food, they will have adverse reactions such as anorexia and vomiting, commonly known as vomitoxin. Some studies have shown that DON participates in signal transduction processes related to cell differentiation and apoptosis, leading to the destruction of normal plant cells. It also acts as a pathogenic factor, accelerating the necrosis of crop tissues and contaminating cereal grains. In recent years, due to the influence of various environmental factors, DON contamination has become increasingly serious around the world. Large amounts of DON toxins have been detected in cereals such as wheat, barley, and corn, and their contents seriously exceed the limit standards, posing a great threat to human and animal health. If the accumulation of DON toxins in wheat can be controlled, it is very likely to reduce the damage of Fusarium head blight in wheat (Mawcha, K. T., Zhang, N., Wang, Y., and Yang, W. Advances in wheat breeding for resistance to Fusarium head blight. Czech J. Genet. Plant. 2022 58 (4), 167 - 188).

[0003] In recent years, the synthesis and regulation mechanisms of DON toxins in wheat have been widely studied. For example, the protein FgRab7 of Fusarium graminearum regulates Fusarium graminearum TriGene expression and DON toxin synthesis, the deletion of which leads to a significant decrease in toxin production; two Sec2 homologs, FgSec2A and FgSec2B, in Fusarium graminearum are located in the apical regions of hyphae and conidia and play crucial roles in DON biosynthesis and secretion in wheat. Their double deletion results in a substantial reduction in DON content (Zheng H, LiL, Miao P, et al., FgSec2A, a guanine nucleotide exchange factor of FgRab8, is important for polarized growth, pathogenicity and deoxynivalenol production in Fusarium graminearum . Environ Microbiol. 2018, 20(9):3378-3392). Meanwhile, plants have also evolved protective mechanisms in response to DON. It has been reported that plant UDP-glucosyltransferase (UGT) can reduce the toxicity of DON and enhance resistance to Fusarium head blight, and a novel gene highly expressed upon Fusarium head blight induction has been successfully cloned and identified in Sumai 3 TaUGT6 . Through enzyme activity assays, it was found that TaUGT6 could glycosylate DON into less toxic D3G, thereby enhancing the plant's tolerance to DON and resistance to Fusarium head blight (He Y, Wu L, Liu X, et al. TaUGT6 , a Novel UDP-Glycosyltransferase gene enhances the resistance to FHB and DON accumulation in wheat. Front Plant Sci. 2020;11:574775.). In addition, a specific glyoxalase in cotton (SPG) can isomerize DON modifiers to reduce DON toxicity (Huang J, Fang X, Tian X, et al. Aromatization of natural products by a specialized detoxification enzyme. Nat Chem Biol, 2020, 16(3):250-256.).

[0004] In addition to the two types of anti-toxin accumulation methods of inhibiting toxin synthesis and toxin chemical modification, a new mechanism was reported in 2021 in which the P4-ATPase-mediated vesicle transport pathway in plant cells transports the mycotoxin DON into vacuoles, thereby enhancing the disease resistance of Arabidopsis plants. In this study, DON was used as a screening agent to identify a detoxification-related protein AtALA1 in the Arabidopsis genome, and it was found that DON targets vacuoles through the AtALA1-mediated vesicle transport pathway; after overexpressing this gene, the efficiency of DON targeting vacuoles increased, and at the same time, the resistance of Arabidopsis plants to Fusarium graminearum was greatly enhanced; more importantly, the content of vomit toxin in transgenic maize and Arabidopsis seeds was significantly reduced (Wang, F., Li, X., Li, Y. etal. Arabidops is P4 ATPase-mediated cell detoxification confers resistance to Fusarium graminearum and Verticillium dahliae . Nat Commun 12, 6426 (2021).). This study uses this vesicle transport-related cell detoxification strategy, which has a significant effect on improving the resistance of plants to toxin-related diseases, and at the same time provides a new idea for exploring how to reduce mycotoxins. The modification and transport process of DON toxin is closely related to the occurrence of Fusarium head blight and grain health in wheat, so it is the focus of attention in the fields of crop disease resistance, food safety and human health. SUMMARY OF THE INVENTION

[0005] Based on the research of the present inventors, it was first discovered that overexpression of the ESCRT-related protein TaALIX can reduce the accumulation of vomit toxin during the occurrence of Fusarium head blight in wheat and enhance the resistance to Fusarium head blight. Thus, the present invention was completed.

[0006] The present invention first provides a key vesicle transport protein TaALIX in the ESCRT system, whose amino acid sequence is as shown in SEQ ID No.2 or its homologous protein sequence, such as the proteins shown in SEQ ID No.4 and SEQ ID No.6.

[0007] The present invention also provides the gene encoding the above TaALIX. Specifically, its nucleic acid sequence is as shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5.

[0008] Furthermore, the present invention provides an expression element, a recombinant vector, and a host cell containing the above gene.

[0009] Preferably, the above gene is overexpressed in plants by transgenic methods.

[0010] The present invention also provides the use of the gene in creating disease-resistant transgenic plants, wherein overexpression is carried out in transgenic plants by transgenic methods.

[0011] Preferably, the plant is a monocotyledonous plant, and preferably the plant is wheat. More preferably, the disease resistance refers to Fusarium head blight caused by Fusarium graminearum.

[0012] Among them, screening for disease-resistant transgenes is carried out by measuring the content of DON in transgenic plants. Specifically, it includes sampling at different times after inoculation with Fusarium graminearum and measuring the DON content.

[0013] The inventors of the present invention have found through research that TaALIX Wheat overexpressing the gene shows resistance to Fusarium graminearum and the toxin content is significantly reduced. Overexpression can be promoted for practical applications. The present invention can provide TaALIX overexpressing plants and homozygous TaALIX mutant plants, which is beneficial to the cultivation of disease-resistant wheat varieties and provides a basis for screening highly resistant wheat varieties in the later stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Showing the genotype identification of wheat overexpressing TaALIX lines.

[0015] Figure 2 Showing the genotype identification of TaALIX knockout lines in wheat.

[0016] Figure 3 Representative images of wheat spikes of WI (Fielder), TaALIX-OE, and TaALIX mutants infected with Fusarium graminearum after 15 days under greenhouse conditions.

[0017] Figure 4 For TaALIX -OE disease index quantitative analysis.

[0018] Figure 5 For the determination and analysis of DON content. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described below through specific examples for better understanding, but it does not limit the present invention.

[0020] Example 1: Genotype identification of wheat overexpressing TaALIX lines For the wheat overexpression construct, the In-Fusion cloning technology (Clontech, catalog number 638910) was used to TaALIX(SEQ ID NO: 1, encoding the amino acid sequence shown in SEQ ID NO: 2, this gene is the toxin target gene screened by one-to-one interaction in the early stage of the laboratory, and is a homologous gene of Arabidopsis thaliana in wheat. Since wheat is an allohexaploid with ABD chromosomes, SEQ ID NO: 3 and SEQ ID NO: 5 are the homologous gene sequences of the ABD chromosomes of this gene) The full-length CDS fragment was inserted into the pUbiGW vector with the Ubiquitin promoter through the BamH I site. All constructs were transformed into the Agrobacterium tumefaciens strain EHA105. Wheat transformation was carried out according to the method described previously (Goetz H., Cornelia M., and Jochen K. Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos. Rom. Agric. Res. 2021, AtALIX , 99-107). Through qPCR identification, a total of 13 positive plants were identified ( 38 ). Figure 1 )

[0021] Example 2: Genotype identification of wheat knockout TaALIX lines Wheat contains three groups of chromosomes, A, B, and D, and is a typical allohexaploid (BBAADD), which was formed by hybridization and genome polyploidization of cultivated tetraploid wheat (BBAA) and diploid Aegilops tauschii (DD). Use the improved CRISPR-Cas9 system to create TaALIX mutants, and design two targets (T1 and T2) on the sgRNA to TaALIX edit the gene. Evaluate the target sequence through CRISPRdirect (https: / / crispr.dbcls.jp), and construct knockout mutants after confirming its targeting specificity in the wheat genome. Genotype identification of mutant plants was carried out by sequencing with gene-specific primers, and TaALIX knockout mutants were identified by PCR technology. A total of one mutant line homozygous on the ABD chromosomes was obtained ( TaALIX ). Figure 2 )

[0022] Example 3: Overexpression in wheat TaALIX can enhance the resistance to Fusarium head blight of wheat and reduce the DON content For the overexpression TaALIX ( TaALIX -OE) positive plants and homozygous TaALIX mutant plant wheat transgenic materials obtained in Example 1 were identified for resistance to Fusarium head blight.

[0023] Under greenhouse conditions, the FHB resistance of transgenic materials was evaluated using the single-flower drip method (Zhang, X., Zhou, M., Ren, L., Bai, G., Ma, H., Scholten, O.E., Guo, P., and Lu, W. Molecular characterization of Fusarium head blight resistance from wheat variety Wangshuibai. Euphytica, 2004, 139, 59–64.).

[0024] TaALIX After the onset of disease in -OE, an average of 9 florets per spikelet were diseased, while the average in the control wild type was 12. The disease severity was significantly lower than that of the wild type, while TaALIX the number of diseased plants in the mutant line was 14, and the disease severity was significantly higher than that of the wild type Fielder ( Figure 3 、 Figure 4 ).

[0025] Subsequently, the diseased grains of the transgenic materials were ground into flour, and the DON content was determined using a rapid detection kit for vomitoxin (Hua'an Maike, product number: HEM1896), TaALIX The DON content in the diseased grains of the -OE line was 6.7×10 3 μg / kg, significantly lower than 6.98×10 3 μg / kg of the wild type, while TaALIX the DON content in the diseased grains of the mutant line increased significantly, with a specific content of 7.35×10 3 μg / kg ( Figure 5 ).

Claims

1. A key vesicle trafficking protein TaALIX in the ESCRT system, characterized in that, Its amino acid sequence is as shown in SEQ ID No. 2 or its homologous protein sequence, such as the proteins shown in SEQ ID No. 4 and SEQ ID No.

6.

2. The gene encoding as claimed in claim 1 TaALIX , specifically, its nucleic acid sequence is as shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.

5.

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

4. As described in claim 1 TaALIX , or its coding gene, the expression element, recombinant vector, recombinant host cell as described in claim 3, and their applications in improving the disease resistance of plants.

5. The application according to claim 4, wherein The plant is a monocotyledonous plant; the disease resistance refers to plant diseases caused by Fusarium spp. (such as Fusarium graminearum).

6. The application according to claim 4, characterized in that, The plant is wheat, and the disease resistance refers to Fusarium head blight of wheat.

7. A method for preparing a transgenic plant with enhanced disease resistance, characterized in that, Including the step of overexpressing, in a transgenic plant by a transgenic method, a gene encoding the one as described in claim 2 TaALIX and screening to obtain a transgenic plant with enhanced disease resistance.

8. The method according to claim 7, wherein The plant is a monocotyledonous plant; the disease resistance refers to plant diseases caused by Fusarium spp.

9. The method according to claim 8, wherein, The plant is wheat, and the disease resistance refers to resistance to Fusarium head blight of wheat.

10. The method according to claim 8, characterized in that, The screening is carried out by measuring the content of DON in transgenic plants. Specifically, it includes sampling at different times after inoculation with Gibberella zeae and measuring the content of DON.

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