Application of maize ZmALIX protein in reducing accumulation of vomitoxin and enhancing plant resistance to disease

Abstract

This invention relates to the fields of biotechnology and plant protection, disclosing the application of maize ZmALIX protein in reducing vomitoxin accumulation and enhancing plant disease resistance. This invention is the first to discover that increasing the expression level of maize ESCRT III protein ZmALIX reduces the DON content in maize and enhances maize's resistance to ear rot, which has practical significance in plant disease control.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of maize ZmALIX protein in reducing vomitoxin accumulation and enhancing plant disease resistance. Background Technology

[0002] Maize ear rot, primarily caused by various Fusarium fungi (such as Fusarium graminearum), is a devastating fungal disease prevalent in major maize-producing regions worldwide. It not only causes ear rot and kernel abortion, leading to a sharp decline in yield, but more seriously, it produces mycotoxins such as fumonisins and deoxynivalenol (DON), which accumulate in large quantities in maize kernels. These toxins not only directly threaten human health (e.g., inducing esophageal cancer and neural tube defects), but also seriously endanger livestock safety through feed chain contamination, posing a triple threat to food security, food safety, and feed safety. Of particular note is that as the world's largest cereal crop, the stability of maize production is crucial to the global food supply structure and the foundation of the bioenergy industry. In recent years, the frequent occurrence of extreme weather events caused by global warming (such as high temperature and humidity during flowering) has greatly exacerbated the risk of corn ear rot and the difficulty of prevention and control. This has made the breeding of corn varieties with high disease resistance and low toxin accumulation a core strategic need to ensure the sustainable development of the global corn industry, and it has irreplaceable urgency and overall significance.

[0003] Currently, identifying disease resistance genetic loci and elucidating their molecular mechanisms are core methods for improving maize resistance. Studies have shown that maize resistance to ear rot is a complex quantitative trait controlled by multiple genes, with moderate to high heritability. With the advancement of multi-omics technologies, researchers have located multiple major-effect QTLs on key chromosomes 1, 5, and 9, and identified the important resistance locus QTL-qRfv2 related to maize resistance to ear rot (FER). These genes play a protective role by enhancing physical barriers or triggering immune signal transduction. However, due to the evolution of pathogen populations and the superposition of environmental stresses, the depth of exploration and utilization efficiency of existing resistance resources are still insufficient to meet the needs of precision breeding.

[0004] In terms of toxin control, traditional methods rely heavily on chemical fungicides, which not only leave pesticide residues but also struggle to penetrate deep into the grains to degrade existing toxins. Currently, the industry is shifting towards a comprehensive control strategy combining "precise disease control and biological detoxification," including targeted gene editing using CRISPR / Cas9 and biological intervention during the silking stage. On the other hand, maize possesses sophisticated detoxification mechanisms; for example, uridine diphosphate glucosyltransferase (UGT) effectively catalyzes the glucosylation modification of toxins, significantly reducing their biotoxicity and serving as a crucial component of endogenous resistance. Beyond traditional toxin synthesis inhibition and chemical modification strategies, recent research (such as the 2021 report) has revealed novel vesicle transport-mediated cellular detoxification pathways, opening revolutionary avenues for enhancing plant tolerance to toxins. While the role of the plant ESCRT system in protein sorting and vacuolar degradation is recognized, whether it can actively recognize and transport small molecule fungal toxins such as fumonisin and DON into vacuolars for isolation or degradation remains a key unresolved scientific question, holding immense potential for mechanistic innovation. In conclusion, in-depth research into the highly effective resistance genes and detoxification mechanisms of maize ear rot, and the development of corresponding green detoxification solutions, have become urgent needs to ensure national food security and sustainable agricultural development.

[0005] Focusing on the functional verification of key resistance genes (such as the ZmALIX gene in maize), this invention employs precise genetic engineering techniques (such as homologous recombination) to construct efficient expression vectors and utilizes a mature Agrobacterium-mediated maize genetic transformation system to create stable transgenic materials. Rigorous artificial inoculation resistance evaluation under greenhouse conditions has confirmed that these materials exhibit significantly enhanced resistance to ear rot and reduced toxin accumulation, representing a solid step towards practical application. However, greenhouse results must undergo rigorous testing in large-scale, multi-ecological-site field trials. Summary of the Invention

[0006] Based on the inventors' research, it was discovered for the first time that overexpression of the ESCRT-related protein ZmALIX can reduce the accumulation of vomitoxin during the disease process of maize ear rot and enhance resistance to ear rot. This invention thus completes the present invention.

[0007] The present invention first provides a key transport protein ZmALIX for the ESCRT system, the amino acid sequence of which is shown in SEQ ID No. 3.

[0008] The gene encoding the protein ZmALIX, specifically, has the nucleotide sequence SEQ ID No. 1.

[0009] Furthermore, the present invention provides an expression element containing the aforementioned gene, a recombinant vector, and a host cell.

[0010] Preferably, the gene is overexpressed in the plant via a transgenic method.

[0011] The present invention also provides the application of the gene in the creation of disease-resistant transgenic plants, wherein the gene is overexpressed in the transgenic plants by a transgenic method.

[0012] Preferably, the plant is a monocotyledonous plant, and more preferably, the plant is maize. More preferably, the disease resistance refers to resistance to maize ear rot caused by Fusarium.

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

[0014] Specifically, the plant is a monocotyledonous plant; the disease resistance refers to resistance to plant diseases caused by Fusarium.

[0015] More preferably, the plant is corn, and the disease resistance refers to corn ear rot.

[0016] Specifically, the screening involves measuring the DON content in the transgenic plants. This includes sampling and measuring the DON content at different times after inoculation with Fusarium. If the accumulation of DON content decreases or increases after inoculation with Fusarium, it indicates enhanced disease resistance.

[0017] This invention relates to the application of an ESCRT system-associated protein (ZmALIX) in disease resistance. Overexpression of the ZmALIX gene does not affect the growth and yield of maize plants; however, transgenic plants overexpressing the ZmALIX gene exhibit significant resistance to maize ear rot and can inhibit the spread of Fusarium within host cells.

[0018] The inventors have discovered that maize overexpressing the ZmALIX gene exhibits resistance to Fusarium wilt and significantly reduced toxin content, suggesting that overexpression can be widely applied in practice. This invention provides ZmALIX overexpressing plants, which is beneficial for the breeding of disease-resistant maize varieties and provides a basis for later screening of highly resistant maize varieties. Attached Figure Description

[0019] Figure 1 Genotyping of maize strains overexpressing ZmALIX.

[0020] Figure 2 Representative images of maize 40 days after infection with Fusarium graminearum by WT (KN5585) and ZmALIX-OE under field conditions.

[0021] Figure 3 Quantitative analysis of the ZmALIX-OE morbidity index.

[0022] Figure 4 For the determination and analysis of DON content. Detailed Implementation

[0023] The present invention will be described below through specific embodiments to better understand the invention, but these embodiments do not constitute a limitation thereof.

[0024] Example 1: Genotyping of maize lines overexpressing ZmALIX

[0025] For the maize overexpression constructs, the full-length CDS fragment of ZmALIX (SEQ ID NO: 1, the wheat TaALIX target gene for vomitoxin DON, which plays an important role in wheat resistance to Fusarium head blight, was inserted into the WMV068 vector with a Ubiquitin promoter using In-Fusion cloning technology (Clontech, catalog number 638910) was inserted into the vector. This was done by first-and-second screening of wheat TaALIX, a homologous gene of wheat TaALIX in maize, which is also a functional gene in the maize ESCRT system; the encoded amino acid sequence is shown in SEQ ID NO: 3; SEQ ID NO: 2 is the corresponding 5' regulatory region sequence) was then transformed into Agrobacterium EHA105 strain. The maize transformation process was as follows: freshly peeled maize embryos (approximately 1 mm in size) were placed in a 2 ml plastic centrifuge tube containing 1.8 mL of suspension. The suspension at the bottom of the tube was removed, and 1.0 ml of Agrobacterium suspension was added. The tube was incubated for 5 min. After suspending the immature embryos, they were poured onto a co-culture medium, and excess Agrobacterium tumefaciens solution was aspirated from the surface. They were then co-cultured at 23°C in the dark for 3 days. After 3 days, the immature embryos were placed on resting medium and cultured at 28°C in the dark for 6 days, then transferred to a selection medium containing diammonium phosphate for two weeks. Resistant callus tissue was transferred to differentiation medium and cultured at 25°C under 5000 lx light for 3 weeks. The differentiated seedlings were then transferred to rooting medium and cultured at 25°C under 5000 lx light until rooting. The seedlings were then transferred to small pots for growth, and after a certain growth stage, they were transplanted into a greenhouse. Seeds were harvested 3-4 months later. Five positive plants were identified by qPCR. Figure 1 ).

[0026] Example 2: Overexpression of ZmALIX in maize enhances resistance to maize ear rot and reduces DON content.

[0027] The transgenic maize material of positive plants obtained in Example 1, which overexpressed ZmALIX (ZmALIX-OE), was used to identify its resistance to ear rot.

[0028] In field trials, FHB resistance in transgenic materials was assessed using an injection method, as follows: First, *Fusarium graminearum* Fg0609 was cultured on potato dextrose agar (PDA) for approximately two weeks. Mycelial blocks were then removed and cultured in mung bean soup at 200 rpm and 28 °C for 2-3 days. The spore suspension was then filtered, counted, and adjusted to a concentration of 1 × 10⁻⁶. 6 A concentration of spores / mL was added, with a final concentration of 0.001% of surfactant Tween-20. For field planting, approximately 10 days after the appearance of maize silks, wounds were created in the middle of the maize ear using a syringe, and 200 µL of the above spore suspension was injected into each wound using a pipette to complete the inoculation (Zhou, G., Ma, L., Zhao, C., Xie, F., Xu, Y., Wang, Q., Hao, D., and Gao, X. (2024). Genome-wide association study and molecular marker development for susceptibility to Gibberella ear rot in maize. Theor. Appl. Genet. 137,222.). After ZmALIX-OE disease onset ( Figure 2 The average diseased area on the ear covered 100 corn kernels, while the average in the wild-type control group was 150 kernels, indicating a significantly lower disease severity in the wild-type. Figure 3 ).

[0029] Subsequently, the diseased grains of the transgenic material were ground into flour, and the DON content was determined using a vomitoxin rapid detection kit (Huaan Maike, catalog number: HEM1896). The DON content in the diseased grains of the ZmALIX-OE strain was 4.0 × 10⁻⁶. 4 μg / kg was significantly lower than the wild type's 6.0×10 μg / kg. 4 μg / kg ( Figure 4 ).

Claims

1. A key transport protein ZmALIX in the endosomal sorting and transport complex, characterized in that, Its amino acid sequence is shown in SEQ ID No.

3.

2. A gene encoding the protein ZmALIX as described in claim 1, specifically, the nucleic acid sequence of which is shown in SEQ ID No.

1.

3. An expression element containing the gene as described in claim 2, a recombinant vector, or a recombinant host cell.

4. The application of the protein ZmALIX as described in claim 1, or its encoding gene, the expression element as described in claim 3, the recombinant vector, and the recombinant host cell in improving plant disease resistance.

5. The application as described in claim 4, characterized in that, The plant is a monocotyledonous plant; the disease resistance refers to resistance to plant diseases caused by Fusarium (e.g., Fusarium graminearum).

6. The application as described in claim 4, characterized in that, The plant in question is corn, and the disease resistance refers to corn ear rot.

7. A method for preparing transgenic plants with enhanced disease resistance, characterized in that, The method includes the steps of overexpressing the gene as described in claim 2 in transgenic plants through transgenic methods, and screening to obtain transgenic plants with enhanced disease resistance.

8. The method as described in claim 7, characterized in that, The plant is a monocotyledonous plant; the disease resistance refers to resistance to plant diseases caused by Fusarium (e.g., Fusarium graminearum).

9. The method as described in claim 8, characterized in that, The plant in question is corn, and the disease resistance refers to corn ear rot.

10. The method as described in claim 8, characterized in that, The screening process involves determining the DON content in transgenic plants. Specifically, this includes sampling and measuring the DON content at different times after inoculation with Fusarium. If the accumulation of DON content decreases after inoculation with Fusarium, it indicates enhanced disease resistance.

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