Use of a myo-inositol phosphate synthase in resistance to corn ear rot

Abstract

This invention discloses the application of inositol phosphate synthase in maize ear rot resistance. Using genetic engineering, the ZmMIPS gene was identified and cloned, confirming its important role in maize ear rot resistance. Experimental verification showed that overexpression of the ZmMIPS gene significantly enhanced maize resistance to ear rot in the field and significantly reduced DON toxin levels.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and plant genetics and breeding, and specifically relates to the application of an inositol phosphate synthase in maize ear rot resistance. Background Technology

[0002] Maize (Zea mays L.) is the highest-yielding and most widely planted grain and forage crop in my country. With the increase in maize planting area, the introduction of new varieties, changes in climate conditions, and continuous cropping for many years, maize diseases have become increasingly serious, hindering the healthy development of the maize industry. Maize ear rot, as one of the important diseases of maize, mainly affects the ears and kernels, and in severe cases can cause significant losses in yield and quality. This disease develops rapidly in the middle and late stages of maize growth, posing a significant challenge to its control. Besides causing direct yield loss due to kernel rot, corn ear rot also produces various toxins, such as DON toxin, caused by pathogens in the kernels. This not only reduces corn quality but also poses a significant threat to food and feed safety, directly endangering human and animal health (Pritsch C, Muehlbauer G, Bushnell W, Somers D, Vance C. 2000. Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum. Molecular Plant-MicrobeInteractions®, 13(2): 159-169.). Therefore, controlling the occurrence and damage of corn ear rot is of great significance.

[0003] In recent years, research on maize ear rot resistance has mainly relied on genome-wide association studies (GWAS) and linkage analysis to identify resistance-related genes and quantitative trait loci (QTLs). However, because ear rot resistance is a typical complex quantitative trait, its genetic basis is regulated by multiple minor-effect genes, and the effect size of a single gene is usually small. Therefore, candidate genes obtained through GWAS and linkage analysis still require extensive follow-up work for functional verification. To improve the accuracy and reliability of resistance locus identification, related studies typically need to integrate multi-year, multi-environmental phenotypic data and continuously expand the size of the analyzed population and the number of molecular markers. To date, functional reports on maize ear rot resistance genes remain very limited, with only a few genes experimentally confirmed to be directly related to resistance. For example, Ye et al. constructed a backcross population using the disease-resistant inbred line 1145 and the disease-susceptible inbred line Y331, and finely mapped and identified a negative regulatory gene for maize ear rot, ZmAuxRP1. This gene not only enhances maize stalk rot resistance but also significantly improves maize ear rot resistance (Ye, J., Zhong, T., Zhang, D., Ma, C., Wang, L., Yao, L., Zhang, Q., Zhu, M., Xu, M. 2019. The auxin-regulated protein ZmAuxRP1 coordinates the balance between root growth and stalk rotdisease resistance in maize. Molecular plant, 12(3), 360-373.). In contrast, in wheat, the major QTL for Fusarium head blight, TaHRC, has been successfully cloned. Further research revealed that knocking out the homologous gene corresponding to the validated resistance gene in wheat in maize also significantly improved maize ear rot resistance, indicating that different cereal crops have certain conserved regulatory mechanisms in resistance to Fusarium diseases (Liu, C., Kong, M., Zhu, J., Qi, X., Duan, C., Xie, C. 2022. Engineering null mutants in ZmFER1 confersresistance to ear rot caused by Fusarium verticillioides in maize. PlantBiotechnology Journal, 20(11), 2045.). It is worth noting that compared with genetic studies related to resistance to infection, there are still relatively few reports on mycotoxin detoxification and metabolism in maize ear rot.Given the central role of toxins such as DON in the occurrence and damage of diseases, a new strategy of "fighting diseases with toxins" by enhancing maize's tolerance, translocation or detoxification ability to toxins is expected to provide a new research direction and important breakthrough for the green control of maize ear rot and the creation of disease-resistant germplasm. Summary of the Invention

[0004] This invention provides a method for enhancing maize ear rot resistance using the ZmMIPS gene. This invention identifies and clones the ZmMIPS gene using genetic engineering techniques, confirming its important role in maize ear rot resistance and providing a solution for breeding ear rot-resistant materials. The technical solution adopted in this invention is as follows.

[0005] This invention first provides a ZmMIPS protein and a ZmMIPS gene related to maize disease resistance, derived from the maize variety KN5585. The ZmMIPS protein has the amino acid sequence shown in SEQ ID No. 2, and the ZmMIPS gene contains the nucleotide sequence encoding the ZmMIPS protein. The application of genes encoding similar domain proteins from other plants in the preparation of disease-resistant transgenic plants is also within the scope of this invention.

[0006] This invention provides an inositol phosphate synthase gene ZmMIPS for enhancing maize disease resistance. The inositol phosphate synthase encoded by ZmMIPS is the rate-limiting enzyme in the inositol synthesis pathway. The amino acid sequence of the inositol phosphate synthase is shown in SEQ ID No. 2.

[0007] Preferably, its nucleotide sequence is shown in SEQ ID No. 1.

[0008] The present invention also provides a gene expression element, recombinant vector, or host cell containing the inositol phosphate synthase gene ZmMIPS described above.

[0009] This invention also provides the application of the aforementioned inositol phosphate synthase gene ZmMIPS in improving plant disease resistance.

[0010] Specifically, the plant is a monocotyledonous plant, more preferably maize, and the disease resistance refers to maize's resistance to ear rot.

[0011] The present invention provides a method for improving plant disease resistance, comprising the following steps: increasing the expression level or enhancing the activity of the protein encoded by the inositol phosphate synthase gene ZmMIPS in the plant.

[0012] Specifically, the plant is a monocotyledonous plant, more preferably maize, and the disease resistance refers to maize's resistance to ear rot.

[0013] More specifically, it involves overexpressing the inositol phosphate synthase gene ZmMIPS in plants through genetic engineering methods; or through gene editing technology to overexpress the inositol phosphate synthase gene ZmMIPS in plants.

[0014] This invention provides the application of the method described in the breeding and improvement of disease-resistant maize varieties. It provides a method for applying the disease-resistant ZmMIPS protein in the breeding and improvement of disease-resistant maize varieties. This invention is beneficial for the breeding of disease-resistant maize varieties and provides a basis for later screening of highly resistant maize varieties. For example, this invention can provide ZmMIPS overexpressing plants. Attached Figure Description

[0015] Figure 1 Detection of ZmMIPS expression levels in overexpressing plants (ZmMIPS-OE).

[0016] Figure 2 Phenotypic analysis of ZmMIPS-OE resistance to maize ear rot under field conditions. A, Representative phenotypes of maize ears before and after inoculation with Fusarium graminearum in control plants (KN5585) and overexpression plants (ZmMIPS-OE); B, Statistical analysis of disease incidence in control plants (KN5585) and ZmMIPS-OE plants after inoculation; C, Thousand-kernel weight of diseased kernels.

[0017] Figure 3 Determination of DON toxin content in seeds of overexpressing plants (ZmMIPS-OE). Detailed Implementation

[0018] The present invention will be described below through specific embodiments in order to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0019] Example 1: Genotyping of ZmMIPS overexpressing lines in maize

[0020] Using small molecule protein-protein interaction technology, a maize protein, ZmMIPS, that binds to DON toxin was screened. To understand the role of ZmMIPS in the response to maize ear rot, a maize strain overexpressing ZmMIPS was generated. For the construction of the maize overexpression vector, the full-length CDS fragment of ZmMIPS (SEQ ID NO: 1; where SEQ ID NO: 3 is the corresponding 5' regulatory region sequence) was inserted into the WMV068 vector with a Ubiquitin promoter using In-Fusion cloning technology (Clontech, catalog number 638910). All constructs were transformed into Agrobacterium EHA105 strain. The maize transformation process was as follows: Freshly peeled immature embryos, approximately 1 mm in size, were placed in 2 mL plastic centrifuge tubes pre-filled with 1.8 mL of suspension. After removing the suspension from the bottom of the tube, 1.0 mL of Agrobacterium suspension was added, and the mixture was allowed to stand for 5 minutes. The immature embryos and bacterial suspension were mixed thoroughly and poured onto the surface of the co-culture medium. Excess bacterial suspension was removed, and the mixture was co-cultured at 23°C in the dark for 3 days. The embryos were then transferred to resting medium and cultured in the dark at 28°C for 6 days, followed by two weeks of culture in a selection medium containing diammonium phosphate. Resistant callus tissue was selected and inoculated into differentiation medium, cultured at 25°C and 5000 lx light for 3 weeks. After seedling differentiation, the embryos were transferred to rooting medium and cultured under the same temperature and light conditions until rooting. Finally, the seedlings were transplanted into small pots and planted in a greenhouse at the appropriate time. Seeds were harvested 3-4 months later. Seven positive plants were identified by qPCR. Figure 1 ).

[0021] Example 2: Overexpression of ZmMIPS in maize enhances its resistance to maize ear rot.

[0022] Fusarium head blight resistance was assessed in maize transgenic materials 1 and 7, which were positive plants overexpressing ZmMIPS (ZmMIPS-OE) in Example 1. In the field, FHB resistance was assessed using an injection method as follows: The strain was first cultured on potato dextrose agar (PDA) for approximately 2 weeks. Mycelial blocks were then removed and the strain was cultured in mung bean soup at 200 rpm and 28 °C for 2-3 days. The spore suspension was filtered, and a spore suspension of *Fusarium graminearum* strain Fg0609 was prepared in 0.001% Tween-20 solution at 1 × 10⁶ spores / mL. Approximately 10 days after the emergence of maize silks, wounds were created in the middle of the maize ear, and 200 µL of the spore suspension was injected into each wound. (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 inmaize. Theor. Appl. Genet. 137, 222.) After inoculation with *Fusarium graminearum*, the wild-type control KN5585 maize ears showed more obvious ear rot symptoms, with a wider distribution of diseased kernels and more severe damage to the ear; in contrast, the disease symptoms of the ZmMIPS-OE transgenic material were significantly reduced, and the diseased area was smaller, indicating that overexpression of ZmMIPS can enhance maize's resistance to *Fusarium graminearum* infection. Figure 2 (A). Statistical analysis of the severity of the disease revealed that the average affected area on the ear of ZmMIPS-OE plants after infection covered approximately 80 corn kernels, while the average for the wild-type control was approximately 170 kernels. The former was significantly lower than the latter, indicating that ZmMIPS overexpression can significantly inhibit the spread of the disease. Figure 2 (Middle B). Further analysis of the thousand-grain weight of diseased ears revealed that the thousand-grain weight of diseased ears in ZmMIPS-OE plants was approximately 120 g, while that in the wild-type control was only approximately 80 g. These results indicate that overexpression of ZmMIPS not only reduced the severity of maize ear rot but also significantly alleviated grain loss caused by the disease. Figure 2 (C)

[0023] In summary, the field inoculation results indicate that ZmMIPS overexpression can significantly improve maize's resistance to ear rot caused by Fusarium graminearum and mitigate the adverse effects of the disease on yield.

[0024] Example 3: Overexpression of ZmMIPS in maize significantly reduces the level of DON toxin produced by maize ear rot.

[0025] The diseased corn kernels from Example 2 were ground into flour, and the DON content was determined using a rapid vomitoxin detection kit (Huaan Maike, catalog number: HEM1896). The DON content in the diseased kernels of the ZmMIPS-OE strain was 25 mg / kg, significantly lower than the 48 mg / kg of the wild type. Figure 3 ).

Claims

1. An inositol phosphate synthase gene ZmMIPS for enhancing maize disease resistance, which encodes inositol phosphate synthase, a rate-limiting enzyme in the inositol synthesis pathway, and the amino acid sequence of inositol phosphate synthase is shown in SEQ ID No.

2.

2. The inositol phosphate synthase gene ZmMIPS as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID No.

1.

3. The promoter of the ZmMIPS gene according to claim 1 or 2, characterized in that, Its nucleotide sequence is shown in SEQ ID No.

3.

4. A gene expression element, recombinant vector, or host cell containing the inositol phosphate synthase gene ZmMIPS as described in claim 1 or 2.

5. The application of the inositol phosphate synthase gene ZmMIPS as described in claim 1 or 2, or the promoter as described in claim 3, in improving plant disease resistance.

6. The application as described in claim 5, characterized in that, The plant is a monocotyledonous plant, more preferably maize, and the disease resistance refers to maize's resistance to ear rot.

7. A method for improving plant disease resistance, characterized in that, The method includes the following steps: increasing the expression level or enhancing the activity of the protein encoded by the inositol phosphate synthase gene ZmMIPS as described in claim 1 or 2 in plants.

8. The method as described in claim 7, characterized in that, The plant is a monocotyledonous plant, more preferably maize, and the disease resistance refers to maize's resistance to ear rot.

9. The method as described in claim 7, characterized in that, This involves overexpressing the inositol phosphate synthase gene ZmMIPS in plants using genetic engineering methods; or using gene editing technology to overexpress the inositol phosphate synthase gene ZmMIPS in plants. Specifically, gene editing technology is used to edit the promoter of the inositol phosphate synthase gene ZmMIPS in plants, thereby significantly increasing the expression level of its encoded protein.

10. The application of the method as described in any one of claims 7 to 9 in the breeding and improvement of disease-resistant maize varieties, specifically, the disease resistance refers to the resistance of maize to ear rot.

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