Application of wheat TaMIPS gene in gibberellic disease and stem rot resistance

Abstract

The invention discloses an application of a wheat gene TaMIPS in regulating and controlling the resistance of gibberellic disease and basal stem rot. Wheat materials are created, it is proved that overexpression of the TaMIPS gene remarkably enhances the resistance of wheat to gibberellic disease and basal stem rot, and the disease resistance of a mutant of the TaMIPS gene is reduced. Meanwhile, the promoter region of the TaMIPS gene is optimized by adopting a gene editing technology, and the inhibition effect of an upstream non-coding open reading frame (uORF) on the expression of the TaMIPS gene is relieved, so that the expression quantity of TaMIPS gene coding protein is remarkably improved, and the disease resistance of wheat is effectively enhanced.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and plant genetic breeding, and particularly relates to the application of a wheat TaMIPS gene in enhancing the resistance of wheat to Fusarium head blight and basal stalk rot. Background Art

[0002] As one of the main food crops in China, the safe production of wheat is of great strategic significance to the national food security. Wheat Fusarium head blight (FHB) and Fusarium crown rot (FCR) caused by Fusarium spp. are devastating and difficult-to-control fungal diseases worldwide, seriously threatening the safe production of wheat. China is one of the countries with the largest affected areas of wheat FHB and FCR in the world. In the past five years, the average annual incidence area nationwide has exceeded 30% of the wheat planting area. Every year, the average yield reduction of wheat in China caused by FHB and FCR is as high as 10%-30%, and even a complete crop failure may occur during a pandemic. More seriously, a series of fungal toxin metabolites, such as deoxynivalenol (DON), are produced during the infection process of the pathogenic bacteria, contaminating wheat grains and further threatening the health and safety of humans and livestock (Mishra, S., Srivastava, S., Dewangan, J., Divakar, A., & Kumar Rath, S. (2020). Global occurrence of deoxynivalenol in food commodities and exposure risk assessment in humans in the last decade: A survey. Critical Reviews in Food Science and Nutrition, 60, 1346–1374.). Since 2010, through the monitoring of wheat production, it has been found that the DON toxin in wheat grains in many major wheat-producing provinces of China seriously exceeds the standard, and the detection rate is as high as 95%.On the other hand, toxins such as DON can act as pathogenic factors, disrupting wheat tissue development and promoting the occurrence of diseases (Desmond, O.J., Manners, J.M., Stephens, A.E., Maclean, D.J., Schenk, P.M., Gardiner, D.M., Munn, A.L., and Kazan, K. (2008). The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat. Mol. Plant Pathol. 9, 435–445; He, Y., Yang, X., Xia, X., Wang, Y., Dong, Y., Wu, L., Jiang, P., Zhang, X., Jiang, C., Ma, H., et al. (2024). A phase-separated protein hub modulates resistance to Fusarium head blight in wheat. Cell Host & Microbe, 32(5), 710-726.).

[0003] In recent years, multiple plant proteins mediating DON modification have been reported to be involved in the resistance to Fusarium head blight in wheat. Wang et al. cloned the major candidate gene Fhb7 for Fusarium head blight resistance using methods such as map-based cloning and genome sequencing. The glutathione S-transferase encoded by this gene can destroy the epoxy group of DON and other trichothecene toxins by catalyzing the conjugation reaction of glutathione with them, thereby conferring the detoxification ability and further improving the resistance to Fusarium head blight in wheat (Wang H, Sun S, Ge W, Zhao L, Hou B, Wang K, Lyu Z, Chen L, Xu S, Guo J, Li M, Su P, Li X, Wang G, Bo C, Fang X, Zhuang W, Cheng X, Wu J, Dong L, Chen W, Li W, Xiao G, Zhao J, Hao Y, Xu Y, Gao Y, Liu W, Liu Y, Yin H, Li J, Li X, Zhao Y, Wang X, Ni F, Ma X, Li A, Xu SS, Bai G, Nevo E, Gao C, Ohm H, Kong L. Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat. Science, 2020, 368(6493):eaba5435.). Additionally, plant UDP-glycosyltransferases (UGTs) can glycosylate DON to reduce its toxicity. Active UGT enzymes have been identified in wheat, barley, and rice and are involved in regulating DON tolerance and Fusarium head blight resistance (Tian, Y., Zhang, D., Cai, P., Lin, H., Ying, H., Hu, Q. N., & Wu, A. (2022). Elimination of Fusarium mycotoxin deoxynivalenol (DON) via microbial and enzymatic strategies: Current status and future perspectives. Trends in Food Science & Technology, 124, 96-107). In summary, the accumulation of DON toxin is closely related to the spread of Fusarium graminearum. Controlling the accumulation of DON toxin in wheat can effectively reduce the damage caused by Fusarium head blight in wheat.However, due to the influence of factors such as genetic background and environmental adaptability, the disease resistance effects of existing resistance genes vary among different wheat varieties. Therefore, screening for new disease resistance genes and achieving stable improvement of disease resistance traits through molecular breeding methods are important strategies for enhancing wheat disease resistance. Summary of the Invention

[0004] The present invention provides a method for enhancing resistance to Fusarium head blight and basal stalk rot in wheat TaMIPS through a gene. Through DON toxin response, a TaMIPS gene in wheat was identified, and the inositol phosphate synthase encoded by it remodels wheat inositol metabolism and weakens the function of DON toxin in the pathogenesis of Fusarium. The present invention confirmed the important role of TaMIPS in wheat resistance to Fusarium head blight and basal stalk rot through genetic engineering means, providing a solution for the cultivation of Fusarium head blight-resistant materials. The present invention adopts the following technical solutions.

[0005] The present invention first provides a TaMIPS protein related to wheat disease resistance and TaMIPS a gene, which is derived from multiple wheat varieties such as Fielder, Ningmai 18, and Yangmai 38. The TaMIPS protein has the amino acid sequence shown in SEQ ID No.2, and the TaMIPS gene contains the nucleotide sequence encoding the TaMIPS protein. The application of genes encoding proteins with the above similar domains in other plants in the preparation of disease-resistant transgenic plants also belongs to the protection scope of the present invention.

[0006] The present invention provides an inositol phosphate synthase gene for enhancing wheat disease resistance TaMIPS , and the inositol phosphate synthase encoded by it 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, SEQ ID No.5, or SEQ ID No.8.

[0007] Preferably, its nucleotide sequence is shown in SEQ ID No.1, SEQ ID No.4, or SEQ ID No.7.

[0008] The present invention also provides the promoter of the TaMIPS gene, and its nucleotide sequence is shown in SEQ ID No.3, SEQ ID No.6, or SEQ ID No.9.

[0009] The present invention further provides a gene expression element, recombinant vector, or host cell containing the inositol phosphate synthase gene TaMIPS .

[0010] The present invention also provides the inositol phosphate synthase gene TaMIPS, or the application of the promoter in improving plant disease resistance.

[0011] Specifically, the plant is a monocotyledonous plant, more preferably wheat, and the disease resistance refers to the resistance of wheat to Fusarium head blight or basal stalk rot.

[0012] The present invention provides a method for improving plant disease resistance, which comprises the following steps: increasing the expression level of the protein encoded by the inositol phosphate synthase gene TaMIPS in plants or enhancing its activity.

[0013] Specifically, the plant is a monocotyledonous plant, more preferably wheat, and the disease resistance refers to the resistance of wheat to Fusarium head blight or basal stalk rot.

[0014] More specifically, it is to overexpress the inositol phosphate synthase gene in plants by genetic engineering methods TaMIPS ; or overexpress the inositol phosphate synthase gene in plants by gene editing technology TaMIPS ; specifically, by using gene editing technology to edit the promoter of the inositol phosphate synthase gene TaMIPS in plants to relieve the inhibition of its expression by uORF, thereby significantly increasing the expression level of the protein it encodes.

[0015] The present invention provides the application of the above method in the improvement of wheat disease-resistant varieties. A method for the application of TaMIPS protein related to wheat disease resistance in the improvement of wheat disease-resistant varieties is provided. The present invention is beneficial to the cultivation of wheat disease-resistant varieties and provides a basis for screening highly disease-resistant wheat varieties in the later stage. For example, the present invention can provide TaMIPS overexpressing plants, mutants, and plants with increased expression by gene editing of their promoter regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is for wheat MIPS is a positive regulator of wheat Fusarium head blight. Figure A shows the determination of the expression level of overexpressing plants ( MIPS -OE). Figure B shows the genotype determination of mips-bb materials. Figure C shows the representative images (left) of wheat spikes and the quantitative analysis of the disease index (right) 13 days after infection with Fusarium graminearum of the control (Fielder), overexpressing plants ( MIPS -OE), and mutant plants (mips-bb) under greenhouse conditions. Figure D shows the representative images (left) of the leaves of the control (Fielder), TaMIPS -OE, and mips-bb infected with Fusarium graminearum at 5 days and the statistical analysis of the lesion area (right).

[0017] Figure 2 is for wheat MIPSRegulate wheat resistance to basal stalk rot. Panel A shows the control (Fielder), overexpressing plants ( MIPS -OE), and mutant plants (mips-bb) inoculated with Fusarium graminearum conidial suspension for 14 days. Panel B shows the quantitative determination of the diseased stem length of wheat basal stalk rot control (Fielder), overexpressing plants ( MIPS -OE), and mutant plants (mips-bb) seedlings.

[0018] Figure 3 By gene editing in wheat TaMIPS The promoter region can enhance its resistance to Fusarium head blight of wheat. Panel A shows the detection of the function of uORF by domain prediction and dual-luciferase methods. Panel B shows the genotype determination of the materials with gene-edited promoter regions. Panel C shows the WB detection of the expression level of MIPS protein in the plants obtained by gene editing. Panel D shows the representative images (upper) of wheat spikes and the quantitative analysis of the disease index (lower) of the control (Fielder) and the materials with gene-edited promoter regions infected with Fusarium graminearum after 15 days under greenhouse conditions. Detailed implementation manners

[0019] The present invention will be described below through specific examples for better understanding of the present invention, but it does not constitute a limitation to the present invention.

[0020] Example 1: Overexpression in wheat TaMIPS Can enhance its resistance to Fusarium head blight of wheat The TaMIPS candidate gene contributing to the resistance to Fusarium head blight of wheat was screened by forward genetics. For the wheat overexpression construct, the full-length CDS fragment of TaMIPS (SEQ ID NO: 1) was inserted into the pUbiGW vector with the Ubiquitin promoter through the BamH I site using the In-Fusion cloning technology (Clontech, catalog number 638910). 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. (2021). Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos. Rom. Agric. Res. 38 , 99 - 107 (2021)). The Basta resistance of transgenic plants was evaluated using the Enviologix QuickStix Kit (Enviologix, catalog number AS013). The overexpression of T1 and T2 generations was further confirmed by qRT-PCR or the homozygous mutation was identified by sequencing with gene-specific primers.

[0021] To understand TaMIPS its role in response to Fusarium head blight in wheat, we generated stable ectopic overexpression TaMIPS ( MIPS -OE) (SEQ ID NO: 1) wheat transgenic materials, and used gene editing methods to generate wheat mutant (mips-bb) plants. Under greenhouse conditions, MIPS after the onset of disease in -OE, the severity of spikelet disease was significantly lower than that of the wild-type Fielder, while the onset of disease in mutant plants was significantly higher than that of the wild-type Fielder. We also tested the disease symptoms caused by Fusarium graminearum in the leaves of the above materials. Notably, compared with the wild type, MIPS -OE showed a significant reduction in lesion size in the leaves, while the onset of disease in the mutants increased ( Figure 1 A, B in

[0022] Among them, the treatment method for detached leaves: Wild-type (Fielder) and transgenic wheat plants were grown in a growth chamber at 22 °C with a photoperiod of 16 h light and 8 h dark. Detached secondary leaves of 2-week-old plants were collected and transferred to a square petri dish containing 1% water agar after treatment with 20 μM DON for 6 h. 10 μL of conidial suspension (about 5×10 4 conidia / mL) was cultured in the center of the wheat leaves. At 5 - 6 days after infection, ImageJ (https: / / www.computerbild.de / download / ImageJ-422527.html) was used to record the infection symptoms to evaluate the necrotic lesions.

[0023] Example 2: Overexpression of TaMIPS in wheat can enhance its resistance to wheat basal stalk rot To understand TaMIPS its role in response to wheat basal stalk rot, we also tested the disease symptoms caused by Fusarium graminearum in the stems of wild-type, overexpressing materials, and mutant materials. Notably, compared with the wild type, MIPS -OE showed a significant reduction in the length of the diseased area at the basal stem region, while the length of the diseased area in the mutants increased ( Figure 2 A, B in

[0024] Among them, the inoculation method for wheat basal stalk rot is as follows: Take wheat seedlings with similar growth vigor, first directly soak them in the Gibberella zeae spore solution for 5 min, then place them in the middle of the wet double-layer kitchen paper, and then cover the wet paper strip on the basal stem position. After rolling it up and wrapping it with plastic wrap, place it upright in a water cup, add water to submerge 1 / 3 to 1 / 2 of the paper roll, and culture it at 25 °C with a 16 h / 8 h light-dark cycle. After 2 days, uncover the plastic wrap to observe the disease situation.

[0025] Example 3: Gene Editing in Wheat TaMIPS The promoter region can enhance its resistance to Fusarium head blight in wheat Through structural prediction, TaMIPS there is a uORF box in the promoter region. At the same time, promoters with deletion or mutation in the uORF region (ATG mutated to AAA) were designed. The above fragments were ligated to a luciferase reporter gene vector and transiently transformed into tobacco to detect the luciferase activity to determine the inhibitory effect of uORF on the translation of MIPS protein. Through sequence analysis, it was found that there are uORFs (uORF-BB, uORF-DD) in the promoter region of MIPS on the B and D chromosomes of hexaploid wheat. The uORF in the promoter region was edited using the CRISPR / Cas9 technology to obtain Crispr-uORF-MIPS plants, and it was found that the protein expression level of TaMIPS was significantly increased. Under greenhouse conditions, after the Crispr-uORF-MIPS plants were diseased, the severity of spikelet disease was significantly lower than that of the wild type Fielder ( TaMIPS in A, B, C, D). Figure 3 ​

Claims

1. An inositol phosphate synthase gene TaMIPS for enhancing wheat disease resistance, and the inositol phosphate synthase encoded by the gene is a rate-limiting enzyme in the inositol synthesis pathway, and the amino acid sequence of the inositol phosphate synthase is shown in SEQ ID No.2, SEQ ID No.5 or SEQ ID No.

8.

2. The inositol phosphate synthase gene TaMIPS according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID No.1, SEQ ID No.4 or SEQ ID No.

7.

3. The promoter of the TaMIPS gene according to claim 1 or 2, characterized in that, Its nucleotide sequence is shown in SEQ ID No.3, SEQ ID No.6 or SEQ ID No.

9.

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

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

6. The application according to claim 5, characterized in that The plant is a monocotyledonous plant, more preferably wheat, and the disease resistance refers to the resistance of wheat to Fusarium head blight or basal stalk rot.

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

8. The method according to claim 7, wherein The plant is a monocotyledonous plant, more preferably wheat, and the disease resistance refers to the resistance of wheat to Fusarium head blight or basal stalk rot.

9. The method according to claim 7, characterized in that It overexpresses the inositol phosphate synthase gene TaMIPS in the plant by genetic engineering methods; or overexpresses the inositol phosphate synthase gene TaMIPS in the plant by gene editing technology; specifically, the promoter of the inositol phosphate synthase gene TaMIPS in the plant is edited by gene editing technology to relieve the inhibition of its expression by uORF, thereby significantly increasing the expression level of the protein encoded by it.

10. The application of the method according to any one of claims 7 to 9 in the improvement of cultivating wheat disease-resistant varieties.

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