Application of glutathione transferase gene EuGST in regulating and controlling ear rot and stem rot resistance of maize fusarium graminearum

Abstract

The invention discloses application of a glutathione transferase gene EuGST in regulating and controlling ear rot and stem rot resistance of maize fusarium graminearum, and belongs to the technical field of gene engineering. The invention discloses an application of a glutathione transferase gene EuGST in regulation and control of corn fusarium graminearum ear rot and stem rot resistance. A nucleotide sequence of the glutathione transferase gene EuGST is shown as SEQ ID NO.1. The invention also discloses an application of the glutathione transferase gene EuGST in regulation and control of corn fusarium graminearum ear rot and stem rot resistance. Fusarium graminearum is inoculated to ears and stems of an overexpressed EuGST transgenic line, so that the ear rot and stem rot resistance of the fusarium graminearum of the maize inbred line is improved; fusarium graminearum is inoculated to ears and stems of the overexpressed EuGST transgenic hybrid, so that the ear rot and stem rot resistance of the fusarium graminearum of the corn hybrid is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, more particularly to the application of glutathione transferase gene EuGST in regulating the resistance of corn to Fusarium graminearum head blight and stalk rot. BACKGROUND

[0002] Glutathione-S-transferase (GST) is a multifunctional enzyme encoded by a multigene family that catalyzes the conjugation of glutathione to hydrophobic and electrophilic compounds through a nucleophilic substitution reaction. It is widely present in plants, animals and microorganisms. At present, researchers have made a relatively extensive study on GST and found that it is involved in reducing the toxicity of heterologous substances, transmembrane transport, protecting cells from oxidative damage, maintaining plant metabolism and responding to various abiotic stresses in different species. In wheat, the GST family member TaGSTU6 interacts with the CBS domain-containing protein TaCBSX3 to regulate the resistance to wheat powdery mildew; the GST encoded by FHB7 gene can bind GSH to the epoxy group of A-type and B-type trichothecenes to detoxify DON, thereby achieving the resistance to wheat scab. In sugarcane, ScOPR1 enhances plant disease resistance by increasing the accumulation of JA, SA and GST and up-regulating the expression of signal pathway-related genes. In summary, glutathione transferase (GSTs) plays a multiple key role in plant immunity, which mainly protects plant cells from oxidative stress by catalyzing the reaction of glutathione (GSH) with various toxins, metabolites and peroxides.

[0003] At present, there is a lack of corn head rot and stalk rot disease-resistant resources, which cannot meet the needs of corn resistance breeding. It is of great significance to mine the genes related to the resistance of corn to head rot and stalk rot caused by Fusarium graminearum. Glutathione-S-transferase is an important disease-resistant resource in plants. Therefore, it is an urgent problem for those skilled in the art to provide the application of glutathione transferase gene EuGST in regulating the resistance of corn to head rot and stalk rot caused by Fusarium graminearum. SUMMARY

[0004] Therefore, the present application provides the application of glutathione transferase gene EuGST in regulating the resistance of corn to head rot and stalk rot caused by Fusarium graminearum.

[0005] The present application provides the application of glutathione transferase gene EuGST in regulating the resistance of corn to head rot and stalk rot caused by Fusarium graminearum by using gene overexpression technology. The glutathione transferase gene EuGST and the encoded protein thereof can be used to improve the resistance of corn to head rot and stalk rot caused by Fusarium graminearum, and provide potential genetic resources for breeding corn varieties resistant to head rot and stalk rot caused by Fusarium graminearum.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] The application of glutathione transferase gene EuGST in regulating the resistance of corn to Fusarium graminearum head rot and stalk rot, wherein the nucleotide sequence of the glutathione transferase gene EuGST is shown as SEQ ID NO. 1.

[0008] Further, the application of overexpressing glutathione transferase gene EuGST in improving the resistance of corn to Fusarium graminearum head rot and stalk rot, wherein the nucleotide sequence of the glutathione transferase gene EuGST is shown as SEQ ID NO. 1.

[0009] Further, the application of biological material overexpressing glutathione transferase gene EuGST in improving the resistance of corn to Fusarium graminearum head rot and stalk rot, wherein the nucleotide sequence of the glutathione transferase gene EuGST is shown as SEQ ID NO. 1.

[0010] The biological material is any one of the following:

[0011] A: an expression cassette capable of overexpressing glutathione transferase gene EuGST with the nucleotide sequence shown as SEQ ID NO. 1;

[0012] B: a recombinant vector containing the expression cassette described in A;

[0013] C: a recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

[0014] Further, a method for improving the disease resistance of corn to Fusarium graminearum head rot and stalk rot, overexpressing glutathione transferase gene EuGST in corn plants, wherein the nucleotide sequence of the glutathione transferase gene EuGST is shown as SEQ ID NO. 1.

[0015] Further, the application of glutathione transferase gene EuGST in breeding corn germplasm with disease resistance to Fusarium graminearum head rot and stalk rot, wherein the nucleotide sequence of the glutathione transferase gene EuGST is shown as SEQ ID NO. 1.

[0016] The application of EuGST gene and its encoded protein in genetic breeding for improving the resistance of corn to Fusarium graminearum head rot and stalk rot.

[0017] Compared with the prior art, the application provides application of glutathione transferase gene EuGST in regulating resistance of corn Fusarium graminearum head rot and stem rot, and the glutathione transferase gene EuGST can significantly improve the resistance of corn to head rot and stem rot, and provides important gene resources for corn breeding against head rot and stem rot. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of the provided drawings.

[0019] Figure 1 A framework diagram of the P3301-Ubi-EuGST vector is shown in Figure 1.

[0020] Figure 2 Identification of the EuGST transgenic lines in Example 3. (A) qPCR was used to identify the transcription level of EuGST. EuGST-42, EuGST-43, EuGST-45, EuGST-46, EuGST-48: different transgenic lines of EuGST gene. (B) PCR method was used to identify the EuGST gene in the transgenic plants. M: Marker; NC: Non-transgenic control; PC: Plasmid control; 1 and 2: two biological replicates of the transgenic line EuGST-42; 3 and 4: two biological replicates of the transgenic line EuGST-45; 5 and 6: two biological replicates of the transgenic line EuGST-46.

[0021] Figure 3Phenotypic identification and analysis of stalk rot of EuGST transgenic lines in Example 4. (A) Phenotype of stalk rot of EuGST transgenic lines in Zhoushan winter sowing in 2022. (B) Analysis of stalk rot grade of EuGST transgenic lines in three different environments. Scale bar, 1.0 cm. Z31: maize inbred line Zeng 31; NT: Non-transgenic; EuGST-42, EuGST-45, EuGST-46: different transgenic lines of EuGST gene; 2022E1: Zhongmu spring sowing in 2022; 2022E2: Zhoushan winter sowing in 2022; 2023E1: Xingyang summer sowing in 2023. a, b and c letters represent significant differences between control Z31, Non-transgenic and EuGST transgenic lines EuGST-42, EuGST-45, EuGST-46, respectively (Duncan’s multiple range test, P=0.05).

[0022] Figure 4 Phenotypic identification and analysis of stalk rot of EuGST transgenic lines in Example 5. (A) Phenotype of stalk rot of EuGST transgenic lines. (B) Statistical analysis of stalk rot grade of EuGST transgenic lines in seedling stage. Scale bar, 1.0 cm. NT: Non-transgenic; EuGST-42, EuGST-45, EuGST-46: different transgenic lines of EuGST gene. a and b letters represent significant differences between control Non-transgenic and EuGST transgenic lines EuGST-42, EuGST-45, EuGST-46, respectively (Duncan’s multiple range test, P=0.05).

[0023] Figure 5Phenotype identification and analysis of ear rot of EuGST transgenic hybrids in Example 6. (A) Phenotype of ear rot of EuGST transgenic hybrids in 2024 Xingyang summer sowing. (B) Statistical analysis of ear rot incidence of EuGST transgenic hybrids in three different environments. Scale bar, 1.0 cm. Z31: corn inbred line Zong 31; NT: Non-transgenic; P138 x Z31: corn inbred line P138 hybridized with Zong 31 F1; P138 x NT: corn inbred line P138 hybridized with NT F1; P138 x EuGST-45, P138 x EuGST-46: corn inbred line P138 hybridized with EsGST gene transgenic lines EuGST-45, EuGST-46 F1; 2024E1: 2024 Zhongmu spring sowing; 2024E2: 2024 Xingyang spring sowing; 2024E3: 2024 Xingyang summer sowing. Letters a and b represent significant differences (Duncan's multiple range test, P = 0.05) between P138 x Z31 and P138 x NT, respectively, and P138 x EuGST transgenic hybrids.

[0024] Figure 6 Phenotype identification and analysis of field stalk rot of EuGST transgenic hybrids in Example 7. (A) Phenotype of field stalk rot of EuGST transgenic hybrids in 2024 Zhongmu spring sowing. (B) Statistical analysis of field stalk rot incidence of EuGST transgenic hybrids in 2024 Zhongmu spring sowing. Scale bar, 1.0 cm. Z31: corn inbred line Zong 31; NT: Non-transgenic; P138 x Z31: corn inbred line P138 hybridized with Zong 31 F1 (Nongda 3138); P138 x NT: corn inbred line P138 hybridized with NT F1; P138 x EuGST-45, P138 x EuGST-46: corn inbred line P138 hybridized with EsGST gene transgenic lines EuGST-45, EuGST-46 F1; Letters a and b represent significant differences (Duncan's multiple range test, P = 0.05) between P138 x Z31 and P138 x NT, respectively, and P138 x EuGST transgenic hybrids. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0026] The test materials used in the embodiments of the present application are all conventional test materials in the art and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional test methods or according to the operation instructions recommended by the suppliers.

[0027] The vector p3301-Ubi-GFP is described in Zhang Xiaofeng (2021), Identification of maize ga20ox5 dwarf mutant and ZmGRF1 gene transformed maize and nitrogen utilization rate research, Chinese Academy of Agricultural Sciences, Master's Thesis, China National Knowledge Network.

[0028] The coding sequence of the EuGST gene is synthesized by using the whole gene synthesis technology (Shanghai Shengong Bioengineering Co., Ltd.), and the length is 843 bp, and the nucleic acid sequence is shown as SEQ ID NO. 1.

[0029] The CDS sequence of EuGST is as follows:

[0030] ATGGCCACCAGCAGCACCAGCACCAGCACCCCGATCATCTTCTACGACATCGCCCAGCGCCCGCCGGTGGCCGAGACCTGCTGCGCCGTGAACCCGTGGAAGAGCCGCCTGGCCCTGAACTTCAAGGCCGTGCCGTACACCACCACCTGGGTGAAGATGCCGGACATCAGCAGCGTGCGCGCCAGCCTGAACGTGCCGGCCTGCCGCAAGTTCGCCGACGGCAGCGACTTCAACACCCTGCCGATCATGCACGACCCGGCCACCGACAGCCTGATCGGCGACAGCTTCGACATCGCCGCCTACCTGCAGCGCACCTACCCGGCCAGCGGCGCCGGCGACCTGTTCCCGCCGCAGAAGCTGGACTACGCCGTGGGCCGCGACATGCAGCTGCTGATCCCGCTGAGCGAGGTGCGCGCCCCGGAGCTGGCCGACTACGCCCGCTTCAACAGCAACGTGGACGCCGCCTTCACCGCCCACGTGGGCCTGATGGTGCACGGCCTGCCGCTGGACCCGGCCACCGCCGACGTGACCAAGGCCGAGTTCGTGCGCCGCGCCGGCCTGAGCAGCTGGGAGGACTTCGAGATGGTGGGCGAGGTGCGCGAGAAGATGATGCAGAGCCTGCGCAACATGCTGGGCGACCTGGCCGCCCTGTTCCGCCGCGACGCCAGCGGCCCGTTCCTGCTGGGCCAGCGCGCCACCTACGCCGACCTGATCGTGGGCGGCTGGCTGCGCATGATGCGCGCCACCCTGCCGGCCAGCGAGTGGCAGGAGGCCCGCGCCTGCCACGGCGCCATCTTCGGCCAGCTGCACGACGCCCTGGACAAGTACGCCGAGGTGAAGTGA; SEQ ID NO. 1.

[0031] The EuGST gene corresponds to a protein sequence of 280 amino acids in length, the amino acid sequence of which is as set forth in SEQ ID NO. 2.

[0032] Amino acid sequence of EuGST:

[0033] MATSSTSTSTPIIFYDIAQRPPVAETCCAVNPWKSRLALNFKAVPYTTTWVKMPDISSVRASLNVPACRKFADGSDFNTLPIMHDPATDSLIGDSFDIAAYLQRTYPASGAGDLFPPQKLDYAVGRDMQLLIPLSEVRAPEL SEQ ID NO.2.

[0034] Example 1

[0035] The coding region of EuGST (as shown in SEQ ID NO.1, or its encoded protein as shown in SEQ ID NO.2) was cloned using a whole-genome synthesis method and constructed into the P3301-Ubi-GFP vector framework.

[0036] The vector construction method is as follows: Using the plasmid vector p3301-Ubi-GFP as a template, the plasmid template was linearized using BamHI and SacI restriction endonucleases (NEB). Then, the EuGST gene CDS sequence synthesized using whole-genome technology was ligated into the p3301-Ubi-GFP vector via BamHI and SacI restriction sites. The resulting p3301-Ubi-EuGST vector has the following structural framework: Figure 1 As shown.

[0037] Example 2 Genetic transformation of maize

[0038] Genetic transformation of maize was performed according to published methods (Ishida, YJ, Hiei, Y. and Komari, T.).

[0039] (2007).Agrobacterium-mediated transformation of maize.Nature protocols 2(7),1614-1621.) The recipient material was maize inbred line 31 (Zong31, Z31), and transgenic lines overexpressing EuGST were obtained.

[0040] Example 3: Identification of transgenic lines

[0041] First, the EuGST transcriptional level of the transgenic plants obtained in Example 2 was identified by qPCR. The PCR conditions used for transcriptional level identification were all in accordance with the nearshore protein qPCR kit (…).

[0042] The assay was performed using SYBR qPCR SuperMix Plus (E096). Primers were used.

[0043] The EuGST gene was identified using 5'-ATGGCCACCAGCAGCACCAGCA-3' (SEQ ID NO.3) and 5'-GCTGCTGATGTCCGGCATCTTCAC-3' (SEQ ID NO.4). Results are as follows... Figure 2 As shown in A, the obtained transgenic lines increased EuGST transcription levels.

[0044] The transgenic lines obtained in Example 2 were identified at the genomic level by PCR. The PCR amplification program used for genomic level identification was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min. The EuGST gene was identified using primers 5'-TATGCAGCAGCTATATGTGGATTT-3' (SEQ ID NO.5) and 5'-GAACTCGGCCTTGGTCACGT-3' (SEQ ID NO.6). The results are as follows. Figure 2 As shown in B, transgenic maize lines with the EuGST gene were successfully obtained.

[0045] Example 4: Overexpression of the EuGST gene in maize can improve the resistance of transgenic maize lines to ear rot caused by Fusarium graminearum.

[0046] (1) Cultivation of Fusarium graminearum

[0047] The cultivation of Fusarium graminearum spore suspension requires two culture media: potato dextrose agar (PDA) and CMC liquid medium. First, the Fusarium graminearum strain is transferred to PDA plates for activation and incubated upside down in a 28°C incubator for 5-7 days until the plates are fully covered with mycelium.

[0048] (2) Collection of Fusarium graminearum conidia

[0049] ① Take approximately 500 mL of CMC liquid culture medium in a 1000 mL sterile Erlenmeyer flask, and use a sterile scalpel to take 6-8 1 cm agar plates covered with mycelia. 2 Place the mycelial blocks in CMC liquid culture medium and incubate at 200 rpm and 28°C for 5-7 days to prepare conidia.

[0050] (2) CMC culture medium preparation ②CMC culture medium was filtered with 4 layers of sterile gauze, and the filtrate was collected in a 50 mL sterile centrifuge tube. The supernatant was discarded after centrifugation at 5000 rpm for 5-10 min, and the conidial pellet was collected.

[0051] (3) Resuspension of conidia ③The conidia were resuspended with sterile water, and the number of conidia was detected under a microscope for standby use.

[0052] (4) Spore dilution ④The F. verticillioides spores were diluted to 2 x 10 6 copies / mL with 0.02% Tween 20 under sterile conditions and stored in a 4°C refrigerator for standby use.

[0053] (3) Fusarium inoculation treatment

[0054] The corn was silked for about two weeks, and 2 mL of F. graminearum spore suspension was injected into the middle of the corn ear with a syringe. The F. verticillioides disease incidence was investigated immediately after harvest (Dong, C. P., Wu, Y. B., Gao, J. Y., Zhou, Z. J., Mu, C., Ma, P. P., Chen, J., F. et al. (2018) Field inoculation and classification of maize ear rot caused by Fusarium verticillioides. Bio-Protocol 8, e3099.).

[0055] (4) Fusarium ear rot resistance identification

[0056] By artificially inoculating F. verticillioides in three different environments, it was found that the control Zong 31 (Z31) and the transgenic negative plants had obvious mold rot on the ears. In contrast, the ear rot disease incidence of the transgenic lines overexpressing EuGST was reduced, and the ear rot disease grade was significantly lower than that of the control (A and B). In summary, overexpression of the EuGST gene in corn can improve the resistance of transgenic lines of corn to F. verticillioides ear rot. Figure 3

[0057] Example 5 Overexpression of EuGST gene in corn can improve the resistance of transgenic lines of corn to F. verticillioides caused stem rot

[0058] The corn plants were cultured in the greenhouse to two-leaf-one-heart stage, and a 1 mm deep wound was made on the stem about 2 cm below the first leaf with a medical syringe needle. After being placed horizontally, 5 microliters of spore suspension (concentration adjusted to 5 x 10 6 ​The plants were inoculated with 20 μL of F. verticillioides spore suspension (1 x 105spores / mL) on the wound. The plants were incubated at room temperature for 1-2 hours, and then placed in a plant incubator for humidification. After 5 days, the disease incidence was investigated (Sun, Y. L., Ruan, X. S., Ma, L., Wang, F. and Gao, X. Q. (2018) Rapid screening and evaluation of maize seedling resistance to stalk rot caused by Fusarium spp. Bio-Protocol 8, e2859.) and the results are shown in Figure 4 Fig. 2. Compared with the transgenic negative plants, the stems of the three transgenic lines EuGST-42, EuGST-45 and EuGST-46 showed weak rotting phenomenon Figure 4 A). The grade statistics showed that the disease grade of the three transgenic lines of EuGST gene was significantly lower than that of the control Figure 4 B). In summary, overexpression of EuGST gene in maize can improve the resistance of transgenic lines of corn to F. verticillioides stalk rot.

[0059] Example 6 Overexpression of EuGST gene in maize can improve the resistance of transgenic hybrid of corn to F. verticillioides ear rot

[0060] (1) Preparation of corn hybrid

[0061] The inbred line P138 was crossed with the EuGST overexpression lines (EuGST-45 and EuGST-46) and the control Zong31 (Z31) and transgenic negative plants to obtain F1 hybrids:

[0062] P138 x EuGST (P138 x EuGST-45 and P138 x EuGST-46), P138 x Z31, P138 x Non-transgenic.

[0063] (2) Identification of F. verticillioides ear rot resistance

[0064] According to the F. verticillioides inoculation method in Example 4, the results are shown in Figure 5 Fig. 4. The results shown in the figure show that the ear rot phenomenon of P138 x EuGST is significantly weaker than that of the controls P138 x Z31 and P138 x NT Figure 5 A); the statistics of the disease grade of F. verticillioides ear rot showed that the disease grade of P138 x EuGST ear rot was significantly lower than that of the controls P138 x Z31 and P138 x NT Figure 5 B). In summary, overexpression of EuGST gene in maize can improve the resistance of transgenic hybrid of corn to F. verticillioides ear rot.

[0065] Example 7 Overexpression of EuGST gene in maize can improve the resistance of maize transgenic hybrid to Fusarium verticillioides stalk rot

[0066] (1) According to the method of configuring maize hybrid in Example 6, about 150 μL of 4 x 10 6 spores / mL of F. verticillioides spore suspension was injected into the middle of the second stem node of the maize field silking for two weeks, the drill hole was sealed with vaseline, and then fixed with pressure-sensitive tape. Two weeks after inoculation, the incidence of field stalk rot was determined (Ye, J. R., Zhong, T., Zhang, D. F., Ma, C. Y., Wang, L. N., Yao, L. S., Zhang, Q. Q. et al. (2019) The auxin-regulated protein ZmAuxRP1 coordinates the balance between root growth and stalk rot disease resistance in maize. Molecular Plant 12, 360-373.).

[0067] (2) Fusarium verticillioides stalk rot resistance identification

[0068] After two weeks of inoculation, the incidence of stalk rot was investigated. The results are shown in Figure 6 the figure, which shows that the investigation of the incidence of corn stalks inoculated with F. verticillioides shows that the stalk rot incidence of P138 x EuGST is significantly lower than that of the controls P138 x Z31 and P138 x NT Figure 6 (A); the statistics of the incidence grade of F. verticillioides stalk rot also show that the stalk rot incidence grade of P138 x EuGST is also significantly reduced compared with the controls P138 x Z31 and P138 x NT Figure 6 (B). In summary, overexpression of EuGST gene in maize can improve the resistance of maize transgenic hybrid to Fusarium verticillioides stalk rot.

[0069] The above description of the disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the glutathione transferase gene EuGST in regulating resistance to Fusarium graminearum ear rot and stalk rot in maize, characterized in that, The nucleotide sequence of the glutathione transferase gene EuGST is shown in SEQ ID NO.

1.

2. The application of overexpression of the glutathione transferase gene EuGST in improving resistance to Fusarium graminearum ear rot and stalk rot in maize, characterized in that, The nucleotide sequence of the glutathione transferase gene EuGST is shown in SEQ ID NO.

1.

3. The application of biomaterials overexpressing the glutathione S-transferase gene EuGST in improving resistance to Fusarium graminearum ear rot and stalk rot in maize, characterized in that, The nucleotide sequence of the glutathione transferase gene EuGST is shown in SEQ ID NO. 1; The biomaterial is any one of the following: A: An expression cassette capable of overexpressing the glutathione transferase gene EuGST with the nucleotide sequence shown in SEQ ID NO.1; B: A recombinant vector containing the expression cassette described in A; C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

4. A method for improving resistance to Fusarium ear rot and stalk rot in maize, characterized in that, The glutathione transferase gene EuGST was overexpressed in maize plants; the nucleotide sequence of the glutathione transferase gene EuGST is shown in SEQ ID NO.

1.

5. The application of the glutathione transferase gene EuGST in the breeding of maize germplasm resistant to Fusarium graminearum ear rot and stalk rot, characterized in that, The nucleotide sequence of the glutathione transferase gene EuGST is shown in SEQ ID NO.1.

Images