Application of TpGST gene in regulating and controlling ear rot and stem rot resistance of maize fusarium graminearum

Abstract

The invention discloses application of a TpGST gene in regulating and controlling ear rot and stem rot resistance of maize fusarium graminearum, and belongs to the technical field of gene engineering. According to the application of the TpGST gene disclosed by the invention in regulating and controlling the ear rot and stem rot resistance of maize fusarium graminearum, the nucleotide sequence of the TpGST gene is shown as SEQ ID NO. 1. Fusarium graminearum is inoculated to ears and stems of an overexpressed TpGST transgenic line, so that the ear rot and stem rot resistance of the fusarium graminearum of the corn transgenic line is improved; fusarium graminearum is inoculated to ears and stems of the corn hybrid agricultural large 3138 overexpression TpGST transgenic line, and the ear rot and stem rot resistance of the fusarium graminearum of the corn transgenic hybrid is improved.

Description

TECHNICAL FIELD

[0001] The application relates to the field of genetic engineering, in particular to application of a TpGST gene in regulating resistance of maize to Fusarium graminearum ear rot and stalk rot. BACKGROUND

[0002] Maize is the world's first grain crop, and the total yield of maize in the world has exceeded 1 billion tons. In China, as the first grain crop, the planting area of maize reached 610 million mu in 2019, and the total yield reached 260 million tons, playing a crucial role in ensuring China's food security.

[0003] Maize diseases and pests have always been a key factor affecting maize yield, causing an annual yield loss of more than 10% of the total yield. Among them, maize ear rot is a common disease in maize planting areas, with an incidence of 5-10% in general years and up to about 50% in severe cases, which is an important factor affecting maize yield. Maize ear rot not only causes significant yield loss, but also produces deoxynivalenol (DON), zearalenol (ZEN) and fumonisins (FB) and other mycotoxins, which not only reduce the quality of maize, but also seriously threaten human and animal health. Studies have shown that although the pathogenic bacteria causing maize ear rot are very complex, Fusarium graminearum and Fusarium verticillioides are the dominant pathogenic bacteria in most disease areas, and with the frequent occurrence of extreme weather, continuous straw return to the field and wheat-maize rotation planting methods, plant disease residues accumulate more and more seriously, pathogenic bacteria are sufficient, disease pressure increases, leading to frequent occurrence of maize ear rot and becoming more and more serious.

[0004] A large number of studies have shown that maize ear rot and stalk rot are diseases regulated by multiple genes, and are affected by a small number of major genes and a large number of microgenes. In recent years, scholars have reported a large number of QTL sites related to maize ear rot and stalk rot resistance, but most of the sites have low genetic effects and poor stability, and only a few related genes have been cloned. Glutathione transferase (GST) has been reported to play an important role in plant resistance to various biological and abiotic stresses, and the use of GST genes to modify maize ear rot and stalk rot resistance has important theoretical significance and application value.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide application of a TpGST gene in regulating resistance of maize to Fusarium graminearum ear rot and stalk rot. SUMMARY

[0006] Therefore, the application provides application of a TpGST gene in regulating Fusarium graminearum head blight and stalk rot resistance of corn.

[0007] The application provides application of a TpGST gene of Thinopyrum ponticum and a coded protein thereof in improving Fusarium graminearum head blight and stalk rot resistance of corn, by using gene overexpression technology, so as to provide potential genetic resources for breeding of corn Fusarium graminearum head blight and stalk rot disease-resistant varieties.

[0008] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions.

[0009] The application of the TpGST gene in regulating Fusarium graminearum head blight and stalk rot resistance of corn, wherein the nucleotide sequence of the TpGST gene is shown as SEQ ID NO. 1.

[0010] Further, the application of the overexpressed TpGST gene in improving Fusarium graminearum head blight and stalk rot resistance of corn, wherein the nucleotide sequence of the TpGST gene is shown as SEQ ID NO. 1.

[0011] Further, the application of the biological material with overexpressed TpGST gene in improving Fusarium graminearum head blight and stalk rot resistance of corn, wherein the nucleotide sequence of the TpGST gene is shown as SEQ ID NO. 1.

[0012] The biological material is any one of the following:

[0013] A: an expression cassette capable of overexpressing the TpGST gene with the nucleotide sequence shown as SEQ ID NO. 1;

[0014] B: a recombinant vector containing the expression cassette of A;

[0015] C: a recombinant microorganism containing the expression cassette of A or the recombinant vector of B.

[0016] Further, a method for improving Fusarium graminearum head blight and stalk rot resistance of corn, overexpressing a TpGST gene in a corn plant, wherein the nucleotide sequence of the TpGST gene is shown as SEQ ID NO. 1.

[0017] Further, the application of the TpGST gene in breeding of Fusarium graminearum head blight and stalk rot disease-resistant germplasm of corn, wherein the nucleotide sequence of the TpGST gene is shown as SEQ ID NO. 1.

[0018] The application of the TpGST gene in improving Fusarium graminearum head blight and stalk rot resistance of corn.

[0019] Compared with the prior art, the application provides application of the TpGST gene in regulating resistance of corn Fusarium moniliforme ear rot and stalk rot, and it is found for the first time that overexpression of TpGST in corn inbred lines can significantly improve the resistance of corn to ear rot and stalk rot, and important gene resources are provided for corn breeding against ear rot and stalk rot. BRIEF DESCRIPTION OF DRAWINGS

[0020] 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.

[0021] Figure 1 A framework diagram of the P3301-Ubi-TpGST vector is constructed.

[0022] Figure 2 TpGST transgenic lines in Example 3 are identified. (A) qPCR is used to identify the transcription level of TpGST. TpGST-10, TpGST-11, TpGST-14, TpGST-16, TpGST-17: different transgenic lines of TpGST gene. (B) PCR method is used to identify TpGST gene of the transgenic plants. M: Marker; NC: Non-transgenic control; PC: Plasmid control; 1 and 2: two biological replicates of the transgenic line TpGST-10; 3 and 4: two biological replicates of the transgenic line TpGST-16; 5 and 6: two biological replicates of the transgenic line TpGST-17.

[0023] Figure 3Phenotypic identification and analysis of TpGST transgenic lines inoculated with Fusarium graminearum at ear in Example 4. (A-B) Phenotype (A) and disease grade statistics (B) of TpGST transgenic lines inoculated with Fusarium graminearum at ear in Zhoushan winter sowing in 2022. Scale bar, 1.0 cm. Z31: maize inbred line Zeng 31; NT: Non-transgenic; TpGST-10, TpGST-16, TpGST-17: different transgenic lines of TpGST gene; 2022E1: Zhongmu summer sowing in 2022; 2022E2: Zhoushan winter sowing in 2022; 2023E1: Xingyang summer sowing in 2023. Different letters represent significant differences between the control Z31, Non-transgenic and TpGST transgenic lines TpGST-10, TpGST-16, TpGST-17, respectively (Duncan’s multiple range test, P=0.05).

[0024] Figure 4 Phenotypic identification and analysis of TpGST transgenic lines inoculated with Fusarium graminearum at stem in seedling stage in Example 5. (A-B) Phenotype (A) and disease grade statistics (B) of TpGST transgenic lines inoculated with Fusarium graminearum at stem. Scale bar, 1.0 cm. NT: Non-transgenic; TpGST-10, TpGST-16, TpGST-17: different transgenic lines of TpGST gene. Different letters represent significant differences between the control Non-transgenic and TpGST transgenic lines TpGST-10, TpGST-16, TpGST-17, respectively (Duncan’s multiple range test, P=0.05).

[0025] Figure 5Phenotypic identification and analysis of TpGST transgenic hybrids for ear inoculation of Fusarium graminearum in Example 6. (A-B) Phenotypes (Fig. A) and disease grade statistics (Fig. B) of TpGST transgenic hybrids for ear inoculation of Fusarium graminearum in 2024 Xingyang summer sowing in three different environments. Scale bar, 1.0 cm. Z31: maize inbred line Zong 31; NT: Non-transgenic; P138 x Z31: F1 hybrid of maize inbred line P138 and Zong 31 (Nongda 3138); P138 x NT: F1 hybrid of maize inbred line P138 and NT; P138 x TpGST-10, P138 x TpGST-16: F1 hybrid of maize inbred line P138 and TpGST gene transgenic line TpGST-10, TpGST-16; 2024E1: 2024 Zhongmu spring sowing; 2024E2: 2024 Xingyang spring sowing; 2024E3: 2024 Xingyang summer sowing. Different letters indicate significant differences between the control P138 x Z31, P138 x NT and P138 x TpGST transgenic hybrids (Duncan’s multiple range test, P = 0.05).

[0026] Figure 6 Phenotypic identification and analysis of TpGST transgenic hybrids for field stalk inoculation of Fusarium graminearum in Example 7. (A-B) Phenotypes (Fig. A) and disease grade statistics (Fig. B) of TpGST transgenic hybrids for field stalk inoculation of Fusarium graminearum in 2024 Zhongmu spring sowing. Scale bar, 1.0 cm. Z31: maize inbred line Zong 31; NT: Non-transgenic; P138 x Z31: F1 hybrid of maize inbred line P138 and Zong 31 (Nongda 3138); P138 x NT: F1 hybrid of maize inbred line P138 and NT; P138 x TpGST-10, P138 x TpGST-16: F1 hybrid of maize inbred line P138 and TpGST gene transgenic line TpGST-10, TpGST-16. Different letters indicate significant differences between the control P138 x Z31, P138 x NT and P138 x TpGST transgenic hybrids (Duncan’s multiple range test, P = 0.05). DETAILED DESCRIPTION

[0027] 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 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 those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0028] 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 not specified in detail are carried out according to the conventional test methods or according to the operation instructions recommended by the suppliers.

[0029] The vector p3301-Ubi-GFP is described in the literature: Zhang Xiaofeng (2021), Identification of maize ga20ox5 dwarf mutant and ZmGRF1 gene maize and nitrogen utilization rate research, Chinese Academy of Agricultural Sciences, Master's thesis, China National Knowledge Network.

[0030] In order to obtain the gene TpGST coding sequence, the CDS sequence coding it is synthesized by using the whole gene synthesis technology (Shanghai Biosynth Biotech Co., Ltd.), and the length is 840 bp, and its nucleic acid sequence is shown as SEQ ID NO. 1.

[0031] CDS sequence of TpGST:

[0032] ATGGCCACCAGCGCCAGCACCA GCACCCCGATCATCTTCTACGACATCGCCCAGCGCCCGCCGGTGGCCGAGACCTGCTGCGCCGTGAACCCGTGGAAGAGCCGCCTGGCCCTGAACTTCAAGGCCGTGCCGTACACCACCACCTGG GTGAAGATGCCGGACATCAGCAGCGTGCGCGCCAGCCTGAACGTGCCGGCCTGCCGCAAGTTCGCCGACGGCAGCGACTTCAACACCCTGCCGATCATCCACGACCCGGCCACCGACAGCCTGGTGGGCGACAGCTTCGACATCGCCGCCTACCTGCAGCGCACCTACCCGGCCAGCGGCGCCGGCGACCTGTTCCCGCCGCAGAAGCTGGACTACGCCGTGGGCCGCGACATGCAGCTGCTGATCCCGCTGAGCGAGATCCGCGCCCCGGAGCTGGCCGACTACGCCCGCTTCAACAGCAACGTGGACGCCGCCTTCACCGCCCACGTGGGCCTGATGGTGCACGGCCTGCCGCTGGACCCGGCCACCGCCGACGTGACCAAGGCCGAGTTCGTGCGCCGCGCCGGCCTGAGCAGCTGGGACGACCTGGAGATGGTGGGCGAGGCCCGCGACAAGATGATGCAGAGCCTGCGCAACATGCTGGGCGACCTGGCCGCCCTGTTCCGCAAGGACGCCAGCGGCCCGTTCCTGCTGGGCCAGCGCGCCACCTACGCCGACATGATCGTGGGCGGCTGGCTGCGCATGATGCGCGCCACCCTGCCGGTGAGCGAGTGGCAGGAGGCCCGCGCCTGCCACGGCGCCATCTTCGGCCAGCTGCACGACGCCCTGGACAAGTACGCCGAGGTGAAGTGA; SEQ ID NO. 1.

[0033] The TpGST gene corresponds to a protein sequence encoding 279 amino acids in length, the amino acid sequence of which is shown as SEQ ID NO. 2.

[0034] Amino acid sequence of TpGST:

[0035] MATSASTSTPIIFYDIAQRPPVAETCCAVNPWKSRLALNFKAVPYTTTWVKMPDISSVRASLNVPACRKFADGSDFNTLPIIHDPATDSLVGDSFDIAAYLQRTYPASGAGDLFPPQKLDYAVGRDMQLLIPLSEIRAPEL SEQ ID NO.2.

[0036] Example 1

[0037] The coding region of the gene TpGST (the gene coding sequence is shown in SEQ ID NO.1, or its encoded protein is shown in SEQ ID NO.2) was cloned by whole gene synthesis and constructed into the P3301-Ubi-GFP vector framework.

[0038] The vector construction method is as follows: using the plasmid vector p3301-Ubi-GFP as a template, the plasmid template is linearized by BamHI and SacI restriction endonucleases (NEB), and then the TpGST gene CDS sequence synthesized by whole gene technology is connected to the P3301-Ubi-GFP vector through the BamHI and SacI restriction sites. The P3301-Ubi-TpGST vector is obtained, and its structural framework is as follows Figure 1 shown.

[0039] Example 2 Genetic transformation of corn

[0040] Genetic transformation of maize was performed according to published methods (Ishida, YJ, Hiei, Y. and Komari, T.

[0041] (2007). Agrobacterium-mediated transformation of maize. Nature protocols 2(7), 1614-1621.) was carried out, and the recipient material was maize inbred line 31 (Zong31, Z31), and a transgenic strain overexpressing TpGST was obtained.

[0042] Example 3 Identification of transgenic lines

[0043] First, the transgenic plants obtained in Example 2 were identified by qPCR for TpGST transcription levels. The PCR conditions used for transcription level identification were all in accordance with the Nearshore Protein qPCR Kit ( SYBR qPCR SuperMix Plus, E096) was performed. Primers 5'-ATGGCCACCAGCGCCAGCACCA-3' (SEQ ID NO. 3) and 5'-GCTGCTGATGTCCGGCATCTTCAC-3' (SEQ ID NO. 4) were used to identify the TpGST gene. The results are shown in Figure 2. Figure 2 As shown in A, the obtained transgenic lines had increased TpGST 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: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 1 min, 35 cycles; 72°C final extension for 10 min. The TpGST gene was identified using primers 5'-TATGCAGCAGCTATATGTGGATTT-3' (SEQ ID NO. 5) and 5'-GAACTCGGCCTTGGTCACGT-3' (SEQ ID NO. 6). The results are shown in Figure 2. Figure 2 As shown in B, a transgenic corn line carrying the TpGST gene was successfully obtained.

[0045] Example 4 Overexpression of the TpGST gene in corn can improve the resistance of corn inbred lines to ear rot caused by Fusarium graminearum

[0046] (1) Cultivation of Fusarium graminearum

[0047] Cultivating a spore suspension of Fusarium graminearum requires two media: potato dextrose agar (PDA) and CMC liquid medium. First, transfer the strain to a PDA plate for activation. Incubate the plate upside down at 28°C for 5-7 days until the plate is covered with hyphae.

[0048] (2) Collection of Fusarium graminearum conidia

[0049] ①Use a 1000mL sterile triangular flask to take about 500mL of CMC liquid culture medium, and use a sterilized scalpel to take 6-8 1cm 2 The bacterial blocks were placed in CMC liquid culture medium and cultured with shaking at 200 rpm and 28°C for 5-7 days to prepare conidia.

[0050] ② Use 4 layers of sterile gauze to filter the CMC culture medium, collect the filtrate in a 50mL sterile centrifuge tube, centrifuge at 5000rpm for 5-10min, discard the supernatant, and collect the conidia precipitate.

[0051] ③ Resuspend the conidia with sterile water, check the number of conidia under a microscope, and set aside.

[0052] ④ Dilute the spores of Fusarium graminearum to 2×10 6 / mL, and store in a 4℃ refrigerator for later use.

[0053] (3) Fusarium graminearum inoculation treatment

[0054] About two weeks after pollination, corn was artificially inoculated with Fusarium graminearum. 2 mL of the prepared Fusarium graminearum spore suspension was injected into the middle of the ear with a syringe. The ears were bagged and kept moist. The incidence of Fusarium graminearum was investigated immediately after harvest (Dong, CP, Wu, YB, Gao, JY, Zhou, ZJ, Mu, C., Ma, PP, Chen, J., F. et al. (2018) Field inoculation and classification of maize earrot caused by Fusarium verticillioides. Bio-Protocol 8, e3099.).

[0055] (4) Identification of resistance to Fusarium graminearum ear rot

[0056] The results of artificial inoculation of Fusarium graminearum in three different environments are as follows: Figure 3 As shown, compared with the control Zong31 (Z31) and transgenic negative plants, the three different transgenic lines overexpressing the TpGST gene had significantly lower ear disease grades ( Figure 3 A and 3B). In summary, overexpression of the TpGST gene in maize can improve the resistance of maize inbred lines to Fusarium graminearum ear rot.

[0057] Example 5 Overexpression of the TpGST gene in corn can improve the resistance of corn inbred lines to stalk rot caused by Fusarium graminearum

[0058] (1) According to the propagation and cultivation method of Fusarium graminearum in Example 4, a 1 mm deep hole was pierced in the stem 2 cm below the first leaf of corn seedlings (cultivated in the greenhouse until they had two leaves and one heart) with a medical syringe needle. After the seedlings were placed horizontally, 5 μl of spore suspension (concentration adjusted to 5×10 6The plants were placed horizontally in a plant incubator for 5 days to investigate the disease (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.).

[0059] (2) Identification of maize seedling resistance to F. verticillioides stalk rot

[0060] The identification results of maize seedling resistance to F. verticillioides stalk rot under greenhouse conditions are shown in FIGS. 4A and 4B. The results show that the three different transgenic lines overexpressing TpGST gene have significantly lower stalk rot disease grade than the transgenic negative plants (FIG. 4B). Figure 4 Figure 4 In summary, overexpression of TpGST gene in maize can improve the resistance of maize inbred lines to F. verticillioides stalk rot.

[0061] Example 6 Overexpression of TpGST gene in maize can improve the resistance of maize hybrids to F. verticillioides ear rot

[0062] (1) Preparation of maize hybrids

[0063] Nongda 3138 is a hybrid from maize inbred Zong 31 (Z31) and P138. The inbred P138 was crossed with TpGST overexpression lines (TpGST-10 and TpGST-16), control Zong 31 (Z31) and transgenic negative plants, respectively, to obtain F1 hybrids: P138 x TpGST (P138 x TpGST-10 and P138 x TpGST-16), P138 x Z31, P138 x Non-transgenic.

[0064] (2) Identification of maize hybrid resistance to F. verticillioides ear rot

[0065] According to the inoculation method of F. verticillioides in Example 4, the results are shown in FIG. 6A and FIG. 6B. The results shown in the figures show that the investigation of the disease of the corn ears inoculated with F. verticillioides shows that the disease area of P138 x TpGST is significantly reduced compared with the controls P138 x Z31 and P138 x NT (FIG. 6A); the statistics of the disease grade of F. verticillioides ear rot also show that the disease grade of P138 x TpGST is significantly less than the controls P138 x Z31 and P138 x NT (FIG. 6B). Figure 5 Figure 5 Figure 5 ​​​B). In conclusion, overexpression of the TpGST gene in maize can improve the resistance of maize hybrids to Fusarium graminearum ear rot.

[0066] Example 7 Overexpression of the TpGST gene in corn can improve the resistance of corn hybrids to Fusarium graminearum stalk rot

[0067] (1) Following the corn hybrid configuration and Fusarium graminearum culture method in Example 6, a handheld micro-electric drill (3 mm drill bit) was used to drill a hole (about 2 cm in depth) at a 45° angle in the middle of the second stem node of corn that had been silking for two weeks in the field, and 4×10 6 Approximately 150 μL of a spore suspension (100 μg / mL) was added to the small wells and sealed with petroleum jelly. Two weeks after inoculation, the incidence of stalk rot in the field was assessed (Ye, JR, Zhong, T., Zhang, DF, Ma, CY, Wang, LN, Yao, LS, Zhang, QQ, 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.).

[0068] (2) Identification of resistance to Fusarium graminearum stem rot

[0069] The results of artificial inoculation of Fusarium graminearum stem rot under natural conditions are as follows Figure 6 The results shown in this figure illustrate that the investigation of the disease incidence of corn stems inoculated with Fusarium graminearum showed that the area of ​​stalk rot in P138×TpGST was significantly reduced compared with the control P138×Z31 and P138×NT ( Figure 6 A); The statistics of the incidence of Fusarium graminearum stem rot also showed that the incidence of P138×TpGST was significantly lower than that of the control P138×Z31 and P138×NT( Figure 6 B). In conclusion, overexpression of the TpGST gene in maize can improve the resistance of maize hybrids to Fusarium graminearum stalk rot.

[0070] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of TpGST gene in regulating resistance to Fusarium graminearum ear rot and stalk rot of corn, characterized in that: The nucleotide sequence of the TpGST gene is shown in SEQ ID NO.

1.

2. Application of overexpressing TpGST gene in improving resistance to Fusarium graminearum ear rot and stalk rot of corn, characterized in that: The nucleotide sequence of the TpGST gene is shown in SEQ ID NO.

1.

3. Use of a biomaterial overexpressing the TpGST gene in improving resistance to Fusarium graminearum ear rot and stalk rot in corn, characterized in that: The nucleotide sequence of the TpGST gene is shown in SEQ ID NO.1; The biological material is any one of the following: A: an expression cassette capable of overexpressing the TpGST gene with the nucleotide sequence shown in SEQ ID NO.1; B: recombinant vector containing the expression cassette described in A; C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

4. A method for improving resistance to Fusarium graminearum ear rot and stalk rot in corn, characterized in that: The TpGST gene is overexpressed in corn plants; the nucleotide sequence of the TpGST gene is shown in SEQ ID NO.

1.

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

Images