Use of an immunization inducing GH20 gene and the protein encoded thereby in the prevention and treatment of corn southern leaf blight

Abstract

The present application relates to the field of genetic engineering, in particular to an immune elicitor GH20 gene and the application of the encoded protein thereof in the prevention and treatment of corn large spot disease. The nucleotide sequence of the immune elicitor GH20 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The present application first discloses the biological function of the immune elicitor GH20 gene and the encoded protein thereof, and further verifies that the immune elicitor GH20 gene and the encoded protein thereof have significant prevention and treatment effects on corn large spot disease through prokaryotic expression, and can be widely applied in the biological prevention and treatment of corn large spot disease. It can be seen that the present application provides a new biological preparation for the prevention and treatment of corn large spot disease, and lays a good foundation for the creation and development of new biological pesticides.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to the application of an immune-inducing GH20 gene and a protein encoded by the same in preventing and controlling corn leaf blight. Background Art

[0002] Fungal plant diseases are among the most common, accounting for approximately 70-80% of all plant diseases. These diseases primarily spread through fungal spores, which, under favorable environmental conditions, invade plant tissues and disrupt normal physiological functions. These diseases pose a serious threat to agricultural production, often manifesting as necrosis and rot of leaves, stems, and fruits, leading to reduced crop yields and quality. Fungal diseases not only affect crop growth and development but can also spread on a large scale, causing long-term damage to agricultural production.

[0003] The pathogenic fungus causing corn leaf blight, Exserohilum turcicum, in its asexual form, belongs to the subdivision Deuteromycotina, the order Hyphomycetales, and the genus Exserohilum. The pathogen produces dark brown, oval, or elongated spores with straight or slightly curved dark brown spore stalks, typically with 3-8 transverse septa. The spore base is narrow, and the umbilicus at the top and bottom of the spore is prominent, protruding from the outer surface and serving as a key morphological hallmark. On host leaves, the pathogen forms oval or spindle-shaped lesions that gradually expand as the disease progresses.

[0004] Plant inducers are agents that use biostimulants to activate a plant's immune system. By administering specific bioactive substances, they stimulate the plant's defense mechanisms, thereby increasing its resistance to disease. This process not only enhances a plant's defenses but also significantly reduces the incidence of disease. Commonly used biostimulants include certain natural compounds and microbial-derived substances, primarily chitosan, plant extracts, and certain probiotics. These substances can activate systemic acquired resistance (SAR) and local acquired resistance (LAR) in plants, thereby enhancing the plant's overall resistance to pathogens. Specifically, the application of biostimulants such as chitosan can trigger plant defense responses and promote the synthesis of defense-related phenolic compounds, antimicrobial proteins, and esters, which can interfere with the growth and spread of pathogens. For example, chitosan can bind to plant cell membranes, triggering intracellular signaling pathways, activating the expression of endogenous defense genes, and enhancing the plant's disease resistance. Furthermore, the effectiveness of immune inducers has been demonstrated in numerous cases. For example, applying chitosan to corn can significantly improve the plant's resistance to the corn leaf blight pathogen. Studies have shown that the area of ​​lesions and the severity of the disease are significantly reduced after the treated plants are infected with the pathogen. Similarly, treatment with certain plant extracts or certain probiotics can also effectively enhance the resistance of crops to various fungal diseases. In general, immune inducers can enhance the plant's natural resistance to disease by activating the plant's own immune system, demonstrating their important potential in plant disease management. This approach can not only effectively control the occurrence of diseases, but also support sustainable agricultural practices.

[0005] As an important member of the glycoside hydrolase family, GH12 is mainly responsible for degrading polysaccharides in plant cell walls, such as cellulose, hemicellulose and pectin. GH12 can hydrolyze glycosidic bonds in polysaccharides (cellulose, hemicellulose and pectin) to release short-chain sugars and oligosaccharides. This process not only helps the infection of pathogens, but also provides nutrients. For example, some members of GH12 can break down pectin and hemicellulose, thereby helping pathogens break through the defenses of plant cell walls during the interaction between plant pathogens and host plants. GH12 has also shown broad potential in industrial applications. Due to its ability to decompose complex sugars, it is widely used in textiles, pulp processing, food processing and other fields. At present, there are no reports that GH20 can prevent and control corn leaf blight. Summary of the Invention

[0006] The present invention aims to provide an immune-inducing GH20 gene and its encoded protein for use in preventing and treating corn leaf blight, so as to solve the problems existing in the above-mentioned prior art. The immune-inducing GH20 gene and its encoded protein provided by the present invention can effectively prevent and treat corn leaf blight.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an immune-inducing GH20 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The present invention provides the protein encoded by the above-mentioned immune-inducing GH20 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The present invention provides a recombinant vector containing the immune-inducing GH20 gene.

[0011] The present invention provides a recombinant bacterium containing the above-mentioned recombinant vector.

[0012] The present invention provides the use of the above-mentioned immune-inducing GH20 gene, the above-mentioned protein, the above-mentioned recombinant vector or the above-mentioned recombinant bacteria in preventing and controlling corn leaf blight.

[0013] The present invention provides the use of the above-mentioned immune-inducing GH20 gene, the above-mentioned protein, the above-mentioned recombinant vector or the above-mentioned recombinant bacteria in the preparation of products for preventing and treating corn leaf blight.

[0014] Preferably, the product includes an immune inducer and a biopesticide.

[0015] The present invention provides a product for preventing and treating corn leaf blight, and the product comprises the above-mentioned protein.

[0016] Preferably, the product includes an immune inducer and a biopesticide.

[0017] The present invention provides a method for preventing and controlling corn leaf blight, comprising the step of spraying the above protein or the above product on corn.

[0018] The present invention discloses the following technical effects:

[0019] The present invention uses the research group's early transcriptome data to screen for GH12-type glycoside hydrolase genes that are induced to express during pathogen infection, and obtains and clones the immune-inducing GH20 gene that is expressed at a high level. The present invention reveals for the first time the sequence of the immune-inducing GH20 gene obtained from the corn leaf blight pathogen and the protein it encodes. In a specific embodiment of the present invention, transient expression in Nicotiana benthamiana was used to analyze the induction effect of the immune-inducing GH20 gene on plant immunity; the immune-inducing GH20 gene was heterologously expressed using a prokaryotic expression system, and the effects of the expression product on the disease resistance and growth and development of the plant were analyzed. The experiment verified that the protein encoded by the immune-inducing GH20 gene can significantly induce the ability of corn to resist the large leaf blight; then, spraying the expression protein of the immune-inducing GH20 gene in prokaryotic cells on the surface of corn leaves can significantly improve the ability of corn to resist infection by the large leaf blight pathogen. It can be seen that the immune-inducing GH20 gene and the protein encoded by it provided by the present invention can be widely used in the biological control of corn leaf blight. The present invention provides a new biological agent for the prevention and control of corn leaf blight and lays a good foundation for the creation and development of new biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The expression level of immune-inducing GH20 gene at different stages of maize infection;

[0022] Figure 2 The results show the ability of immune-induced GH20 protein to utilize disaccharides. YTK12 is the wild-type yeast strain YTK12; Avr1b is the yeast strain YTK12 carrying the positive control pSUC2-Avr1b; mg87 is the yeast strain YTK12 carrying the negative control pSUC2-Mg87; GH20 is the yeast strain YTK12 carrying the recombinant plasmid pSUC2-GH20-SP.

[0023] Figure 3The results are the results of the immune-induced GH20 protein signal peptide secretion function verification (TTC colorimetric ability verification); among them, YTK12 is the wild-type yeast strain YTK12; Avr1b is the yeast strain YTK12 carrying the positive control pSUC2-Avr1b; mg87 is the yeast strain YTK12 carrying the negative control pSUC2-Mg87; GH20 is the yeast strain YTK12 carrying the recombinant plasmid pSUC2-GH20-SP;

[0024] Figure 4 The electrophoresis diagram of the CDS of the immune-induced GH20 gene; wherein, M is a marker, and from top to bottom, they are 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp and 2000 bp; lane 1 is the PCR product of the immune-induced GH20 gene;

[0025] Figure 5 The following are the enzyme digestion results of the immune-induced GH20 gene prokaryotic expression recombinant vector pET-28a-GH20-1; where M is the DL2503 marker, and from top to bottom are 10000bp, 5000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; Lane 1 is the immune-induced GH20 gene prokaryotic expression recombinant vector pET-28a-GH20-1;

[0026] Figure 6 The following are the enzyme digestion results of the immune-induced GH20 gene transient expression vector Super1300-GH20-1; M is the DL2503 marker, and from top to bottom are 10000bp, 5000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; Lane 1 is the immune-induced GH20 gene transient expression vector sSuper1300-GH20-1;

[0027] Figure 7 Immunization against GH20 gene-induced tobacco PCD reaction; a and b are two replicates of the same experiment, with the left side being empty vehicle injection, the upper side of the right side being gene injection, and the lower side being Bax injection;

[0028] Figure 8 The burst of ROS in tobacco induced by immune-induced GH20 gene; the left side of a and b is the leaf after EV treatment, and the right side is the leaf after immune-induced GH20 gene treatment;

[0029] Figure 9Figure 2: The deposition of callose in tobacco leaves induced by immune-induced GH20 gene. Figure a shows leaves injected with pSuper1300 vector; Figure b shows leaves injected with immune-induced GH20 gene; Figure c shows a comparison of the callose deposition phenomenon. * indicates 0.01 < p < 0.05, ** indicates p < 0.01, and ** indicates p < 0.001.

[0030] Figure 10 is the effect of immune-induced GH20 gene on PTI maker gene expression; among them, a is the effect of immune-induced GH20 gene on NbPR1 gene expression; b is the effect of immune-induced GH20 gene on NbPR4 gene expression;

[0031] Figure 11 Screening for the optimal temperature for prokaryotic expression of immune-induced GH20 gene; wherein, a is the SDS-PAGE electrophoresis detection diagram of the target protein; M is Maker, 10-280kd; 1 is the Pet-28a empty supernatant; 2 is the Pet-28a empty precipitate; 3 is the bacterial supernatant after induction at 20℃; 4 is the bacterial precipitate after induction at 20℃; 5 is the bacterial supernatant after induction at 25℃; 6 is the bacterial precipitate after induction at 25℃; 7 is the bacterial supernatant after induction at 30℃; 8 is the bacterial precipitate after induction at 30℃; 9 is the bacterial supernatant after induction at 37℃; 10 is the bacterial precipitate after induction at 37℃; b is the relative expression level of immune-induced GH20 gene;

[0032] Figure 12 Screening for the optimal time of prokaryotic expression of immune-induced GH20 gene; wherein, a is the SDS-PAGE electrophoresis detection diagram of the target protein; M is Maker, 10-280kd; 1 is the pET-28a empty supernatant; 2 is the pET-28a empty precipitate; 3 is the bacterial supernatant after 8h induction; 4 is the bacterial precipitate after 8h induction; 5 is the bacterial supernatant after 12h induction; 6 is the bacterial precipitate after 12h induction; 7 is the bacterial supernatant after 16h induction; 8 is the bacterial precipitate after 16h induction; 9 is the bacterial supernatant after 24h induction; 10 is the bacterial precipitate after 24h induction; b is the relative expression level of immune-induced GH20 gene;

[0033] Figure 13The purification of immune-induced GH20 protein; wherein, a is the electrophoresis test of the purification effect of different imidazole concentrations, M is Maker, 10-280kd; 1 is the immune-induced GH20 protein solution after elution with a buffer of 30mM imidazole concentration; 2 is the immune-induced GH20 protein solution after elution with a buffer of 50mM imidazole concentration; 3 is the immune-induced GH20 protein solution after elution with a buffer of 100mM imidazole concentration; 4 is the immune-induced GH20 protein solution after elution with a buffer of 350mM imidazole concentration; b is the electrophoresis test of the purification effect of protein, M is Maker, 10-280kd; 1 is the crude protein solution of unpurified immune-induced GH20 protein; 2 is the purified immune-induced GH20 protein solution;

[0034] Figure 14 The effect of immune-induced GH20 crude protein on the resistance of Nicotiana benthamiana to Botrytis cinerea; a is the tobacco leaf treated with immune-induced GH20 crude protein; the left part of the leaf is the untreated part; the right part of the leaf is the part treated with the induced protein; b is the comparison of the diseased area between different treatments;

[0035] Figure 15 The effect of immune-induced GH20 crude protein on the expression levels of NbPR1 and NbPR4 in Nicotiana benthamiana; a is the expression level of NbPR1; b is the expression level of NbPR4;

[0036] Figure 16 The effect of immune-induced GH20 crude protein on corn resistance to northern leaf spot disease; a is an untreated corn leaf; b is a corn leaf treated with immune-induced GH20 crude protein; c is a comparison of diseased areas between different treatments;

[0037] Figure 17 The effect of immune-induced GH20 protein on the expression levels of maize ZmPR4b and ZmPR5; a is the expression level of ZmPR4b; b is the expression level of ZmPR5;

[0038] Figure 18 The effect of immune-induced GH20 protein on the growth and development of maize seedlings; a is the statistical graph of plant height; b is the statistical graph of stem diameter; c is the statistical graph of root length; d is the statistical graph of fresh weight; e is the statistical graph of dry weight;

[0039] Figure 19 For immune-induced GH20 protein powder; wherein, a is immune-induced GH20 protein dry powder; b is 1 mg immune-induced GH20 protein dry powder dissolved in 1 mL PBS;

[0040] Figure 20The effect of immune-induced GH20 protein on corn resistance to northern leaf spot disease; a is a corn leaf without protein solution treatment; b is a corn leaf treated with 50 μg / mL protein solution; c is a corn leaf treated with 100 μg / mL protein solution; d is a corn leaf treated with 250 μg / mL protein solution; e is 500 μg / mL; f is a comparison of diseased areas among different treatments;

[0041] Figure 21 The figure shows the effect of immune-induced GH20 protein on the expression levels of maize ZmPR4b and ZmPR5; wherein, a is the effect of different concentrations of immune-induced GH20 protein solution on the expression level of ZmPR4b; b is the effect of different concentrations of immune-induced GH20 protein solution on the expression level of ZmPR5. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The nucleotide sequence of the immune elicitor GH20 gene is shown in SEQ ID NO.1, specifically: ATGAAGGTCGTATCTCTCATCCTTGGTGCTCTCGCCTCCGTGGGAGTTGCACAGCAAGCCACTCTCTGCCAGGACTTCCAGTACTTTTCCAGCAACGGCTACGAGCTCAACAACAACATCTGGGGCCGCGGCTCCGCCACCTCTGGCTCTCAGTGCACTTATGTCGACAGTGTCAGCT CGACTGGCGCCAAGTGGCACTCCAACTGGCAGTGGCAGGGTGGCAAGGACAACGTCAAGAGCTACGTCTACTCTGGACGTCAGATCACCAAGAAGCCCGTCACCCAGTACAGCAACCTCGAGACCGAGGCCTACTGGGTCTATGATACGACTAACATTCGCTGCAATGTTGCATATGATTTGTTCACATCTGCCAATGTGAACCATGACACCAGCAGTGGTGACTACGAGCTCATGGTTTGGCTCGCCAGGTACGATGTGTACCCCATTGGATCCTCGCAAGGGATGGTCAACGTCGCCGGACATACCTGGGAACTCTTCTACGGCCTCAACGGCTCCATGAAGGTGTACAGCTTCGTCACTCCTTCCGGCCCCATCTACAACTTCAAGGCCAGCATGAAGGACTTCTTCACCTACCTCCAGAACAACAAGGGCTTCCCTGCTTCCAGCCAGAACCTCATTACCTACCAATTCGGTACCGAGGCTTTCACCGGTGGCCCAGCCAAGTTCACTGTTAACCAGTGGTCTGCCAACGCCTACTAA; The amino acid sequence of the immune elicitor GH20 protein encoded by it is as shown in SEQ ID NO.2, specifically: MKVVSLILGALASVGVAQQATLCQDFQYFSSNGYELNNNIWGRGSATSGSQCTYVDSVSSTGAKWHSNWQWQGGKDNVKSYVYSGRQITKKPVTQYSNLETEAYWVYDTTNIRCNVAYDLFTSANVNHDTSSGDYELMVWLARYDVYPIGSSQGMVNVAGHTWELFYGLNGSMKVYSFVTPSGPIYNFKASMKDFFTYLQNNKGFPASSQNLITYQFGTEAFTGGPAKFTVNQWSANAY*. The first 19 amino acids at the N-terminus of the amino acid sequence are a signal peptide with a molecular weight of 26.6 kDa.

[0048] Example 1

[0049] 1. Discovery of the immune-inducing GH20 protein

[0050] Based on the genome data of the corn leaf blight pathogen, a candidate effector factor database was established. Combined with the transcriptome data of susceptible corn B73 infected by the pathogen, an effector protein gene (immune-induced GH20 gene ( Figure 1 Cloning and structural analysis revealed that the nucleotide sequence of the immune-inducing GH20 gene is shown in SEQ ID NO. 1. The protein encoded by the gene has a 19-aa signal peptide at its N-terminus, followed by a 180-aa conserved glycoside hydrolase family 12 domain. Based on its gene ID, the protein encoded by the gene was named immune-inducing GH20 protein.

[0051] 2. Demonstration of secretory activity of immune-induced GH20 protein in yeast secretion system

[0052] The yeast strain YTK12 is a defective strain that cannot grow normally on CMD-W medium. The signal peptide fragment amplified using a primer pair (upstream primer: GAATTCATGAAGGTCGTATCTCTCATCCT, SEQ ID NO. 3; downstream primer: CTCGAGCGAGCTGACACTGTCGACATAA, SEQ ID NO. 4) was cloned into pSUC2T7M13ORI between the EcoRI and XhoI restriction sites. The effector signal peptide fragment was ligated with the sucrase gene SUC2 lacking the start codon and signal peptide fragment for fusion expression, generating the recombinant plasmid pSUC2-GH20-SP.

[0053] If the effector signal peptide fragment is successfully cloned into the pSUC2 vector and transformed into the yeast strain YTK12, it will grow normally on CMD-W medium. If the signal peptide is secretory, the enzyme activity will be detected by TTC, indicating a brick-red color. Avr1b, an effector from Phytophthora sojae, has a strong secretory function and serves as a positive control. Mg87, a signal peptide fragment from Magnaporthe oryzae lacking secretory function, serves as a negative control.

[0054] The yeast strain YTK12 carrying the recombinant plasmid pSUC2-GH20-SP, the positive control pSUC2-Avr1b, and the negative control pSUC2-Mg87 grew normally on CMD-W medium, while YTK12 could not grow normally ( Figure 2 ). From the above results, it can be seen that the recombinant vector successfully transformed the yeast strain YTK12. The results of TTC color development test are as follows Figure 3 As shown, the results showed that the yeast strain carrying the recombinant plasmid pSUC2-GH20-SP turned the colorless sucrose solution red, consistent with the results obtained with the yeast strain carrying the positive control pSUC2-Avr1b. These results indicate that the effector signal peptide GH20-SP has secretory function and, combined with the predicted subcellular localization results, is located in the extracellular matrix. Therefore, the candidate effector GH20-SP is an extracellular secretory protein.

[0055] 3. Cloning of the Immunity-Inducing GH20 Gene

[0056] Using cDNA from the wild-type strain 01-23 of Setosphaeria turcica as a template, the CDS of the immune-inducible GH20 gene was amplified using the following PCR amplification procedure: 94°C denaturation, 55°C annealing, 72°C extension, and 34 cycles. The system consisted of 1 μL of cDNA, 1 μL of each primer, 10 μL of 2× PerfectStart Green qPCR SuperMix, and 7 μL of ddH2O. The nucleotide sequence of the upstream primer was ATGAAGGTCGTATCTCTCATCCTTGGT, SEQ ID NO. 5; the nucleotide sequence of the downstream primer was TTAGTAGGCGTTGGCAGACCAC, SEQ ID NO. 6. The electrophoresis pattern of the CDS of the immune-inducible GH20 gene is shown in Figure 2. Figure 4 The results showed that the target band size was 720 bp.

[0057] 4. Prokaryotic and transient expression of immune-induced GH20 genes

[0058] The prokaryotic expression recombinant vector of the immune-inducible GH20 gene was constructed using pET-28a as the backbone. The CDS sequence of the effector gene immune-inducible GH20 gene, after removing the signal peptide sequence and the stop codon, was amplified using the successfully constructed recombinant plasmid pSUC2-GH20-SP as the template. The target gene fragment was obtained using 1 μL of the upstream primer (nucleotide sequence: AAGCTTATGGCCACTCTCTGCCAG, SEQ ID NO. 7), 1 μL of the downstream primer (nucleotide sequence: GCGGCCGCGTAGGCGTTGGCAGACCA, SEQ ID NO. 8), 2 μL of the cDNA template, 10 μL of 2×RapaidTaq MasterMix, and 7 μL of ddH2O. The target gene fragment was ligated between BamHI and NotⅠ of pET-28a to obtain the prokaryotic expression recombinant vector pET-28a-GH20-1 for the immune-inducible GH20 gene. Figure 5 The results of enzyme digestion of the prokaryotic expression recombinant vector pET-28a-GH20-1 for the immune-inducing GH20 gene show that the target band is 1089 bp in size.

[0059] The immune-induced GH20 gene transient expression recombinant vector was constructed using pSuper1300 as the backbone. The immune-induced GH20 gene was integrated between the SmalⅠ and XbaⅠ sites of the pSuper1300 vector to obtain the immune-induced GH20 gene transient expression vector pSuper1300-GH20-1. Figure 6 The results of enzyme digestion of the immune-induced GH20 gene transient expression vector pSuper1300-GH20-1 show that the target band is 1089 bp in size.

[0060] 5. Verification of the immune-stimulating function of immune-induced GH20 gene and immune-induced GH20 protein

[0061] A single clone of the target gene (immune-inducing GH20 gene) was integrated between the SmalⅠ and XbaⅠ sites of the pSuper1300 vector and transformed into Agrobacterium GV3101. The clones were injected into the back of 4-week-old Nicotiana benthamiana leaves using the Agrobacterium system, with Bax as the positive control and the empty vector (pSuper1300 vector, EV) as the negative control, to obtain GH20 Nicotiana benthamiana, positive control Nicotiana benthamiana, and negative control Nicotiana benthamiana. The lesions were observed after one week of culture.

[0062] The results showed that leaves in the region injected with Bax and immune-induced GH20 gene showed necrosis, while leaves in the region injected with EV showed no significant changes ( Figure 7), which indicates that the function of immune-induced GH20 gene is similar to that of Bax and can induce tobacco allergic necrosis reaction; it is preliminarily speculated that immune-induced GH20 gene may have elicitor activity.

[0063] After transient expression of GH20 in Nicotiana benthamiana for 48 hours, DAB tissue staining was used. After removing chlorophyll with alcohol, it was found that reddish-brown spots appeared in the leaves where the immune-induced GH20 gene was transiently expressed, and there was no obvious phenomenon when EV was used ( Figure 8 ), so it was concluded that the immune-induced GH20 gene and immune-induced GH20 protein can promote the accumulation of reactive oxygen species in Nicotiana benthamiana leaves.

[0064] After transient expression of GH20 in Nicotiana benthamiana for 48 h, 50 randomly selected 1 mm 2 The accumulation of callose in the field of view was observed. The results showed that the tobacco leaves treated with EV had a small amount of fluorescence, with the smallest area, while the tobacco leaves treated with immune-induced GH20 gene had a large area of ​​fluorescence ( Figure 9 ), indicating that the immune-induced GH20 gene and immune-induced GH20 protein have a more obvious promoting effect on callose accumulation in Nicotiana benthamiana.

[0065] The effect of immune-induced GH20 gene on the expression of pathogenesis-related genes (NbPR1 and NbPR4) was detected. The results showed that compared with EV, the relative expression of NbPR1 and NbPR4 increased significantly 12 hours after immune-induced GH20 gene injection into Nicotiana benthamiana leaves, and NbPR4 reached about 2.3 times that before treatment ( Figure 10 ), indicating that the immune-inducible GH20 gene and immune-inducible GH20 protein can trigger PTI response in Nicotiana benthamiana.

[0066] 6. Prokaryotic Expression of Immune-Induced GH20 Gene and Purification of Immune-Induced GH20 Protein

[0067] The prokaryotic expression recombinant vector of the immune-inducing GH20 gene was constructed using pET-28a as the backbone and transformed into the Escherichia coli strain BL21 (DE3) for prokaryotic expression. The induction conditions were explored and it was found that when the induction temperature was 20°C ( Figure 11 ), the final concentration of inducer IPTG was 0.1 mM, the rotation speed was 150 rpm, and the induction time was 16 h ( Figure 12 ) and induced the recombinant immune-induced GH20 protein (immune-induced GH20 crude protein), whose molecular weight was 44.5kDa. The target protein was purified by nickel ion affinity chromatography ( Figure 13), the immune-induced GH20 soluble protein (immune-induced GH20 protein) obtained had a protein concentration of up to 1419 μg / mL.

[0068] 7. Analysis of plant resistance induced by immune-induced GH20 protein

[0069] The leaves of Nicotiana benthamiana were smeared with immune-induced GH20 crude protein, and crude protein containing an empty (pSuper1300 vector) receptor strain was used as a control. One day later, Botrytis cinerea was inoculated in the corresponding area, and the disease was observed after nine days. The results showed that compared with the control, the area of ​​lesions on the leaves treated with immune-induced GH20 crude protein was smaller. The effect of the part treated with immune-induced GH20 crude protein was obvious, and the area of ​​lesions on the leaves was only 1 / 3 of the control part ( Figure 14 ).

[0070] Similarly, the effects of immune-induced GH20 crude protein treatment on the expression of NbPR1 and NbPR4 in Nicotiana benthamiana leaves were analyzed. The results showed that after 1 day of immune-induced GH20 protein treatment, the relative expression of NbPR1 and NbPR4 increased significantly, increasing the expression level of NbPR1 by about 2.8 times and that of NbPR4 by about 3 times ( Figure 15 a and b).

[0071] In order to further determine whether immune-induced GH20 protein can enhance disease resistance to pathogens by inducing an immune response in corn, this example used immune-induced GH20 crude protein to smear corn leaves, and used crude protein containing an empty receptor strain as a control. One day later, corn leaf blight pathogens were inoculated in the corresponding areas, and the disease was observed 12 days later. The results showed that compared with the control, the area of ​​lesions on leaves treated with immune-induced GH20 crude protein was significantly smaller, only 1 / 3 of the area of ​​lesions in the control group ( Figure 16 ).

[0072] In addition, this example also tested the effects of different treatments on the expression levels of ZmPR4b and ZmPR5 in corn leaves. The results showed that compared with the control, leaves treated with immune-induced GH20 crude protein could induce the upregulation of ZmPR4b and ZmPR5 genes. The immune-induced GH20 protein had a significant induction effect, increasing the expression of ZmPR4b by about 4.1 times; the expression of ZmPR5 by about 3.6 times ( Figure 17 ).

[0073] In order to explore whether immune-induced GH20 protein can also affect the development of corn, its crude protein solution was used to smear the leaves of corn seedlings at the 3-leaf stage, and crude protein containing an empty receptor strain was used as a control. The growth of corn seedlings was observed and the effects on seedling height, stem diameter, root length, fresh weight and dry weight were recorded after 10 days. The results showed that, except for stem diameter, immune-induced GH20 protein had a significant promoting effect on plant height, fresh weight, dry weight and root length of corn seedlings ( Figure 18 ).

[0074] 8. Preliminary Study on the Application of Immune-Inducing Anti-GH20 Protein

[0075] The purified protein was desalted and concentrated using an ultrafiltration centrifuge tube. After treatment, the liquid immune-inducing GH20 protein solution was placed in a freeze dryer to prepare a powder. The results showed that the freeze-dried immune-inducing GH20 protein appeared as a fine white powder with a light and uniform texture and no obvious crystallization. The powder could be dissolved in PBS buffer in a short time without obvious precipitation by simply shaking or gently vortexing. Figure 19 ).

[0076] The immune-induced GH20 protein mother powder was diluted into 50, 100, 250, and 500 μg / mL immune-induced GH20 protein solutions, and the immune-induced GH20 protein solutions of different concentrations were applied to corn leaves. One day later, the corn leaf blight pathogen was inoculated in the corresponding area, and the disease was observed after 12 days. The results showed that compared with the control, the lesion area of ​​the different concentrations was significantly reduced, and generally the higher the concentration, the smaller the lesion area. Further analysis found that there was no significant difference in the lesion area between the parts treated with 250 and 500 μg / mL ( Figure 20 ).

[0077] RT-qPCR was used to detect the expression levels of ZmPR4b and ZmPR5 in corn leaves treated with different protein concentrations. The results showed that compared with the control, the expression levels of ZmPR4b and ZmPR5 in leaves treated with different concentrations of immune-induced GH20 protein solution were significantly increased. Among them, the effect of leaves treated with 500 μg / mL immune-induced GH20 protein solution was the most significant, which could increase the expression levels of ZmPR4b and ZmPR5 to 4.9 and 3.7 times respectively. Although the effect of 250 μg / mL immune-induced GH20 protein solution was not as good as that of 500 μg / mL immune-induced GH20 protein solution, it could also increase the expression levels of ZmPR4b and ZmPR5 to 4.6 and 3.5 times at 12 hours. Therefore, considering the comprehensive factors of cost and effect, 250 μg / mL immune-induced GH20 protein solution was selected as the optimal application concentration of immune-induced GH20 protein powder ( Figure 21 ).

[0078] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a protein encoded by an immune-inducing GH20 gene in increasing plant height, fresh weight, dry weight and root length of corn seedlings, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.2.

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