A fusion polypeptide CT01 having both induction of plant hypersensitive response and antibacterial effect

Abstract

The application discloses a fusion polypeptide CT01 with the functions of inducing plant hypersensitive response and inhibiting bacteria. The amino acid sequence of the fusion polypeptide CT01 is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. After injecting the fusion polypeptide CT01 with a concentration of 15 μg / mL into leaves of Nicotiana benthamiana, obvious cell death can be observed; treating Magnaporthe grisea conidia on a hydrophobic glass slide with the fusion polypeptide CT01 with a concentration of 30 μg / mL can significantly inhibit the germ tube emergence of the Magnaporthe grisea conidia; adding the fusion polypeptide CT01 into a Magnaporthe grisea conidia suspension to a final concentration of 30 μg / mL and spraying rice seedlings can significantly inhibit the occurrence of rice blast. Therefore, the fusion polypeptide CT01 can play a role in preventing and controlling rice blast from the aspects of inducing rice disease resistance and inhibiting Magnaporthe grisea infection.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticides, specifically involving the design, expression, preparation, and application of a novel fusion polypeptide CT01 that can induce plant resistance by causing hypersensitivity reactions and effectively inhibit the germination of rice blast fungus conidia. Background Technology

[0002] Pesticides play a crucial role in the control of crop diseases and pests. However, traditional chemical pesticides have drawbacks such as high toxicity, high residue, and environmental instability, seriously affecting food safety and causing environmental pollution. Therefore, traditional chemical pesticides can no longer meet the needs of modern green agriculture. With the increasing awareness of ecological agriculture, the development of low-toxicity, environmentally friendly new green biological pesticides has become the mainstream trend in agricultural disease control worldwide. Short peptides / polypeptides are protein molecules composed of multiple amino acids. Different types of short peptides are produced in both plants and animals, participating in the regulation of various life activities. Hypersensitive short peptides are peptide segments in Harpin proteins secreted by plant bacterial pathogens through the type III secretion system that can exogenously induce cell death in plants. Hypersensitive short peptides enhance the plant's defense against pathogens by activating the plant's immune response, thus achieving disease control. However, hypersensitive short peptides themselves do not have a direct inhibitory effect on pathogens. Fusion peptides designed by combining functional short peptides from different organisms have low harm to humans, animals, and the ecological environment, providing a direction for the development of new green pesticides. In the medical field, researchers have designed a variety of short peptides that can perform different functions in clinical practice. However, fusion peptides have not yet been developed and applied in agricultural production and the control of crop diseases and pests.

[0003] Rice is one of the most important food crops in my country and even the world. However, throughout its growth cycle, rice is susceptible to various pathogenic microorganisms, leading to a variety of rice diseases that severely impact yield stability. Rice blast, caused by Magnaphalium oryzae, has long been considered the most serious fungal disease affecting rice production. In my country, rice blast directly causes a loss of up to 3 billion kilograms of rice yield annually, and in severe cases, it can lead to a 40%-50% reduction in yield, or even total crop failure. Currently, commonly used pesticides for rice blast control include tricyclazole, isoprothiolane, pyraclostrobin, and isoprothiolane, among others. The excessive use of these chemical pesticides can affect agricultural ecological health and food safety. In recent years, several biological pesticides have been reported to be effective in controlling rice blast, such as kasugamycin and Bacillus subtilis.

[0004] This invention successfully designed and developed a fusion polypeptide that links the two ends of a short peptide with antibacterial activity from barnyard grass to the Harpin peptide segment of pear blight pathogen, which induces hypersensitivity in plants, via flexible linkers. This design enhances the water solubility of the barnyard grass antibacterial short peptide, enabling the fusion polypeptide to simultaneously possess the hypersensitivity protein properties of the Harpin peptide segment and the function of the antibacterial peptide in inhibiting the germination of conidia of rice blast fungus, thus effectively controlling the occurrence of rice blast. Summary of the Invention

[0005] The purpose of this invention is to provide a fusion polypeptide (named CT01) that can be used to control the occurrence of rice blast disease, as well as its design concept and expression preparation method. CT01 can achieve the effect of controlling the occurrence of rice blast disease by activating the immune defense response of rice and inhibiting the germination of rice blast fungus conidia.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A fusion polypeptide CT01, which can induce plant hypersensitivity and inhibit the germination of rice blast fungus conidia, has the amino acid sequence shown in SEQ ID NO.1.

[0007] A nucleic acid molecule encoding the fusion polypeptide CT01 of claim 1, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0008] A recombinant vector for expressing the fusion polypeptide CT01 is obtained by inserting the nucleic acid molecule encoding the fusion polypeptide CT01 into the prokaryotic expression vector pET-28a.

[0009] A recombinant bacterium expressing the fusion polypeptide CT01 was obtained by introducing the above-mentioned recombinant vector into Escherichia coli BL21 competent cells.

[0010] The application of the aforementioned fusion polypeptide CT01 in inducing tobacco hypersensitivity.

[0011] The application of the aforementioned fusion polypeptide CT01 in inhibiting the germination of rice blast fungus conidia.

[0012] The application of the aforementioned fusion polypeptide CT01 in the prevention and control of rice blast disease.

[0013] The beneficial effects of this invention are as follows: The fusion polypeptide CT01 provided by this invention has the ability to induce plant hypersensitivity response and inhibit the germination of rice blast fungus conidia, and can effectively control rice blast disease. Attached Figure Description

[0014] Figure 1 The images show the Coomassie Brilliant Blue staining of the control supernatant and the supernatant of the fusion peptide CT01 on SDS-PAGE gel.

[0015] Figure 2 The image shows the hypersensitive response induced in tobacco leaves by the supernatant of the fusion peptide CT01.

[0016] Figure 3 Image showing the inhibition of blast fungus conidia germination by the supernatant of the fusion peptide CT01 (scale bar is 30 μm).

[0017] Figure 4 The supernatant of the fusion peptide CT01 inhibits the infection of rice leaves by rice blast fungus. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of the present invention. Unless otherwise specified, the molecular biological and biochemical methods used in the following embodiments of the present invention are conventional methods well known to those skilled in the art. Example 1: Obtaining the fusion peptide CT01 The amino acid sequence of the fusion peptide CT01 (N-terminus-C-terminus) is as follows: MGHHHHHHMQQPIDRQTIEQMAQLLAELLKSLLSPQGGGGSGGGGSGGGGSGSGRGSCR The sequence SQCMRRHEDEPWRVQECVSQCRRRRGGGDGGGGSGGGGSGGGGSQQPIDRQTIEQMAQLLAELLKSLLSPQ contains a 6×His tag from the N-terminus, amino acid sequences from the 10th to the 36th position (Hypericum pyrifolium Harpin peptide), amino acid sequences from the 37th to the 51st position (Hypericum pyrifolium var. pyrifolium), amino acid sequences from the 52nd to the 88th position (Hypericum pyrifolium var. pyrifolium), amino acid sequences from the 89th to the 103rd position (Hypericum pyrifolium var. pyrifolium), and amino acid sequences from the 104th to the 130th position (Hypericum pyrifolium Harpin peptide).

[0019] The nucleic acid sequence encoding the fusion polypeptide CT01 is as follows: 5'-CCATGGGCCATCATCATCATCACATGCAACAACCCATCGATCGGCAAACCATTGAGCAAATGGCTCAATTATTGGCGGAACTGTTAAAGTCACTGCTATCGCCACAAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCTCCGGCAGGGGCTCGTGCCGGAGCCAGTGCATGCGCCGCCACGAGGACGAGCCG TGGCGGGTCCAGGAGTGCGTCAGCCAGTGCCGCCGCCGTCGTGGCGGCGGCGACGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAACAACCCATCGATCGGCAAACCATTGAGCAAATGGCTCAATTATTGGCGGAACTGTTAAAGTCACTGCTATCGCCACAATGACTCGAG-3'; In this sequence, 1bp to 6bp from the 5' end are Nco The Xho I restriction site consists of the following sequences: 9 bp to 26 bp are the 6×His tag-encoded nucleotide sequence; 27 bp to 29 bp are the start codon; 30 bp to 110 bp are the Harpin peptide-encoded nucleotide sequence from *Pyracantha fortuneana*; 111 bp to 155 bp are the flexible linker-encoded nucleotide sequence; 156 bp to 266 bp are the barnyardgrass antimicrobial peptide-encoded nucleotide sequence; 267 bp to 311 bp are the flexible linker-encoded nucleotide sequence; 312 bp to 392 bp are the Harpin peptide-encoded nucleotide sequence from *Pyracantha fortuneana*; 393 bp to 395 bp are the stop codon; and 396 bp to 401 bp are the Xho I restriction site.

[0020] The nucleic acid sequence encoding the fusion polypeptide CT01 was artificially synthesized. This nucleic acid sequence and the prokaryotic expression vector pET-28a were digested using restriction endonucleases Nco I and XhoI. The digestion products were ligated using T4 ligase to obtain the recombinant vector pET-28a-CT01. The recombinant vector pET-28a-CT01 and the empty vector pET-28a (used as a control for subsequent treatment) were transformed into *E. coli* BL21 competent cells. Small amounts of PCR-verified BL21 monoclonal cells were picked and cultured in liquid LB medium, then transferred to 150 mL of liquid LB medium at a 1:100 v / v inoculation rate. The cells were cultured at 37°C with shaking until the OD600 value reached 0.4–0.6. IPTG was added to a final concentration of 0.5 mM, and the cells were cultured at 28°C with shaking for 16 hours to induce expression of the fusion polypeptide and the control empty vector. The bacterial cells were collected by centrifugation, and the supernatant was discarded. The bacterial pellet was resuspended in 20 mL of ST buffer (10 mM Tirs-HCl pH 8.0, 150 mM NaCl), and then sonicated before centrifugation. The supernatant containing the fusion peptide CT01 and the control supernatant were collected. The concentration of CT01 in the supernatant was determined using a BSA protein gradient loading method.

[0021] 25 μL of the control supernatant and the fusion peptide CT01 supernatant were respectively subjected to SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining. Figure 1 The molecular weight of the fusion peptide CT01 is 14 kDa, and the concentration of the fusion peptide CT01 supernatant is 300 μg / mL.

[0022] Example 2: Hypersensitivity response of tobacco leaves induced by fusion peptide CT01 The control supernatant and the fusion peptide CT01 supernatant were diluted 20-fold with water (final CT01 concentration: 15 μg / mL) and then injected into tobacco leaves. One week later, tissue necrosis was observed in the tobacco leaf areas injected with the fusion peptide CT01 supernatant. Figure 2 The control group showed no significant changes in the tobacco leaf area after injection of the supernatant, indicating that the fusion peptide CT01 can induce hypersensitivity in plants.

[0023] Example 3: Fusion peptide CT01 inhibits the germination of rice blast fungus conidia. To verify the inhibitory effect of the fusion peptide CT01 on the conidia of *Blastoma orientalis*, *Blastoma orientalis* strain Guy11 was cultured in rice bran medium (40 g / L rice bran, 20 g / L agar powder, pH = 6.5) under light for 10 days. Conidia were washed off with 0.02% Tween 20 aqueous solution and filtered through double-layered filter paper. The supernatant of the fusion peptide CT01 was added to the *Blastoma orientalis* strain Guy11 conidia suspension to a final concentration of CT01 of 30 μg / mL. An equal volume of control supernatant was added to the conidia suspension as a reference. The experimental and control suspensions were dropped onto hydrophobic glass slides and placed in a humidified, dark environment at 25°C for 4 hours. The germination rate of conidia was then counted. Three sets of data were recorded for each treatment, with at least 100 conidia counted in each set. Compared with the control supernatant, the germination rate of *Blastoma orientalis* conidia was significantly reduced after treatment with the fusion peptide CT01 supernatant. Figure 3 ** indicates a highly significant difference. This result shows that the fusion peptide CT01 has a significant inhibitory effect on the germination of rice blast fungus conidia.

[0024] Example 4: The fusion peptide CT01 has an inhibitory effect on rice infected with rice blast fungus. To further verify the control effect of the fusion peptide CT01 on rice blast, the supernatant of the fusion peptide CT01 was added to the conidial suspension of the rice blast strain Guy11 to achieve a final concentration of 30 μg / mL. An equal volume of control supernatant was added to the conidial suspension as a reference. Three-week-old rice seedlings were sprayed with the above suspension, and the incidence of rice blast on the leaves was observed and recorded after 5 days. Compared with the control group, the lesion area in the fusion peptide CT01 treatment group was significantly reduced. Figure 4 * indicates a significant difference. This result shows that the fusion peptide CT01 can effectively inhibit rice blast infection and has a significant control effect on rice blast disease.

[0025] The above embodiments are merely illustrative of the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of this invention should be covered within the scope of protection of this invention.

Claims

1. A fusion polypeptide CT01, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the fusion polypeptide CT01 of claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant vector expressing the fusion polypeptide CT01, characterized in that: It is obtained by inserting the nucleic acid molecule encoding the fusion polypeptide CT01 as described in claim 2 into the prokaryotic expression vector pET-28a.

4. A recombinant bacterium expressing the fusion polypeptide CT01, characterized in that: It is obtained by introducing the recombinant vector described in claim 3 into Escherichia coli BL21 competent cells.

5. The application of the fusion polypeptide CT01 as described in claim 1 in inhibiting the germination of rice blast fungus conidia.

6. The application of the fusion polypeptide CT01 as described in claim 1 in the prevention and control of rice blast disease.

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