Antibacterial small peptide CsCAPE9 and application thereof in prevention and control of citrus huanglongbing

Abstract

The invention discloses an antibacterial small peptide CsCAPE9 and application of the antibacterial small peptide CsCAPE9 in prevention and control of citrus huanglongbing. The amino acid sequence of the antibacterial small peptide CsCAPE9 is as shown in SEQ ID NO.1; the antibacterial small peptide CsCAPE9 provided by the invention is a small peptide synthesized in a plant body, and the antibacterial small peptide CsCAPE9 can activate biosynthesis of salicylic acid and induce systematic defense reaction of citrus. The antibacterial small peptide CsCAPE9 has a good inhibition effect on pathogenic bacteria of citrus huanglongbing in greenhouses and fields, and has a wide application prospect in comprehensive prevention and control of the huanglongbing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antimicrobial peptide CsCAPE9 and its application in the control of citrus Huanglongbing (HLB). Background Technology

[0002] Citrus is one of the world's most important economic crops. In recent years, China's citrus industry has developed rapidly, currently accounting for about one-third of the world's output and ranking first in the world in terms of industry scale. Bacterial diseases, including Huanglongbing (HLB), pose a serious threat to the green and sustainable development of citrus. HLB is a devastating global disease and is the most damaging, difficult to control, and costly disease in my country's citrus industry. In production, chemical pesticides are mainly used to control HLB by killing the psyllid, the vector. However, due to the high reproductive rate and rapid mutation rate of psyllids in southern my country, production costs are high and there are potential ecological risks. Currently, the industry urgently needs green and efficient control technologies.

[0003] The pathogen causing Huanglongbing of citrus is a phloem-restricted Gram-negative bacterium belonging to the phylum Proteobacteria, class Alpha proteobacteria, order Rhizobiales, family Rhizobiaceae, and genus Bacillus phloem. Candidatus Liberibacter). It can be divided into Asian strains. C. Liberibacter asiatium ( C Las), African strains C. Liberibacter africanus ( C Laf (including a new variant), and American strains C. Liberibacteramercanus (C Lam ) The Asian species is the most widely distributed and damaging globally. The citrus Huanglongbing (HLB) outbreak in China is also caused by this species. Citrus fruits infected with HLB exhibit abnormal coloring (commonly known as "red-nosed fruit"), are deformed, and have poor flavor; the root system is poorly developed, with fewer fibrous roots, and in severe cases, root rot occurs; significant growth retardation and reduced yield occur, ultimately leading to tree death. Traditional control methods mainly rely on felling diseased trees and controlling vector insects, which are not only costly and environmentally stressful but also difficult to eradicate. This has caused devastating damage to major citrus-producing regions worldwide, resulting in billions of dollars in annual economic losses and seriously threatening the sustainable survival and development of the industry.

[0004] Antimicrobial peptides are small polypeptide molecules composed of 7 to 100 amino acids, widely present in the innate immune defense system of organisms. Compared with traditional antibiotics, antimicrobial peptides have key advantages such as low molecular weight, high specificity, unique mechanism of action (multi-target membrane disruption), low likelihood of inducing drug resistance, and low / non-toxicity. These characteristics not only endow them with strong broad-spectrum or specific antimicrobial activity, but also demonstrate great potential in developing novel targeted therapeutic strategies, making them a current research hotspot in the fields of biomedicine and agricultural disease control.

[0005] Therefore, developing novel, efficient, safe, and environmentally friendly control strategies for Huanglongbing (HLB), often referred to as "citrus cancer," is urgently needed. Control methods based on antimicrobial peptides are considered a crucial entry point in combating HLB due to their low likelihood of developing resistance, potential systemic translocation, ability to directly target pathogens in the phloem, and low risk to the environment and non-target organisms. This patent focuses on developing novel antimicrobial peptides or optimized derivatives of the HLB pathogen, aiming to support the development of green products for HLB control. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an antimicrobial peptide CsCAPE9 and its application in the control of citrus Huanglongbing (HLB). The antimicrobial peptide CsCAPE9 of this invention is a small peptide synthesized within the plant itself, which is green and highly effective. This antimicrobial peptide CsCAPE9 can activate the biosynthesis of salicylic acid and induce a systemic defense response in citrus. The antimicrobial peptide CsCAPE9 exhibits good inhibitory effects on the pathogen of citrus HLB (a Gram-negative bacterium, *Bacillus phloem*) in both greenhouse and field conditions, and has broad application prospects for the integrated control of HLB.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides an antimicrobial peptide CsCAPE9, the amino acid sequence of which is shown in SEQ ID NO.1: PPGNFVGEKPY.

[0008] The gene CsCAPE9 encodes the antimicrobial peptide CsCAPE9 described above, and the nucleotide sequence of the gene CsCAPE9 is shown in SEQ ID NO.2: CCCCCAGGCAACTTTGTTGGGGAAACCTTAC.

[0009] The present invention also provides the application of the above-mentioned antimicrobial peptide CsCAPE9 in inhibiting the proliferation of citrus Huanglongbing pathogen.

[0010] The present invention also provides the application of the above-mentioned antimicrobial peptide CsCAPE9 in the preparation of products that inhibit Huanglongbing bacteria.

[0011] The present invention also provides an antibacterial agent for inhibiting Huanglongbing bacteria, wherein the antibacterial agent contains the antimicrobial peptide CsCAPE9.

[0012] Furthermore, the concentration of the antimicrobial peptide CsCAPE9 in the antibacterial agent is 1~10 μM.

[0013] Furthermore, the concentration of the antimicrobial peptide CsCAPE9 in the antibacterial agent is 1~10uM.

[0014] The present invention also provides a method for inhibiting Huanglongbing (HLB), wherein the above-mentioned antibacterial agent is injected into the diseased tree by leaf injection.

[0015] Furthermore, the specific steps of the method are as follows: when the citrus leaves are diseased, a small hole is punctured on the back of the diseased leaf with a needle, and an antibacterial agent is injected into the mesophyll cells through a syringe, wherein the concentration of CsCAPE9 in the antibacterial agent is 1~10uM.

[0016] Furthermore, the concentration of the antimicrobial peptide CsCAPE9 in the antibacterial agent is 1~10uM.

[0017] The beneficial effects of this invention are: This invention marks the first application of the antimicrobial peptide CsCAPE9 for the control of citrus Huanglongbing (HLB). It effectively inhibits the proliferation of HLB pathogens, exhibits low toxicity, is easily absorbed, and possesses broad-spectrum antimicrobial activity. Furthermore, CsCAPE9 is a naturally occurring peptide synthesized within the plant itself to maintain systemic immunity, demonstrating significantly higher safety than other antibiotics that inhibit HLB pathogens. Attached Figure Description

[0018] Figure 1 Figure showing the enrichment of differentially expressed genes upregulated 3 days after injecting Huanglongbing leaf with antibacterial agent 3.

[0019] In the figure, A and B are the GO enrichment and KEGG enrichment maps, respectively.

[0020] Figure 2 A schematic diagram showing the salicylic acid content in citrus leaves 3 days after injecting antibacterial agent 3 into leaves infected with Huanglongbing.

[0021] Figure 3 The graph shows the changes in Huanglongbing fungus 3 days before and after injecting CsCAPE9 into Huanglongbing leaves, which are respectively the control group 1 and the antibacterial agents 1-3. In the picture, This indicates a significant difference at the P<0.01 level. P<0.001 indicates a significant difference, while ns indicates no significant difference. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0023] Example 1 Synthesis of antimicrobial peptide CsCAPE9 The antimicrobial peptide CsCAPE9 was synthesized by Genscript Biotech; among which, The amino acid sequence of the antimicrobial peptide CsCAPE9 is shown in SEQ ID NO.1: PPGNFVGEKPY.

[0024] The gene encoding the antimicrobial peptide CsCAPE9 as described above CsCAPE9 The nucleotide sequence of the gene CsCAPE9 is shown in SEQ ID NO.2: CCCCCAGGCAACTTTGTTGGGGAAACCTTAC.

[0025] Example 2: Antibacterial agent 1 for inhibiting Huanglongbing bacteria The preparation of antibacterial agent 1 for inhibiting Huanglongbing bacteria is as follows: The antimicrobial peptide CsCAPE9 was chemically synthesized and dissolved in secondary pure water to prepare a 1 μM diluted injection solution, which is the antimicrobial agent 1.

[0026] Example 3: Antibacterial agent 2 for inhibiting Huanglongbing bacteria The preparation of antibacterial agent 2, which inhibits Huanglongbing bacteria, is as follows: The antimicrobial peptide CsCAPE9 was chemically synthesized and dissolved in secondary pure water to prepare a diluted injection solution with a concentration of 5 μM, which is the antimicrobial agent 2.

[0027] Example 4: Antibacterial agent 3 for inhibiting Huanglongbing bacteria The preparation of antibacterial agent 3, which inhibits Huanglongbing bacteria, is as follows: The antimicrobial peptide CsCAPE9 was chemically synthesized and dissolved in secondary pure water to prepare a diluted injection solution with a concentration of 10 μM, which is the antibacterial agent 3.

[0028] Comparative Example 1 The diluted injection solution is prepared by dissolving BSA in dilute water to a concentration of 1 μM.

[0029] I. Antimicrobial peptide CsCAPE9 enhances salicylic acid biosynthesis and activates citrus immunity. 1. Experimental materials: 1) Compound: the above-mentioned antibacterial agent 3.

[0030] 2) Biological material: sweet orange plants infected with Huanglongbing.

[0031] 2. Experimental Procedure The antibacterial agent 3 was injected into citrus leaves, with BSA as a control. Three trees were injected for each treatment, totaling more than eight leaves. The salicylic acid content and transcriptome data were analyzed in the citrus leaves three days after treatment.

[0032] The results showed that CsCAPE9 in antibacterial agent 3 could significantly activate the biosynthesis of salicylic acid and increase the salicylic acid content in citrus leaves. Figure 2 ).

[0033] Transcriptome data analysis showed that after CsCAPE9 treatment, the differentially expressed genes upregulated were mainly enriched in biotic and abiotic stress response pathways (i.e., MAPK signaling and disease resistance-related pathways), indicating that CsCAPE9 can activate the systemic immune response of citrus against Huanglongbing (HLB). Figure 1 ).

[0034] Therefore, CsCAPE9 fights Huanglongbing by activating the citrus immune response by increasing salicylic acid biosynthesis.

[0035] II. Analysis of the antimicrobial peptide CsCAPE9 against Huanglongbing (HLB) 1. Experimental materials: 1) Compounds: the above-mentioned antibacterial agents 1, 2, and 3, and the diluted injection solution.

[0036] 2) Biological material: sweet orange plants infected with Huanglongbing.

[0037] 2. Experimental Procedure Stable Huanglongbing-infected trees were selected as test materials (the pathogen causing Huanglongbing is...). Candidatus Liberibacter asiaticus). Bacterial load was detected using real-time qPCR, with primers from... C The 16S rRNA gene of Las, the internal reference gene is the mitochondrial gene of citrus: cytochrome oxidase ( COX CT values ​​reflect C For Las biomass, a smaller CT value indicates a larger biomass. COX It is used to test whether DNA quality and initial DNA concentration are comparable.

[0038] 3. Injecting antibacterial metabolites into infected leaves Three stable, bacteria-carrying sweet orange plants were selected, and four leaves from each plant were chosen from different locations. Before injection, two holes were punched on each side of the leaf vein using a puncher. Two of these leaves were cut off and used as a sample, placed in a 2 mL centrifuge tube containing a sampling ball, serving as a day 0 control. Afterward, a small peptide was injected into one side of the vein of the selected leaves. Similar leaves were selected for the control and treated with BSA. On the third day after injection, two holes were punched on each side of the leaves used for the control and the small peptide injection. Two of these leaves were cut off and used as a replicate, placed in a 2 mL centrifuge tube containing a sampling ball.

[0039] 4. Detection of antibacterial effect of small peptides DNA was extracted from the samples collected in step 3) at 0 days and 3 days post-injection using the CTAB method. After extraction, the DNA concentration was adjusted to a range of 100-150 ng / μL. Subsequently, quantitative detection was performed using a probe-based quantitative mix (HieffUnicon® qPCR TaqMan Probe Master Mix, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) to obtain the CT values ​​of the samples at 0 days and 3 days post-treatment.

[0040] 5. Statistics on the antibacterial effect of small peptides like Figure 3 As shown in Table 1: In HLB leaves treated with bacteriostatic agents 1, 2, 3, and diluted injection solutions, the CT value increased significantly, the content of Huanglongbing fungus decreased significantly, and a dose-effect relationship was observed; COX The relative bacterial counts calculated as internal references showed that the relative bacterial count decreased by 32.71% after treatment with bacteriostatic agent 1; by 56.14% after treatment with bacteriostatic agent 2; and by 81.33% after treatment with bacteriostatic agent 3.

[0041] The results showed that CsCAPE9 treatment had a significant antibacterial effect, and the higher the concentration, the better the effect.

[0042] As can be seen from the above, the antibacterial effect of antibacterial agent 3 is related to concentration compared with antibacterial agents 2 and 1.

[0043] Table 1 All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide CsCAPE9, characterized in that, The amino acid sequence of the antimicrobial peptide CsCAPE9 is shown in SEQ ID NO.

1.

2. A gene CsCAPE9 encoding the antimicrobial peptide CsCAPE9 as described in claim 1, characterized in that, The nucleotide sequence of the gene CsCAPE9 is shown in SEQ ID NO.

2.

3. The application of the antimicrobial peptide CsCAPE9 according to claim 1 in inhibiting the proliferation of citrus Huanglongbing pathogen.

4. The use of the antimicrobial peptide CsCAPE9 according to claim 1 in the preparation of a product inhibiting Huanglongbing bacteria.

5. A bacteriostatic agent for inhibiting Huanglongbing bacteria, characterized in that: The antibacterial agent contains the antimicrobial peptide CsCAPE9.

6. The antibacterial agent according to claim 5, characterized in that: The concentration of the antibacterial peptide CsCAPE9 in the antibacterial agent is 1~10uM.

7. The antibacterial agent according to claim 5, characterized in that: The concentration of the antibacterial peptide CsCAPE9 in the antibacterial agent is 10 μM.

8. A method for suppressing Huanglongbing (HLB), characterized in that: The method involves injecting the antibacterial agent described in claim 5 into the diseased tree via leaf injection.

9. The method for inhibiting Huanglongbing according to claim 8, characterized in that: The specific steps of the method are as follows: when citrus leaves are infected, a small hole is punctured on the back of the infected leaf with a needle, and an antibacterial agent is injected into the leaf mesophyll cells through a syringe. The concentration of CsCAPE9 in the antibacterial agent is 1~10uM.

10. The method for inhibiting Huanglongbing according to claim 8, characterized in that: The concentration of the antibacterial peptide CsCAPE9 in the antibacterial agent is 10 μM.

Images