Antibacterial peptide Cscape9 and its application in prevention and control of citrus huanglongbing

Abstract

The application discloses an antibacterial small peptide CsCAPE9 and application thereof in prevention and control of citrus Huanglongbing, and the amino acid sequence of the antibacterial small peptide CsCAPE9 is shown as SEQ ID NO. 1. The antibacterial small peptide CsCAPE9 is a small peptide synthesized in a plant body, and the antibacterial small peptide CsCAPE9 can activate biosynthesis of salicylic acid and induce a systemic defense response of citrus. The antibacterial small peptide CsCAPE9 has a good inhibiting effect on a pathogenic bacterium of citrus Huanglongbing in a greenhouse and in a field, and has a wide application prospect in comprehensive prevention and control of 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] Antibacterial peptides are small molecular polypeptides composed of 7 to 100 amino acids, which widely exist in the natural immune defense system of organisms. Compared with traditional antibiotics, antibacterial peptides have the core advantages of low molecular weight, high specificity, unique action mechanism (multi-target membrane destruction), low risk of inducing drug resistance, and low / zero toxicity. These characteristics not only endow them with strong broad-spectrum or specific antibacterial activity, but also make them show great potential in developing new targeted treatment strategies, becoming a research hotspot in the fields of biological medicine and agricultural disease control.

[0005] Therefore, it is urgent to develop a new prevention and control strategy with a completely new mechanism, high efficiency, safety and environmental friendliness for citrus Huanglongbing, which is called "citrus cancer". The prevention and control method based on antibacterial peptides is considered as an important breakthrough point for combating Huanglongbing, because of its characteristics of not being prone to drug resistance, potential systemic transmission ability, possible direct targeting of pathogenic bacteria in the phloem, and low risk to the environment and non-target organisms. The present patent focuses on the development of new antibacterial peptides or optimized derivatives of citrus Huanglongbing bacteria, aiming to provide support for the research and development of green products for Huanglongbing prevention and control. SUMMARY

[0006] The present application aims to overcome the shortcomings of the prior art and provides an antibacterial small peptide CsCAPE9 and its application in preventing and controlling citrus Huanglongbing. The antibacterial small peptide CsCAPE9 is a small peptide synthesized in the body of plants, which is green and efficient. The antibacterial small peptide CsCAPE9 can activate the biosynthesis of salicylic acid and induce the systemic defense response of citrus. The antibacterial small peptide CsCAPE9 has good inhibitory effect on the pathogen of citrus Huanglongbing (gram-negative bacteria - Liberibacter) in greenhouse and field, and has a wide application prospect for the comprehensive prevention and control of Huanglongbing.

[0007] To achieve the above-mentioned purpose, the technical solutions designed by the present application are as follows:

[0008] The present application provides an antibacterial small peptide CsCAPE9, the amino acid sequence of which is shown in SEQ ID NO. 1: PPGNFVGEKPY.

[0009] The gene CsCAPE9 encoding the above-mentioned antibacterial small peptide CsCAPE9 has the nucleotide sequence shown in SEQ ID NO. 2:

[0010] CCCCCAGGCAACTTTGTTGGGGAGAAACCTTAC.

[0011] The present application also provides the application of the above-mentioned antibacterial small peptide CsCAPE9 in inhibiting the proliferation of the pathogen of citrus Huanglongbing.

[0012] The application further provides application of the antibacterial small peptide CsCAPE9 in the preparation of a product for inhibiting Candidatus Liberibacter.

[0013] The application further provides a bacteriostatic agent for inhibiting Candidatus Liberibacter, wherein the bacteriostatic agent contains the antibacterial small peptide CsCAPE9.

[0014] Further, in the bacteriostatic agent, the concentration of the antibacterial small peptide CsCAPE9 is 1-10 uM.

[0015] Further, in the bacteriostatic agent, the concentration of the antibacterial small peptide CsCAPE9 is 1-10 uM.

[0016] The application further provides a method for inhibiting Huanglongbing, wherein the method is to inject the bacteriostatic agent into a diseased tree through leaf injection.

[0017] Further, the method comprises the following specific steps: when a citrus leaf is diseased, a small hole is punctured on the back of the diseased leaf by using a needle, and the bacteriostatic agent is injected into and fills the mesophyll cells through a syringe, wherein the concentration of CsCAPE9 in the bacteriostatic agent is 1-10 uM.

[0018] Further, in the bacteriostatic agent, the concentration of the antibacterial small peptide CsCAPE9 is 1-10 uM.

[0019] Advantages of the application:

[0020] The application first applies the antibacterial small peptide CsCAPE9 to the prevention and treatment of Huanglongbing of citrus, effectively inhibits the proliferation of Candidatus Liberibacter, has low toxicity, is easy to absorb, and has wide antibacterial property. In addition, CsCAPE9 is a natural small peptide synthesized in a plant body to maintain the immune system, and has obviously higher safety than other antibiotics for inhibiting Candidatus Liberibacter. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 FIG. 1 shows the up-regulated differential gene enrichment after the Huanglongbing leaf is injected with the bacteriostatic agent 3 for 3 days.

[0022] In the figure, A and B are GO enrichment and KEGG enrichment diagrams, respectively.

[0023] Figure 2 FIG. 4 shows the content of salicylic acid in the citrus leaf after the Huanglongbing leaf is injected with the bacteriostatic agent 3 for 3 days.

[0024] Figure 3 FIG. 5 shows the change of Candidatus Liberibacter before and after the Huanglongbing leaf is injected with the bacteriostatic agent 1-3.

[0025] In the figure, indicates that there is a significant difference at the level of P<0.01, Significant difference at the P < 0.001 level, n.s. indicates no significant difference. DETAILED DESCRIPTION

[0026] The application will be further described in conjunction with specific examples so that those skilled in the art will understand.

[0027] Example 1 Synthesis of antibacterial small peptide CsCAPE9

[0028] The antibacterial small peptide CsCAPE9 was synthesized by the Jinshui Biological Company; wherein,

[0029] The amino acid sequence of the antibacterial small peptide CsCAPE9 is shown in SEQ ID NO. 1: PPGNFVGEKPY.

[0030] Gene encoding the antibacterial small peptide CsCAPE9 as described above CsCAPE9 The nucleotide sequence of the gene CsCAPE9 is shown in SEQ ID NO. 2:

[0031] CCCCCAGGCAACTTTGTTGGGGAGAAACCTTAC.

[0032] Example 2 Bacteriostatic agent 1 for inhibiting Candidatus Liberibacter

[0033] The bacteriostatic agent 1 for inhibiting Candidatus Liberibacter is prepared as follows:

[0034] After the antibacterial small peptide CsCAPE9 is chemically synthesized, it is dissolved with secondary pure water to prepare a dilution injection agent with a concentration of 1 uM, which is bacteriostatic agent 1.

[0035] Example 3 Bacteriostatic agent 2 for inhibiting Candidatus Liberibacter

[0036] The bacteriostatic agent 2 for inhibiting Candidatus Liberibacter is prepared as follows:

[0037] After the antibacterial small peptide CsCAPE9 is chemically synthesized, it is dissolved with secondary pure water to prepare a dilution injection agent with a concentration of 5 uM, which is bacteriostatic agent 2.

[0038] Example 4 Bacteriostatic agent 3 for inhibiting Candidatus Liberibacter

[0039] The bacteriostatic agent 3 for inhibiting Candidatus Liberibacter is prepared as follows:

[0040] After the antibacterial small peptide CsCAPE9 is chemically synthesized, it is dissolved with secondary pure water to prepare a dilution injection agent with a concentration of 10 uM, which is bacteriostatic agent 3.

[0041] Comparative Example 1

[0042] Dilution injection solution is to dissolve BSA with secondary pure water to prepare a solution with a concentration of 1 uM.

[0043] I. Antimicrobial peptide CsCAPE9 enhances salicylic acid biosynthesis and activates citrus immunity

[0044] 1. Experimental materials:

[0045] 1) Compound: the above-mentioned bacteriostatic agent 3.

[0046] 2) Biological material: sweet orange plants infected with huanglongbing.

[0047] 2. Experimental operation process

[0048] The bacteriostatic agent 3 was injected into the citrus leaves, with BSA as a control, 3 trees were injected for each treatment, and a total of more than 8 leaves. After 3 days of treatment, the salicylic acid content of the citrus leaves was detected and the transcriptome data was analyzed.

[0049] The results showed that CsCAPE9 in the bacteriostatic agent 3 can significantly activate the biosynthesis of salicylic acid and increase the content of salicylic acid in citrus leaves (P < 0.05). Figure 2 ).

[0050] The transcriptome data analysis results showed that the differentially expressed genes up-regulated after CsCAPE9 treatment were mainly enriched in the response to biotic and abiotic stress pathways (i.e. MAPK signal transduction and disease resistance related pathways), indicating that CsCAPE9 can activate the systemic immune response of citrus against huanglongbing (P < 0.05). Figure 1 ).

[0051] Therefore, CsCAPE9 resists huanglongbing by activating the immune response of citrus through increasing the biosynthesis of salicylic acid.

[0052] II. Analysis of antimicrobial peptide CsCAPE9 against huanglongbing

[0053] 1. Experimental materials:

[0054] 1) Compound: the above-mentioned bacteriostatic agent 1, bacteriostatic agent 2, bacteriostatic agent 3 and dilution injection solution.

[0055] 2) Biological material: sweet orange plants infected with huanglongbing.

[0056] 2. Experimental operation process

[0057] Stable huanglongbing-infected trees were selected as test materials (the pathogen of huanglongbing is Candidatus Liberibacter asiaticus). The amount of bacteria was detected by Real-time qPCR method, and the detection primers were from CLas's 16S rRNA gene, the internal reference gene is citrus mitochondria gene: cytochrome oxidase (COII) COX ). The CT value reflects C The smaller the CT value of Las's biomass, the greater the biomass. COX To test the DNA quality and the initial DNA concentration is comparable.

[0058] 3. Injection of bacteriostatic metabolites into diseased leaves

[0059] Select 3 stable sweet orange plants with bacteria, and select a total of 4 leaves in different directions. Before injection, use a punch to make two holes on both sides of the leaf vein, and cut two leaves as a sample and put them into a 2 mL centrifuge tube containing a punch steel ball, as a 0d control. Then, inject small peptides into the selected leaf veins, and select similar leaves for BSA treatment. On the 3rd day after injection, make two holes on both sides of the injection control and small peptide leaves, and cut two leaves as a repeat and put them into a 2 mL centrifuge tube containing a steel ball.

[0060] 4. Detection of bacteriostatic effect of small peptides

[0061] DNA extraction was performed on the 0d and 3d post-injection samples collected in step 3, using the CTAB method. After extraction, the DNA concentration was adjusted to a concentration range of 100-150 ng / μL. Then, quantitative detection was performed using a quantitative Mix with a probe (HieffUnicon® qPCR TaqMan Probe Master Mix, purchased from Yisen Biotechnology (Shanghai) Co., Ltd.), and the CT values of the 0d and 3d post-treatment samples were obtained.

[0062] 5. Statistical analysis of bacteriostatic effect of small peptides

[0063] As Figure 3 and Table 1 show: the CT values of HLB leaves treated with bacteriostatic agent 1, bacteriostatic agent 2, bacteriostatic agent 3 and dilution injection solution increased significantly, the content of Candidatus Liberibacter asiaticus decreased significantly, and there was a dose effect; using COX as an internal reference to calculate the relative bacterial content showed that the relative bacterial content after treatment with bacteriostatic agent 1 decreased by 32.71%; the relative bacterial content after treatment with bacteriostatic agent 2 decreased by 56.14%; and the relative bacterial content after treatment with bacteriostatic agent 3 decreased by 81.33%.

[0064] The results show that CsCAPE9 treatment has a significant bacteriostatic effect, and the higher the concentration, the better the effect.

[0065] As can be seen from the above: bacteriostatic agent 3 has a bacteriostatic effect related to concentration compared with bacteriostatic agent 2 and bacteriostatic agent 1.

[0066] Table 1

[0067]

[0068] Other parts not described in detail are prior art. Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiment of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, which all belong to the protection scope of the present application.

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, characterized in that: The pathogen causing Huanglongbing is the Huanglongbing pathogen. Candidatus Liberibacter asiaticus.

4. The application of the antimicrobial peptide CsCAPE9 according to claim 1 in the preparation of a product inhibiting Huanglongbing (HLB) of citrus, characterized in that: The Huanglongbing bacterium is the pathogen causing Huanglongbing. Candidatus Liberibacter asiaticus.

5. A bacteriostatic agent for inhibiting Huanglongbing (HLB) of citrus, characterized in that: The antibacterial agent contains the antimicrobial peptide CsCAPE9 as described in claim 1.

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 citrus Huanglongbing (HLB), characterized in that: The method involves injecting the antibacterial agent described in claim 5 into the diseased tree via leaf injection, wherein the pathogen causing Huanglongbing (HLB) in the diseased tree is the Huanglongbing pathogen Candidatus Liberibacter asiaticus.

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

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