Lyase preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of agricultural biology, in particular to a lyase preparation and a preparation method and application thereof, the lyase preparation comprises two high-efficiency broad-spectrum lyase: LysPSA2 and LysPSA3, the amino acid sequences of the lyase are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, or variants with at least 95% homology with the lyase are shown as SEQ ID NO: 1 and SEQ ID NO: 2. The lyase preparation disclosed by the invention has the advantages of efficient sterilization, good stability, safety in use, environmental friendliness and the like, and a novel effective solution is provided for green prevention and control of the kiwifruit canker.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a lysin preparation, its preparation method, and its application. Background Technology

[0002] Kiwifruit (Actinidia chinensis) is an important economic crop, favored by consumers for its rich nutrition and unique flavor. However, diseases severely impact its yield and quality during cultivation. Kiwifruit canker, a devastating bacterial disease primarily caused by *Pseudomonas syringae*, infects the branches, leaves, and fruits of kiwifruit, leading to lesions, cankers, branch dieback, and even the death of the entire plant, causing significant economic losses to the kiwifruit industry. Currently, the main methods for controlling kiwifruit canker include chemical spraying and orchard sanitation. However, chemical pesticides suffer from unstable efficacy, environmental pollution, and the accumulation of resistance, while orchard sanitation cannot fundamentally control the disease. Less researched biological control methods primarily focus on biocontrol agents.

[0003] Lyases are a class of enzymes that specifically hydrolyze the polysaccharide components of bacterial cell walls, leading to cell lysis and death by disrupting the integrity of the bacterial cell wall. Some studies have shown that lyases have potential applications in controlling bacterial plant diseases. However, the development and application of lyase preparations targeting *Pseudomonas syringae*, the pathogen of kiwifruit bacterial canker, are currently relatively limited. Therefore, developing a highly efficient, safe, and environmentally friendly lyase preparation for controlling kiwifruit bacterial canker is of significant practical importance. Summary of the Invention

[0004] In view of this, the present invention proposes a lysin preparation, its preparation method and application, to effectively prevent and control kiwifruit canker. The lysin preparation can specifically lyse the pathogen of kiwifruit canker, *Pseudomonas syringae*, thereby inhibiting its growth and reproduction, and achieving the purpose of disease prevention and control.

[0005] The technical solution of this invention is implemented as follows:

[0006] On one hand, the present invention provides a lysin preparation comprising lysin LysPSa2 and lysin LysPSa3, wherein the amino acid sequence of lysin LysPSa2 is shown in SEQ ID NO.1: MSLRSRLINLGAAGAVLAAAAFLGPTEELRTVPYADIGGVSTWCYGQTVGIPKARFSVAECDADLLRSLSRYTEAIKPATVGAPASVVAAMVSVQYNTQGNGVPHAVFLKPLAERDWRAAKDAIVDPWQGKYGVSKGFKATVQCRPLRGLENRRAKEFDYCVSGL;

[0007] The amino acid sequence of the lysin LysPSa3 is shown in SEQ ID NO.2: MSVLQRLAALGLTSALVLAGTTLVAPWEGKENKAYKDVVGVWTQCEGDTHDVNRTRAKTDEECADSLAKQLVKHNVSMKQYVIVPLTDYQEVAFTSLVYLEGAGNWKNSTALKLLNKGLRREACLQLPRWNKAGGQVYRGLTNRRLSNLEVCLGNNKQAIEEARRAVAAYRDVDFIDPLIGEVKSEK.

[0008] The preparation method of the lysin comprises: amplifying gene fragments encoding LysPSa2 and LysPSa3 by PCR, wherein the LysPSa2 gene is derived from *Pseudomonas syringae* phage GRNPSaP2 and the LysPSa3 gene is derived from *Pseudomonas syringae* phage GRNPSaP3; designing primers according to the genes to ensure specific amplification; cloning the amplified gene fragments into the BamHI / SalI site of the expression vector pET-28a(+) using Gibson assembly, adding an N-terminal His tag, and constructing recombinant plasmids pET28a-LysPSa2 and pET28a-LysPSa3; verifying the correctness of the recombinant plasmids by sequencing; then introducing the recombinant plasmids into *Escherichia coli* BL21(DE3) host bacteria by chemical transformation, and screening for positive clones; culturing the recombinant bacteria to OD28a. 600 When the concentration reached 0.6, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and expression was induced at 15°C for 12 hours to obtain the lyase protein. The lyase was then purified by Ni-NTA affinity chromatography, and impurities were removed by dialysis after elution to obtain a functional lyase with a purity ≥95%.

[0009] Based on the above scheme, preferably, the mass ratio of the lysin LysPSa2 to the lysin LysPSa3 is (0.2-1):(0.2-1); more preferably, the mass ratio of the lysin LysPSa2 to the lysin LysPSa3 is 1:1.

[0010] Based on the above scheme, preferably, the total concentration of the lysin LysPSa2 and lysin LysPSa3 is 9-11 mg / mL; more preferably, the total concentration is 10 mg / mL.

[0011] Secondly, a method for preparing the lysin preparation as described above is provided, comprising the following steps:

[0012] S1, mix the above lyases LysPSa2 and LysPSa3 thoroughly;

[0013] S2, add buffer, stabilizer, surfactant, preservative, penetrant and binder, mix well to obtain lysin preparation.

[0014] Based on the above scheme, preferably, the stabilizer is one or more of glycerol and sorbitol; the surfactant is one or more of Tween series and Span series; the penetrant is one or more of dimethyl sulfoxide and ethanol; the adhesive is one or more of polyvinyl alcohol and sodium carboxymethyl cellulose; and the preservative is one or more of sodium benzoate and potassium sorbate.

[0015] Among them, stabilizers are used to improve the stability of lysin during storage and use; surfactants are used to improve the wettability and spreadability of the formulation on plant surfaces; penetrants are used to promote the penetration of lysin into plant tissues or bacterial cell walls; adhesives are used to improve the adhesion of the formulation to plant surfaces and reduce rainwater erosion; and preservatives are used to prevent microbial contamination and extend the shelf life of the formulation.

[0016] Based on the above scheme, preferably, the amount of stabilizer added is 5-10 wt%, the amount of surfactant added is 0.1-0.5 wt%, the amount of penetrant added is 1-5 vol%, the amount of adhesive added is 0.1-0.3 wt%, and the amount of preservative added is 0.05-0.1 wt%.

[0017] Based on the above scheme, preferably, the buffer is one or more of phosphate buffer and citrate buffer. The buffer is used to maintain the preparation within a suitable pH range to ensure enzyme activity.

[0018] Based on the above scheme, preferably, the pH of the buffer is between 6.5 and 7.5.

[0019] Based on the above scheme, a further optimized method is to add 8 wt% sorbitol, 0.3 wt% Tween 80, 3 wt% dimethyl sulfoxide (DMSO), 0.05 wt% potassium sorbate, 0.2 wt% polyvinyl alcohol (PVA), and phosphate buffer at pH 7.0 to a final lyase concentration of 10 mg / mL. Mix thoroughly, filter sterilize, dispense into 1L sterile bottles, and store at room temperature.

[0020] Thirdly, the application of the lyase preparation as described above in the prevention and control of kiwifruit canker is provided. Preferably, the preparation is administered by foliar spraying or root irrigation, once every 7 days, for a total of 3 consecutive applications, at the early stage of the disease or for prevention.

[0021] The lysin preparation of the present invention has the following advantages over the prior art:

[0022] (1) The lysin preparation of the present invention contains two highly efficient and broad-spectrum lysins, LysPSa2 and LysPSa3, which can effectively prevent and control kiwifruit canker and provide a new and effective solution for the prevention and control of kiwifruit canker.

[0023] (2) The lyase preparation of this application has the advantages of high efficiency sterilization, safe use, and environmental friendliness, and has good stability. After being stored at room temperature for 6 months, the lysing activity did not change significantly. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The diagram shows the lysis zones of the lysin preparation of the present invention against three strains of Pseudomonas syringae.

[0026] Figure 2 The in vitro bactericidal effect curves of the lysin preparation of the present invention against three strains of Pseudomonas syringae are shown.

[0027] Figure 3 This is a graph showing the stability determination of the lysin preparation of the present invention. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] Construction of lyase gene

[0031] LysPSa2, a lyase derived from GRNPSaP2 phage of Pseudomonas syringae, and LysPSa3, a lyase derived from GRNPSaP3 phage of Pseudomonas syringae, were amplified by PCR. The amino acid sequences encoded by the genes of lyases LysPSa2 and LysPSa3 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0032] Subsequently, the gene was cloned into the BamHI / SalI site of the expression vector pET28a(+) using Gibson assembly, and an N-terminal His tag was added. The recombinant plasmids were named pET28a-LysPSa2 and pET28a-LysPSa3. After sequencing verification, the constructed recombinant plasmids were transformed into Escherichia coli BL21(DE3) host bacteria to facilitate subsequent expression and purification experiments.

[0033] Example 2

[0034] Expression and purification of lysin

[0035] Positive clones were picked from LB agar plates containing 50 mg / L kanamycin and inoculated onto LB agar (50 mg / L kanamycin) and cultured until OD. 600 Once the concentration reached 0.6, IPTG was added to a final concentration of 0.5 mM for induction, and the cells were incubated at 15°C for 12 hours. After incubation, the cells were collected and lysed by sonication, followed by Ni... 2+ His-tagged purification was performed using affinity chromatography. The eluted protein was dialyzed to remove impurities, and the purity and molecular weight of the protein were analyzed using SDS-PAGE. Simultaneously, the protein expression level was verified using a Nanodrop instrument, ultimately yielding high-purity LysPSa2 and LysPSa3 lyases.

[0036] Example 3

[0037] Preparation of lysin preparations

[0038] The purified lyase from Example 2 was mixed to a final concentration of 10 mg / mL, with a mass ratio of LysPSa2 to LysPSa3 of 1:1. Then, 8 wt% sorbitol, 0.3 wt% Tween 80, 3 wt% dimethyl sulfoxide (DMSO), 0.05 wt% potassium sorbate, 0.2 wt% polyvinyl alcohol (PVA), and phosphate buffer at pH 7.0 were added. The mixture was thoroughly mixed, filtered sterilely, and dispensed into 1L sterile bottles for storage at room temperature.

[0039] Example 4

[0040] Preparation of lysin preparations

[0041] The purified lyase from Example 2 was mixed to a final concentration of 9 mg / mL, with a mass ratio of LysPSa2 to LysPSa3 of 1:5. Then, 5 wt% sorbitol, 0.1 wt% Tween 20, 1 wt% ethanol, 0.08 wt% potassium sorbate, 0.1 wt% polyvinyl alcohol (PVA), and citrate buffer at pH 6.5 were added. The mixture was thoroughly mixed, filtered sterilely, and dispensed into 1L sterile bottles for storage at room temperature.

[0042] Example 5

[0043] Preparation of lysin preparations

[0044] The purified lyase from Example 2 was mixed to a final concentration of 11 mg / mL, with a mass ratio of LysPSa2 to LysPSa3 of 5:1. Then, 10 wt% glycerol, 0.5 wt% Span 20, 5 wt% DMSO, 0.1 wt% sodium benzoate, 0.3 wt% sodium carboxymethyl cellulose (CMC-Na), and phosphate buffer at pH 7.5 were added. The mixture was thoroughly mixed, filtered sterilely, and dispensed into 1L sterile bottles for storage at room temperature.

[0045] Comparative Example 1

[0046] The comparative example is prepared using the same method as the lyase preparation in Example 3, except that only the lyase LysPSa2 is added.

[0047] Comparative Example 2

[0048] The comparative example is prepared using the same method as the lyase preparation in Example 3, except that only the lyase LysPSa3 is added.

[0049] Comparative Example 3

[0050] The comparative example is prepared using the same method as the lyase preparation in Example 3, except that the mass ratio of lyase LysPSa2 to lyase LysPSa3 is 0.4:3.

[0051] Comparative Example 4

[0052] The comparative example is prepared using the same method as the lyase preparation in Example 3, except that the mass ratio of lyase LysPSa2 to lyase LysPSa3 is 3:0.4.

[0053] Example 6

[0054] Lysis assay of lysin preparations

[0055] The lytic effect of the lysin preparation on *Pseudomonas syringae* strains GRNPSa002, GRNPSa004, and GRNPSa005 was determined using the double-layer agar plate method. 10 μL of the lysin preparation prepared in Example 3 was spotted onto an agar plate containing bacterial growth. After incubation at 28°C for 24 hours, the lysis zone was observed to evaluate the lytic activity of the lysin preparation. The results showed that the lysin preparation had a significant lytic effect on all three *Pseudomonas syringae* strains, with lysis zones reaching 12-15 mm in diameter. Figure 1 As shown.

[0056] Example 7

[0057] In vitro bactericidal activity test of lysin preparation against Pseudomonas syringae

[0058] Pseudomonas syringae GRNPSa002 was cultured in 200 ml of NB liquid medium and diluted to OD. 600 The concentration of the lyase was 0.05 mg / L. Lyase preparations prepared in Examples 3-5 and the comparative examples were added to bring the lyase concentration to 100 mg / L. An equal volume of physiological saline was added to the blank control group. The cultures were incubated at 28°C and 200 rpm, with OD values ​​measured at 1-hour intervals. 600 Values. This experiment involved double-well calculations to determine the average value, which was then plotted to present the bacterial growth curve. Results are as follows: Figure 2 As shown, the lysin preparation can effectively inhibit the growth of Pseudomonas syringae GRNPSa002, and the preparation method in Example 3 exhibits the best bactericidal ability.

[0059] Example 8

[0060] Storage stability of lysin preparations

[0061] The storage stability of the lyase preparation was determined using the double-layer agar plate method. The lyase preparation from Example 3, stored at room temperature (20–25°C), was tested every month. 10 μL of the lyase preparation was spotted onto an agar plate containing GRNPSa002 bacterial growth, incubated at 28°C for 24 hours, and the lysis zone was observed to assess its size. The results showed no significant difference in the lysis zones produced by the lyase preparations over 6 months. Figure 3 As shown.

[0062] Example 9

[0063] Field trials (Zhejiang)

[0064] This experiment was conducted in a plantation in Zhejiang Province, China, using the 'Jinyan' variety, and covering an area of ​​1 hectare. The lysin preparation described in Example 3 of this invention was applied starting from the kiwifruit budding stage, with two application methods employed:

[0065] Spray treatment group: Dilute the lysin preparation 1000 times, that is, the concentration is 10μg / mL, and spray 20L per acre;

[0066] Root irrigation treatment group: The lyase preparation was diluted 500 times, that is, the concentration was 20μg / mL, and 1L was irrigated per plant.

[0067] Spraying frequency is once every 7 days, for a total of 3 consecutive sprays. Set up a control area and apply an equal amount of water.

[0068] The severity of the disease is recorded using a grading system, with the grading criteria as follows:

[0069] Level 0: No disease;

[0070] Grade 1: The area of ​​lesions accounts for less than 5% of the total leaf area;

[0071] Level 3: The area of ​​lesions accounts for less than 6%-10% of the total leaf area;

[0072] Level 5: The area of ​​lesions accounts for less than 11%-20% of the total leaf area;

[0073] Level 7: The area of ​​lesions accounts for less than 21%-50% of the total leaf area;

[0074] Level 9: The area of ​​lesions accounts for more than 50% of the total leaf area.

[0075] Methods for calculating the effectiveness of prevention

[0076] Disease incidence rate (%) = (Number of diseased leaves / Total number of leaves surveyed) × 100%

[0077] Disease index = ∑(number of diseased leaves at each level × relative grade value) / (total number of leaves surveyed × 9) × 100

[0078] Control efficacy (%) = [1 - (disease index before treatment in blank control area × disease index after treatment in treatment area) / (disease index after treatment in blank control area × disease index before treatment in treatment area)] × 100%.

[0079] The experimental results are shown in Table 1: the incidence of diseases in the treated kiwifruit was significantly reduced, the lesion area was significantly reduced, and the control efficacy reached over 75%. Regarding plant safety, no adverse reactions such as yellowing of kiwifruit leaves or growth inhibition were observed during the experiment, indicating that the formulation has good plant compatibility at this dilution concentration, further verifying its safety for field application.

[0080] Table 1. Field efficacy statistics of lyase preparations

[0081]

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lysin preparation, characterized in that, It includes lyases LysPSa2 and LysPSa3, the amino acid sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.

2.

2. The lysin preparation according to claim 1, characterized in that, The mass ratio of the lyase LysPSa2 to the lyase LysPSa3 is (0.2–1): (0.2–1).

3. The lysin preparation according to claim 2, characterized in that, The total concentration of the lyases LysPSa2 and LysPSa3 was 9–11 mg / mL.

4. The method for preparing the lysin preparation as described in claim 1, characterized in that, Includes the following steps: S1, mix the lyases LysPSa2 and LysPSa3 thoroughly; S2, add buffer, stabilizer, surfactant, preservative, penetrant and binder, mix well to obtain lysin preparation.

5. The method for preparing the lysin preparation as described in claim 4, characterized in that, The stabilizer is one or more of glycerol and sorbitol; the surfactant is one or more of Tween 20, Tween 80, Span 20, and Span 40; the penetrant is one or more of dimethyl sulfoxide and ethanol; the adhesive is one or more of polyvinyl alcohol and sodium carboxymethyl cellulose; and the preservative is one or more of sodium benzoate and potassium sorbate.

6. The method for preparing the lysin preparation as described in claim 5, characterized in that, The amount of stabilizer added is 5-10 wt%, the amount of surfactant added is 0.1-0.5 wt%, the amount of penetrant added is 1-5 vol%, the amount of adhesive added is 0.1-0.3 wt%, and the amount of preservative added is 0.05-0.1 wt%.

7. The method for preparing the lysin preparation as described in claim 4, characterized in that, The buffer is one or more of phosphate buffer and citrate buffer.

8. The method for preparing the lysin preparation as described in claim 7, characterized in that, The pH of the buffer is between 6.5 and 7.

5.

9. The application of the lysin preparation as described in claim 1 in the preparation of an agent for preventing and treating kiwifruit canker.

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