Engineering strain for preventing and controlling pine wood nematodes as well as construction method and application of engineering strain

Abstract

The invention discloses an engineering strain for preventing and controlling pine wood nematodes as well as a construction method and application of the engineering strain, and relates to the technical field of microorganisms. The invention discloses a construction method of an engineering strain for preventing and treating pine wood nematodes, which comprises the following steps: constructing an oprL promoter, an SD sequence and a Peptidase03 gene sequence into a modified vector pBBR1MCS2-Tac-EGFP (Enhanced Green Fluorescent Protein), so as to obtain a recombinant plasmid pBBR1MCS2-P03; the method comprises the following steps: transforming a plasmid pBBR1MCS2-P03 into a recipient bacterium BM06, so as to obtain an engineering bacterium BM06-P03; the sequence of the Peptidase03 gene is as shown in SEQ ID NO. 1; the sequence of the oprL promoter is as shown in SEQ ID NO. 4; and the SD sequence is as shown in SEQ ID NO.5. The engineering strain BM06-P03 constructed by the invention delays the morbidity speed of masson pine and plays a role in preventing and controlling pine wood nematodes.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to an engineering strain for preventing and controlling pine wood nematodes, and a construction method and application thereof. Background Art

[0002] Pine wilt disease (Bursaphelenchus xylophilus) is a major forest disease that has caused great economic losses and environmental damage worldwide. The use of chemical pesticides can lead to environmental pollution, and nematodes are prone to develop drug resistance. The pathogenic mechanism of pine wilt disease has not yet been clarified, and prevention and control are very difficult. Once the disease occurs, the entire pine forest will suffer devastating damage. The disease has been studied for many years at home and abroad, and the main focus has been on the exploration of scientific issues related to pathogens, such as pathogen types, pathogenic mechanisms, ecological characteristics of pathogenic nematodes, pathogen transmission vectors, disease diagnosis and prevention, etc. Due to the complexity of the occurrence and development of the disease, the pathogenic mechanism of the pathogen is not very clear, and there is still a lack of simple and effective prevention and control methods.

[0003] In recent years, high-efficiency and low-toxic biological control has become a research hotspot. Introducing exogenous resistance genes into plant endophytic bacteria through genetic means can not only enhance the biological control potential of endophytic bacteria, but also enable the host plant to acquire a certain degree of stress resistance. A large number of studies have been conducted on the genetic engineering of endophytic bacteria at home and abroad. For example, after the Bt gene was introduced into endophytic bacteria such as sugarcane, corn, cotton, and rice, the host plants all acquired strong insecticidal ability, and the modified endophytic bacteria still maintained their inherent nitrogen fixation, growth promotion and disease resistance capabilities.

[0004] Pine wood nematodes are the pathogen of pine wood nematode disease and the direct cause of the disease. The cuticle is a barrier between pine wood nematodes and the external environment, and plays a vital role in maintaining the body shape and integrity of the nematodes. The cuticle of pine wood nematodes is composed of a variety of proteins, which are connected by peptide bonds. How to break through the barrier of the nematode body wall is of great significance for the search for nematode-killing factors.

[0005] Peptidase 03 (NlpC / P60) comes from Pseudomonas syringae MB03, contains the protease catalytic triad Cys-His-His, and has the activity of catalyzing peptide bonds. The hydrolysis of peptide bonds leads to the decomposition of proteins, which can seriously interfere with the normal physiological activities of nematodes and cause their death. The NlpC / P60 domain is similar to papain in structure. It can exist alone or in combination with other domains. Its catalytic function changes with structural changes, and it can form proteins with diverse functions. For example, the SH3 domain in the NlpC / P60 endopeptidase domain participates in the process of hyaluronidase anchoring to the cell wall, regulates the concentration and anchoring position of hyaluronic acid, forms an effective enzyme-substrate complex and exerts its function. Peptidoglycan (PG) is the main component of bacterial cell walls and is essential for maintaining structural integrity and internal osmotic pressure and shaping bacterial morphology. However, peptidoglycan is also highly dynamic, and its glycosidic bonds and amide bonds can be broken or formed by different enzymes, one of which is NlpC / P60 endopeptidase. The degradation products produced by the catalytic activity of NlpC / P60 endopeptidase can be recycled for re-biosynthesis of peptidoglycan, or can act as signal molecules to trigger the regrowth of antibiotic-resistant or dormant cells, or as effector molecules in immune responses. NlpC / P60 endopeptidase can also be secreted into the environment, or enter the periplasm of other bacteria through the type VI secretion system to affect their survival, giving bacteria that secrete NlpC / P60 endopeptidase a competitive advantage.

[0006] Therefore, developing a strain that can efficiently colonize in Masson pine and has strong indoor nematode effect and outdoor biocontrol potential is of great significance for the prevention and control of pine wilt disease. Summary of the invention

[0007] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an engineered strain for controlling pine wood nematodes. Another technical problem to be solved by the present invention is to provide a construction method of an engineered strain for controlling pine wood nematodes. Another technical problem to be solved by the present invention is to provide an application of an engineered strain for controlling pine wood nematodes for controlling pine wood nematodes.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for constructing an engineered strain for controlling pine wood nematodes, comprising constructing an oprL promoter, an SD sequence and a Peptidase03 gene sequence into a transformation vector pBBR1MCS2-Tac-EGFP to obtain a recombinant plasmid pBBR1MCS2-P03; transforming the plasmid pBBR1MCS2-P03 into a recipient bacterium Pseudomonas koreensis BM06 to obtain an engineered bacterium BM06-P03;

[0010] The Peptidase03 gene sequence is shown in SEQ ID NO.1;

[0011] The sequence of the oprL promoter is shown in SEQ ID NO.4;

[0012] The SD sequence is shown as SEQ ID NO.5.

[0013] The amplification primers for the oprL promoter are as follows:

[0014] oprL-F: 5'-ACTACAACGCCAACCCGAAGCTGTC-3',

[0015] oprL-R: 5'-TGAAACTCCTAATGAACCCCAGTGT-3'.

[0016] The construction method comprises the following specific steps:

[0017] 1) Primers oprL-F: 5'-ACTACAACGCCAACCCGAAGCTGTC-3', oprL-R: 5'-TGAAACTCCTAATGAACCCCAGTGT-3' were designed with reference to the oprL gene sequence of Pseudomonas putida KT2440 strain GenBank AAN66847, genomic DNA of Korean Pseudomonas was extracted, and the oprL gene promoter was obtained by PCR;

[0018] The sequence of the oprL promoter is shown in SEQ ID NO.4;

[0019] 2) Insert the oprL promoter, SD sequence and Peptidase03 gene sequence into the EcoRI and HindIII restriction sites of the transformation vector pBBR1MCS2-Tac-EGFP to obtain the vector pBBR1MCS2-P03, and extract the plasmid pBBR1MCS2-P03;

[0020] The SD sequence is shown in SEQ ID NO.5;

[0021] The Peptidase03 gene sequence is shown in SEQ ID NO.1;

[0022] 3) The plasmid pBBR1MCS2-P03 was transformed into DH5α, and the positive clones were picked and the pBBR1MCS2-P03 plasmid was extracted again and transformed into the recipient bacterium Pseudomonas koreensis BM06 to obtain the recombinant engineering strain BM06-P03.

[0023] Recombinant engineered strain BM06-P03 for controlling pine wood nematodes.

[0024] Application of the recombinant engineered strain BM06-P03 in controlling pine wood nematodes.

[0025] The method for controlling pine wood nematodes is to slow down the incidence of Masson pine.

[0026] Application of the fermentation filtrate of the recombinant engineered strain BM06-P03 in killing pine wood nematodes.

[0027] The method for preparing the fermentation filtrate is as follows: taking the recombinant engineering strain BM06-P03, activating it on an LB plate containing knna, and culturing it at 30°C overnight; picking a single clone with an inoculation loop, inoculating it into 20mL NB liquid culture medium containing 50μg / mL kanna antibiotic, and culturing it at 30°C and 200r / min for 12h, so as to prepare the fermentation filtrate of the recombinant engineering strain BM06-P03.

[0028] The amino acid sequence of the protein expressed by the recombinant engineering strain BM06-P03 is shown in SEQ ID NO.3.

[0029] Application of the protein expressed by the recombinant engineering strain BM06-P03 in the prevention and control of pine wood nematode disease.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1) The invention discloses a method for constructing an engineered strain for controlling pine wood nematodes, comprising constructing an oprL promoter, an SD sequence and a Peptidase03 gene sequence into a transformation vector pBBR1MCS2-Tac-EGFP to obtain a recombinant plasmid pBBR1MCS2-P03; transforming the plasmid pBBR1MCS2-P03 into a recipient bacterium Korean Pseudomonas Pseudomonaskoreensis BM06 to obtain an engineered bacterium BM06-P03; the Peptidase03 gene sequence is shown in SEQ ID NO.1; the oprL promoter sequence is shown in SEQ ID NO.4; and the SD sequence is shown in SEQ ID NO.5.

[0032] 2) The results of the present invention show that when the purified Peptidase03 (120.00 μg / mL) protein was used to treat nematodes for 24 hours, the mortality rate of the nematodes was 100%, the nematode intestine was damaged, vesicles appeared, and the worm body was broken. As the concentration of Peptidase03 increased, the mortality rate of pine wood nematodes also increased. The EC of the purified Peptidase03 50 The value is 25.73μg / mL. Peptidase03 is highly toxic to pine wood nematodes and is an important factor in killing pine wood nematodes.

[0033] 3) The results of the examples of the present invention show that when the fermentation filtrate of the BM06-P03 strain is uniformly diluted to 5 times, 10 times or 20 times, it has a nematicidal effect, has a higher application potential, and provides an effective strategy for the prevention and control of pine wood nematode disease.

[0034] 4) The results of the examples of the present invention show that the engineered strain BM06-P03 slows down the disease rate of Masson pine and has a control effect on pine wood nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the structural diagram of the recombinant vector pET-32a(+)-P03;

[0036] Figure 2 This is the result of SDS and Western blot detection of the expression of purified protein Peptidase 03;

[0037] Figure 3 This is a picture of the effect of Peptidase03 treatment on the body surface of pine wood nematode observed by scanning electron microscopy;

[0038] Figure 4 This is a transmission electron microscopy observation of the effect of Peptidase03 treatment on the intestine of pine wood nematode;

[0039] Figure 5 The figure shows the mortality rate of purified Peptidase03 protein to pine wood nematodes at different concentrations;

[0040] Figure 6 is a structural diagram of the vector pBBR1MCS2-P03;

[0041] Figure 7 The results of SDS and Western blot detection of the expression of protein Peptidase 03 in BM06 strain;

[0042] Figure 8The fermentation filtrates of the wild-type strain BM06 and the engineered strain BM06-P03 were cultured for 12h, 24h, 36h, and 48h, respectively, and diluted 5, 10 times, and 20 times, respectively. The corrected mortality rate of pine wood nematodes after 24h of treatment (A is the corrected mortality rate of nematodes when the fermentation filtrate was diluted 5 times; B is the corrected mortality rate of nematodes when the fermentation filtrate was diluted 10 times; C is the corrected mortality rate of nematodes when the fermentation filtrate was diluted 20 times);

[0043] Fig. 9 The colonization dynamic diagram of the engineering strain BM06-P03 in the leaves, branches and roots of Masson pine for 15d, 30d, 45d and 60d;

[0044] Fig.10 This is a graph showing the control effect of the engineered strain BM06-P03 on pine wood nematode disease in Masson pine seedlings. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0046] The host bacterium Pseudomonas koreensis BM06 used in this application is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20241926, a deposit date of September 5, 2024, and a deposit address of Wuhan University, Bayi Road, Wuchang District, Wuhan City. The host bacterium Pseudomonas koreensis BM06 has a type VI secretion system, which provides favorable conditions for the extracellular secretion of Peptidase 03 (NlpC / P60).

[0047] Example 1

[0048] 1. Construction of vector

[0049] The pET-32a(+) vector used in this example is 5887 bp in length and contains multiple restriction sites such as EcoRI, HindIII, NotI and XhoI. The vector has an Amp resistance gene, which plays a screening role when constructing the plasmid.

[0050] The target gene sequence Peptidase 03 (SEQ ID NO.1) was inserted into the EcoRI and HindIII restriction sites of the vector pET-32a(+), and the target gene sequence was fused with the vector sequence for expression (synthesized by GenScript), to construct the vector pET-32a(+)-P03. The plasmid was extracted using a plasmid extraction kit (Ecoray, Nanjing), and the target gene was PCR-generated and sequenced (Sipgen, Nanjing). The sequence of the vector pET-32a(+)-P03 is shown in SEQ ID NO.2, and the vector structure is shown in Figure 1 shown.

[0051] 2. Protein expression and purification

[0052] Plasmid pET-32a(+)-P03 was transformed into E.coil BL21(DE3) cells, and positive clones were picked to verify the recombinant plasmid by PCR and sequencing (Sipogen, Nanjing).

[0053] The recombinant plasmid glycerol bacteria pET-32a(+)-P03 were inoculated into LB liquid medium (Amp, 50 μg / mL) and shaken at 37°C for 12 h to obtain seed culture solution. The seed culture solution was then inoculated into LB liquid medium (Amp, 50 μg / mL) at a ratio of 1:100 and cultured until OD 600 =0.6, IPTG (0.5mM) was added to induce growth, and the cells were shaken at 200r / min at 28°C and incubated for 8h. Finally, the induced cells were collected by centrifugation, resuspended with phosphate buffered saline (1×PBS) buffer, broken with a cell crusher, centrifuged at 10000r / min at 4°C, and the supernatant was collected. Peptidase03 protein (SEQ ID NO.3) was purified from the supernatant using His-Bind column chromatography (Sangon Biotech) according to the instructions, and the purified product was placed in a dialysis bag containing 0.01M phosphate buffered saline and incubated overnight at 4°C. The protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as the standard.

[0054] SDS-PAGE molecular weight analysis was performed using 10% polyacrylamide gel. Western blot analysis was performed using anti-his-tag primary antibody and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Abmart Shanghai, China). First, the purified protein samples were separated on SDS-PAGE gel and then physically transferred to a nitrocellulose membrane. The membrane was immersed in a 1:5000 dilution of anti-his-tag primary antibody overnight. After washing, the membrane was incubated with a 1:6000 dilution of enzyme-labeled secondary antibody. Then, the signal was observed using an enhanced chemiluminescence (ECL) substrate (BioRAD).

[0055] The results are as follows Figure 2 As shown, both SDS and Western blot results indicated that the purified protein Peptidase 03 was successfully expressed.

[0056] 3. Effect of protein Peptidase 03 on pine wood nematodes

[0057] The effect of pure protein Peptidase 03 on nematodes was tested by immersion method. The specific steps are as follows: add 200 μL pure protein extract to 200 μL worm liquid as the experimental group; add 200 μL BSA to 200 μL worm liquid as the control group. Observe the changes of nematodes after 24 hours of protein treatment. Dilute the pure protein extract of Peptidase 03 to 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL, and determine the EC of Peptidase 03. 50 value.

[0058] The results are as follows Figure 3 As shown, when the purified Peptidase03 (120.00 μg / mL) protein was used to treat nematodes for 24 hours, the mortality rate of nematodes was 100%. Scanning electron microscopy was used to observe the epidermis of nematodes. After treatment with purified Peptidase03, the nematodes were swollen, broken, and the surface of the body was dissolved. The surface of the control CK nematodes was intact and smooth.

[0059] The results are as follows Figure 4 As shown, transmission electron microscopy was used to observe the intestine of nematodes. After treatment with purified Peptidase03, nematodes showed vacuolization, disordered cells, damaged muscle fibers, exudation of contents, and incomplete intestinal structure. The control CK had a dense structure, neatly arranged muscle fibers, and complete intestinal structure.

[0060] The results are as follows Figure 5 As shown in Figure 2, with the increase of Peptidase03 concentration, the mortality rate of pine wood nematodes also increased. 50 The value is 25.73μg / mL. Peptidase03 is highly toxic to pine wood nematodes and is an important factor in killing pine wood nematodes.

[0061] Example 2

[0062] 1. Construction of BM06-P03 engineering strain

[0063] The modified vector pBBR1MCS2-Tac-EGFP used in this example is 5134 bp in length. The original vector is pBBR1MCS2-Tac-EGFP, and the fluorescent tag fragment of the original vector pBBR1MCS2-Tac-EGFP is removed from the modified vector pBBR1MCS2-Tac-EGFP. The modified vector pB BR1MCS2-Tac-EGFP contains restriction sites such as EcoRI, HindIII, SalI, and KpnI. The Kana resistance gene on the vector plays a screening role when constructing the plasmid.

[0064] Primers oprL-F: 5'-ACTACAACGCCAACCCGAAGCTGTC-3' and oprL-R: 5'-TGAAACTCCTAATGAACCCCAGTGT-3' were designed with reference to the oprL gene sequence of Pseudomonas putida KT2440 strain (GenBank AAN66847). Genomic DNA of Korean Pseudomonas was extracted and the oprL gene promoter (SEQ ID NO. 4) was obtained by PCR.

[0065] The oprL promoter, SD sequence (SEQ ID NO.5) and Peptidase03 gene sequence were inserted into the EcoRI and HindIII restriction sites of the modified vector pBBR1MCS2-Tac-EGFP, named pBBR1MCS2-P03, and synthesized by GenScript. The plasmid was extracted using a plasmid extraction kit (Ecoray, Nanjing), and the target gene was PCR-confirmed and sequenced (Sipgen, Nanjing). The sequence of the vector pBBR1MCS2-P03 is shown in SEQ ID NO.6, and the vector structure is shown in Figure 6 shown.

[0066] The plasmid pBBR1MCS2-P03 was transformed into DH5α, and the positive clones were picked for verification. The pBBR1MCS2-P03 plasmid was extracted using a plasmid extraction kit (Acroray, Nanjing) and transformed into the recipient bacterium Pseudomonas koreanus BM06 to obtain the recombinant engineered strain BM06-P03.

[0067] 2. Protein expression and purification

[0068] The glycerol bacteria BM06-P03 was inoculated into LB liquid medium (kana, 50 μg / mL) and shaken at 30°C for 12 h to obtain a seed culture solution. The seed culture solution was then inoculated into LB liquid medium (Amp, 50 μg / mL) at a ratio of 1:100 and cultured until OD 600=0.6, IPTG (0.5mM) was added to induce growth, and the cells were shaken at 200r / min at 28°C and incubated for 8h. Finally, the induced cells were collected by centrifugation, resuspended with phosphate buffered saline (1×PBS) buffer, broken with a cell crusher, centrifuged at 10000r / min at 4°C, and the supernatant was collected. Peptidase03 protein was purified from the supernatant using His-Bind column chromatography (Sangon Biotech) according to the instructions. The protein concentration was determined by Bradford method using bovine serum albumin (BSA) as the standard.

[0069] SDS-PAGE molecular weight analysis was performed using 10% polyacrylamide gel. Western blot analysis was performed using anti-his-tag primary antibody and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Abmart Shanghai, China). First, the purified protein samples were separated on SDS-PAGE gel and then physically transferred to a nitrocellulose membrane. The membrane was immersed in a 1:5000 dilution of anti-his-tag primary antibody overnight. After washing, the membrane was incubated with a 1:6000 dilution of enzyme-labeled secondary antibody. Then, the signal was observed using an enhanced chemiluminescence (ECL) substrate (BioRAD).

[0070] The results are as follows Figure 7 As shown, SDS and Western blot analysis detected that Peptidase03 was correctly secreted and expressed in Pseudomonas koreensis BM06.

[0071] 3. Nematicidal activity of engineered strains against pine wood nematodes

[0072] 1) Cultivation of pine wood nematodes

[0073] The test nematode AMA3 was inoculated into a PDA medium plate full of Botrytis cinerea and placed in a constant temperature incubator at 25°C until the Botrytis cinerea hyphae were completely eaten. The nematodes were collected using the Berman funnel method, and the collected nematodes were washed 3 times with sterile water to prepare a nematode suspension with a concentration of 3000 nematodes / mL for later use.

[0074] 2) Preparation of bacterial fermentation filtrate

[0075] The wild-type strain Pseudomonas koreensis BM06 and the engineered strain BM06-P03 were taken out at -80℃, activated on LB (antibiotic-free) plates and LB (containing knna) plates, respectively, and cultured at 30℃ overnight. Single clones were picked with an inoculation loop and inoculated into 100mL triangular flasks containing 20mL NB liquid culture medium and 20mL NB liquid culture medium containing kanna antibiotic (50μg / mL), respectively, and cultured at 30℃ and 200r / min for 12h as the original bacterial liquid. The original bacterial liquid was inoculated into a 500mL triangular flask containing 200mL NB liquid culture medium at a concentration of 1:100, and cultured at 30℃ and 200r / min for 12h, 24h, 36h, and 48h, 10000r / min, 10min, centrifuged, and the supernatant was filtered with a 0.22um filter membrane. The fermentation filtrate was diluted 5 times, 10 times, and 20 times with sterile water for standby use.

[0076] 3) Nematicidal activity of engineered strains against pine wood nematodes

[0077] Take 100 μL of fermentation filtrate of wild-type strain Korean Pseudomonas skoreensis BM06 and engineered strain BM06-P03 cultured for different time and diluted at different times, add them to 1.5mL centrifuge tubes, then add 100 μL of nematode suspension (about 500 nematodes) and mix them. After 24 hours of nematode treatment, take 20 μL of the mixed solution (about 50 nematodes) and observe the number of dead pine wood nematodes under a microscope (nematode body stiff, "J" or "C" shaped, body surface dull, considered dead, count the total number and death of pine wood nematodes, calculate the mortality rate and corrected mortality rate of pine wood nematodes, with sterile NB culture medium as control, and each treatment is repeated 3 times. Determine the nematicidal activity of the fermentation filtrate of the strain at different culture times and different dilution times at 24 hours.

[0078] Mortality rate (%) = number of nematodes killed / total number of nematodes × 100,

[0079] Corrected mortality rate (%) = (treatment mortality rate - blank control mortality rate) / (100 - blank control mortality rate) × 100.

[0080] The results are as follows Figure 8As shown, when the fermentation filtrate was uniformly diluted 5 times, the nematicidal activity of the fermentation filtrates of BM06 and BM06-P03 strains at 12h, 24h, 36h and 48h was significantly different (P < 0.05). When the fermentation filtrate was uniformly diluted 10 times, the corrected mortality of pine wood nematodes when the fermentation filtrates of BM06 and BM06-P03 strains were cultured for 24h, 36h, and 48h was significantly different (P < 0.05). When the fermentation filtrate was uniformly diluted 20 times, the fermentation filtrates of BM06 and BM06-P03 strains were cultured for 36h and had significant differences (P < 0.05).

[0081] In summary, the insertion of exogenous insecticidal genes enhanced the nematicidal effect of the wild-type strain, which has a higher application potential and provides an effective strategy for the prevention and control of pine wood nematode disease.

[0082] 4. Colonization of engineered strains in Masson pine seedlings

[0083] The engineered strain BM06-P03 was inoculated into LB medium (kanamycin 50 μg / mL) and shaken at 30°C for 12 hours to obtain a seed culture solution. The seed culture solution was then inoculated into LB liquid medium (kanamycin 50 μg / mL) at a ratio of 1:100, 30°C, 200 rpm, 24 hours, centrifuged at 10000 r / min for 10 minutes, and suspended in sterile water to generate a concentration of about 10 8 cfu / mL of inoculant. 20mL of inoculant was sprayed on Pinus massoniana and placed in a greenhouse. Subsequently, leaves, stems, and roots of pine seedlings in the treatment and control groups were collected at 0d, 15d, 30d, 45d, 60d, and 90d after inoculation and stored at -80℃. The total genomic DNA of each sample was extracted using the Plant Genome Kit, and the concentration and quality of the extracted genomic DNA were determined using Nanodrop. The DNA of each sample was diluted to a concentration of less than 100ng / μL and stored at -20℃.

[0084] The copy number of the specific target gene of the engineered strain BM06-P03 was determined by absolute quantification. First, the whole genome of nine Pseudomonas (P.koreensis BM06 CP155621, P.abietaniphila BHJ04CP155619, P.abietaniphila ATCC 700689, P.abietaniphila KF701, P.abietaniphilaKF717, P.aerμginosa NC_002516.2, P.fluorescens NZ_LT907842.1, P.kribbensis NZ_CP029608.1, P.putida NC_021505.1) was analyzed for colinearity using Mauve software, and the BM06 specific gene was selected for comparison on NCBI to ensure its specificity. Bacterial DNA was extracted by freeze-thaw method. The target gene PCR amplification product was collected and tested at a concentration of 80 ng / μL. Then, the 10-fold diluted product was used as a template for fluorescence quantitative PCR amplification. 2 , slope and amplification efficiency (E) were used to test the effectiveness of the established fluorescence quantitative PCR method.

[0085] Real-time fluorescence quantitative PCR was performed using the extracted genomic total DNA as a template. Four technical replicates were established for each sample, and the amplification reaction was performed using two-step amplification.

[0086] The reaction conditions were 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, for 40 cycles.

[0087] The conditions for melt curve analysis were 95°C for 15 s; 60°C for 60 s; and 95°C for 30 s.

[0088] The results are as follows Fig. 9 As shown in Figure 2, the number of colonization of the engineered strain BM06-P03 in the branches of Pinus massoniana was the highest, with the colonization numbers being 4.82×10 at 0d, 15d, 30d, 45d, and 60d, respectively. 2 fu / g、5.88×10 4 fu / g、3.49×10 4 fu / g, 1.02×10 4 fu / g、1.67×10 3 The number of engineered strain BM06-P03 colonized in the roots, stems and leaves of Masson pine for 15 days, reaching the highest level.

[0089] 5. Outdoor biocontrol experiments with engineered strains

[0090] 1) Prepare the engineering strain suspension (1×10 8 cells / mL)

[0091] The engineered strain BM06-P03 was inoculated into LB medium (kana, 50 μg / mL) and shaken at 30°C for 12 h. 1:100 seed culture was added to inoculate LB broth (kana, 50 μg / mL) and incubated at 30°C, 200 rpm, for 24 h. Centrifuged at 4°C, 10000 rpm for 10 min. The supernatant was discarded and the precipitate was suspended with sterile water to obtain a concentration of about 10 8 cfu / mL of inoculum.

[0092] 2) Inoculation of two-year-old Masson pine seedlings with engineered strains

[0093] The engineered strain suspension was inoculated into two-year-old Masson pine seedlings by spraying, with 15-20 mL inoculated per Masson pine plant. Three days later, 3,000 pine wood nematodes AMA3 were injected into the middle of the stem of the Masson pine seedlings. The following treatment groups were set up:

[0094] ① No strain + no nematodes; ② Inoculation with sterile water + inoculation with nematodes; ③ Inoculation with wild-type strain BM06 + nematodes; ④ Inoculation with engineered strain BM06-P03 + nematodes.

[0095] The disease symptoms of the inoculated host were observed every 7 days, and the disease rate and disease index were calculated. The disease index was calculated according to the disease classification standard. The severity of the disease of Masson pine is divided into 5 levels, Level I: the plant needles are green and growing healthily, with a representative value of 0; Level II: 0-25% of the needles turn green and yellow, with a representative value of 1; Level III: 25%-50% of the needles turn green and yellow, and the branches are bent, with a representative value of 2; Level IV: 50%-70% of the needles turn green and yellow, and the branches droop, with a representative value of 3; Level V: 75%-100% of the needles turn green and brown, and the plants wither and die, with a representative value of 4.

[0096] The results are as follows Fig.10 As shown, 14 days after inoculation with the AMA3 insect strain, the wild-type strain and the engineered strain had the same disease rate, with 2 diseased strains and a grade of 1; all the controls became ill; 30 days after inoculation, all the wild-type strains died, and the number of dead engineered strains was 1.

[0097] In summary, the engineered strain BM06-P03 slowed down the disease rate of Masson pine and played a role in the prevention and control of pine wood nematodes.

[0098] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method for constructing an engineered strain for controlling pine wood nematodes, characterized in that: The oprL promoter, SD sequence and Peptidase03 gene sequence were constructed into the transformation vector pBBR1MCS2-Tac-EGFP to obtain the recombinant plasmid pBBR1MCS2-P03; the plasmid pBBR1MCS2-P03 was transformed into the recipient bacterium Pseudomonas koreensis BM06 to obtain the engineered bacterium BM06-P03; The Peptidase03 gene sequence is shown in SEQ ID NO.1; The sequence of the oprL promoter is shown in SEQ ID NO.4; The SD sequence is shown as SEQ ID NO.

5.

2. The construction method according to claim 1, characterized in that: The amplification primers for the oprL promoter are as follows: oprL-F: 5'-ACTACAACGCCAACCCGAAGCTGTC-3', oprL-R: 5'-TGAAACTCCTAATGAACCCCAGTGT-3'.

3. The construction method according to claim 1, characterized in that: The specific steps are: 1) Primers oprL-F: 5'-ACTACAACGCCAACCCGAAGCTGTC-3', oprL-R: 5'-TGAAACTCCTAATGAACCCCAGTGT-3' were designed with reference to the oprL gene sequence of Pseudomonas putida KT2440 strain GenBank AAN66847, genomic DNA of Korean Pseudomonas was extracted, and the oprL gene promoter was obtained by PCR; The sequence of the oprL promoter is shown in SEQ ID NO.4; 2) Insert the oprL promoter, SD sequence and Peptidase03 gene sequence into the EcoRI and HindIII restriction sites of the transformation vector pBBR1MCS2-Tac-EGFP to obtain the vector pBBR1MCS2-P03, and extract the plasmid pBBR1MCS2-P03; The SD sequence is shown in SEQ ID NO.5; The Peptidase03 gene sequence is shown in SEQ ID NO.1; 3) The plasmid pBBR1MCS2-P03 was transformed into DH5α, and the positive clones were picked and the pBBR1MCS2-P03 plasmid was extracted again and transformed into the recipient bacterium Pseudomonas koreensis BM06 to obtain the recombinant engineering strain BM06-P03.

4. The recombinant engineered strain BM06-P03 for controlling pine wood nematodes constructed by the method according to any one of claims 1 to 3.

5. Use of the recombinant engineered strain BM06-P03 according to claim 4 in controlling pine wood nematodes.

6. The use according to claim 5, characterized in that: The method for controlling pine wood nematodes is to slow down the incidence of Masson pine.

7. Use of the fermentation filtrate of the recombinant engineered strain BM06-P03 according to claim 4 in killing pine wood nematodes.

8. The use according to claim 7, characterized in that: The method for preparing the fermentation filtrate is as follows: taking the recombinant engineering strain BM06-P03, activating it on an LB plate containing knna, and culturing it at 30°C overnight; picking a single clone with an inoculation loop, inoculating it into 20mL NB liquid culture medium containing 50μg / mL kanna antibiotic, and culturing it at 30°C and 200r / min for 12h, so as to prepare the fermentation filtrate of the recombinant engineering strain BM06-P03.

9. The protein expressed by the recombinant engineered strain BM06-P03 according to claim 4, whose amino acid sequence is shown in SEQ ID NO.

3.

10. Use of the protein according to claim 9 in preventing and treating pine wood nematode disease.

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