An engineered bacterium of Serratia marcescens with stable inheritance of exogenous genes and its application

Abstract

The present invention specifically relates to an engineered bacterium of Serratia marcescens with stable genetic exogenous genes, BRC-CXG2-pCHSW, and its application, belonging to the field of microbial technology. The strain is classified and named as Serratia marcescens Serratia marcescens , deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee, with the deposit number CGMCC NO.23125 and the deposit date August 6, 2021. The Serratia marcescens engineered bacteria can stably nptII-cry3AaT-hasA The exogenous gene can secrete and express Cry3AaT protein. The engineered Serratia marcescens bacteria can be used in the prevention and treatment of coleopteran pests and their offspring, and has high insecticidal activity.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an engineering bacterium BRC-CXG2-pCHSW of Serratia marcescens with stable genetic exogenous genes and an application thereof. Background Art

[0002] Monochamus alternatus, Anoplophora sutchuenensis, Anoplophora glabripennis, Anoplophora glabripennis, Red palm weevils, Tribolium castaneum, etc. are important Coleoptera pests that cause serious damage to many types of agricultural and forestry plants in my country. The study and establishment of a long-lasting and efficient biological control method for the above pests has important ecological and economic significance. It has been found that the biological insecticide toxin Cry3AaT protein has insecticidal activity against a variety of Coleoptera pests, but there are still some important problems in its application in production practice. Its insecticidal mechanism is mainly that after the insects eat the toxin, the toxin binds to the surface receptors of the insect intestinal epithelial cells to form membrane perforations, thereby causing the death of the insects; while the boring pests such as Monochamus alternatus, Anoplophora sutchuenensis, Anoplophora glabripennis, and Red palm weevils are mainly hidden in the trunks, resulting in the toxin being unable to directly enter the pest intestine, thereby affecting the application of the toxin. Therefore, screening and obtaining symbiotic bacteria between the plant and the pest intestine as toxin carriers, and constructing engineered bacteria that can express and secrete toxins through genetic engineering is an ideal way to solve this problem.

[0003] Serratia marcescens is a symbiotic bacterium commonly found in the intestines of coleopteran insects and plant tissues. The bacterium can be enriched in the intestines of coleopteran insects and play a role in degrading and digesting lignin, and it itself is not pathogenic to insects. At the same time, the bacterium can exist in plants for a long time, is widely distributed in the soil, and can persist in the natural environment for a long time. Combining the characteristics of the strain's enrichment in the intestines of coleopteran insects with the insecticidal activity of Cry3AaT toxin, constructing an engineered strain, and then enriching the insecticidal toxin protein in the intestines of pests will help greatly improve the application efficiency of the toxin. At the same time, due to the problems of easy loss of plasmids and low genetic stability in traditional engineered bacteria, and the insecticidal toxin protein can only be expressed intracellularly, the protein cannot be secreted outside the cell. Therefore, it is of great practical significance to construct a Serratia engineered bacterium that can stably inherit exogenous genes and can secrete toxin proteins exogenously. Summary of the invention

[0004] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide an engineered Serratia marcescens bacteria with stable inheritance of exogenous genes and its application.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A Serratia marcescens engineered bacterium BRC-CXG2-pCHSW with stable genetic exogenous genes, the strain is classified and named Serratia marcescensSerratia marcescens It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 6, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.23125.

[0007] Furthermore, the above-mentioned Serratia marcescens engineered bacteria BRC-CXG2-pCHSW can stably nptII- cry3AaT-hasA The exogenous gene can secrete and express Cry3AaT protein.

[0008] A method for constructing the above-mentioned Serratia marcescens engineered bacteria is to transfer the gene carrying the insecticide toxin protein exocrine expression by conjugation transfer technology. nptII-cry3AaT-hasA The pCHSW plasmid was transferred into the Serratia marcescens strain BRC-CXG2, and the foreign gene was transferred into nptII-cry3AaT-hasA The pCHSW was inserted into the genome of Serratia marcescens BRC-CXG2, thereby obtaining the Serratia marcescens engineered bacterium BRC-CXG2-pCHSW.

[0009] Wherein, the nucleotide sequence of the pCHSW plasmid is shown as SEQ ID NO.1.

[0010] The application of the above-mentioned engineered Serratia marcescens bacteria BRC-CXG2-pCHSW in controlling coleopteran pests.

[0011] The above-mentioned engineered Serratia marcescens bacterium BRC-CXG2-pCHSW is used in controlling the offspring of coleopteran pests.

[0012] Among them, the above-mentioned Coleopteran pests are one or more of the pine sawyer, starry-shaped longhorn beetle, clouded longhorn beetle, glabripennis longhorn beetle, red palm weevil, and red flour beetle.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] The engineered Serratia marcescens bacteria constructed by the present invention selects the plant and coleopteran intestinal symbiotic strain Serratia marcescens BRC-CXG2 as a toxin carrier, and uses conjugation transfer technology and transposon to transfer the gene capable of secreting insecticidal toxin protein. nptII-cry3AaT-has A was inserted into the genome of Serratia marcescens BRC-CXG2, making the foreign gene nptII- cry3AaT-hasA can be stably inherited. At the same time, by utilizing the characteristic that Serratia marcescens BRC-CXG2 can be enriched in the intestine of the pest, combined with the HasA heme transport system, the insecticidal toxin is effectively secreted from the bacterial body to the outside of the bacterial body, thereby enriching the insecticidal toxin and directly acting on the intestine, thereby achieving the purpose of killing coleopteran pests. The Serratia marcescens engineered bacteria constructed by the present invention can persist in the plant body and the environment for a long time, can transport toxins to the trunk and enrich in the intestine of the pest, and has a certain poisoning effect on the offspring of the pest, can play a good control role on borer pests, and provides an important solution to the problem of the difficulty of controlling borer pests in production practice, and has significant technical progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the pCHSW plasmid map.

[0016] Figure 2 The figure is the electrophoresis diagram of the genomic DNA and PCR product of the engineered bacteria BRC-CXG2-pCHSW of Serratia marcescens; wherein, lane M is the DNA marker, lane 1 is the genomic DNA of the engineered bacteria BRC-CXG2-pCHSW of Serratia marcescens, and lane 2 is the PCR product.

[0017] Figure 3 SDS-PAGE and Western blotting were used to analyze the expression of Cry3AaT; among them, lane M is a protein marker, lane 1 is the extracellular protein of BRC-CXG2-pCHSW strain (SDS-PAGE), lane 2 is the intracellular protein of BRC-CXG2-pCHSW strain (SDS-PAGE), lane 3 is the extracellular protein of BRC-CXG2-pCHSW strain (Western blotting), and lane 4 is the intracellular protein of BRC-CXG2-pCHSW strain (Western blotting).

[0018] Figure 4 The toxic effect of the engineered bacteria BRC-CXG2-pCHSW of Serratia marcescens on the offspring of Monochamus alternatus.

[0019] Figure 5 The results of colonization and passage of Serratia marcescens in Monochamus alternatus.

[0020] Figure 6 The results of colonization and migration of Serratia marcescens in pine trees. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The materials and reagents used in the following examples are as follows:

[0023] The pine beetle, star beetle, clouded beetle, star beetle, red palm weevil, red flour beetle larvae, and Serratia marcescens BRC-CXG2 involved in the present invention are all provided by the Key Laboratory of Major Pest Control in Ecological Public Welfare Forests of Fujian Province, Fujian Agriculture and Forestry University. E. coli S17-1λpir was purchased from Biovector NTCC Plasmid Vector Bacteria Cell Gene Collection Center. The pCHSW plasmid involved in the present invention was synthesized by GenScript Biotech Co., Ltd. Other conventional reagents can be obtained from commercial sources.

[0024] Embodiment 1:

[0025] A method for constructing an engineered Serratia marcescens bacteria capable of stably inheriting exogenous genes, the specific steps are as follows:

[0026] (1) Build and carry nptII-cry3AaT-has A Donor strain for expression element

[0027] The present invention uses the pBAM1 plasmid (GenBank: HQ908071) as a reference. The pBAM1 plasmid is a suicide plasmid that can only grow in specific strains. The plasmid components include: Kanna resistance gene Kan ; Transposable gene tnp ; Ampicillin resistance gene promoter AmpR promoter ; Ampicillin resistance gene AmpR ; Conjugative transfer gene oriT ; Replication origin R6K ; Mosaic ME-O and ME-I Since the test strain of the present invention, Serratia marcescens BRC-CXG2, is resistant to ampicillin (Amp), it is not possible to select AmpR gene as a resistance gene. However, the test strain BRC-CXG2 has no resistance to spectinomycin (Spec). Therefore, the present invention replaces the pBAM1 plasmid with AmpR Gene replacement with spectinomycin resistance gene ( S m ) for subsequent donor strain screening and Kan Gene upstream insertion by nptII Promoter, cry3AaT Genes and hasA Expression elements composed of sequential gene splicing—— nptII-cry3AaT-hasAGene, thereby constructing the pCHSW plasmid (see the plasmid map for details) Figure 1 ), and commissioned GenScript Biotech Co., Ltd. to synthesize the pCHSW plasmid, the nucleotide sequence of which is shown in SEQ ID NO.1. nptII Promoter and hasA The gene comes from pGHKW4 plasmid (Genbank: MK562404). cry3AaT The nucleotide sequence of the gene is identical to the nucleotide sequence of the gene encoding the BRC1912 toxin protein in patent CN111647058A [Bt toxin highly toxic to Monochamus alternatus and other Monochamus alternatus insects].

[0028] Heat shock transformation was used to transform the pCHSW plasmid into E. coli S17-1λpir (transformation method refers to the literature: Guo Peiyi, Chen Xiangdong, Xie Zhixiong, et al. A simple and rapid Escherichia coli plasmid transformation method [J]. Heredity, 1999, 21(4): 4.), and the transformation product was obtained. The transformation product was spread on LB solid medium containing Spec antibiotics (final concentration of 100 μg / mL) and cultured in a 37 ℃ constant temperature incubator for 12 h. The positive transformants on the plate were picked and inoculated into LB liquid medium containing Spec antibiotics (final concentration of 100 μg / mL), and cultured at 150 r / min and 37 ℃ for 12 h. Subsequently, the plasmid was extracted according to the instructions of the plasmid extraction kit Plasmid Mini Protocol I of OMEGA, and the selection Bm I and Sac I restriction endonuclease double digestion, and finally sequencing verification, to obtain the pCHSW plasmid E. coli S17-1λpir strain, carrying the pCHSW plasmid E. coli The S17-1λpir strain was used as the donor strain in the next conjugative transfer experiment.

[0029] (2) Co-cultivation to obtain the engineered strain of Serratia marcescens BRC-CXG2-pCHSW

[0030] The donor bacteria carrying the pCHSW plasmid E. coli S17-1λpir and the recipient bacterium Serratia marcescens BRC-CXG2 were cultured to the logarithmic growth phase and 10 mM MgSO 4 Wash the cells with the solution and resuspend them to OD 600The volume ratio of the donor bacterial suspension and the recipient bacterial suspension was 0.1, and then the donor bacterial suspension and the recipient bacterial suspension were mixed at a volume ratio of 1:1, and spread on an LB solid medium plate without any antibiotics, and cultured at 37°C for 5 hours. The bacterial lawn on the plate was scraped and cultured at 30°C for 12 hours in an LB liquid medium containing Amp antibiotics (final concentration of 200 μg / ml), and then streaked on an LB solid medium plate containing Kan antibiotics (final concentration of 200 μg / ml) and cultured at 30°C for 12 hours, and a single colony was picked to obtain the engineered Serratia marcescens.

[0031] (3) DNA extraction and detection of engineered strains

[0032] The single colony of the engineered Serratia marcescens obtained above was inoculated into LB liquid culture medium containing Kan antibiotics and Amp antibiotics (the final concentration of Kan and Amp antibiotics were 100 μg / mL) and cultured at 150 r / min and 37°C for 12 h. Subsequently, the engineered bacteria DNA was extracted according to the Bacterial DNA Kit D3350 of OMEGA Company, and finally PCR verification was performed using the extracted DNA as a template. PCR verification nptII-cry3AaT-hasA The primers required for the gene sequence were 5′-CGAGCTCgtcaggctgttacagctcagagaaa-3′ (SEQ ID NO.2), 3′-GCggatccGGATACGGCACCGGCGCAC-5′ (SEQ ID NO.3). The total PCR reaction system was 25 μL: 2 μL template DNA, 1.25 μL each of forward and reverse primers (10 mM), 12.5 μL of Q5® Hot Start Ultra-Fidelity Enzyme (NEB) 2× Master Mix, and ddH 2 O 8 μL. PCR amplification conditions were: 98℃ 5 min; 98℃ 1 min, 66.1℃ 30 s, 72℃ 2 min, 30 cycles; 72℃ 10 min. The electrophoresis results were as follows Figure 2 As shown, a 2000 bp target band can be observed, indicating that the exogenous gene nptII-cry3AaT-hasA The gene was successfully inserted into the genome of Serratia marcescens BRC-CXG2, and the engineered Serratia marcescens strain was successfully constructed, which was named BRC-CXG2-pCHSW.

[0033] Example 2

[0034] Take the BRC-CXG2-pCHSW strain (bacterial solution OD 600The value was 0.6) and the inoculation volume was transferred to LB liquid medium without antibiotics at a volume of 1% (v / v), and the culture was continuously shaken at 37℃ and 150 r / min, and the tube was transferred every 12h. The bacterial liquid after the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, 19th, and 21st transfers was diluted and spread on LB solid medium without antibiotics, and cultured at 30℃ for 24 h. 100 single colonies were picked and inoculated in LB solid medium containing Kan antibiotics (final concentration of 100 μg / ml) and LB solid medium without antibiotics, and the number of colonies was counted after culturing for 12 h. Then the gene carrying rate was calculated by dividing the number of colonies in LB solid medium containing Kan antibiotics (final concentration of 100 μg / ml) by the number of colonies in LB solid medium without antibiotics, so as to detect the genetic stability of the recombinant plasmid under non-selective pressure, and the BRC-CXG2 strain treated with the same conditions was used as a negative control.

[0035] The results are shown in the following table. Under the condition of no antibiotic selection pressure, the gene carrying rate of BRC-CXG2-pCHSW strains of 148 generations was 100%, indicating that the exogenous gene nptII-cry3AaT-hasA The invention can stably inherit in the host strain and solve the problem of genetic instability existing in traditional transgenic engineering bacteria.

[0036] Table 1 Genetic stability test of strains

[0037]

[0038] The above-mentioned recombinant Serratia marcescens engineered bacteria BRC-CXG2-pCHSW was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 6, 2021, with the deposit number CGMCC NO.23125.

[0039] Example 3

[0040] The steps for extraction and verification of the exocrine insecticidal toxin protein of the engineered strain BRC-CXG2-pCHSW of Serratia marcescens are as follows:

[0041] (1) Pipette 30 μL of the engineered Serratia marcescens BRC-CXG2-pCHSW bacterial solution (OD 600 The bacterial suspension was inoculated into 3 mL of LB liquid medium containing Kan antibiotic (final concentration was 100 μg / mL) at 150 r / min at 30°C for 12 h, and then transferred into 3 mL of new LB liquid medium containing Kan antibiotic (final concentration was 100 μg / mL) at a 1 vol% inoculum, and then cultured at 30°C at 150 r / min for 24 h.

[0042] (2) Centrifuge the cultured bacterial solution in step (1) at 4°C and 8000 r / min for 10 min, collect the supernatant and slowly add it to an ice-pretreated ultrafiltration tube (Amicon Ultra-4 centrifugal filter devices), and centrifuge it at 4°C and 5000 r / min for 20 min. Repeat this operation until all the supernatant is concentrated.

[0043] (3) After the centrifugation in step (2) is completed, the protein solution in the ultrafiltration tube is gently mixed with a pipette and collected into a centrifuge tube. This is the extracellular protein and is stored at -20°C for later use.

[0044] (4) Resuspend the cells centrifuged in step (2) above in PBS buffer (1.0 M, pH = 7.3), centrifuge at 4°C and 8000 r / min for 8 min, and discard the supernatant. Repeat this step 3 times.

[0045] (5) The precipitate obtained in step (4) was placed on ice for ultrasonic disruption (50 Hz) at a frequency of 10 s and an interval of 5 s. The disruption time for each tube of bacteria was approximately 30 min. The disrupted cell solution was centrifuged at 4°C and 8000 r / min for 8 min. The supernatant obtained was the intracellular protein and was stored at -20°C for later use.

[0046] (6) Prepare SDS-PAGE gel according to the method of molecular cloning experiment guide, and prepare samples of extracellular protein and intracellular protein obtained in steps (3) and (5) for SDS-PAGE electrophoresis. Place the SDS-PAGE gel in R250 Coomassie brilliant blue staining solution and decolorizing solution for slow shaking for staining and decolorization, and finally analyze the SDS-PAGE situation using a two-dimensional electrophoresis scanning imager. At the same time, perform Western blotting analysis, first perform SDS-PAGE, then place the SDS-PAGE gel at 200 mA for 1 h to transfer the protein to the NC membrane, and then perform conventional 5wt% skim milk powder blocking, primary antibody incubation and secondary antibody incubation operations, wherein the primary antibody solution is Cry3Aa-Tag Rabbite PolycionalAntibody diluted 1:4000, and the secondary antibody solution is goat anti-rabbit IgG diluted 1:5000 by volume. Finally, a multifunctional imaging system (FluorChem Q, ProteinSimple) was used to detect the fluorescent bands on the NC membrane and analyze the expression of Cry3AaT protein.

[0047] The results are as follows Figure 3As shown: SDS-PAGE results show that the engineered Serratia marcescens BRC-CXG2-pCHSW can secrete proteins both intracellularly and extracellularly; Western blotting results show that the engineered Serratia marcescens BRC-CXG2-pCHSW has secretory expression of insecticidal toxin protein Cry3AaT both intracellularly and extracellularly. The above results show that the HasA heme transport system can function in the engineered Serratia marcescens BRC-CXG2-pCHSW and can successfully perform exocrine expression of the target protein.

[0048] Example 4

[0049] The toxicity of the engineered strain of Serratia marcescens BRC-CXG2-pCHSW to Monochamus alternatus adults and subcultures is determined as follows:

[0050] Thirty newly emerged female Monochamus alternatus adults and 30 male adults were raised in plastic fresh-keeping boxes of 19.7 m*13.4 m*7.3 m, and placed in a container evenly sprayed with OD 600 6. Thirty newly emerged female adults and 30 male adults of Monochamus alternatus were raised in plastic fresh-keeping boxes of 19.7m*13.4m*7.3m, and then placed in a uniformly sprayed OD 600 The pine branches were inoculated with the bacterial liquid of Serratia marcescens (BRC-CXG2) with a value of 0.6. 30 female adults and 30 male adults fed with the pine branches sprayed with sterile water were used as the blank control (CK). After culturing in the fresh-keeping box at 28°C, relative humidity above 70%, and a photoperiod of 8 h / 16 h (day / night) for 10 days, male and female pine beetles were placed in pairs in a fresh-keeping box of 22.4m*15.4m*8.2m for mating, and fed with branches sprayed with sterile water. The number of deaths of pine beetle adults was observed and counted every day, and LC was performed after 10 days of observation. 50 Calculation. After feeding for 3 days, place a tree of equal size in each fresh-keeping box for the female adults of Monochamus alternatus to lay eggs. Take out the tree after 6 days and place it in an environment with a temperature of 28°C and a relative humidity of more than 70% for 10 days. Cut the bark of the tree and count the number of surviving offspring in the tree.

[0051] The results are as follows: Figure 4As shown, the toxicity of the modified engineered bacteria is significantly improved compared with the wild-type Serratia marcescens, and the mortality rate of the adult Monochamus alternatus fed with the engineered bacteria is significantly increased, indicating that the engineered bacteria has a high insecticidal activity against the adult Monochamus alternatus. There is no significant difference in the number of offspring survival between the offspring fed with Serratia marcescens (BRC-CXG2) and sterile water (CK), while the number of offspring survival of the offspring fed with the engineered Serratia marcescens (BRC-CXG2-pCHSW) is significantly lower than that of the offspring fed with Serratia marcescens (BRC-CXG2) and sterile water (CK). This shows that the engineered Serratia marcescens BRC-CXG2-pCHSW constructed by the present invention has a toxic effect on the offspring of Monochamus alternatus.

[0052] Table 2 Insecticidal activity of BRC-CXG2-pCHSW strain against adults of Monochamus alternatus

[0053]

[0054] Example 5

[0055] The toxicity of the engineered strain of Serratia marcescens BRC-CXG2-pCHSW to larvae of the family Cerambycidae (Monochromis alternatus, Anoplophora spheniscus, Anoplophora glabripennis), the steps are as follows:

[0056] The second instar Cerambycidae larvae were placed in a 1.5 mL centrifuge tube containing sterilized, dried and single-distilled water-soaked paper towels. They were initially raised for 3 days at a temperature of 28°C, a relative humidity of more than 70%, and a light cycle of 8 h / 16 h (day / night) to ensure the vitality of the larvae. After 3 days, the Cerambycidae larvae with good vitality were transferred to a new 1.5 mL centrifuge tube sterilized by high pressure. 100 mL of the bacterial solution of Serratia marcescens BRC-CXG2 and the engineered bacteria of Serratia marcescens BRC-CXG2-pCHSW, which had been activated and cultured for 24 hours, were centrifuged at 4°C and 8000 r / min for 8 minutes, and the supernatant was removed. After resuspending with sterile water, the supernatant was removed after centrifugation at 4°C and 8000 r / min for 8 minutes. This step was repeated 3 times, and then 5 mL of sterile water was added to resuspend the suspension to obtain the bacterial suspension, which was then spread and counted. According to the concentration of the bacterial suspension, 10 were diluted respectively. 2 times, 10 4 times, 10 6 Each gram of paper was fed with 100 μL of a concentration gradient of bacterial suspension for feeding Cerambycidae larvae. Each concentration gradient was repeated 15 times. The same amount of sterile water was used as a control. The number of deaths of Monochamus alternatus, Anoplophora spheniscus, Anoplophora spheniscus, and Anoplophora glabripennis was observed and counted every day. After 10 days of observation, LC was performed. 50 calculate.

[0057] The results are shown in Table 3. The Serratia marcescens BRC-CXG2 had no insecticidal activity against Monochamus alternatus, Anoplophora sutchuenensis, Anoplophora glabripennis, and Anoplophora glabripennis. In comparison, the LC of the engineered Serratia marcescens BRC-CXG2-pCHSW 50 All of them decreased exponentially, indicating that the virulence of the constructed Serratia engineering bacteria BRC-CXG2-pCHSW was greatly improved compared with that of Serratia marcescens BRC-CXG2, and also indicating that the Serratia engineering bacteria BRC-CXG2-pCHSW had extremely high insecticidal activity against the larvae of Cerambycidae.

[0058] Table 3 Insecticidal activity of BRC-CXG2-pCHSW strain against Monochamus alternatus larvae

[0059]

[0060] Example 6

[0061] The toxicity test of the engineered Serratia strain BRC-CXG2-pCHSW against red palm weevils was carried out as follows:

[0062] The artificial diet of red palm weevils was added to a 24-well plate, and a second-instar red palm weevil larva was placed in each well, and the plate was placed in an artificial climate chamber for culture. Culture conditions: temperature of 28°C, relative humidity of more than 70%, and photoperiod of 8 h / 16 h (day / night). The larvae were initially fed for 3 days to ensure the vitality of the larvae. After 3 days, the red palm weevil larvae with better vitality were transferred to a new 24-well plate containing artificial diet for red palm weevils, and then the bacterial liquid was activated and cultured for 24 hours (Serratia BRC-CXG2 and Serratia engineered bacteria BRC-CXG2-pCHSW), and the bacterial suspension was prepared and diluted according to the method described in Example 5, and then added to the 24-well plate to feed the red palm weevils. 15 replicates were prepared for each concentration gradient, and a blank control was used for sterile water feeding. The number of deaths of red palm weevil larvae was observed and counted every day, and LC was performed after 10 days of observation. 50 calculate.

[0063] The results are shown in Table 4. The LC of the engineered Serratia marcescens strain BRC-CXG2-pCHSW 50 is 6.815×10 7 CFU / mL. Compared with Serratia marcescens BRC-CXG2, the virulence of the engineered Serratia marcescens BRC-CXG2-pCHSW was greatly improved, which also showed that the engineered Serratia marcescens BRC-CXG2-pCHSW had extremely high insecticidal activity against the larvae of red palm weevils.

[0064] Table 4 Insecticidal activity of BRC-CXG2-pCHSW strain against red palm weevil larvae

[0065]

[0066] Example 7

[0067] The toxicity test of the engineered Serratia strain BRC-CXG2-pCHSW against Tribolium castaneum was carried out as follows:

[0068] The artificial diet of red flour beetles was added to a 24-well plate, and a red flour beetle larva with good vitality in the first age was placed in each well. Then, the bacterial liquid was activated and cultured (Serratia BRC-CXG2 strain and Serratia engineered bacteria BRC-CXG2-pCHSW) and the bacterial suspension was prepared and diluted according to the method described in Example 5 and added to the 24-well plate to feed the red flour beetles. 15 replicates were prepared for each concentration gradient, and a blank control was performed using sterile water feeding. The culture conditions were: temperature of 28°C, relative humidity of more than 70%, and a photoperiod of 8 h / 16 h (day / night). The number of dead red flour beetle larvae was observed and counted every day, and LC was performed after 10 days of observation. 50 calculate.

[0069] The results are shown in Table 5. The LC of the engineered Serratia strain BRC-CXG2-pCHSW 50 is 1.583×10 6 CFU / mL. Compared with Serratia marcescens BRC-CXG2, the virulence of the engineered Serratia BRC-CXG2-pCHSW was greatly improved, which also showed that the engineered Serratia BRC-CXG2-pCHSW had extremely high insecticidal activity against the larvae of Tribolium castaneum.

[0070] Table 5 Insecticidal activity of BRC-CXG2-pCHSW strain against Tribolium castaneum larvae

[0071]

[0072] Example 8

[0073] The colonization and subculture assay of Serratia marcescens on Monochamus alternatus is as follows:

[0074] Thirty newly emerged female Monochamus alternatus adults and 30 male adults were raised in plastic fresh-keeping boxes of 19.7 m*13.4 m*7.3 m, and placed in a container evenly sprayed with OD 600The pine branches were fed with the GFP fluorescently labeled bacterial solution of Serratia marcescens with a value of 0.6. 30 female adults and 30 male adults fed with the pine branches sprayed with sterile water were used as blank controls (CK). The fresh-keeping boxes were placed at a temperature of 28°C, a relative humidity of more than 70%, and a photoperiod of 8 h / 16 h (day / night) for cultivation. Male and female pairs of Monochamus alternatus were placed in a fresh-keeping box of 22.4m*15.4m*8.2m for mating, and fed with branches sprayed with sterile water. Subsequently, the intestines and reproductive organs were dissected, and the homogenate with 10-fold continuous gradient dilutions was spread on LB plates containing 100 μg / ml kanamycin. After culturing until single colonies were generated, fluorescence observation and counting were performed under a fluorescence microscope. At the same time, DNA was extracted from the intestines and reproductive organs, and conventional PCR verification was performed using the primers of the GFP sequence using the extracted total DNA as a template. The primers required for PCR verification are: 5′-CCCCGGGgtcaggctgttacagctcaga-3′ (SEQ ID NO.4),

[0075] 5′-CctcgagTTatttgtatagttcatccat-3′ (SEQ ID NO.5). The total PCR reaction system was 25 uL: template DNA 1μL, 12×TransTaq® High Fidelity (HiFi) PCR SuperMix I 12.5 μL, ForwardPrimer 0.5 μL, ReversePrimer 0.5 μL, ddH2O 10.5 μL. The PCR reaction conditions were: 94℃ 5 min; 95℃ 30 s, 55℃ 30 s, 72℃ 1min 15s, 30 cycles; 72℃ 10 min.

[0076] The results are as follows Figure 5 As shown, Serratia was detected in the intestines and reproductive organs of the pine beetle. At the same time, the intestines of the offspring of insects carrying the labeled strain were dissected and the labeled strain was also detected, indicating that Serratia can colonize the intestines and reproductive organs of the pine beetle and can pass it to the offspring larvae.

[0077] Example 9

[0078] The colonization and migration assay of Serratia marcescens in Pinus massoniana was carried out as follows:

[0079] Select Masson pine seedlings with similar growth, trunk diameter and well-developed root system. Prepare 500 ml of purified fluorescent labeled bacterial solution and irrigate the roots of Masson pine seedlings at a concentration of 10 5CFU / ml. After inoculation, continue to observe the culture. Inoculate an equal amount of water as a blank control, and extract the total DNA of the samples from the phloem, roots and rhizosphere soil of pine trunks and branches on the 7th, 14th, 30th, 60th, 120th, 150th and 180th days. Use PCR to detect whether the gfp marker gene exists in the samples, and recover and count the strains of pine trunks, branches, roots and surrounding soil. Samples taken at different time periods were treated with 3 groups of replicates.

[0080] The results are as follows Figure 6 As shown, Serratia can colonize in pine trees. 180 days after inoculation with the labeled strain, the strain can still be detected in various tissues of the pine trees, indicating that Serratia can stably exist in pine trees and can migrate to various tissues of pine trees.

Claims

1. An engineered strain of Serratia marcescens with stable inheritance of exogenous genes, BRC-CXG2-pCHSW, characterized in that: The engineering bacteria of Serratia marcescens BRC-CXG2-pCHSW is named as Serratia marcescens ( Serratia marcescens ), was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 6, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.23125.

2. The use of the engineered Serratia marcescens BRC-CXG2-pCHSW according to claim 1 in controlling coleopteran pests, characterized in that: The coleopteran pests are one or more of Monochamus alternatus, Anoplophora anterior, Anoplophora glabripennis, Red palm weevil and Red flour beetle.

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