A non-target intestinal lactobacillus strain with improved insecticidal activity of Cry protein and its application

Abstract

The present invention relates to a non-target intestinal lactobacillus strain with improved Cry protein insecticidal activity and application thereof. The lactobacillus strain is numbered R-8 and is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC No.31544.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a non-target intestinal lactobacillus strain capable of improving the insecticidal activity of Cry protein and its application. Background Art

[0002] Borer borer ( Chilo suppressalis Walker) is an insect of the genus Graminus in the family Pyralidae of the order Lepidoptera. The adult is yellow-brown or gray-brown, with females larger than males; the forewings are slightly rectangular, and the hindwings are all white. The forewings of male moths are yellow-brown to gray-brown, with irregular brown spots densely distributed on the wing surface. There is a gray-black spot in the center of the forewing, and there are three gray-black spots arranged obliquely below it. The abdomen is thin and cylindrical. The Chilo suppressalis is widely distributed in China and is one of the important pests of rice. It is common in northern rice-growing areas.

[0003] Bacillus thuringiensis ( Bacillus thuringiensis Insecticidal proteins expressed by Bacillus thuringiensis (Bt) can effectively control target pests and reduce the use of chemical pesticides. However, the effect of using insecticidal proteins alone to control target pests is limited. Summary of the invention

[0004] One of the present invention provides a strain of Lactobacillus ( Lactiplantibacillus sp. ) strain R-8, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 31544.

[0005] The second aspect of the present invention provides an engineered bacterium obtained by genetically improving the lactobacillus strain R-8 as described in one of the present invention. Wherein, since the engineered bacterium is targeted by the lactobacillus CGMCC No. 31544 of the present invention, and the means generally adopted are to transfer and / or knock out specific genes and / or DNA fragments therein, the engineered bacterium is still lactobacillus. In addition, the engineered bacterium can be an engineered bacterium that improves the insecticidal activity of the insecticidal protein of Bacillus thuringiensis against the striped stem borer. It can also be an engineered strain having insecticidal activity against the striped stem borer, concurrently having other pest control activities and / or being endowed with other beneficial properties.

[0006] In a specific embodiment, the engineered bacteria obtained after genetic improvement are engineered strains obtained by introducing plasmids carrying functional genes, or engineered strains obtained by recombining functional genes into the genome of wild strains.

[0007] In a specific embodiment, the functional gene is at least one of a gene for controlling pests, a gene for controlling plant pathogenic microorganisms, and a gene that enhances the pest control effect of the Lactobacillus strain R-8 on the insecticidal protein from Bacillus thuringiensis.

[0008] The third aspect of the present invention provides a composition, which comprises the Lactobacillus plantarum strain R-8 as described in the first aspect of the present invention or the engineered bacteria as described in the second aspect of the present invention, and an insecticidal protein from Bacillus thuringiensis.

[0009] In a specific embodiment, the insecticidal protein is Cry1Ca protein.

[0010] In a specific embodiment, the genbank accession number of the insecticidal protein is AAM00264.1.

[0011] In one embodiment, the composition further comprises an acceptable carrier.

[0012] In a specific embodiment, the dosage form of the composition is one of a suspension, a powder and a granule.

[0013] In a specific embodiment, the composition is in the form of an oil suspension or a wettable powder.

[0014] The fourth aspect of the present invention provides the use of the Lactobacillus strain R-8 according to the first aspect of the present invention, the engineered bacteria according to the second aspect of the present invention or the composition according to the third aspect of the present invention in controlling borer pests.

[0015] In a specific embodiment, the Pyralidae pest is Chilo suppressalis.

[0016] Beneficial effects of the present invention: The present invention found that the Lactobacillus plantarum strain R-8 can improve the insecticidal activity of the insecticidal protein from Bacillus thuringiensis against Pyralidae pests, especially Chilo suppressalis, which provides a new biological control resource for controlling Chilo suppressalis.

[0017] Strain preservation: The strain of the present invention is named R-8, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC No. 31544 and a deposit date of August 5, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Lactobacillus Lactiplantibacillus sp. . BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The bioactivity analysis of the R-8 strain against Chilo suppressalis is shown.

[0019] Figure 2 The colony morphology of the R-8 strain is shown.

[0020] Figure 3 The Gram staining results of the R-8 strain are shown.

[0021] Figure 4 The phylogenetic tree of the R-8 strain is shown. DETAILED DESCRIPTION

[0022] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0023] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0024] LB solid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 15.0 g agar, 1000 ml distilled water. No agar was added to LB liquid medium.

[0025] The artificial feed for Chilo suppressalis larvae is prepared according to the method in invention patent CN200910080336.2.

[0026] The insecticidal protein was a Cry1Ca protein solution with a purity of 90% obtained after affinity chromatography purification, purchased from Beijing Meiyan Agricultural Co., Ltd. Its amino acid genbank accession number is AAM00264.1.

[0027] Example 1: Isolation of strains.

[0028] The 4th instar larvae of Chilo suppressalis were dissected in a clean bench and the midgut contents were removed. They were ground with an electric pestle under sterile conditions, diluted 10 times with sterile water into 5 concentration gradients, and then spread on LB plates and cultured in a constant temperature incubator at 37 degrees Celsius. After a single colony grew in the culture medium, a single colony was picked according to the color, size and morphology of the colony. Each single colony was streaked and purified on the LB plate for at least 3 times to obtain a purified isolated strain, and the isolated strains were numbered.

[0029] Example 2: Analysis of the biological activity of the R-8 isolate against Chilo suppressalis.

[0030] The isolate numbered R-8 was streaked on an LB medium plate and cultured at 37 degrees Celsius for 72 hours. A single colony was picked and transferred to 5 ml of liquid culture medium and cultured overnight in a constant temperature shaker at 37 degrees Celsius and 200 rpm; the overnight culture was transferred to 500 ml of liquid LB culture medium and cultured until the logarithmic growth phase. The bacterial solution was centrifuged in a large-capacity centrifuge and the supernatant was discarded; then the precipitate was rinsed with sterile 1×PBS and diluted, and the OD of the bacterial solution was measured with an ELISA instrument after dilution. 600 The bacterial solution was diluted to 2.5×10 10 The colony forming units / ml (CFU / mL) were used to obtain the bacterial suspension of the R-8 isolate.

[0031] The Cry1Ca activated protein was prepared into a Cry1Ca protein solution with a final concentration of 2.50 mg / ml using a 20 mmol / L Na2CO3-NaHCO3 aqueous solution (pH 9.8).

[0032] Add penicillin, streptomycin, rifampicin, chlortetracycline and gentamicin at a final concentration of 500 μg / ml to the artificial diet of the Chilo suppressalis larvae and treat the newly hatched Chilo suppressalis larvae for 7 days to remove bacteria in the intestines of the Chilo suppressalis and obtain sterile intestinal larvae. Before the bioassay, the larvae were picked into an empty bioassay box and starved for 7 to 8 hours.

[0033] The artificial diet of the Chilo suppressalis larvae was heated to dissolve, cooled to 50 to 60 degrees Celsius, and the Cry1Ca protein solution and an equal amount of PBS buffer solution to the test sample of the R-8 isolate were added thereto, mixed evenly to make the final concentration of the Cry1Ca protein in the artificial diet to be 240 micrograms / gram, and cooled to room temperature to solidify the feed to prepare the Cry1Ca test sample.

[0034] The artificial diet of the Chilo suppressalis larvae was heated and dissolved, cooled to 50 to 60 degrees Celsius, and a 20 mmol / L Na2CO3-NaHCO3 aqueous solution equal to the Cry1Ca test sample was added thereto, and the diet was cooled to room temperature to solidify, and then 200 μL of the bacterial solution of the R-8 isolate was evenly added and mixed, so that the final concentration of the R-8 isolate in the artificial diet was 5×10 9 The colony forming units / gram were mixed evenly to prepare the test sample of the R-8 isolate.

[0035] The artificial diet of the Chilo suppressalis larvae was heated and dissolved, cooled to 50 to 60 degrees Celsius, and the Cry1Ca protein solution was added thereto and mixed evenly to make the concentration of Cry1Ca protein in the artificial diet 240 micrograms / gram. The diet was cooled to room temperature to solidify, and then 200 microliters of the bacterial solution of the R-8 isolate was evenly added to make the concentration of the R-8 isolate in the artificial diet 5×10 9 The colony forming units / gram were mixed evenly to prepare the test samples of Cry1Ca and R-8 isolates.

[0036] The artificial feed for the larvae of Chilo suppressalis was heated to dissolve, cooled to 50 to 60 degrees Celsius, and a 20 mmol / L Na2CO3-NaHCO3 aqueous solution in an amount equal to that of the Cry1Ca test sample and a PBS buffer in an amount equal to that of the R-8 isolate test sample were added thereto, mixed evenly, and cooled to room temperature to solidify the feed, thereby preparing a negative control test sample.

[0037] Weigh 1 gram of each sample to be tested and spread it on the bottom of a plastic bioassay box. Then, inoculate the Chilo suppressalis larvae that have been treated with antibiotics for 7 days and starved for 8 hours into the bioassay box. Set up 4 replicates, with 15 Chilo suppressalis larvae in each replicate (each bioassay box). After inoculation, place the bioassay box in a light incubator with a temperature of 27 to 28 degrees Celsius, a humidity of 70% to 80%, and a photoperiod of 16 L:8 D. After 7 days, count the mortality rate of the larvae or the weight of the live larvae.

[0038] The statistical results showed that the mortality rate of the larvae of Chilo suppressalis treated with the strain numbered R-8 was not significantly different from that of the negative control; compared with Cry1Ca alone, the combination of Cry1Ca and R-8 significantly increased the mortality rate of the larvae. The specific results are shown in Figure 1 .

[0039] Example 3: Taxonomic identification of R-8 isolates with biological activity.

[0040] Colony morphology of R-8 isolate: R-8 isolate was streaked on LB solid medium and incubated upside down at 37 degrees Celsius for 72 hours. The colony morphology was observed and photographed. Figure 2 .

[0041] according to Figure 2 It can be seen that the colonies of the R-8 isolate are round in shape, moist, smooth in surface, and milky white in color.

[0042] Gram identification of R-8 isolates: drop a drop of sterile water on the slide, use an inoculation loop to gently pick up a single colony of R-8 and put it into sterile water, gently stir to make the bacterial solution uniform, stain and decolorize according to the Gram staining solution operation method, and then observe under an oil microscope. The staining results of R-8 are all blue, indicating that it is a Gram-positive bacterium. The staining results are shown in Figure 3 .

[0043] Determination of physiological and biochemical characteristics of R-8 isolate: Physiological and biochemical tests were performed with reference to the Manual of Identification of Common Bacteria. The results of physiological and biochemical identification are as follows: esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose and raffinose were all positive, indicating that R-8 isolate can utilize esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose and raffinose, and indicating that R-8 isolate belongs to the genus Lactobacillus ( Lactiplantibacillus ) species.

[0044] Molecular identification of R-8 isolates - 16S rDNA identification: According to the operation steps of the bacterial DNA genome kit, the total DNA of each isolate was extracted, and 27F (as shown in SEQ ID No. 1) / 1492R (as shown in SEQ ID No. 2) was used as primers, and the total DNA of R-8 isolates was used as a template to perform PCR amplification of 16S rDNA. After the amplified PCR product was detected by gel electrophoresis, the amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. (Tsingke Biotechnology) for bidirectional sequencing. Among them, the sequence of 16S rDNA measured by R-8 strain is shown in SEQ ID No. 3.

[0045] The 16S rDNA sequences were compared and analyzed at NCBI. The results showed that the R-8 strain was similar to Lactobacillus plantarum ( Lactiplantibacillus plantarum )、Lactobacillus pentosus( Lactiplantibacillus pentosus )、Lactobacillus argentinus( Lactiplantibacillus argentoratensis ) are closely related. The phylogenetic tree was constructed using the maximum likelihood method (Construct / Maximum Likelihood Tree) under the Phylogeny algorithm using MEGA7.0. The phylogenetic tree of the R-8 strain is shown in Figure 4 .according to Figure 4 The phylogenetic tree shows that the R-8 strain is Lactiplantibacillus plantarum _JCM 1149, Lactiplantibacillus plantarum _NRRLB-14768, Lactiplantibacillus pentosus _124-2, Lactiplantibacillus argentoratensis _DKO 22, Lactiplantibacillus plantarum _CIP 103151 and so on are clustered into one branch, so its lactobacillus ( Lactiplantibacillus sp ) strains are most closely related.

[0046] In summary, the R-8 strain is systematically classified as Lactobacillus ( Lactiplantibacillus sp ).

[0047] The R-8 strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration with the deposit number CGMCC No. 31544 and the deposit date August 5, 2024 at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Lactobacillus Lactiplantibacillus sp. .

Claims

1. A composition comprising Lactobacillus ( Lactiplantibacillus sp .) strain R-8, and an insecticidal protein from Bacillus thuringiensis; the Lactobacillus plantarum ( Lactiplantibacillus sp .) strain R-8 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 31544; the insecticidal protein is Cry1Ca protein.

2. The composition according to claim 1, characterized in that The genbank accession number of the insecticidal protein is AAM00264.

1.

3. The composition according to claim 1, characterized in that The composition also includes an acceptable carrier.

4. The composition according to claim 1, characterized in that The dosage form of the composition is one of a suspension, a powder and a granule.

5. The composition according to claim 1, characterized in that The dosage form of the composition is oil suspension or wettable powder.

6. Use of the Lactobacillus strain R-8 in the composition according to any one of claims 1 to 5 for improving the insecticidal activity of Cry1Ca protein against Pyralidae pests.

7. The use according to claim 6, characterized in that: The Pyralidae pest is Chilo suppressalis.

8. Use of the composition according to any one of claims 1 to 5 for controlling Pyralidae pests.

9. The use according to claim 8, characterized in that: The Pyralidae pest is Chilo suppressalis.

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