WO2022095290A1 - Bacillus sj110, insecticidal protein, vip3-like insecticidal gene and use thereof - Google Patents

Abstract

Disclosed are Bacillus SJ110, an insecticidal protein, a vip3-like insecticidal gene and the use thereof. The Bacillus SJ110 belongs to Bacillus toyonensis, and is deposited at the China Center for Type Culture Collection with a deposit number of CCTCC NO: M2020524. A bioactivity assay shows that the strain SJ110, the insecticidal protein and the vip3-like insecticidal gene have a very high toxicity for beet armyworm, cotton bollworm and Caenorhabditis elegans, and can be used for preventing and controlling agricultural pests.

Description

A kind of Bacillus SJ110, insecticidal protein, vip3-like insecticidal gene and application technical field

The invention relates to the technical field of biological control, in particular to a Bacillus SJ110, an insecticidal protein, a vip3-like insecticidal gene and applications.

Background technique

Bacillus (such as Bacillus thuringiensis) can produce a variety of insecticidal factors that are biologically active against a variety of insects, nematodes, protozoa and cancer cells during the growth process. Such as insecticidal crystal proteins (Insecticidal Crystal Proteins, ICPs), chitinase, thuringiensis and vegetative insecticidal proteins (Vegetative Insecticidal Proteins, VIPs). Different from traditional chemical pesticides, these insecticidal factors are non-toxic to humans and animals, are environmentally friendly, have a wide insecticidal spectrum and are easy to ferment, which will be very beneficial to the control of agricultural and forestry pests and the application of genetically modified crops.

Insecticidal crystal proteins (ICPs) are delta-endotoxins produced by Bt during the growth process. They are one of the main insecticidal factors of Bt and accumulate in bacteria in the form of paracellular crystals. ICPs are mainly divided into crystal protein (Cry) and extracellular soluble protein (Crtolytic protein, Cyt). Among them, Cry toxin protein is the earliest Bt toxin protein used in crop pest control.

Vegetative Insecticidal Proteins VIPs are produced in the logarithmic phase of the growth of Bt strains, and have no homology to the amino acid sequence of ICPs proteins. They are a new type of insecticidal protein. Vip3Aa and Vip3Ab proteins were first discovered in 1996 by Estruch et al. The proteins begin to be secreted during the vegetative growth stage of bacteria. Among the more than 100 discovered in the four Vip protein families, Vip3 is the most extensively studied, and only Vip3 has been used in transgenic crops.

At present, few studies have reported the application of the novel Bacillus toyonensis and its insecticidal proteins and genes in insecticidal.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a Bacillus SJ110, an insecticidal protein, a vip3-like insecticidal gene and applications.

The technical scheme of the present invention is as follows:

The present invention obtains a novel Bacillus SJ110, its insecticidal protein and vip3-like insecticidal gene, the Bacillus SJ110 belongs to Bacillus toyonensis, and its preservation number CCTCC NO: M2020524 in the China Center for Type Culture Collection, preservation date 2020.09.21, the deposit address is Wuhan University, Wuhan, China. The amino acid sequence of the insecticidal protein is shown in SEQ ID NO.1. The nucleotide sequence of the vip3-like insecticidal gene is shown in SEQ ID NO.2.

On the other hand, the present invention also provides the application of the Bacillus SJ110, the insecticidal protein and the vip3-like insecticidal gene in insecticidal.

Further, the insects are Lepidoptera insects and/or Lepidoptera insects.

Further, the insects are Lepidoptera Noctuidae insects and/or Lepidoptera Lepidoptera insects.

Further, the insect is Helicoverpa armigera, Spodoptera exigua and/or Caenorhabditis elegans.

The beneficial effects of the present invention are:

The invention provides a Neobacillus SJ110, an insecticidal protein and its vip3-like insecticidal gene. The biological activity assay shows that the strain SJ110, the insecticidal protein and the vip3-like insecticidal gene are effective against Spodoptera littoralis, Helicoverpa armigera and Caenorhabditis elegans Nematodes are highly virulent. It shows that the strain SJ110 and its insecticidal protein and vip3-like insecticidal gene can be used for the control of agricultural pests.

Description of drawings

Figure 1: A is the observation of Coomassie brilliant blue staining light microscope; B is the observation of scanning electron microscope;

Figure 2: SDS-PAGE analysis of strain SJ110 protein; Note: PM is protein molecular weight standard, Lane1 and Lane2 are strain SJ110 protein, and Lane3 is Bacillus thuringiensis YBT1520 protein;

Figure 3: The 2832bp target fragment was cloned from the vip3-like gene by PCR; Note: Lane1, Lane2, Lane3 and Lane4 are the vip3-like gene fragments, and M is the nucleic acid molecular standard quantity.

Figure 4: Purification results of Vip3-like expressed protein.

Detailed ways

In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

Example 1 Screening and identification of strain SJ110

Soil samples were collected from places in the tropical rainforest of Hainan Island where there was little human intervention in the ecological environment and rich soil humus. Using the sodium acetate-temperature screening method of Bt strains, weigh about 5 g of soil and put it into 20 mL of BPA (beef extract 0.3%, peptone 0.5%, sodium chloride 0.5%) medium, fully shake, and culture at 30°C for 4- 5h, 75℃ water bath for 15min, take 1mL of supernatant and dilute to 10 ^-3 , 10 ^-4 and 10 ^-5 , draw 200μL of each and spread evenly on NB (beef extract 0.5%, peptone 1%, sodium chloride 3.4%) for culture Plate, invert at 30°C for 3 days, pick out single colonies with different shapes and plate them. Bacillus spores were formed after culturing for 3-5 days, and further observed by Coomassie brilliant blue staining and light microscope, colorless spores and blue paracellular crystals were observed. The homology analysis of the 16S rDNA sequence confirmed that it was a Bacillus toyonensis strain, and the strain number was SJ110. (figure 1)

Example 2 Scanning electron microscope observation of crystal protein

Strain SJ110 was cultured in G-Tris medium at 30°C until spores were fully formed, 500 μL of cells were collected by centrifugation at 12,000 g for 10 min at 4°C, and then washed with 1M ice-cold NaCl three times, and finally the cells were suspended in 500 μL of ice-cold ultrapure water. Take 10 μL of the mixture of spores and parasporal crystals and carefully spread them on a clean cover glass, dry them in a vacuum at 4°C, and then fix the samples with 1% osmium tetroxide (OsO ₄ ). The samples are placed on a metal sample stage and placed in A metal film with a thickness of about 20 nm is sprayed in a vacuum evaporator. The prepared samples were placed in a S3-400N scanning electron microscope (HITACHI Ltd, Japan), under the condition of 20kV voltage for image observation and recording.

Example 3 Analysis of strain crystal protein by SDS-PAGE electrophoresis

Generally, 7.5% or 12% SDS-PAGE can be used to analyze the crystal protein composition, 5 μL of the prepared paracytosine crystal protein and 5 μL of 2×SDS-PAGE sample Buffer [0.075M Tris·Cl, (pH 6.8); 0.02M EDTA ; 2% SDS; 0.02% bromophenol blue; 0.25M dithiothreitol (DTT); 50% glycerol] mixed, boiled in boiling water at 100 °C for 5 min, then centrifuged at 12,000 g for 5 min, the supernatant was loaded immediately, and at 100 V, the Mini- Protein II, cell slab vertical apparatus device (BioRad, USA) was electrophoresed for about 1 h, and then stained with Coomassie blue R250. The protein size was judged according to the standard protein quality standard.

Example 4 Extraction of genomic DNA of strain SJ110

A single colony was inoculated into 7mL of LB liquid medium, cultivated overnight at 30°C, 220rpm, 1% was transferred to a 250mL conical flask containing 50mL of LB medium, and continued to culture at 30°C, 220rpm for 4 hours to OD ₆₀₀ =1.0～2.0; centrifuge to collect 10 mL of bacterial cells, wash the precipitate with 2 mL of J Buffer (0.1M Tris HCl (pH 8.0), 0.1M EDTA (pH 8.0), 0.15M NaCl); lightly suspend the precipitate in 2 mL of J Buffer and add 80 μL Freshly prepared lysozyme (50mg/mL), mix well, incubate at 37°C for 45min; then add 15μL RNase (10mg/mL), act at 50°C for 15min; then add 200μL SDS (20%), treat at 70°C for 20min; slowly Cool to 37°C, extract once each with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1), then add an equal volume of isoamyl alcohol and mix well for -20 The supernatant was pelleted at °C for 20 min, and centrifuged at 12,000 rpm for 10 min. The precipitate was washed twice with 70% ethanol, air-dried and dissolved in 100 μL TE Buffer (10 mM Tris HCl (pH 8.0), 1 mM EDTA (pH 8.0)), and 5 μL DNA samples were taken for electrophoresis on a 0.8% agarose gel and stained with EB. After UV analyzer detection.

Example 5 Genome sequence analysis of strain SJ110 and identification of novel toxin proteins

The SJ110 genome sequence was obtained by combining Illumina and Pacbio sequencing technology, and the filtered subreads were assembled using Canu v1.5 software to obtain the final genome sequence with higher accuracy. Further through local blast, a new type of vegetative insecticidal protein (Vip) was found with an open reading frame (ORF) size of 2832bp, encoding 939 amino acids and a molecular weight of 105kDa, named Vip3-like protein.

Example 6 Preparation of recombinant protein PB6012

According to the genome sequence, primers 6012F (ttccaggggccccctgggatccATGGTTACTACAAAACTGACACCT)/6012R (gtcacgatgcggccgctcgagTTAAATTTGTTCCGATTCGTAAAG) were designed to clone and amplify the complete coding sequence of the vip3-like gene. High-fidelity ExTaq enzyme was used for PCR amplification. The correct recombinant PB6012 was transferred into the expression host cell E.coli BL21(DE3) to induce protein expression. 4 °C, 12,000 rpm centrifugation for 10 min to collect bacterial protein, fully suspended in an equal volume of TE solution, add lysozyme to a final concentration of 20ug/mL, 37 °C, 220 rpm shaking culture for 30 min, centrifuge to collect bacterial cells and proteins, use an equal volume of The cells were washed 3 times with ice-cold 1M NaCl, and then sonicated for 20 min (model VC-130, Sonics and Materials Inc, USA). Since the expressed fusion protein contained a GST tag, the recombinant protein PB6012 was purified with reduced glutathione at a final concentration of 10 mM using the Fusion Protein Purification Kit (Sangon, Shanghai, China). The BSA protein was used as the standard concentration, and the lowry method was used to determine the concentration of the expressed protein. The expressed protein was dissolved in PBS buffer and then diluted to 8 concentrations for bioactivity assay.

Example 7 Toxicity determination of Vip3-like protein to Helicoverpa armigera/Spodoptera exigua

The recombinant protein PB6012 was mixed with artificial feed, air-dried naturally, and placed in a 24-well cell culture plate. Select fast crawling and active newly hatched larvae (egg hatched for 5 days) and put them into the cell culture plate, 1 head per well. Investigate the number of dead and live insects on the 2nd and 4th days, and use the corrected mortality rate=[(the mortality rate of the treatment group-the mortality rate of the control group)/(1-the mortality rate of the control group)]×100% to obtain the expression of the recombinant protein PB6012 The Vip3-like protein at a concentration of 0.09 mg/mL had an insecticidal activity-corrected mortality rate of 43.33% against the second instar Spodoptera littoralis.

Example 8 Toxicity assay of Vip3-like protein to Caenorhabditis elegans

Inoculate Escherichia coli OP50 bacterial solution in LB medium at a ratio of 1:100, and culture at 37°C for 6-8h. OP50 cells were collected, washed three times with sterile water, and an appropriate amount of S medium solution (10 mL of 1 M potassium citrate buffer (pH 6.0) 10 mL, 3 mL of 1 M CaCl ₂ solution, 3 mL of 1 M MgSO ₄ was added to 1 L of S basal solution), The bacterial cells were suspended, and the final bacterial solution OD ₆₀₀ =3. Synchronized treatment of Caenorhabditis elegans to obtain homogenized 4th instar larvae (adding 0.1% TritonX-100 at 1% to the collected nematodes can prevent nematodes from adhering to the pipette tip or centrifuge tube in subsequent experiments). Take 40 μL of E. coli OP50 suspended in S medium, 5 μL of 4th instar larvae suspended in M9 buffer, 48 μL of S medium, 5 μL of 5-fluoro-2’-deoxyurea nucleoside, and 2 μL of chloramphenicol, and aliquot them into 48-well plates. Another 400 μL of PB6012 recombinant protein with a concentration of 0.09 mg/mL was placed in the positive sample well of a 48-well plate, and the plasmid pET30 was transfected into BL21 (DE3) as a negative control. Finally, the well-mixed 48-well plate was placed in a sealed plastic box with wet paper towels to provide humidity. Incubate at 25°C for 5 days, observe and record growth and death every day, and determine larval poisoning or death according to whether the larvae move. Examine the worms in each well under a dissecting microscope. Worms that move visibly are marked alive. Worms that do not move are gently touched with platinum and observed for movement. Worms that fail to respond after a few touches are marked dead. The proportion of dead larvae in each well was recorded, and the corrected mortality rate of the expressed Vip3-like protein against C. elegans at a concentration of 0.072 mg/mL was 100%.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (7)

1. Bacillus SJ110, characterized in that the Bacillus SJ110 belongs to Bacillus toyonensis, and its deposit number CCTCC NO: M2020524 in the China Type Culture Collection.
2. An insecticidal protein, characterized in that the amino acid sequence of the insecticidal protein is as shown in SEQ ID NO.1.
3. A vip3-like insecticidal gene, characterized in that the nucleotide sequence of the vip3-like insecticidal gene is shown in SEQ ID NO.2.
4. Application of Bacillus SJ110 of claim 1, insecticidal protein of claim 2 and/or vip3-like insecticidal gene of claim 3 in insecticidal.
5. The application according to claim 4, wherein the worms are Lepidopteran insects and/or Lepidoptera insects.
6. The application according to claim 5, wherein the worms are Lepidoptera Noctuidae insects and/or Lepidoptera Noctuidae insects.
7. The application according to claim 5, wherein the worm is Helicoverpa armigera, Spodoptera exigua and/or Caenorhabditis elegans.

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