CN116676321A - Insecticidal gene orf5878 of bacillus new strain HSY204 and application thereof - Google Patents

Abstract

The present invention provides an insecticidal gene orf5878 of a new Bacillus strain HSY204 and its application. The DNA sequence of the insecticidal gene orf5878 is as shown in SEQ ID NO. The insecticidal protein gene orf5878 of the (Bacillus cereus) HSY204 strain is highly toxic to Aedes aegypti larvae and has a significant impact on the growth of Aedes aegypti. It can be used to control mosquitoes and effectively reduce the risk of large-scale transmission of mosquito-borne diseases.

Description

An insecticidal gene orf5878 of a new Bacillus strain HSY204 and its application

technical field

The invention relates to the field of environmental microbes and the technical field of molecular biology, in particular to an insecticidal gene orf5878 of a new bacillus strain HSY204 and its application.

Background technique

Mosquitoes are a kind of pests we are familiar with. There are many kinds of them. It is known that there are more than 3,200 species in the world. Among them, 18 genera, 48 subgenera and 371 species or subspecies of mosquitoes are known in my country. Mosquitoes not only seriously affect our lives, but more importantly, they are the vectors of spreading various diseases. At present, there are more than 10 species of mosquitoes that suck blood and transmit diseases in our country. Diseases transmitted by mosquitoes occupy an important position in preventive medicine, because mosquitoes have strong transmission power, high incidence and great harm. The diseases it spreads mainly include malaria, dengue fever, filariasis, Japanese encephalitis and Zika, etc., which seriously endanger the health of the people.

In recent years, some old mosquito-borne diseases in our country have been recurring, and new mosquito-borne diseases have appeared from time to time, so the prevention and control of mosquitoes has received widespread attention. The main strategies of mosquito control include environmental control, chemical control and biological control. The efficiency of environmental control is low; the efficiency of chemical control is high and quick, but it pollutes the environment, which will lead to resistance, high disability, and even directly cause human and animal poisoning in a short period of time. However, biological control is to use one kind of organism to control another kind of organism or to use the characteristics of the organism itself to control. Its biggest advantage is that it is more friendly to the environment and lower in cost.

At present, with the continuous extensive and in-depth research on the crystal protein structure and related molecular mechanism of mosquito-killing bacterial strains, the mosquito-killing effect is improved through the use of modern biological technology. Undoubtedly, the study of highly efficient strain bioinsecticides has very important practical significance for controlling mosquitoes. The key to studying the mechanism of killing mosquitoes is the receptor. Pootanakit et al. have identified two crystal protein receptors, aminopeptidase N isoforms, from the midgut of Aedes aegypti. Charles et al. found that the crystal protein of the Israeli subspecies acted on the intestinal epithelial cells of Aedes mosquitoes, resulting in histopathological changes similar to those in Lepidoptera insects. Bacillus is a general term for a class of facultative anaerobic or aerobic, Gram-positive bacilli. It produces spores, has rich and diverse physiological characteristics, and is extremely widely distributed. It is an important microbial population on the plant surface, rhizosphere, soil, and air. Bacillus is a species that produces a large number of polypeptide antibacterial substances of different chemical structures. Bacillus cereus is one of the common food-borne pathogens in the environment, facultatively aerobic. The growth temperature ranges from 20 to 45°C. It grows slowly or does not grow below 10°C. It exists in soil, water, air, and animal intestines. It can produce antibacterial substances, inhibit the reproduction of harmful microorganisms, degrade nutrients in the soil, and improve Ecological environment, but there is no report on the application of the insecticidal gene of Bacillus cereus in killing Aedes aegypti.

Contents of the invention

In view of this, the present invention proposes an insecticidal gene orf5878 of a new bacillus strain HSY204 and its application.

Technical scheme of the present invention is realized like this:

The invention provides an insecticidal gene orf5878 of a new bacillus strain HSY204, the DNA sequence of the insecticidal gene orf5878 is shown in the nucleotide sequence of SEQ ID NO.1.

Further description, the amino acid sequence of the insecticidal gene orf5878 is shown in SEQ ID NO.2.

Further explanation, the cloning method of the insecticidal gene orf5878 is as follows: the genome of Bacillus cereus HSY204 is used as a template, and the complete insecticidal gene fragment of orf5878 is obtained by PCR amplification.

Further illustrate, the primer sequence and restriction endonuclease of described PCR amplification comprise:

orf5878 primer F: taagaaggagatatacatatgaaaataatgggggaatatatat, R: gtggtggtggtggtgctcgagccaccaaactccctcttttac, restriction enzymes XhoI and NdeI.

The application of the insecticidal gene orf5878 of a new Bacillus strain HSY204 in killing Aedes mosquitoes.

The application of the insecticidal gene orf5878 of a new bacillus strain HSY204 in the preparation of mosquitoicide.

Wherein, the new strain of Bacillus HSY204 is classified as Bacillus cereus (Bacillus cereus) HSY204, and is preserved in the China Center for Type Culture Collection with the preservation number: CCTCC NO.M2020523.

Compared with the prior art, the beneficial effect of the present invention is: the present invention passes through Bacillus cereus (Bacillus cereus) HSY204 to screen, identify, the insecticidal activity of vegetative period insecticidal protein and the clone expression of its gene etc. experiment, through Bioactivity assays showed that the insecticidal protein gene orf5878 of Bacillus cereus (Bacillus cereus) HSY204 strain was highly toxic to Aedes aegypti larvae and had significant effects on the growth of mosquitoes, but had no effect on the growth of diamondback moth and cotton bollworm , can be applied to prevent and control mosquitoes, and effectively reduce the risk of large-scale transmission of mosquito-borne diseases.

Description of drawings

Fig. 1 is the optical microscope figure under the different field of view multiples of bacillus cereus (Bacillus cereus) HSY204 of the present invention;

Fig. 2 is the scanning electron micrograph under different field of view multiples when Bacillus cereus (Bacillus cereus) HSY204 of the present invention is cultivated to the spore stage;

Fig. 3 is the SDS-PAGE analysis of Bacillus cereus (Bacillus cereus) HSY204 of the present invention, in the figure, 1: HSY204; 2: Bti (Bacillus thuringiensis israelensis)

Fig. 4 is the agarose electrophoresis analysis figure of the PCR amplification product of Bacillus cereus (Bacillus cereus) HSY204 insecticidal gene orf5878 of the present invention;

Fig. 5 is an SDS-PAGE analysis chart of the insecticidal gene 5878 protein of Bacillus cereus (Bacillus cereus) HSY204 of the present invention.

Detailed ways

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

The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.

The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

Screening and identification of embodiment 1-bacterial strain HSY204

(1) Soil sample collection: The sampling site was selected in the tropical rainforest of Hainan Province. In order to increase the probability of obtaining new strains, the sampling site was selected in a place with complete vegetation protection, no human intervention in the ecological environment, and rich soil humus;

(2) Screening of strain HSY204: Use sodium acetate-temperature screening method, weigh about 5g of soil and put it into 20ml of BPA medium, shake fully, culture on a shaker at 30°C for 4-5h, bathe in 75°C for 15min, take 1ml of supernatant to dilute To 10 ^-3 , 10 ^-4 and 10 ^-5 , pipette 200 μL NB plates each, incubate at 30°C for 3 days, and pick single colonies with different shapes to draw plates.

(3) After the bacillus was cultured for 3-5 days to form spores, further use carbofusin staining and optical microscope to isolate whether the strain contained parasporal crystal protein. Under the optical microscope, it was observed that the colorless spores were bacillus and produced parasporal crystals. The bacillus is named as Bacillus cereus HSY204, and then the shape of the spores is further observed with an electron microscope, as shown in Figure 1 and Figure 2, the specific shape of the bacteria and crystals can be obtained. In order to clearly screen the insecticidal activity of wild strains, Aedes aegypti was used as the target insect for bioassay, and a strain HSY204 was screened to show a good insecticidal effect on Aedes mosquitoes, named Bacillus cereus (Bacillus cereus) HSY204 . In order to further identify whether the strain is a new spore strain, the parasporal crystal band pattern was analyzed by SDS-PAGE electrophoresis, and it was found that the strain HSY204 had similar bands to the standard strain, as shown in Figure 3.

DNA extraction and insecticidal gene identification of embodiment 2-bacterial strain HSY204

The genome sequence of strain HSY204 was analyzed molecularly and bioinformatically by using MiSeq Illumina next-generation sequencing and PacBio technology.

1. Genomic DNA extraction of strain HSY204

(1) A single colony of HSY204 was inoculated in about 2-5 mL of LB liquid medium, and cultured at 28°C and 220 rpm for overnight activation;

(2) The next day, transfer 1% to fresh LB liquid medium, continue culturing at 28°C, 220rpm for 4-6 hours until OD600=2.0;

(3) Collect 1-3mL bacteria by centrifugation at 8000rpm for 2min;

(4) Wash the precipitate with 1mL J Buffer (0.1M Tris HCl (pH 8.0), 0.1M EDTA (pH 8.0), 0.15M Nacl), centrifuge at 8000rpm for 2min, discard the supernatant;

(5) Pipette to resuspend the pellet in 500 μL J Buffer (lysozyme was added to 20 mg/mL before J Buffer is used), incubate at 37°C for 30 minutes, and shake 3-5 times during the period;

(6) Add 15 μL RNase (10 mg/mL), and act at 50 ° C for 15 min;

(7) Add 80 μL of SDS (10%), treat at 70°C for 20 minutes, and cool to room temperature;

(8) Equal volumes of phenol: chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) were extracted once each (inverted vigorously up and down 10-30 times, centrifuged at 12,000rpm for 10min, carefully pipette clear);

(9) Add an equal volume of isopropanol to mix, centrifuge at 12,000rpm for 10min, and pour off the supernatant;

(10) Add 500 μL of 70% ethanol (do not shake vigorously), and centrifuge at 12,000 rpm for 2 min;

(11) After air-drying, dissolve in 50 μL TE Buffer, and take 5 μL DNA solution for detection by 1.0% gel electrophoresis.

2. Identification of insecticidal genes of strain HSY204

The Illumina pair-end library and SMRT bell library were constructed with the genomic DNA fragments of strain HSY204, which were used for whole-genome sequencing by Illumina Hiseq and PacBio RSII, respectively. The original sequencing data of PacBio was assembled with Canu software, and the software Pilon corrected the assembly results of Canu with the original sequencing data of Illumina, and finally obtained the whole genome of HSY204 with a length of 6,035,572bp and 0 gaps, consisting of one chromosome and four Plasmid composition. Using Glimmer software for gene prediction, HSY204 contains 6102 CDS. These genes were annotated in six databases (NR, Swiss-Prot, Pfam, EggNOG, GO and KEGG) to further explore potential functions.

rRNA and tRNA were predicted by software RNAmmer-1.2 and tRNAscan-SE, respectively.

Embodiment 3-cloning and expression of bacterial strain HSY204 insecticidal gene

The genomic DNA of the strain HSY204 was extracted by alkaline lysis, primers were designed according to the coding sequence of the gene orf5878, and the gene was inserted into the expression vector pET-30a by means of seamless cloning technology, and the successful recombinant was constructed and transferred into BL(DE3) , for inducible expression.

The SeamLess cloning technology is specifically:

A. Linearize the vector using double enzyme digestion or PCR amplification

(1) After enzyme digestion, the linear vector is recovered by gel, and both blunt ends and sticky ends are acceptable;

(2) PCR amplification to obtain a linear vector, if it is a single band, the PCR product is directly purified, otherwise, gel recovery and purification are performed.

B. Use the designed primers to perform PCR amplification of the target DNA fragment;

Table 1: The orf5878 gene primer sequence information table in HSY204 bacteria

(1) The primer cannot be phosphorylated;

(2) The amplification product is a high-fidelity DNA polymerase with blunt ends;

(3) Electrophoresis to detect the integrity of the amplified product, if there is a band, the gel recovers the target fragment;

(4) Cloning primers include target fragment-specific primer sequences (22nt) and vector overlapping sequences (15-25nt; for recombination of more than 3 fragments, increase the homologous region to 30nt);

(5) There are strict requirements on the enzyme cutting site, select the appropriate enzyme cutting site, and the forward and reverse primers increase the lack of bases to restore the original enzyme cutting site;

(6) Design primers according to the end structure of the linear vector with 5' overhang, 3' overhang, and blunt end.

C. Cloning reaction system

Component Volume

PCR product 1-4μL

Linearized Vector 1-2μL

2×Assembly Mix 5μL

H ₂ O xμL

Total 10μL

(1) Add 0.01-0.25 pmol of the target DNA fragment and linearized carrier (estimated 1kb 20ng, 1.5kb 30ng), the optimal molar ratio is 2:1, add to the centrifuge tube and mix well;

(2) Recombination reaction at 50°C for 15 minutes, after the reaction, place on ice for a few seconds (for the recombination of more than 3 fragments, the reaction time is extended to 60 minutes);

(3) Store the recombinant product at -20°C or directly use it for transformation.

Recombinant products are transformed into E.coli competent cells by rapid or traditional methods

(4) After the competent cells were taken out from the -70°C refrigerator, they were placed in an ice bath to melt;

(5) Add 5 μL of recombinant product to 50 μL of competent cells, gently flick the wall of the centrifuge tube to mix (do not vortex), then place it on ice for 30 minutes, and do not shake the centrifuge tube during this period;

(6) Place the centrifuge tube in a constant temperature water bath at 42°C for heat shock for 30-90 sec, and immediately place the tube in an ice bath for 2-3 minutes after taking it out;

(7) Add 500 μL of 37°C preheated SOC or LB medium (without antibiotics) to the centrifuge tube, shake at 200 rpm and 37°C for 1 hour;

(8) Gently flick or pipette the bacterial solution in the centrifuge tube to mix, pipette 200 μL and add it to LB solid medium (preferably preheated at 37°C) containing the corresponding antibiotic for overnight culture.

Positive clone identification

(9) Colony PCR or PCR verification of extracted recombinant plasmids;

(10) Recombinant plasmid digestion verification;

(11) Sequencing identification. After preliminary identification, select 1-2 positive clones from all newly constructed vectors for sequencing identification.

Embodiment 4-preparation of toxin protein

The protein expression was induced by IPTG, expressed in a small amount, ultrasonically crushed, and the expressed protein was analyzed by SDS-PAGE electrophoresis. A large amount of expression uses a metal chelated nickel column for affinity chromatography, and uses a high-affinity Ni-NTA purification medium to covalently couple the chelating agent (nitrotriacetic acid or NTA) to the agarose medium (4% cross-linking), Then it is prepared by chelating Ni ²⁺ . NTA can firmly chelate Ni ²⁺ through four sites, thereby reducing the leakage of Ni ²⁺ into protein samples during the purification process.

The expression method of bacterial strain HSY204 insecticidal gene in Escherichia coli is as follows:

(1) The constructed expression engineering strain was inoculated into 2-5mL LB medium (Amp100Kan30μg/mL) containing the corresponding antibiotic to activate the expression strain overnight;

(2) The next day, transfer 1% to fresh LB medium containing corresponding antibiotics and culture for 2 hours until the OD600 is about 0.6, add 2-8μl IPTG (500mM) to a final concentration of 0.1-0.5mM and culture for 8- 20h (wherein, the empty load was induced with IPTG as a control; the expression strain was transferred in 2 copies, one was added with IPTG, and the other was used as a negative control without IPTG);

(3) Optimize the expression system with different temperatures, IPTG solubility and expression time to achieve soluble expression;

(4) Centrifuge at 8000rpm, collect the bacteria for 5min, and wash 1-3 times with 0.5M NaCl or ddH2O or PBS buffer;

(5) Add an appropriate amount of 800 μl PBS buffer solution, ultrasonically crush for 5-20 minutes, until the liquid is clarified (Sonics VCX750S parameters: small probe power 20%, work 10s, interval 10s);

(6) Centrifuge at 10000rpm for 5min, collect the supernatant respectively, wash the precipitate with 1mL PBS buffer 1-2 times, and then blow and suspend with 50μl PBS buffer;

(7) SDS-PAGE analysis was performed on the protein product supernatant and precipitate of the expression strain and the reference strain, and the results are shown in FIG. 5 .

Embodiment 5-HSY204 protein bioactivity assay

The prepared HSY204 protein was used for the determination of biological activity, and the Bio-Rad densitometric scanning method was used to quantitatively analyze the expressed protein. The protein samples were serially diluted, and 2-3 instar target insects were prepared. The number of worms, using SPSS13.0 software for probability regression analysis, to obtain LC50 value and 95% confidence interval value, clear insecticidal activity.

1. Artificial bioassay of cotton bollworm and Spodoptera litura

(1) Weigh 10 g of artificial feed and place it in a sterilized petri dish, add 1000 μl of the sample solution to be tested, fully stir evenly with a medicine spoon, and place it at room temperature to evaporate excess water in the feed;

(2) Dispense it in a sterilized 24-well cell culture plate, gently insert the newly hatched larvae with a brush, one head in each well, repeat 3 times for each treatment, and strictly seal to prevent the larvae from escaping;

(3) Place them in a light incubator (25°C, 65% humidity, 12h:12h photoperiod) for culture, investigate the number of dead and alive insects on the 4th or 6th day, and calculate the mortality and LC50.

2. Aedes aegypti Rearing and Mosquito Bioassays

(1) Mosquito larvae are collected from small, still streams in summer;

(2) In an artificial indoor environment, the temperature is 26 degrees, the relative air humidity is 80% and the photoperiod is 14 hours;

(3) The growth cycle of mosquitoes is 4 stages: eggs, larvae, larvae and adults; larvae grow in dechlorinated tap water and are fed with artificial feed (flour 9: yeast powder 1) continuously;

(4) Adults are placed in glass worm cages, put into 10% sucrose solution (or honey solution) absorbent cotton balls to feed adults, put into small mice to allow mosquitoes to suck blood and enter the breeding stage;

(5) Because female mosquitoes lay eggs in still water, put an open container filled with water in the imago cage to collect eggs;

(6) 3-4 instar larvae are used for bioassay experiments, observe every day, record the number of deaths, and calculate mortality and LC50;

(7) The mosquitoes used for the determination of protein toxicity are sensitive strains artificially raised in the laboratory, and the calibrated protein solution is diluted with dechlorinated tap water to a certain concentration, and placed in sterilized transparent drinking water plastic cups;

(8) Each plastic cup holds 10ml of protein solution, and transfers 10 third-instar mosquito larvae with a pasteur pipette;

(9) Dechlorinated tap water without adding any protein was used as a negative control, and three replicates were set for each protein concentration.

3. The biological activity assay results of strain HSY204 insecticidal protein are as follows:

Table 2 Determination of biological activity of HSY204 insecticidal protein

(Wherein, ND: not detected. >300: No lethal effect and body weight suppression at 300 μg/g.) As can be seen from the above table, HSY204 insecticidal protein has a significant impact on the growth of mosquitoes, and has a significant effect on the growth of diamondback moth and cotton bollworm. Growth is not affected.

4. The results of the determination of the insecticidal activity of the protein of the insecticidal gene in the bacterial strain HSY204 against Aedes aegypti are as follows:

Table 3 Determination of insecticidal activity of HSY204 insecticidal gene orf5878 against Aedes aegypti

insecticidal gene protein Molecular weight of insecticidal protein insecticidal protein solubility Aedes aegypti fatality rate orf5878 45KDa ≥200mg/mL 100%

It can be seen from the above table that the protein of the insecticidal gene orf5878 has a relatively high lethality to Aedes aegypti, and the lethality to Aedes aegypti can reach 100%.

5. The test results of the insecticidal activity of HSY204 insecticidal gene protein 5878 against Aedes mosquitoes at different concentrations are as follows:

Table 4 Test of the insecticidal activity of HSY204 insecticidal gene 5878 against Aedes mosquitoes at different protein concentrations

It can be seen from the above table that as the protein concentration of the insecticidal gene 5878 increases, its lethality to Aedes mosquitoes increases. When the protein concentration of the insecticidal gene 5878 is 22.845 μg/mL, its lethality to Aedes mosquitoes can reach 40%; calculated by SPSS, the LC50 of insecticidal gene 5878 to Culex mosquitoes is 27.115 μg/m (19.381-61.199 μg/mL).

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 (6)

1. An insecticidal gene orf5878 of a new bacillus strain HSY204, which is characterized in that: the DNA sequence of the insecticidal gene orf5878 is shown as a nucleotide sequence of SEQ ID NO. 1.

2. The pesticidal gene orf5878 of a new strain of bacillus HSY204 of claim 1, wherein: the amino acid sequence of the insecticidal gene orf5878 is shown as SEQ ID NO. 2.

3. The pesticidal gene orf5878 of a new strain of bacillus HSY204 of claim 1, wherein: the cloning method of the insecticidal gene orf5878 comprises the following steps: the complete insecticidal gene fragment of orf5878 is obtained by using the genome of Bacillus cereus (HSY 204) as a template and adopting a PCR amplification method.

4. The pesticidal gene orf5878 of a new strain of bacillus HSY204 of claim 3, wherein: the primer sequences and restriction enzymes for PCR amplification include:

orf5878 primer F: taagaaggagatatacatatgaaaataatgggggaatatatat, R: gtggtggtggtggtgctcgagccaccaaactccctcttttac, restriction enzymes XhoI and NdeI.

5. The use of the insecticidal gene orf5878 of a new strain of bacillus HSY204 as claimed in claim 1, wherein: the application is in the aspect of killing aedes.

6. The use of the insecticidal gene orf5878 of a novel strain of bacillus HSY204 as claimed in claim 1 for the preparation of a mosquito killer.