CN101984045B - Bacillus thuringiensis cry8Na1 gene, expressed protein and application thereof - Google Patents

Abstract

The invention relates to "bacillus thuringiensis cry8Na1 gene, expressed protein and application thereof", belonging to the technical field of biological control. Bacillus thuringiensis strain BTQ52-7, its preservation number is CGMCC No.4188. The insecticidal protein has the amino acid sequence shown in SEQ ID NO 2, and the gene encoding the insecticidal protein, preferably the nucleotide sequence of the gene is shown in SEQ ID NO 1. The above-mentioned genes have high toxicity to coleopteran pests, and can be applied to transform microorganisms and plants, so that they can show toxicity to related pests, and overcome and delay the generation of drug resistance of pests to engineering bacteria and transgenic plants.

Description

Bacillus thuringiensis cry8Na1 gene, expressed protein and its application

technical field

The present invention relates to the technical field of biological control, in particular further, the present invention relates to the Bt cry8Nal gene with high toxicity to coleopteran pests and the protein encoded by the gene.

Background technique

Bacillus thuringiensis (Bt) is a widely distributed Gram-positive bacterium, an entomopathogenic microorganism with strong toxicity to pests and no toxicity to natural enemies, and no toxicity to higher animals and humans. It is currently the most in-depth study and the most widely used microbial insecticide, and it is active against more than 3000 kinds of pests in 16 orders. Bt can form insecticidal crystal proteins (Insecticidal CrystalProteins, ICPs), also known as delta-endotoxin (delta-endotoxin) in the sporulation stage, its shape, structure and size are closely related to its virulence [Schnepf.E, Crickmore .N, Van Rie.J., Lereclus.D, Baum.J, Feitelson.J, Zeigler.D.R., Dean.D.H. Bacillus thuringiensis and its pesticide crystalproteins.Microbiol.Mol.Biol.Rev, 1998, 62(3): 775-806.]. Since Schnepf etc. cloned the first ICPs gene of Bt in 1981, and published its DNA base sequence and the amino acid sequence of its encoded protein in 1985, at present (July 2010) a kind of insecticidal crystal protein has been found There are 530 species, which are divided into 67 types of cry proteins and 2 types of cyt proteins. Today, the use of chemical pesticides to prevent and control the pests can reduce the damage to crops, but chemical pesticides cause environmental pollution. For a long time, a large number of chemical pesticides sprayed will not only enhance the resistance of pests, but also make beneficial insects and other ecological areas The system is destroyed, and it seriously pollutes the environment, increases production costs, and destroys the ecological balance. Bacillus thuringiensis insecticidal crystal protein is widely used in pest control because of its good insecticidal effect, safety and high efficiency. In addition to being directly used as a biological pesticide, the world's first transgenic insect-resistant plant was approved for use in the United States in 1996. The gene it uses comes from Bt cry1Ac. In the next few years, insect-resistant maize with the cry1Ab gene and potato with the cry3Aa gene will be released. In China, insect-resistant cotton containing the cry1Ac/cry1Ab gene has been widely planted since 1998 when it was officially promoted. In the first 12 years (1996-2007) of the commercialization of transgenic crops, the amount of farmers planting transgenic crops increased year by year due to the continuous and stable income. In 2009, 25 countries planted 134 million hectares of biotech crops, an increase of 7% over 2008. The United States remains the largest grower of biotech crops, with 64 million hectares, and China, with 3.7 million hectares. In the first 12 years, the commercialization of GM crops brought economic and environmental benefits to farmers in both industrialized and developing countries. Bacillus thuringiensis and its gene discovery have become an important topic in sustainable agriculture.

The cry8 gene of Bacillus thuringiensis has specific insecticidal activity against various Coleoptera pests such as Scarabidae, Weevilidae and Chrysoopteridae. The cry8 gene is composed of 1160-1210 amino acids, and its molecular weight is between 128-137kDa. In 1992, Ohba et al screened out a new strain (B.thuringiensis subsp.japonensis BuiBui) with specific insecticidal activity to scarab larvae from Bt strains for the first time (Ohba M., Iwahana H., Asano S., Sato R.and Hori H..A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeidbeetles, Letters in Applied Microbiology 1992, 14:54～57), this strain is mainly effective against scarabaeidbeetles such as red copper beetle and Japanese scarab Pests are poisonous. In 1994, Hori et al. proved that the toxicity of the bacterial strain came from the 130kDa protein in the bacteria, from which Sato et al. cloned a new insecticidal gene cry8Ca1 (Sato R., Takeuchi K., Ogiwara K., Masayosi Minami, Kaji Y., Suzuki N., Hor H.i, Asan S.O, Ohba M.and Iwahana H..Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensisserovar japonensis strain Buibui toxic to Scarabaeid insects. 15), the gene has been used in the development of biopesticides. Due to the great potential application value of cry8 genes, most genes have already applied for patents. The cry8Aa1 and cry8Ba1 isolated by Mycogen Company of the United States have obvious insecticidal activity against various pests of the Scarabidae; cry8Bb1 and cry8Bc1 cloned by DuPont Company of the United States Gene has significant insecticidal effect on Western corn rootworm (Abad, Andre, R., Duck Nicholas, B., Feng, Xiang, FlannaganRonald, D., Kahn, Theodore, W., Sims, Lynne, E..Genes encoding novel proteins with pesticidal activity against Coleopterans.2002, WO 02/34774A2), and have been used in the development of transgenic insect-resistant corn; cry8Da1, cry8Da2 and cry8Da3 cloned by Asano in Japan have also applied for patents in Japan (Asano S ., Yamashita C., Iizuka T., Takeuchi K., Yamanaka S., Cerf D. and T. Yamamoto.. A strain of Bacillus thuringiensis subsp. Galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae). Biological Control 2003, 28: 191-196). At present, the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences and Hebei Agricultural University have successively screened and obtained a number of Bt strains with specific insecticidal activity against the larvae of Anomala exoleta and A. corpulenta ( Feng Shuliang et al. A new isolate of Bacillus thuringiensis with insecticidal activity against scarab larvae. China Biological Control, 2000, 16(2): 74-78). In 2006, the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences discovered the first Bt strain Bt185 with insecticidal activity against the black beetle, and then they cloned the Bt strain Bt185 against the dark beetle (Holotrichia parallela) and the large black beetle (Holotrichia oblita) respectively. cry8Ea1 and cry8Fa1 with specific insecticidal activity (Yu H., Zhang J., Huang D., Gao J. and Song F.. Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holotichia parallela. Current Microbiology 2006, 53: 13～17. Shu C., Yu H., Wang R., Fen S., Su X., Huang D., Zhang J., Song F.. Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185. Current Microbiology 2009, 58(4):389-92.) and cry8Ga1 gene (Shu C.L., Yan G.X., Wang R.Y., ZhangJ., Feng S.L., Huang D.F., Song F.P..Characterization of first cry8 gene specific to Melolonthidaepests:Holotrichia Holotrichia and oblita parallela. Applied Microbiology and Biotechnology. 2009.3.17). At present, there are 33 cry8 genes cloned in the world.

Since the current commercialized transgenic insect-resistant crops have a single type of insect-resistant gene, such large-scale promotion of planting has the risk of reducing pest refuges and increasing pest resistance. Therefore, it is necessary to continuously isolate highly virulent or new gene combinations to avoid the risk of increasing pest resistance. Therefore, screening and cloning new and highly virulent Bt insecticidal genes can enrich insecticidal gene resources, provide new gene sources for transgenic crops and engineered strains, improve the insect-resistant effect of Bt transgenic products, and reduce the impact of pests on The risk of resistance to Bt toxins can avoid new ecological disasters and have important economic, social and ecological benefits.

Contents of the invention

The present invention provides a highly toxic Bacillus thuringiensis Q52-7 to the beetle family pest, the dark beetle beetle, and its new insecticidal gene cry8Na1 gene and its crystal insecticidal protein, so as to be applied to the transformation of microorganisms and plants, so that they can express Toxicity to related pests, and to overcome and delay the development of resistance of pests to engineering bacteria and transgenic plants.

Bacillus thuringiensis strain BTQ52-7, its preservation number is: CGMCC No.4188.

Application of Bacillus thuringiensis strain BTQ52-7 in killing coleopteran pests.

A cry8Na1 gene isolated from Bacillus thuringiensis strain BTQ52-7, the nucleotide sequence of which is shown in SEQ ID NO1.

The insecticidal protein encoding the gene has an amino acid sequence as shown in SEQ ID NO 2.

An expression vector is characterized by containing the above-mentioned gene.

Described expression vector is pEB 8, and its backbone vector is pEB, and its structure is as shown in Figure 6.

A microbial transformant is characterized by containing the above-mentioned gene.

The application of the above-mentioned genes in resisting coleopteran pests.

The application is that the protein expressed by the gene is used as an effective component of a biopesticide.

The application is to transfer the gene into microorganisms or plants to express the toxic protein to Coleoptera.

The present invention isolates a Bacillus thuringiensis strain BTQ52-7 from the soil near Yuantong Temple, Qianshan Mountain, Liaoning Province, and its preservation number is CGMCC No.4188. The parasporal crystals that kill coleopteran pests have a strong ability to kill the black beetle; a positive clone of a new gene, pEB8 (see Figure 6), is obtained from the strain, and it is sequenced and analyzed, and it is found that Clone pEB 8 contains 3519 bases, see SEQ ID NO1, and encodes 1174 amino acids, see SEQ ID NO2. Compared with the published gene, it has the highest similarity with cry8Ca, with a similarity of 70%. It is a new gene; extract the plasmid from the positive clone above, transfer it into the recipient bacteria, obtain the expression strain, and measure the activity of the expressed protein of the gene. The protein expressed by the gene cry8Na1 has a primary screening bioassay result on the black beetle larvae, the corrected mortality rate is 100%, as shown in Table 1, and it has certain activity on the larvae of the North China black beetle, as shown in Table 2. See Table 3 and Table 4 for the no insecticidal activity of the larvae of C. aeruginosa.

The cry8Na1 gene can transform microorganisms and plants according to conventional methods of biotechnology, and exhibit toxicity to related coleopteran pests.

The above-mentioned gene is transformed into a bacterial strain, and the expressed protein can be made into a biopesticide for killing coleopteran pests. At the same time, plants can be transferred to construct insect-resistant transgenic plants for pest control.

The isolated and cloned Bt cry8Na1 gene sequence of the present invention and its gene expression product can produce strong toxicity to coleopteran pests, especially have high activity to black beetles, are good biological insecticidal genes, and have very wide application prospects. By combining the cry8Na1 gene with gene expression products such as cry1Ab, cry1Ba, cry2Ab, etc., the insecticidal spectrum against Lepidoptera and Coleoptera pests can be expanded. By applying it to transforming microorganisms and plants, making them exhibit toxicity to related pests, it can overcome or delay insect resistance to engineering bacteria and transgenic plants.

Strain preservation information:

Bacteria classification and naming: Bacillus thuringiensis

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms

Date of preservation: September 20, 2010

Deposit number: CGMCC No.4188

Description of drawings

Observe the morphology of bacterial strain BTQ52-7 thalline under the optical microscope of Fig. 1,

Figure 2 Morphology of bacteria strain BTQ52-7 under the electron microscope

Figure 3 Genomic PCR identification containing the target gene,

1: Negative control 2: PCR amplification product of strain BTQ52-7S5un8/S3un8

M: Marker (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500bp

Figure 4 PCR results of full-length cry8Na1 gene

1: Negative control M: Marker (300, 500, 800, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 8000, 10000bp) 2: Full-length amplification product of cry8 gene

Figure 5 SDS-PAGE of protein expressed by cry8Na1 gene in Escherichia coli

M: protein high molecular weight marker (200, 116, 97, 66, 44kDa) 1: crystal protein of BTQ52-7 strain 2: pEB8 protein expressed in E. coli 3: pEB empty vector

Figure 6 Schematic diagram of pEB8 structure,

Figure 7 Homology analysis diagram.

Detailed ways

Embodiment 1, isolate Bacillus thuringiensis bacterial strain BTQ52-7

The applicant's laboratory staff obtained a strain of Bacillus thuringiensis isolated from the soil near Yuantongguan, Qianshan, Liaoning. The spores of Bacillus thuringiensis are the outer wall of the spore, the coat of the spore, the cortex, the inner wall of the spore, the plasma membrane and the inner wall of the spore. protoplast. The main component of the cortex is peptidoglycan, a polysaccharide teichoic acid that does not contain vegetative cells, and it maintains the dehydration state and heat resistance of the spores. On the other hand, during the formation of the spores, a large amount of DPA-Ca will be produced. thuringiensis spores will not die and dormant spores are treated at a sub-lethal temperature of 75°C for 15 minutes, and the activation effect is the best , not only promote its rapid germination, but also improve the survival rate of spores (Yu Ziniu 1990). According to this characteristic, temperature screening can be implemented (Knowles B H, EllarD J. Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillusthuringiensis d-endotoxins with different specificity [J]. Biochimica et biophysicaacta, 1987, 924: 509-518.; Dai Lianyun, Wang Xuepin, etc. Distribution of Bacillus thuringiensis in forest soil of eight nature reserves in China [J]. Acta Microbiology, 1994, 30(2)117-121).

1.1 Isolation of strains

1) Take the aliquoted soil sample and add it to a 50ml large centrifuge tube until it reaches the conical point of the conical tube.

2) Add sterilized water to 15ml, and put in 5-10 glass beads.

3) Break the soil sample with a vibrator.

4) Put it in a water bath at 80°C for 20 minutes.

5) Take 1.5ml of EP tubes, add 1ml of sterilized water to each tube, then take 10 microliters of bacteria solution from the 50ml tubes and add them to the EP tubes and mix well.

6) Spray 100 microliters from the EP tube into 1/2 LB medium and spread evenly.

7) Put it into a 30°C incubator and cultivate it for 2 to 3 days.

8) Microscopic observation.

crystal observation

Optical microscope:

Drop the mixture of cell crystals on the glass slide, smear evenly, dry and fix, stain with phenolic fuchsin staining solution for 3 minutes, rinse with water, and perform microscopic examination with a 100x oil microscope. For the preparation method of phenolic acid fuchsin staining solution, please refer to the literature (Baroy F, Lecadet M M, Deleluse A. Cloning and sequencing of three new putative toxin genes from Clostridium bifermentans [J]. Gene, 1998, 211: 293-295). See Figure 1. After being cultured on 1/2LB medium for 48 hours, a single colony was formed. The strain BTQ52-7 was observed under an optical microscope as a long rod, spores as oblong rods, and crystals as spherical.

Electron Microscopic Observation:

Scanning electron microscope sample preparation: the spore crystal mixture was dropped on a glass slide, dried, fixed by osmic acid, then dehydrated by alcohol gradient, critical point drying, ion sputtering gold (2nm thick), New Bio-TEM H-7500 scanning electron microscope Observe and take pictures. as shown in picture 2.

Biological measurements showed that the results of the primary screening bioassay on the black beetle were 100% corrected mortality, as shown in Table 1, and that it had certain biological activity on the larvae of the North China black beetle, as shown in Table 2. It has no insecticidal activity against Coleoptera pests Ulmus indigo beetle and Aeruginosa beetle.

Example 2. Obtaining new genes

2.1 Detection of strain BTQ52-7 by using cry8 gene universal primers, the primers are as follows

Amplification cycle: denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 4 minutes, 25 cycles, and finally extension at 72°C for 10 minutes.

The results are shown in Figure 3. After sequencing, nucleic acid and amino acid comparisons revealed that it was a new gene.

2. Cloning of cry8 gene in 1BTQ52-7 strain

The new cry gene in the strain was isolated and cloned by rapid cloning method.

A pair of primers for amplifying the full-length cry8 gene was designed with reference to the 5' and 3' sequences of the cry8 gene coding region published in GenBank. The primer sequences are as follows:

cry8N5: 5′-3′: TTACTCTTTCTTCTAACACGAGTTTCTACAC

cry8N3: 5′-3′: ATGAGTCCGAATAATCAAAACGAAT

Using the plasmid DNA of bacterial strain BTQ52-7 as a template, PCR full-length amplification was carried out with the full-length primers designed above, and a full-length 3.5kb cry8 full-length gene was amplified (see Figure 4), which is similar to the known cry8 class The gene profiles were different, suggesting that the strain may contain a new cry8 insecticidal gene.

After DNA recovery and connection with the expression vector pEB, the obtained recombinant plasmid was named pEB8, and its structure is shown in Figure 6.

Using pfuDNA polymerase, PCR amplification was performed with the following system.

10×PCR buffer 5μL

dNTP(10mM) 1μL

Primer pair (10mM) 1μL/pc

Template 1uL

pfuDNA polymerase (5U/μL) 0.5μL

Make up to 50 μL with ultrapure water, mix well and centrifuge.

Amplification cycle: denaturation at 94°C for 1 minute, annealing at 54°C for 1 minute, extension at 72°C for 4 minutes, 25 cycles, and finally extension at 72°C for 10 minutes.

The purified fragment was ligated with the vector pEB to transform Escherichia coli JM109 to obtain a positive transformant. The transformants were digested with enzymes, and a band of about 3.5kb was obtained, indicating that the new cry8 gene sequence had been successfully inserted. Sequencing analysis was performed on the inserted fragment, and the sequence SEQ ID NO 1 was obtained, with a full length of 3522bp, encoding a protein consisting of 1173 amino acids. After determination, its amino acid sequence is shown in SEQ ID NO 2.

2.2 Connection scheme

Carrier 0.1-0.2μg

Target fragment DNA 0.5-1.0μg

5×Ligation Buffer 2μL

T4 DNA Ligase 1μL

Make up the volume to 10 μL with ultrapure water, mix thoroughly, and connect at 16°C for 4 hours or overnight at 4°C.

2.3 Transformation plan

1. Pick a single colony and culture overnight in 5ml LB with shaking;

2. Inoculate in LB liquid medium according to 1% inoculum amount, culture at 37°C, 230rpm for 2-2.5hr, (OD ₆₀₀ =0.5-0.6);

Centrifuge at 4,000 rpm for 10 minutes at 3.4°C;

4. Discard the supernatant, add 50ml of pre-cooled 0.1M CaCl ₂ to suspend the cells, and place on ice for more than 30 minutes;

Centrifuge at 5.4°C, 4,000rpm for 10min, and recover the cells;

6. Resuspend the cells with 2-4ml of ice-cold 0.1M CaCl ₂ , aliquot into 200μl/0.5mL centrifuge tubes, and store at 4°C (it can be stored for one week).

7. Take 200 μl of competent cells and 5 μL of the ligation product, mix well, and ice-bath for 30 minutes.

8. Heat shock at 42°C for 1.5 minutes and ice bath for 3 minutes.

9. Add 800μl LB medium and incubate at 37°C for 45min.

10. Take 200 μl to smear the plate, add corresponding antibiotics, and IPTG, X-gal, and culture at 37°C.

2.4 Homology analysis

Genetic distance and homology analysis of SEQ ID NO2 and cry8 class genes: It shows that the genetic distance of SEQ ID NO2 and cry8Ca is the nearest 0.297, and the amino acid homology is 70.3%. The data are as follows, according to the classification standard of Bacillus thuringiensis (Crickmore N, D R Zeigler, J Feitelson, et al.1998.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.Microbiol.Mol Biol Rev.62:807～813) is a new gene, named cry8Na1 gene by the Bt insecticidal crystal protein nomenclature committee. The protein properties are as follows:

Protein Length Protein Length=1173,

The protein molecular weight is MW=132697.5,

The isoelectric point pI=4.74.

cry8Aa.txt 0

cry8Ab.txt 0.233 0

cry8Ba.txt 0.334 0.345 0

cry8Bb.txt 0.311 0.336 0.175 0

cry8Bc.txt 0.313 0.332 0.175 0.089 0

cry8Ca.txt 0.475 0.485 0.476 0.464 0.464 0

cry8Da.txt 0.356 0.393 0.456 0.441 0.442 0.353 0

cry8Ea.txt 0.340 0.323 0.281 0.260 0.266 0.459 0.432 0

cry8Fa.txt 0.349 0.338 0.354 0.349 0.353 0.481 0.452 0.307 0

cry8Ga.txt 0.435 0.454 0.436 0.448 0.452 0.346 0.373 0.447 0.436 0

cry8Ka.txt 0.372 0.374 0.376 0.363 0.363 0.446 0.461 0.371 0.369 0.388 0

cry8Ma.txt 0.464 0.477 0.507 0.482 0.490 0.513 0.490 0.491 0.486 0.514 0.498 0

SEQ.txt 0.453 0.456 0.467 0.449 0.450 0.297 0.371 0.453 0.449 0.320 0.425 0.509 0

Homology matrix of 13 sequences

cry8Aa.txt 100%

cry8Ab.txt 76.7% 100%

cry8Ba.txt 66.6% 65.5% 100%

cry8Bb.txt 68.9% 66.4% 82.5% 100%

cry8Bc.txt 68.7% 66.8% 82.5% 91.1% 100%

cry8Ca.txt 52.5% 51.5% 52.4% 53.6% 53.6% 100%

cry8Da.txt 64.4% 60.7% 54.4% 55.9% 55.8% 64.7% 100%

cry8Ea.txt 66.0% 67.7% 71.9% 74.0% 73.4% 54.1% 56.8% 100%

cry8Fa.txt 65.1% 66.2% 64.6% 65.1% 64.7% 51.9% 54.8% 69.3% 100%

cry8Ga.txt 56.5% 54.6% 56.4% 55.2% 54.8% 65.4% 62.7% 55.3% 56.4% 100%

cry8Ka.txt 62.8% 62.6% 62.4% 63.7% 63.7% 55.4% 53.9% 62.9% 63.1% 61.2% 100%

cry8Ma.txt 53.6% 52.3% 49.3% 51.8% 51.0% 48.7% 51.0% 50.9% 51.4% 48.6% 50.2% 100%

SEQ.txt 54.7% 54.4% 53.3% 55.1% 55.0% 70.3% 62.9% 54.7% 55.1% 68.0% 57.5 49.1% 100%

Embodiment 3, gene expression and activity assay

3.1 Plasmid DNA was extracted from the above clone, and transformed into the recipient strain Rosetta (DE3) to obtain an expression strain.

After IPTG induced expression, SDS-PAGE protein electrophoresis was performed.

The process of inducing expression is as follows:

1) Activated strains (37°C, 12hr);

2) 10% inoculated in LB medium (37°C, 2hr);

3) Add the inducer IPTG, 150rpm, and induce at 18-22°C for 4-20h at low temperature;

4) Collect the bacteria by centrifugation, add 10mM Tris Cl (pH 8.0) to suspend;

5) Broken bacteria (ultrasonic crushing is complete);

Centrifuge at 12,000rpm for 10min at 4°C;

Collect 10-15 μL each of the supernatant and the precipitate, and detect them by electrophoresis.

The polyacrylamide gel configuration is as follows.

8 % (ml) accumulating glue 5 % (ml)

Distilled water 4.6 2.7

30%Degassed Acrylamide 2.7 0.67

1.5M Tris(pH8.8) 2.5

1.0M Tris(pH6.8) 0.5

10%SDS 0.1 0.04

10%APS 0.1 0.04

TEMED 0.006 0.004

Sample loading: 10-15μl sample loading, electrophoresis: 130-150V constant voltage.

Staining and decolorization: Take out the gel after electrophoresis, wash it with distilled water, put it into the staining solution, shake it at 60 rpm for about 1 hour, decolorize it in the decolorization solution for about 2 hours, decolorize until the background of the gel is transparent, and rinse in water until the protein band is clear.

The detection results showed that the molecular weight of the expressed protein was about 130kD, and the results are shown in FIG. 5 .

3.2 Determination of the insecticidal activity of the protein encoded by the gene

The cry8Na1 gene expression protein is diluted with water to different concentrations, and the insecticidal activity on coleopteran pests is determined.

Determination of biological activity of dark beetle (H.parallela), large black beetle (H.oblita) and aeruginosa beetle (A.corpulenta):

According to the amount of protein contained in Bt dry powder, suspend in sterile water in a certain amount, dilute the above-mentioned suspension according to the 2-fold proportional gradient concentration, add it to the sterilized fine soil of potato shreds of uniform thickness, and mix evenly, with 5-7 Day-old black beetle and large black beetle larvae were used as the tested insect species, each treatment was inoculated with 30 worms, repeated three times, and the treatment of adding water was used as a blank control, and the number of dead insects was checked for 7 days and 14 days after infection, and calculated Adjusted for mortality.

Determination of indoor insecticidal activity against elm blue leaf beetle (P.aenescens):

Cut suitable elm leaves, wash, dry, infiltrate with the pre-diluted Bt bacterial solution for 10 seconds, take it out, dry naturally, put it into a petri dish, and lay a layer of soaked filter paper on the bottom of the petri dish in advance for Moisturize. Water treatment was used as a control. Inject 30 2-3 instar elm beetle larvae (dark brown) into each petri dish, repeat each treatment three times, cover it with a petri dish, place it at room temperature (25-28°C), and investigate for 96 hours Larval mortality.

Table 1 The detection results of Cry8Na1 protein on the black beetle

Table 2 The detection results of Cry8Na1 protein on the black beetle

Table 3 Cry8Na1 protein detection results on Ulmus indigo

Table 4 Cry8Na1 protein detection results on the A. aeruginosa beetle

Beneficial effects of the present invention: the Bt cry8Na1 gene sequence and its gene expression products isolated and cloned in the present invention can produce strong toxicity to coleopteran pests, and the combination of cry8Na1 gene and cry8Na1, cry8Na1, cry8Na1 and other gene expression products can expand the resistance to lepidoptera. Insecticidal spectrum of insect pests of order and Coleoptera. By being applied to transform microorganisms and plants to make them exhibit toxicity to related pests, it can overcome or delay insect resistance to engineering bacteria and transgenic plants.

sequence listing

Claims (8)

1. Bacillus thuringiensis strain BTQ52-7, its preservation number is CGMCC No.4188. 2. The application of the Bacillus thuringiensis bacterial strain BTQ52-7 described in claim 1 in killing the Coleopteran pest, and the Coleopteran pest is the black beetle or the North China large black beetle. 3. a cry8Nal gene that is isolated from the bacillus thuringiensis bacterial strain BTQ52-7 described in claim 1, its nucleotide sequence is as shown in SEQ ID NO1. 4. the insecticidal protein of cry8Nal gene coding described in claim 3, its aminoacid sequence is as shown in SEQ ID NO 2. 5. An expression vector, characterized in that it contains the cry8Nal gene as claimed in claim 3. 6. the expression carrier described in claim 5 is pEB 8, and its backbone carrier is pEB, and the structure of pEB 8 is as shown in Figure 6. 7. A microbial transformant, characterized in that it contains the cry8Nal gene according to claim 3. 8. The application of the insecticidal protein according to claim 4 in resisting coleopteran pests, wherein the coleopteran pests are black beetle or North China large black beetle.

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