WO2006053473A1 - A bacillus thuringiensis and genes with high larvicidal activity to coleopterans - Google Patents

Abstract

A Bacillus thuringiensis and genes with high larvicidal activity to coleopterans, belongs to the Biological Control field. The invention provides A Bacillus thuringiensis with high larvicidal activity to coleopterans, the nucleotide sequence of cry 8E and cry 8F as well as the amino acid sequence of the protein which the cry 8E and cry 8F encode for. The invention also provides the express production of the Bacillus thuringiensis cry8E and cry8F genes and both of them. Using or cooperative using the genes above or the combination of them, makes them have higher toxicity to coleopterans than the origin strains and the express productions of single gene, and expands the larvicidal spectra to lepidopterans, coleopterans, dipterans, and makes it show the toxicity to related pests, and overcomes or delays the resistance generation of the engineering strains and transgenic plants by using them to transform the microbe and plants. At the same time, the biological larvicidal agents preparing from the proteins which the cry 8E and cry 8F gene encode for, expand the application range of the protein.

Description

 High-efficiency Bacillus thuringiensis strains and genes for coleopteran pests

 The invention belongs to the technical field of biological control. The present invention relates to a strain of Bacillus thuringiensis which is active against a coleopteran pest, and relates to a nucleotide sequence of a highly virulence CA§£ and ctySF gene of a coleopteran pest and an amino acid sequence of a protein encoded thereby, involving synergy The cry8E and cr?F gene expression products were used in combination.

Background technique

 The scarab belongs to the genus Scarabaeidae, and its larvae (commonly known as 蛴螬, hereinafter referred to as "蛴螬") are an important class of underground pests that can harm food, cotton, oil crops, vegetables, Sugar crops, tobacco, pasture, flowers, turfgrass, fruit trees and many other plants. A large number of investigations have shown that the hazards of cockroaches in underground pests are the highest, among which larvae are mainly larvae and larvae, accounting for the total amount of underground pests.

70-80% or more. According to statistics, the annual occurrence of cockroaches in the country is about 100 million mu. In severe years, it has reached 300 thousand ounces of 'mu, and the loss of production is as high as 20%. Some plots are even out of production. In recent years, the area with the largest area and the largest amount of occurrence is the Huanghuaihai area, which mainly harms crops such as grain and oilseeds. The damage in other areas is also very serious, such as the damage to sugarcane, which is common in Guangdong, Guangxi, Yunnan, Sichuan, Fujian and other places. Occurred; in the western regions of Tibet, Qinghai, Gansu, Xinjiang, etc., the occurrence of cockroaches is also very serious (Wei Hongjun et al., "China Underground Pests", Shanghai: Shanghai Science and Technology Press, 1989, 1 -41; Wang Yongxiang, etc., "冀中"Types and Integrated Control Techniques in the Plain Area", Journal of Hebei Normal University (Natural Science Edition), 1998, 22(2): 268-270. The peanut planted in China's oil crops is second only to the rapeseed. For example, China's peanut production accounts for about 35% of the world's total peanut production, ranking first in the world, with an annual export revenue of 20.7 billion US dollars. In 2001, the national peanut area (5 million hectares) and total output (1450 ton tons) all reached history. The highest level. But the damage to peanuts is very serious. In order to control the hazards of cockroaches, agriculture, chemistry and materials are generally used. The comprehensive prevention and control strategy, although it has certain effects, is difficult to achieve the effect of continuous control. Therefore, finding new effective prevention methods has become a top priority.

 On the basis of obtaining the highly virulent Bt gene, the cultivation of acaricidal transgenic plants is a new way to prevent and cure.

 BaciU thw'ingie is, referred to as Bt), is an extremely widely distributed Gram-positive bacterium. It forms a protein-like parasporal crystal while forming spores, for Lepidoptera, Diptera, Coleoptei'a, Hymenoptera. , Homoptera, Orthoptera, various insects such as Mallophaga, and specific insecticidal activities of nematodes, mites and protozoa (Schnepf, EN et al, Microbiol. And Molecular Biology Review, 1998, 62:3 775-806). Insecticidal Crystal Proteins (ICPs), also known as delta-endotoxins, are harmless to humans and animals and do not pollute the environment. Therefore, Bt has been widely used in the biological control of pests.

More than 300 Bt insecticidal genes encoding insecticidal crystal proteins have been cloned, belonging to 141 model genes. In recent years, the research trend of genes in the world has attracted people's attention. Studies have shown that these genes have insecticidal effects on various coleoptera II pests such as the tortoise family, the genus Aphididae, and the genus. In 1992, Ohba et al. screened for the first time in the world a new strain (./. subsp. Japonensis BuiBui) with specific insecticidal activity against the larvae of the chafer (Ohba, M. et al., A unique isolate of Bacillus thuringiensis serovar). Japonensis with a high larvicidal activity specific for scarabaeid beetles, Letters in Applied Microbiology, 1992.14:54-57), in 1994, Sato et al. cloned a new insecticidal gene o Sa Sato, R. et al, Cloning, heterologous expression , and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeid insects, Cun: Microbiol. 1 994. 28: 1 5- 19.4). Nine genes have been discovered, and the encoded protein consists of 1 1 60-121 amino acids with a molecular weight between 12 and 1371 Da. For detailed information, see Table 1 (Asano, S., Yamanaka, S. and Takeuchi. . , Protein having insecticidal activity, DA encoding the protein, and controlling agent and controlling method of noxious organisms, 2002, JP 2002045186-A and JP 20020451 86-A/2)). Among them Mycogen's isolated Cry8Aa l and Cry8Bal have significant insecticidal activity against a variety of pests of the family Turtle (Tracy E. Michaels, et al., Bacillus (huringiensis toxins active against scarab pests, 1994, USP 5554534). The two genes, ciy8l3bl and crySBc!, were isolated and found to have significant insecticidal effects on Western com rootworm and have been used in transgenic insect-resistant maize (Abad, Andre, R. Duck Nicholas). , B. Feng, Xiang, Flannagan Ronald, D., ahn, Theodore, W., Sims, Lynne, E. Genes encoding novel proteins with pesticidal activity against coleopterans, 2002, WO 02/34774 A2). In China, Hebei Province In recent years, the Institute of Plant Protection of the Academy of Agricultural Sciences and the Agricultural University of Hebei have successively screened and obtained a number of Bt strains with specific insecticidal activity against the larvae of the yellow-breasted tortoise (Acw ex/ca) and the larvae of the genus A cw w/c /fl. The mortality rate of the test is 100% (Feng Shuliang et al., "A new strain of Bacillus thuringiensis with insecticidal activity against the larvae of the chafer", "Chinese Biological Control", 2000 16(2): 74-78 ).

 Table 1 Bacillus thuringiensis Cry8 insecticidal crystal protein

Summary of the invention

 The purpose of the invention:

基因所编码的蛋白质 Cry8Eal和 Cry8Fal， 与上述蛋白的氨基酸序列同源性不低于 83. 5%的氨基酸序列， 以及 含有 Cry8Eal蛋白或 /和 Cry8Fal蛋白的生物制剂， 扩大了该蛋白的应用范围。 The object of the present invention is to provide a Bacillus thuringiensis strain having high virulence to important coleopteran pests such as Diptera, Coleoptera, and two novel "≠E and a' _y 8F pattern gene sequences, and a pair of Diablo The combination of the cr≠E and cr _y 8F genes with significant synergistic effects, and the microorganisms and plants transformed with the genes a'y8E, crySF and their genes, which show toxicity to related pests and overcome and delay pests Resistance to engineered bacteria and transgenic plants is produced. At the same time, the present invention also provides a separate ώ

The proteins encoded by the genes, Cry8Eal and Cry8Fal, have an amino acid sequence homology of not less than 83.5%, and a biological preparation containing Cry8Eal protein or/and Cry8Fal protein, which expands the application range of the protein.

 The technical solution of the invention:

 1. Screening and identification of Bt strain 1 85 effective against underground pests of the golden tortoise

The present invention isolates Bt strain 1 85 by itself. The soil was collected from the apple orchard in Shunping County, Baoding City, Hebei Province. 0.1-0.2 g of soil sample was placed in a test tube containing 10 ml of sterilized water and glass beads, shaken on a vortex shaker for 3 minutes, the soil particles were broken, and then placed on a 200 rp _m shaker for 10 minutes, 75 In a water bath of °C water bath for 1 7 minutes, fully kill the non-spore bacteria. After a little rest, select three dilutions of I f) - ² 10- ³ 10_ ⁴ and draw 100 ul of bacterial suspension on the BP plate. On the top, the coating was uniform, and cultured at 37 ° C for three days, picking up a Bt colony smear microscopy. A strain of Bt containing a spherical body was found (see Figure 1).

The culture condition of the strain was 30, ordinary LB medium, pH 7.0. The strain is of the genus Bacillus (fi"c/7/wy) and Bacillus thuringiensis, and has been preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee. The retention period is January 1, 2004, and the preservation number is For CGMCC NO. I 242. The information about this strain has been identified as follows: It can form buds and form spherical parasporal crystals. SDS-PAGE electrophoresis indicates that the insecticidal crystal protein is about 130kDa (see Figure 2); its crystal protein is expressed at 14 hours, and the growth curve indicates that it has entered a stagnation period of 14 hours (see Figure 3), indicating that the crystal protein promoter may be spore-dependent; The test showed that the strain had obvious insecticidal effect on the underground pest of the genus Diptera, and the TF mortality rate was 90% in 7 days, and it also had certain insecticidal activity against the leaf beetle (see Table 2).

Table 2 Insecticidal activity of Bt strain 185

 A pair of universal arches were designed based on the conserved regions of the c y<? gene:

 S5un8: 5-' CGGCAAACTTAGTAGAATGC-3 '

 S3un8: 5-' CTGACTGATTTCCACCATCACG-3.

 Table 3 shows the homologous sequences of these genes and primers. Table 4 shows the amplification products of the ^.^ gene predicted by the pair of primers, and the size of the fragment, and these genes can be identified by this PCR-RFLP method.

 Table 3 Pairing of the conserved regions of the primers and cry8 genes and the location of the paired regions in the gene

 Pairing of S5un8/S3un8 with Conserved Regions of Genes

 Primer S5un8: Primer S3un8:

 Genotype

 5, -cggcaaacttagtagaat location 5, -gcactaccacc tttagtcagt gc-3, c-3

 crySAa cggcaaacttagtGgaatgc 2101-2120 cgtgatggtggaaatcaAtcag 3273-3294 crySBa cggcCaacttagtGgaatgc 2089-2108 cgtgatggtggaaatcaAtcag 3261-3282 crySBb cggcaaacttagtGgaatgc 2104-2123 cgtgatggtggaaacaAAcag 3276-3297 crySBc cggcaaacttagtGgaatgc 2116-2135 cgtgatggtggaaatcaAAcag 3288-3309 crySCa cggcaaacttaAtagaatgc 2092-2111 cgtgatggtgcaaatcagAcag 3282-3303 crySDo TAAAaaacttagtagaatgc 2044-2063 cgtgatggtgCGaatcagTcag 3234-3255 Note: "N" is an unpaired base.

 Table 4 PCR amplification products and restriction length polymorphisms

 PCR (S5un8/S3un8)

 Base product size &o0109I and digestion results

 Size (bp) Digested wil;h EccO\09l and Oral (bp) crySAa 1194 197, 42, 555

 crySBa 1194 639, 555,

 crySBb 1J94 639, 555

 crySBc 1194 639, 555

 crySCa 1212 197,92, 310, 613

 crySDa 1212 197, 92, 310, 613 Bt strain 185 was identified using the following PCR reaction system (50 ί):

 Add ultra-pure water to 50μί, mix and centrifuge, add paraffin oil 30μί.

Amplification cycle: 94 Ό denaturation for 1 minute, annealing at 54 ° C for 1 minute, 72 ° C for 4 minutes, 25 cycles, most Extend at 72 ° C for 10 minutes.

 The results (see Figure 4) show a different map from the known c'S gene, indicating that strain 185 may contain a new o insecticidal gene.

 3. Cloning of the gene in strain 185

 The total DNA of Bt strain was digested with Pstl and Kpnl, and two DNA fragment libraries were constructed with vector pBluescript SK(+). Then, two DA libraries were detected by primer S5un8/S3un8 (see Table 3) and PCR method to obtain two DNA libraries. The positive clones pSS.3612 and pSS162 were inserted into the lpkb fragment of llkb and the Kpnl fragment of 2. Okb, respectively (see Figure 5). The pSS3612 was digested with restriction enzymes, and the 7 kb Pstl and Kpnl double-cleavage fragments contained the full-length gene of cry8Eal. The fragment was subcloned with pBluescript SK(+) to obtain pSS3612-7, and the 4 kb Kpnl fragment was subcloned to obtain PSS3612- 4 (see Figure 6). The inserts of pSS36l2 4 and pSS3612-7 were sequenced. The sequence SEQ ID NO 1 was obtained.

 4. Sequence analysis of the crySEal gene

The sequence SEQ ID NO 1 is Pst and l ( _p n double-cut fragment in pSS3612, the full length of the sequence is 7276bps, and the analysis indicates that it contains two large open reading frames, the position of 0RF1 is 3658-7152, and the position of 0RF2 is 2799 — 3377.

 The position S of 0RF1 is 3658-7152, (; C content is 38.03%, encoding a protein consisting of 1164 amino acids. The amino acid sequence is determined as shown in SEQ ID NO 2. Homology analysis indicates that the protein has a Cry8 protein Higher homology, Table 5 is its homology data. The amino acid homology with the known Cry8 protein is less than 78%, and the highest is only 58.2% (Cry8Bbl), which is named by the Bt insecticidal crystal protein nomenclature committee. Cry8EaL.

 Table 5 Cry8 protein homologous comparison data

 The present invention further analyzed the amino acid composition of the CrySEal protein (see Table 6, Figure 7), and found that its molecular weight was 131.56 kDa and the isoelectric point was pH 4.735 (see Figure 8).

 Table 6 Amino acid composition of CrySEal protein

 5. CT. '/3⁄4/gene cloning

The inserted sequence of PSS162 plasmid was analyzed. The fragment was 2.3 kb in length and had a complete 3' end sequence. It was completely homologous to the C <3⁄43⁄47 sequence, but the 5' end was quite different, and the complete reading frame was lacking. Sequence specificity Section design 7 1 pair of primers (5-185-Kpn 1: TTGGTATGGCGTTTCGTTG; and 3-185-Kpnl: TATTGCAGGTCCAGGATTCAC) for cloning the full-length gene, and the plasmid DNA of strain 185 was digested with Xbal, and the vector pBluescript SK (+) ligation, screening for a positive clone _P SS266 inserted into an exogenous fragment of about 9Kb (see Figure 9), and further digestion analysis showed that the 3. Okb fragment produced by Clal digestion contains a 5' reading frame (see attached Figure 10), the fragment was subcloned with pBluescript SK (+), the positive clone was named pSS266-3, the fragment was sequenced, and the resulting sequence was spliced with the inserted sequence in PSS162 to obtain a 3.9 kb fragment.

 6. Sequence analysis of c ^ genes

 The above 3.9 kb nucleic acid fragment was sequenced, and the obtained nucleotide sequence was as shown in SEQ [D NO 3 ]. Analysis of this sequence revealed that the sequence contains a large open reading frame 357-3878: GC content of 36.88%: a protein encoding 1174 amino acids (the amino acid sequence of the encoded protein is set forth in SEQ ID NO: 4 Show). Further homology analysis indicated that the protein has high homology with Cry8 proteins (see Table 5 above for homology data). Since the amino acid homology with the known C.ry8 protein is less than 78% and the highest is only 64.8% (Cry8Eal), the protein is named Cry8Fal by the Bt insecticidal crystal protein nomenclature committee.

 The amino acid composition of the Cry8Fal protein was further analyzed by the bioanalytical software Bioedit, and the results are shown in Table 1 and Figure 11. The results showed that the protein had a molecular weight of 133.08 kDa and an isoelectric point of pH 4.565 (see Figure 12).

 Table 7 Amino acid composition of CrySEal protein

 7. 0 (5 £ and cr^ gene expression

According to the full-length sequence of the cloned cr _y 8EaJ and crySFal genes, the present invention designed a primer for expressing two genes, and the sequence is as follows:

 8E1: CGCGGATCC (Bam HI) GATGAGTCCAAATAATCAAAATG

 8E2: ACGCGTCGAC (Sal I) CTCTACGTCAACAATCAATCAATTC

 8F1: CGCGGATCC (Ba HI) GATGAGTCCAAATAATCAAAATG 8F4: CCGCTCGAG (Xlw I) CTCTACGTCAACAATCAATCAATTC

 Primers 8E1 and 8E2 were introduced into the B-Hi and Sal\ loci, respectively. The full-length gene was amplified by using the full-length crySEal pSS3612 plasmid DNA as a template, inserted into the Bt expression vector pSXY422b, transformed into Escherichia coli SCS11O, and the plasmid was extracted. The Bt crystalless mutant HD-73-transformed from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, which can be supplied to the public, was obtained to obtain the engineering strain BioT8E.

因均获得了表达， 表达物的分子量为 130kDa左右。 Since there is a β-continuation HI tangent point in the full-length gene of crySFcd, this site was mutated by overlapping PCR, and point mutations (lined part) were introduced in overlapping primers 8F2 and 8F3. Items 8F1 and 8F4 were introduced into BamHi and Xhoi, respectively. The pSS266 plasmid DNA containing the full-length ay 8 Fa! gene was used as a template to amplify 0.3 kb and 3.1 kb products with 8F1 and 8F2, 8F3 and 8F4, respectively, and then use them as templates to amplify with primers 8F1 and 8F4, respectively. A full-length gene of 3.4 kb was obtained, inserted into the Bt expression vector pSXY422b, transformed into E. coli SCSU 0, and the plasmid was extracted and transformed into Bt crystalless mutant HD-73 by electroporation to obtain the engineering strain BioT8F. The above two engineering bacteria 3CTC were cultured for 30 hours in beef extract medium (peptone 5 g, beef extract 3 g, glucose 10 g, water 1000 mL, 121 ° C, 20 minutes autoclaving), and 500 μί bacteria solution was taken. To Eppendorf, ultrasonic disruption for 30 seconds (B. Braun U Labsonic, 230V, T«, =0.5 seconds); Add ίμί to 25μί with 0.5Ν NaOH, 25 °C for 5 minutes: add 65μί 3 X sample buffer Solution (925 μί loading buffer + 75 β-mercaptoethanol), boil for 5 minutes at 100 。. The precipitate was removed by centrifugation. The lOuL was loaded for SDS-PAGE electrophoresis analysis (for a method, see Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989). The results (see Figure 13) indicate that the engineered bacteria Biot8E and Biot8F

Since the expression was obtained, the molecular weight of the expression was about 130 kDa.

 8. Determination of activity of Cry8E and Cry8F proteins

 Bt engineering strains were inoculated on common bacterial agar medium flask for 3 days. The recipient strain HD-73_ was inoculated on a common bacterial agaric flask medium for 4 days. The culture was washed, diluted 2 times in gradient concentration, and 40 ml of the bacterial suspension was added to 200 g of fine soil (ultraviolet sterilized) having uniform thick and thin potato silk, and mixed to maintain the soil water content at 18% - 20%. The dark scorpion golden tortoise was connected to 20 larvae of 15 days old. The treatment with adding clear water was used as a blank control, and 28 Ό was infected. The number of dead insects was checked in 14 days, and LC was calculated. The results are shown in Table 8, indicating that the engineered strain has a very high toxicity to the dark scorpion golden tortoise. Both the Cry8E and CrySF proteins expressed have the activity of killing the larvae of the black scorpion.

 Table 8 Toxicity determination of Bt engineering bacteria and 185 strains against the larvae of the dark cockroach

 Culture preservation information:

 Name of the strain: Bacillus, Bacillus thuringiensis, Bacillus thuringiensis

 Depository: China Microbial Culture Collection Management Committee General Microbiology Center

 Date of deposit: November 2004 5曰

 Deposit number: CGMCCN0.1242

DRAWINGS

 Figure 1: Spores and crystal morphology of 185 strains.

 Figure 2: Crystalline protein of 185 strain SDS-PAGE analysis.

 Figure 3: Growth curve of 185 strain.

 Figure 4: PCR-RFLP map of strain 185. among them:

 M. DNA molecular weight standard

 1. PCR product, 2. PCR product digestion

Figure 5: PCR amplification products and restriction analysis of recombinant plasmids pSS 162 and pSS3162. among them:

 M. λ XMKIEco 1301

 1. pSS162 PCR product, 2. pSS3162 PCR product, 3. pBIueScript SK (+) / Kpnl

4. pSS162/ Kpnl, 5. pSS3l62/ 3⁄4iI

Figure 6: Subcloning analysis of the pSS3612 insert. among them:

 M. XOHMEco 1301

 1. pBIueScript S (+) /Kpnl , 2. pSS3162/ Pstl+ Kpnl, 3. pSS3612/ Kpnl

4. pSS3: 162 / / /; I + / w7: [

Figure 7: Amino acid composition of the CrySEal protein.

Figure 8: Titration curve of Cry8Eal protein.

Figure 9: PCR amplification product and restriction analysis of recombinant plasmid pSS266. among them:

 M. λ U MEco 1301

1. pS$266 PCR product, 2. pSS162/ Xbal, 3. pBIueScript SK(+)/M Figure 10: Enzyme digestion analysis of recombinant plasmid PSS266. among them:

1. Xbal, 2. Notl, 3. Pstl, 4. Ndel, 5. Clal, 6. Sad, 7. Kpnl

Figure 11: Amino acid composition of the Cry8Fal protein.

Figure 12: Titration curve of Cry8Fal protein.

Figure 13: Expression of the cry8Eal and crySFaJ genes in Bt amorphous mutants. among them:

 M. Protein molecular weight standard

 1. Biot8E, 2. Biot8F, 3. Bt 185, 4. HD-73-

 Embodiments in accordance with embodiments of the present invention are described below. It should be noted that the embodiments of the present invention are illustrative only and not limiting.

 Example 1. Screening and Identification of Alfalfa Strain 185 Effective for Underground Pests

The soil was collected from the apple orchard in Shunping County, Baoding City, Hebei Province. 0.1-0.2 g of soil sample was placed in a test tube containing 10 ml of sterilized water and glass beads, shaken on a vortex shaker for 3 minutes, the soil particles were broken, and then placed on a 200 rpm shaker for 10 minutes, 75 ° C Water bath in a water bath for 17 minutes, fully kill the non-spore bacteria, wait for a little static _, then select 10 - ² , 10_ ³ , 10- ⁴ three dilutions, respectively, take lOOul bacterial suspension on the BP plate, painted The cloth was evenly distributed and cultured at 37 ° C for three days, and a Bt colony smear microscopy was picked. A strain of Bt containing spherical crystals was found (see Figure 1).

 The strain is a species of Bacillus, Bacillus thuringiensis. The strain was submitted to the General Microbiology Center of the China Microbial Culture Collection and Management Committee for preservation. The date of deposit is November 5, 2004, and the deposit number is CGMCCN0.1242. The information about the strain was identified as follows: It can form spores and form spherical parasporal crystals. SDS-PAGE electrophoresis showed that the insecticidal protein was about 130kDa (see Figure 2), and its crystal protein was in 14 hours. The initial expression, and the growth curve indicates that it has entered the stagnation period for 14 hours (see Figure 3), indicating that the proteoprotein promoter may be spore-dependent; biological assays indicate that the strain is against the underground pest of the tortoise family. The golden tortoise has obvious insecticidal effect, and the corrected mortality rate is 90% in 7 days, and it also has certain insecticidal activity against the coconut leaf beetle.

 Example 2. Identification of cr_y gene in strain 185

 A pair of universal bows are designed according to the conserved regions of the gene-like genes.

 S5un8: 5-' CGGCAAACTTAGTAGAATGC-3 '

 S3un8: 5-' CTGACTGATTTCCACCATCACG- 3.

， 最 后 72°C延伸 10分钟。

, last 10 minutes at 72 ° C.

 The results (see Figure 4) show a different map from the known TJ^ gene, indicating that strain 185 may contain new insecticidal genes.

 Example 3, cloning of cry8E gene in strain 185

The total DNA of Bt strain was digested with Pstl and Kpnl, and two DNA fragment libraries were constructed with vector pBIuescript SK (+). Then, two DNA pools were detected by primer S5un8/S3un8 and PCR, and two positive clones pSS3612 and pSS162 were obtained. , insert a lpkb fragment of llkb and a Kpnl fragment of 2. Okb, respectively (see Figure 5). The pSS3612 was digested with restriction enzymes, and the 7 kb Pstl and Kpnl double-cleavage fragments contained the full-length gene of cry8Eal. The fragment was subcloned with pBIuescript S (+) to obtain PSS3612-7, and the 4 kb Kpnl fragment was subcloned to obtain pSS3612- 4 (such as Figure 6). The inserted sequences of pSS3612-4 and pSS3612-7 were sequenced to obtain the sequence SEQ ID NO 1. The sequence was a double-cut fragment of Pst. and Kpnl in pSS3612. The full length of the sequence was 7276 bp _{S. The} analysis indicated that it contained two large open reading frames. The position of 0RF1 was 3658-7152, and the position of 0RF2 was 2799-3377.

 The position of 0RF1 is 3658-7152, the GC content is 38.03%, and the protein of 1164 amino acids is encoded, and the amino acid sequence thereof is determined as SEQ ID NO 2. Homology analysis indicated that the protein has high homology with Cry8 proteins (see Table 5). Since the amino acid homology with the known Cry8 protein is less than 78%, the highest is only 58.2% (Cry8Bbl), and it is named Cry8Eal by the Bt insecticidal crystal protein nomenclature committee.

 The present invention further analyzed the amino acid composition of the Cry8Eal protein (see Table 6 and Figure 7) and found that its molecular weight was 131.56 kDa and the isoelectric point was pH 4.735 (see Figure 8).

 Example 4. Cloning of cr. <?/3⁄47 gene

The inserted sequence of PSS162 plasmid was analyzed. The fragment was 2.3 kb in length and had a complete 3' end sequence. It was completely homologous to the _C <3⁄43⁄47 sequence, but the 5' end was quite different, and the complete reading frame was lacking. A pair of primers (5-185-KpnI: TTGGTATGGCGTTTCGTTG; and 3-185-Kpnl: TATTGCAGGTCCAGGATTCAC) were designed to clone the full-length gene, and 185 plasmid DNA was digested with Xbal, and the vector pBluescript SK was designed. (+) ligation, screening for a positive clone pSS266 inserted into an exogenous fragment of about 9Kb (see Figure 9). A further restriction analysis revealed that the 3.0 kb fragment generated by Clal digestion contained a 5' reading frame (see Figure 10). The fragment was subcloned with pBluescript SK (+), and the positive clone was named pSS266-3. The fragment was sequenced, and the resulting sequence was spliced with the inserted sequence in PSS162 to obtain a 3.9 kb fragment. The nucleic acid fragment was sequenced to obtain a nucleotide sequence as shown in SEQ ID NO 3. Analysis of the sequence revealed that the sequence contains a large open reading frame 357-3878; GC content is 36.88%; a protein encoding 1174 amino acids (the amino acid sequence of the protein is shown in SEQ ID NO 4) . A further homologous analysis indicated that the protein has high homology to the Cry8 class of proteins (see Table 5). The amino acid homology with the known Cry8 protein is less than 78%, and the highest is only 64.8% (Cry8Eal). This protein is named Cry8Fal by the Bt insecticidal protein nomenclature committee.

The present invention further analyzed the amino acid composition of the Cry8Fal protein using bioanalysis software Bioedit (see Table 7 and Figure 11). The results showed that the protein had a molecular weight of 133.08 kDa and an isoelectric point of pH 4.565 (see Figure 12). · Example 5, £^ and expression of the CTW gene'· Based on the full-length sequences of the cloned c'y8Ea and c'≠F _a genes, primers for expression of the two genes were designed. The sequences are as follows:

 8E I: CGCGGATCC (Bam HI) GATGAGTCCAAATAATCAAAATG

 8F1: CGCGGATCC (Bam HI) GATGAGTCCAAATAATCAAAATG

8F4: CCGCTCGAG (Xho I) CTCTACGTCAACAATCAATCAATTC

 Primers 8E1 and 8E2 were introduced into the ZtonHI and Sal\ sites, respectively, to the full-length ay8Eal pSS3612 plasmid.

DNA was used as a template to amplify the full-length gene, inserted into the Bt expression vector _P SXY422b, transformed into Escherichia coli SCS110, and the plasmid was extracted and electroporated into Bt crystalless mutant HD-73 to obtain the engineering strain BioT8E.

 Since there is a Z^roHI cut-point in the full-length gene of ay8Fal, this site was mutated by overlapping primer PCR, and point mutations (lined part), primers 8F1 and 8F4 were introduced in overlapping primers 8F2 and 8F3. Introduced BatnHl and Xhol, respectively. Using the pSS266 plasmid DNA containing the full-length 'ySFal gene as a template, the 0.3 kb and 3.1 kb products were amplified with 8F1 and 8F2, 8F3 and 8F4, respectively, and they were used as templates, respectively, and amplified by primers 8F1 and 8F4, respectively. The full-length gene of kb was inserted into the Bt expression vector pSXY422b, transformed into Escherichia coli SCSI 10, and the plasmid was extracted and electroporated into Bt-free mutant strain HD-73 to obtain the engineering strain BioT8F.

 The above two engineering bacteria were cultured in beef extract medium for 30 hours at 30 ° C, and 500 μί bacteria liquid was taken until

In an Eppendorf tube, ultrasonically disrupted for 30 seconds (B. Braun U Labsonic, 230V, T _f ,, _ll = 0.5 sec); ΙΟΟμ added 25μί with 0.5Ν NaOH, 25°C for 5 minutes; add 65 iL 3 X Sample buffer (925 L loading buffer - 5 μL β-mercaptoethanol), boil at 100 ° C for 5 minutes. The precipitate was removed by centrifugation. 10μL for SDS-PAGE electrophoresis analysis The result. The results (see Figure 13) indicate that both the _C r_y Ea7 and c ^ genes in the engineered organisms Biot8E and Biot8F were expressed, and the molecular weight of the expression was about 130 kDa.

 Example 6. Determination of activity of Cry8E and Cry8F proteins

The Bt engineering strain was inoculated on a common bacterial agar medium flask for 3 days. The recipient strain HD-73- was inoculated on a common bacterial agaric flask medium for 4 days. The culture was washed, diluted 2 times in gradient concentration, and 40 ml of the bacterial suspension was added to 200 g of fine soil (ultraviolet sterilized) having uniform thick and thin potato silk, and mixed to maintain the soil water content at 18% to 20%. Twenty-five-day-old larvae of the dark-spotted golden tortoise were connected to the larvae, and the treatment with adding clear water was used as a blank control, and 28 Ό was infected, and the number of dead insects was checked 4 days, and LC _{5 was} calculated. The results (see Table 8) indicate that the engineered strain has extremely high toxic activity against the dark scorpionfish. Both of the expressed Cry8E and CrySF proteins have the activity of killing the larvae of the black cockroach.

Claims

Claim I. A Bacillus thuringiensis strain BaclUus thuringie is ), which is deposited under the Chinese Microbial Culture Collection Management Committee General Microbiology Center as CGMCC No. 1242. 2. Use of the strain of claim 1 for killing coleopteran pests. 3. A _C A§E gene isolated and cloned from the Bacillus thuringiensis strain of claim 1, wherein the gene has the nucleotide sequence shown in SEQ ID NO: 1.  A Cry8Eal protein characterized in that the protein is encoded by the gene of claim 3 and has the amino acid sequence of SEQ ID NO: 2. 5%。 The amino acid sequence of the amino acid sequence of the CrySEa protein of claim 4 having an amino acid sequence of not less than 83.5%.  A c ^ F gene isolated and cloned from the B. thuringiensis strain according to claim 1, wherein the gene has the nucleotide sequence shown in SEQ ID NO: 3. An Oy8F _a l protein characterized in that the protein is encoded by the gene of claim 5 and has the amino acid sequence of SEQ ID NO: 4.  A 5% homology of the amino acid sequence of the Cry8Fal protein of claim 7 is not less than 83.5%.  A method for enhancing the toxicity to a coleopteran pest, characterized in that the method uses the cry8E. gene of claim 3 in combination with the gene of claim 5. 10. A transgenic microorganism characterized by the transformation of the gene of claim 3 into a microorganism.  I I. A transgenic microorganism characterized by the transformation of the gene of claim 6 into a microorganism.  12. A transgenic microorganism characterized by the transformation of the y8E gene of claim 3 and the cr^F gene of claim 6 into a microorganism.  13. A transgenic plant characterized by the transformation of the rySE gene of claim 3 into a plant. A transgenic plant characterized in that the plant is transformed with the method of claim 6.

gene.  15. A transgenic plant characterized by the transformation of the gene of claim 3 and the c^F gene of claim 6 into a plant.  16. A biological preparation comprising the C:ry8Eal protein of claim 4.  A biological preparation comprising the CrySFal protein of claim 7. A biological preparation comprising the Cry8Eal protein of claim 4 and the Cry8Fa1 protein of claim 7.