WO2006053473A1 - Souche de bacillus thuringiensis et genes a haute activite larvicide sur les coleopteres - Google Patents

Souche de bacillus thuringiensis et genes a haute activite larvicide sur les coleopteres Download PDF

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WO2006053473A1
WO2006053473A1 PCT/CN2005/001133 CN2005001133W WO2006053473A1 WO 2006053473 A1 WO2006053473 A1 WO 2006053473A1 CN 2005001133 W CN2005001133 W CN 2005001133W WO 2006053473 A1 WO2006053473 A1 WO 2006053473A1
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gene
protein
amino acid
genes
strain
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French (fr)
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Fuping Song
Jie Zhang
Shuliang Feng
Dafang Huang
Rongyan Wang
Zhihong Lang
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Biotechnology Research Institute of CAAS
Institute of Plant Protection of CAAS
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Biotechnology Research Institute of CAAS
Institute of Plant Protection of CAAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • 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.
  • the scarab belongs to the genus Scarabaeidae, and its larvae (commonly known as ⁇ , hereinafter referred to as " ⁇ ") 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.
  • ⁇ larvae
  • 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.
  • 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.
  • 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.
  • 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.
  • ICPs Insecticidal Crystal Proteins
  • 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.
  • the present invention also provides a separate ⁇
  • 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 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.
  • 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).
  • 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.
  • 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
  • crySDa 1212 197, 92, 310, 613 Bt strain 185 was identified using the following PCR reaction system (50 ⁇ ):
  • Amplification cycle 94 ⁇ 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.
  • results show a different map from the known c'S gene, indicating that strain 185 may contain a new o insecticidal gene.
  • 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.
  • 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.
  • 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).
  • PSS162 plasmid 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.
  • 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.
  • 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).
  • the present invention designed a primer for expressing two genes, and the sequence is as follows:
  • 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.
  • 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 ⁇ bacteria solution was taken. To Eppendorf, ultrasonic disruption for 30 seconds (B.
  • 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 ⁇ 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.
  • 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:
  • Figure 5 PCR amplification products and restriction analysis of recombinant plasmids pSS 162 and pSS3162. among them:
  • 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:
  • Figure 11 Amino acid composition of the Cry8Fal protein.
  • Figure 12 Titration curve of Cry8Fal protein.
  • 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.
  • a pair of universal bows are designed according to the conserved regions of the gene-like genes.
  • S3un8 5-' CTGACTGATTTCCACCATCACG- 3.
  • results show a different map from the known TJ ⁇ gene, indicating that strain 185 may contain new insecticidal genes.
  • 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 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).
  • 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.
  • 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:
  • 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.
  • primers 8F1 and 8F4 were introduced in overlapping primers 8F2 and 8F3. Introduced BatnHl and Xhol, respectively.
  • 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 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 ⁇ was infected, and the number of dead insects was checked 4 days, and LC 5 was calculated.
  • the results 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.

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Description

对鞘翅目害虫高效的苏云金芽孢杆菌菌株和基因 技术领域
本发明属于生物防治技术领域。本发明涉及一种对鞘翅目害虫具有活性的苏云金芽孢 杆菌菌株， 涉及对鞘翅目害虫高毒力的 C A§£和 ctySF基因的核苷酸序列和分别由其编 码的蛋白质的氨基酸序列， 涉及协同组合使用 cry8E与 cr ?F基因表达产物。
背景技术
金龟子属于鞘翅目余龟总科 （Scarabaeidae ) , 其幼虫 （俗称蛴螬， 本发明以下也简 称为 "蛴螬"）是一类重要的世界性分布地下害虫， 可危害粮食、 棉花、 油料作物、 蔬菜、 糖料作物、 烟草、 牧草、 花卉、 草坪草、 果树等多种植物。 大量调查表明， 蛴螬在地下 害虫中的危害居首位， 其中主要以鳃金龟科和丽金龟科幼虫为主， 占总地下害虫量的
70- 80%以上。 据统计每年全国蛴螬发生面积约 1亿亩， 严重年份曾达 3亿 2千力'亩， 产 量损失高达 20%以上， 有些地块甚至绝产。 近年发生面积最大、 发生量最多的为黄淮海 地区， 主要危害粮食、 油料等作物； 其它地区的危害情况也很严重， 如危害甘蔗的蛴螬， 在广东、 广西、 云南、 四川、 福建等地普遍发生； 在西藏、 青海、 甘肃、 新疆等西部地 区， 蛴螬的发生也很严重 （魏鸿钧等， 《中国地下害虫》， 上海： 上海科学技术出版社， 1989 , 1 -41 ; 王永祥等， "冀中平原区蛴螬种类及综合防治技术"， 《河北师范大学学报》 (自然科学版）， 1998 , 22(2):268-270 以在我国油料作物中种植面积仅次于油菜居第 二位的花生为例。 我国的花生产量占世界花生总产量的 35%左右， 居世界首位， 年出口 收入达 207亿美元， 2001年全国花生面积 （500万公顷） 和总产 （1450力吨） 均达到历 史最高水平。 但蛴螬对花生的危害十分严重。 为控制蛴螬的危害， 一般采用农业、 化学、 物理等综合防治策略， 这虽有一定成效， 却难以达到持续控制的效果。 因此， 寻找新的 有效防治方法， 己成为当务之急。
在获得对蛴螬高毒力 Bt基因的基础上， 培育杀蛴螬的转基因植物是一条值得探索的 新的防治途径。
苏云金芽孢杆菌 BaciU thw'ingie is, 简称 Bt )是一种分布极其广泛的革兰氏阳性 细菌。 它在形成芽孢的同时， 能产生蛋白性质的伴孢晶体（parasporal crystal ) , 对鳞翅目 ( Lepidoptera)、 双翅目 （Diptera)、 鞘翅目 （ Coleoptei'a )、 膜翅目 .（Hymenoptera)、 同翅 目 （Homoptera)、 直翅目 C Orthoptera ) 食毛目 ( Mallophaga ) 等多种昆虫， 以及线虫、 螨类和原生动物具有特异性的杀虫活性 (Schnepf, E.N. et al , Microbiol. And Molecular Biology Review , 1998, 62:3 775-806)。这种杀虫晶体蛋白（ Insecticidal Crystal Proteins, ICPs ) 又称 δ-内毒素 （delta-endotoxin ) , 对人畜无害， 不污染环境， 因而 Bt在害虫的生物防治 中得到了最广泛的应用。
目前人们已经克隆了 300多种编码杀虫晶体蛋白的 Bt杀虫基因， 它们分属 141种模 式基因。近年国际上 类基因的研究动向引人瞩目。研究表明， 这类基因对余龟子科、 象甲科、 叶甲科等多种鞘翅 II害虫具有杀虫作用。 1992年， Ohba等在世界上首次从 菌株中筛选出对金龟子幼虫具有特异杀虫活性的新菌株 （ ./. subsp. Japonensis BuiBui ) (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) , 1994年 Sato等从中克隆出一种新的杀虫基因 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)。 目前已发现 9种基因， 编码的蛋白由 1 1 60- 1 21 0个氨基酸组成， 分子量在 l 28- 1371 Da之间。 详细的信息见表 1 ( Asano, S., Yamanaka,S. and Takeuchi. ., Protein having insecticidal activity, D A encoding the protein, and controlling agent and controlling method of noxious organisms, 2002, JP 2002045186-A and JP 20020451 86-A/2) )。 其中奕国 Mycogen 公司分离的 Cry8Aa l 和 Cry8Bal 对余龟科的多种害虫具有明显的杀虫活性 (Tracy E. Michaels, et al., Bacillus (huringiensis toxins active against scarab pests, 1994, USP5554534)。 美国从 Bt菌株中分离了两种基因 ciy8l3bl和 crySBc!基因， 发现对西方 玉米根叶甲 （Western com rootworm )具有显著的杀虫效果并已用于转基因抗虫玉米的丌 发 (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)。 在我国， 河北省农业科学院植物保护研究所和河北 农业大学近年来先后筛选获得多株对黄褐丽金龟 akt exokta)和铜绿丽余龟 (A cw w/c /fl)幼虫具有特异杀虫活性的 Bt菌株，室内生测死亡率均达 100 % (冯书亮等， "一 株对金龟子类幼虫具有杀虫活性的苏云金杆菌新分离株"， 《中国生物防治》， 2000 16(2)： 74-78)。
表 1 苏云金芽孢杆菌 Cry8类杀虫晶体蛋白
Figure imgf000004_0002
发明内容
本发明的目的：
本发明的目的是提供对暗黑鳃金龟、椰心叶甲等鞘翅目重要害虫具有高毒力的一种苏 云金芽孢杆菌菌株和两种新的 "≠E和 a'y8F模式基因序列， 以及一种对暗黑鳃金龟具 有明显增效作用的 cr≠E和 cry8F基因组合， 以及基因 a'y8E、 crySF及其基因组合转化 的微生物和植物， 使之表现出对相关害虫的毒性， 并克服、 延缓害虫对工程菌和转基因 植物的抗药性产生。 同时本发明还提供一种分别 ώ
Figure imgf000004_0001
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。
本发明的技术方案：
1 . 对鳃金龟地下害虫有效的 Bt菌株 1 85的筛选与鉴定
本发明自行分离了 Bt菌株 1 85。 土壤采自河北省保定市顺平县苹果园。 取 0.1— 0.2g 土样放入装有 10ml灭菌水和玻璃珠的试管中，在旋涡振荡器上振荡 3分钟，将土粒打碎， 然后放于 200rpm摇床上振荡 1 0分钟， 75 °C水浴锅中水浴 1 7分钟，充分将非芽孢菌杀死， 待稍静置后， 选取 I f)-2 10-3 10_4三个稀释度， 分别吸取 l OOul菌悬液于 BP平板上， 涂布均匀， 于 37°C培养三天， 挑取似 Bt菌落涂片镜检。 发现一株含有球形品体的 Bt菌 株 （见附图 1 )。
该菌株的培养条件为 30 ，普通 LB培养基， pH7.0。该菌株为芽孢杆菌属（fi"c/7/w.y)、 苏云金芽孢杆菌种， 并已在中国微生物菌种保藏管理委员会普通微生物中心保藏， 保藏 闩期为 2004年 1 1月 5日， 保藏编号为 CGMCC NO. I 242。经鉴定， 关于该菌株的信息包 括： 能形成芽抱， 同时能形成球形伴孢晶体， SDS-PAGE 电泳表明其杀虫晶体蛋白约为 130kDa (见附图 2); 其晶体蛋白在 14小时幵始表达， 而生长曲线表明其 14小时己进入 停滞期 （见附图 3)， 表明该晶体蛋白启动子可能为依赖芽孢形成的； 生物学测定表明， 该菌株对金龟科地下害虫暗黑鳃金龟具有明显杀虫作用， 7天校 TF死亡率达 90%， 对椰 心叶甲也具有一定的杀虫活性 （见表 2)。
表 2 Bt菌株 185的杀虫活性
Figure imgf000005_0001
根据 c y<?类基因保守区设计了一对通用弓 I物：
S5un8: 5-' CGGCAAACTTAGTAGAATGC-3 '
S3un8: 5-' CTGACTGATTTCCACCATCACG-3。
表 3是这些基因与引物的同源序列， 表 4是用这对引物预测的 ^.^基因扩增产物， 以及酶切片段大小， 通过这种 PCR— RFLP方法可以分别鉴定出将这些基因。
表 3 引物与 cry8各基因的保守区配对情况及配对区在基因上的位置
引物 S5un8/S3un8与基因的保守区配对情况
引物 S5un8: 引物 S3un8:
基因型
5、 -cggcaaacttagtagaat 位置 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 注： "N" 为不配对碱基。
表 4 的 PCR扩增产物和限制性酶切长度多态性
PCR (S5un8/S3un8)
基 型 产物大小 &o0109I和 酶切结果
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 用下列 PCR反应体系 （50 ί) 鉴定了 Bt菌株 185:
Figure imgf000005_0002
超纯水补至 50μί， 混匀离心， 加石蜡油 30μί。
扩增循环： 94Ό变性 1 分钟， 54°C退火 1 分钟， 72°C延伸 4分钟， 25个循环， 最 后 72°C延伸 10分钟。
结果 （见附图 4) 显示与己知 c' S类基因的图谱不同， 表明菌株 185中可能含有新 的 o 杀虫基因。
3. 菌株 185中 基因的克隆
用 Pstl和 Kpnl酶切 Bt菌株总 DNA,用载体 pBluescript SK(+)分别建立了两个 DNA 片段库， 然后用引物 S5un8/S3un8(见表 3)和 PCR方法检测两个 D A库， 得到两个阳性克 隆 pSS.3612和 pSS162, 分别插入 llkb的 pstl片段和 2. Okb的 Kpnl片段 (见附图 5)。 酶切分析 pSS3612， 7kb 的 Pstl 和 Kpnl 双酶切片段含有 cry8Eal 的全长基因， 用 pBluescript SK(+)亚克隆该片段， 得到 pSS3612-7, 同时亚克隆了 4kb的 Kpnl片段中， 得到 PSS3612- 4(见附图 6)。将 pSS36l2 4和 pSS3612-7的插入序列测序。得到序列 SEQ ID NO 1。
4. crySEal基因的序列分析
序列 SEQ ID NO 1为 pSS3612中 Pst 和 l(Pn 双酶切片段，序列全长 7276bps, 分析 表明其含有两个较大的开放阅读框， 0RF1 的位置是 3658-7152， 0RF2 的位置是 2799— 3377。
0RF1 的位 S是 3658-7152， (； C含量为 38.03%, 编码 1164个氨基酸组成的蛋白。 经 测定， 其氨基酸序列为 SEQ ID NO 2所示。 同源分析表明该蛋白与 Cry8类蛋白具有较高 同源性， 表 5为其同源性数据。 ώ于与已知的 Cry8类蛋白氨基酸同源性均低于 78%， 最 高只有 58.2%(Cry8Bbl)， 被 Bt杀虫晶体蛋白命名委员会命名为 Cry8EaL。
表 5 Cry8蛋白同源比较数据
Figure imgf000006_0001
本发明进一歩分析了 CrySEal蛋白的氨基酸组成 （见表 6， 附图 7) ，得知其分子量 为 131.56kDa, 等电点为 pH4.735 (见附图 8)。
表 6 CrySEal蛋白的氨基酸组成
Figure imgf000006_0002
5. CT. '/¾/基因的克隆
对 PSS162质粒中插入序列进行了分析， 片段长 2.3kb， 具有完整的 3' 端序列， 与 C <¾¾7序列完全同源， 但 5' 端差异较大， 并且缺少完整的读码框， 根据该序列的特异 区段设计 7 1 对引 物 ( 5- 185-Kpn 1： TTGGTATGGCGTTTCGTTG； 和 3- 185-Kpnl: TATTGCAGGTCCAGGATTCAC), 用于克隆该全长基因， 将菌株 185的质粒 DNA用 Xbal酶切， 与载体 pBluescript SK(+)连接， 筛选得到插入约 9Kb外源片段的阳性克隆 PSS266 (见 附图 9)，进一歩酶切分析表明 Clal酶切产生的 3. Okb片段含有 5' 端读码框（见附图 10)， 用 pBluescript SK (+)亚克隆得到该片段， 阳性克隆命名为 pSS266- 3， 对该片段进行了 测序，把得到的序列与 PSS162中的插入序列进行拼接得到 3.9kb片段。
6. c ^ 基因的序列分析
对上述 3.9kb的核酸片段进行测序， 得到的核苷酸序列如 SEQ【D NO 3所示。 对该 序列进行分析表明： 该序列含有 1个较大的丌放阅读框 357- 3878: GC含量为 36.88%: 编 码 1174个氨基酸组成的蛋白 （其编码的蛋白质的氨基酸序列如 SEQ ID NO 4所示）。 进 一步的同源分析表明，该蛋白质与 Cry8类蛋白有较高的同源性（同源性数据见以上表 5)。 由于与己知的 C.ry8类蛋白氨基酸同源性均低于 78%， 最高只有 64.8%(Cry8Eal)， 该蛋白 被 Bt杀虫晶体蛋白命名委员会命名为 Cry8Fal。
本发明用生物分析软件 Bioedit进一步分析了 Cry8Fal蛋白的氨基酸组成， 结果见 表 1和附图 11。结果表明，该蛋白的分子量为 133.08kDa,等电点为 pH4.565(见附图 12)。
表 7 CrySEal蛋白的氨基酸组成
Figure imgf000007_0001
7. 0 (5£和 cr^ 基因的表达
本发明根据克隆的 cry8EaJ和 crySFal基因的全长序列， 设计了用于表达两种基因 的引物， 序列如下：
8E1: CGCGGATCC (Bam HI) GATGAGTCCAAATAATCAAAATG
8E2: ACGCGTCGAC (Sal I) CTCTACGTCAACAATCAATCAATTC
8F1: CGCGGATCC (Ba HI) GATGAGTCCAAATAATCAAAATG 8F4: CCGCTCGAG (Xlw I) CTCTACGTCAACAATCAATCAATTC
引物 8E1和 8E2分别引入 B画 Hi和 Sal\位点， 以含全长 crySEal的 pSS3612质粒 DNA为模板，扩增得到全长基因，插入 Bt表达载体 pSXY422b中，转化大肠杆菌 SCSllO, 提取质粒， 电击转化 Bt无晶体突变株 HD-73—中（该突变株来源于中国农业科学院植物保 护研究所生物技术实验室， 可以向公众提供）， 得到工程菌 BioT8E。
由于 crySFcd全长基因内存在 1个 β續 HI切点，用重叠弓 I物 PCR的方法对这一位点 进行了突变， 重叠引物 8F2和 8F3中引入了点突变（划线部分）、 弓 i物 8F1和 8F4分别引 入 BamHi和 Xhoi。 以含全长 ay 8 Fa!基因的 pSS266质粒 DNA为模板， 分别用 8F1和 8F2、 8F3和 8F4扩增得到 0.3kb和 3.1kb产物， 再分别以它们为模板， 分别利用引物 8F1 和 8F4扩增得到 3.4kb的全长基因，插入 Bt表达载体 pSXY422b中，转化大肠杆菌 SCSU 0， 提取质粒， 电击转化 Bt无晶体突变株 HD-73—中， 得到工程菌 BioT8F。 分别将上述两株工程菌 3CTC于牛肉膏培养基（蛋白胨 5克， 牛肉膏 3克， 葡萄糖 10克， 水 lOOOmL, 121°C， 20分钟高压蒸汽灭菌） 中培养 30小时， 取 500μί菌液至 Eppendorf皆中， 超声波破碎 30秒钟 (B. Braun U Labsonic, 230V, T«,=0.5秒）； 取 ΙΟΟμί加入 25μί新配 0.5Ν NaOH, 25 °C作用 5分钟：加入 65μί 3 X样品缓冲液 (925μί上样缓冲液 + 75 β-巯基乙醇)， 100Ό煮沸 5分钟。 离心除去沉淀。 上样 lOuL进行 SDS-PAGE电泳分析结果 （方法参见 Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.1989)。结果 (见附图 13)表明，工程菌 Biot8E 禾口 Biot8F中
Figure imgf000008_0001
因均获得了表达， 表达物的分子量为 130kDa左右。
8. Cry8E和 Cry8F蛋白的活性测定
将 Bt工程菌株接种在普通细菌琼脂克氏瓶培养基上培养 3天。 将受体菌株 HD- 73_ 接种在普通细菌琼脂克氏瓶培养基上培养 4天。 将培养物洗下， 2倍梯度浓度稀释， 将 40ml菌悬液加入到 200g有均匀粗细土豆丝的细土（紫外线灭菌） 中， 混匀， 使土壤含水 量保持在 18%- 20%。 接入暗黑鳃金龟 15天龄幼虫 20头， 以加入清水的处理为空白对照， 28Ό感染饲养， .14天检査死虫数， 计算 LC。 结果见表 8， 表明工程菌株对暗黑鳃金龟具 有极高的毒杀活性。 其表达的 Cry8E和 CrySF蛋白均具有杀暗黑鳃金龟幼虫的活性。
表 8 Bt工程菌和 185菌株对暗黑鳃金龟幼虫的毒力测定
Figure imgf000008_0002
菌种保藏信息：
菌种名称： 芽孢杆菌属、 苏云金芽孢杆菌， Bacillus thuringiensis
保藏机构： 中国微生物菌种保藏管理委员会普通微生物中心
保藏日期： 2004年 11月 5曰
保藏编号： CGMCCN0.1242
附图说明
图 1： 185菌株芽孢和晶体形态。
图 2： 185菌株晶体蛋白 SDS-PAGE分析。
图 3: 185菌株生长曲线。
图 4: 菌株 185的 PCR-RFLP图谱。 其中：
M. DNA分子量标准
1. PCR产物， 2. PCR产物酶切
图 5： 重组质粒 pSS 162和 pSS3162的 PCR扩增产物和酶切分析。 其中：
M. λ XMKIEco 1301
1. pSS162 PCR产物， 2. pSS3162 PCR产物， 3. pBIueScript SK (+) / Kpnl
4. pSS162/ Kpnl, 5. pSS3l62/ ¾iI
图 6: pSS3612插入片段的亚克隆酶切分析。 其中：
M. XOHMEco 1301
1. pBIueScript S (+) /Kpnl ， 2. pSS3162/ Pstl+ Kpnl, 3. pSS3612/ Kpnl
4. pSS3:162// /;I+/w7:[
图 7： CrySEal蛋白的氨基酸组成图。
图 8: Cry8Eal蛋白的滴定曲线。
图 9: 重组质粒 pSS266的 PCR扩增产物和酶切分析。 其中：
M. λ U MEco 1301
1. pS$266 PCR产物， 2. pSS162/ Xbal , 3. pBIueScript SK(+)/M 图 10: 重组质粒 PSS266的酶切分析。 其中：
1. Xbal, 2. Notl, 3. Pstl, 4. Ndel, 5. Clal , 6. Sad， 7. Kpnl
Figure imgf000009_0001
图 11: Cry8Fal蛋白的氨基酸组成图。
图 12: Cry8Fal蛋白的滴定曲线。
图 13: cry8Eal和 crySFaJ基因在 Bt无晶体突变株中的表达。 其中：
M. 蛋白质分子量标准
1. Biot8E， 2. Biot8F, 3. Bt 185, 4. HD-73- 具体实施方式
以下叙述根据本发明实施方案的实施例。应该说明的是，本发明的实施例对于本发明 只有说明作用， 而没有限制作用。
实施例 1、 对鳃余龟地下害虫有效的 ΒΓ菌株 185的筛选与鉴定
土壤采自河北省保定市顺平县苹果园。 取 0.1— 0.2g土样放入装有 10ml灭菌水和玻 璃珠的试管中， 在旋涡振荡器上振荡 3分钟， 将土粒打碎， 然后放于 200rpm摇床上振荡 10分钟， 75°C水浴锅中水浴 17分钟， 充分将非芽孢菌杀死， 待稍静 _實后， 选取 10—2 、 10_3、 10-4三个稀释度， 分别吸取 lOOul菌悬液于 BP平板上， 涂布均匀， 于 37°C培养三 天， 挑取似 Bt菌落涂片镜检。 发现一株含有球形晶体的 Bt菌株 （见附图 1)。
该菌株为芽孢杆菌属 Bacillus)、 苏云金芽孢杆菌种。 将该菌株在中国微生物菌种 保藏管理委员会普通微生物中心提交保藏， 保藏日期为 2004年 11 月 5日， 保藏编号为 CGMCCN0.1242。 经鉴定， 关于该菌株的信息包括： 能形成芽孢， 同时能形成球形伴孢 晶体， SDS-PAGE电泳表明其杀虫品体蛋白约为 130kDa (见附图 2)， 其晶体蛋白在 14 小时丌始表达， 而生长曲线表明其 14小时己进入停滞期（见附图 3)， 表明该品体蛋白启 动子可能为依赖芽孢形成的； 生物学测定表明， 该菌株对余龟科地下害虫暗黑鳃金龟具 有明显杀虫作用， 7天校正死亡率达 90%, 对椰心叶甲也具有一定的杀虫活性。
实施例 2、 菌株 185中 cr_y基因鉴定
根据 类基因保守区设计了一对通用弓 I物
S5un8: 5-' CGGCAAACTTAGTAGAATGC-3 '
S3un8: 5-' CTGACTGATTTCCACCATCACG- 3。
Figure imgf000009_0003
Figure imgf000009_0002
， 最 后 72°C延伸 10分钟。
结果 （见附图 4) 显示与已知 TJ^类基因的图谱不同， 表明菌株 185中可能含有新 的 杀虫基因。
实施例 3、 菌株 185中 cry8E基因的克隆
用 Pstl和 Kpnl酶切 Bt菌株总 DNA, 用载体 pBIuescript SK (+)分别建立了两个 DNA 片段库，然后用引物 S5un8/S3un8和 PCR方法检测两个 DNA库，得到两个阳性克隆 pSS3612 和 pSS162, 分别插入 llkb的 pstl片段和 2. Okb的 Kpnl 片段 （见附图 5)。 酶切分析 pSS3612,7kb的 Pstl和 Kpnl双酶切片段含有 cry8Eal的全长基因，用 pBIuescript S (+) 亚克隆该片段， 得到 PSS3612- 7， 同时亚克隆了 4kb的 Kpnl片段中， 得到 pSS3612-4 (如 附图 6)。 将 pSS3612-4和 pSS3612- 7的插入序列测序， 得到序列 SEQ ID NO 1。 该序列 为 pSS3612中 Pst.和 Kpnl双酶切片段，序列全长 7276bpS， 分析表明其含有两个较大的 开放阅读框， 0RF1的位賈是 3658-7152, 0RF2的位置是 2799— 3377。
0RF1的位置是 3658- 7152， GC含量为 38.03%， 编码 1164个氨基酸组成的蛋白， 经 测定， 其氨基酸序列为 SEQ ID NO 2所示。 同源分析表明该蛋白与 Cry8类蛋白具有较高 同源性 （见表 5)。 由于与己知的 Cry8 类蛋白氨基酸同源性均低于 78%, 最高只有 58.2%(Cry8Bbl)， 被 Bt杀虫晶体蛋白命名委员会命名为 Cry8Eal。
本发明进一歩分析了 Cry8Eal蛋白的氨基酸组成 （见表 6和附图 7) ,得知其分子量 为 131.56kDa, 等电点为 pH4.735 (见附图 8)。
实施例 4、 cr. <?/¾7基因的克隆
对 PSS162质粒中插入序列进行了分析， 片段长 2.3kb， 具有完整的 3' 端序列， 与 C <¾¾7序列完全同源， 但 5' 端差异较大， 并且缺少完整的读码框， 根据该序列的特异 区段设计 了 1 对引 物 ( 5-185-KpnI： TTGGTATGGCGTTTCGTTG； 和 3- 185- Kpnl: TATTGCAGGTCCAGGATTCAC), 用于克隆该全长基因， 将 185质粒 DNA用 Xbal酶切， 与载体 pBluescript SK(+)连接， 筛选得到插入约 9Kb外源片段的阳性克隆 pSS266 (见附图 9), 进一歩酶切分析表明 Clal 酶切产生的 3.0kb 片段含有 5' 端读码框 （见附图 10)， 用 pBluescript SK (+)亚克隆得到该片段，阳性克隆命名为 pSS266- 3，对该片段进行了测序， 把得到的序列与 PSS162中的插入序列进行拼接得到 3.9kb片段。 对核酸片段进行测序， 得到如 SEQ ID NO 3所示的核苷酸序列。 对该序列进行分析表明： 该序列含有 1个较大 的开放阅读框 357-3878； GC含量为 36.88%； 编码 1174个氨基酸组成的蛋白 (该蛋白质. 的氨基酸序列如 SEQ ID NO 4所示）。 进一歩的同源分析表明， 该蛋白质与 Cry8类蛋白 有较高的同源性（见表 5)。 ώ于与已知的 Cry8类蛋白氨基酸同源性均低于 78%， 最高只 有 64.8%(Cry8Eal)， 该蛋白被 Bt杀虫品体蛋白命名委员会命名为 Cry8Fal。
本发明用生物分析软件 Bioedit进一歩分析了 Cry8Fal 蛋白的氨基酸组成 （见表 7 和附图 11)。结果表明，该蛋白质的分子量为 133.08kDa,等电点为 pH4.565 (见附图 12)。· 实施例 5、 £^£和 CTW 基因的表达 '· 根据克隆的 c'y8Ea】和 c'≠Fa】基因的全长序列， 设计了用于表达两种基因的引物，. 序列如下：
8E I： CGCGGATCC (Bam HI) GATGAGTCCAAATAATCAAAATG
8F1: CGCGGATCC (Bam HI) GATGAGTCCAAATAATCAAAATG
8F4: CCGCTCGAG (Xho I) CTCTACGTCAACAATCAATCAATTC
引物 8E1和 8E2分别引入 ZtonHI和 Sal\位点， 以含全长 ay8Eal的 pSS3612质粒
DNA为模板，扩增得到全长基因，插入 Bt表达载体 PSXY422b中，转化大肠杆菌 SCS110, 提取质粒， 电击转化 Bt无晶体突变株 HD-73—中， 得到工程菌 BioT8E。
由于 ay8Fal全长基因内存在 1个 Z^roHI切点，用重叠引物 PCR的方法对这一位点 进行了突变， 重叠引物 8F2和 8F3中引入了点突变（划线部分）、 引物 8F1和 8F4分别引 入 BatnHl和 Xhol。 以含全长 'ySFal基因的 pSS266质粒 DNA为模板， 分别用 8F1和 8F2、 8F3和 8F4扩增得到 0.3kb和 3.1kb产物， 再分别以它们为模板， 分别利用引物 8F1 和 8F4扩增得到 3.4kb的全长基因，插入 Bt表达载体 pSXY422b中，转化大肠杆菌 SCSI 10， 提取质粒， 电击转化 Bt无品体突变株 HD-73—中， 得到工程菌 BioT8F。
分别将上述两株工程菌 30°C于牛肉膏培养基中培养 30小时， 取 500μί菌液至
Eppendorf管中， 超声波破碎 30秒钟 (B. Braun U Labsonic, 230V, Tf,,ll =0.5秒）； 取 ΙΟΟμ 加 入 25μί新配 0.5Ν NaOH, 25°C作用 5分钟； 加入 65 iL 3 X样品缓冲液 (925 L上样缓冲液-卜 ^5μL β-巯基乙醇)， 100°C煮沸 5分钟。 离心除去沉淀。 上样 10uL进行 SDS- PAGE电泳分析 结果。 结果 （见附图 13 ) 表明， 工程菌 Biot8E和 Biot8F中的 Cr_y Ea7和 c ^ 基因均获得 了表达， 表达物的分子量为 130kDa左右。
实施例 6、 Cry8E和 Cry8F蛋白的活性测定
将 Bt工程菌株接种在普通细菌琼脂克氏瓶培养基上培养 3天。 将受体菌株 HD-73— 接种在普通细菌琼脂克氏瓶培养基上培养 4天。 将培养物洗下， 2倍梯度浓度稀释， 将 40ml菌悬液加入到 200g有均匀粗细土豆丝的细土（紫外线灭菌） 中， 混匀， 使土壤含水 量保持在 18%-20%。 接入暗黑鳃金龟 15天龄幼虫 20头， 以加入清水的处理为空白对照， 28Ό感染饲养， ] 4天检査死虫数， 计算 LC5,,。 结果 （见表 8 )表明工程菌株对暗黑鳃金龟 具有极高的毒杀活性。 其表达的 Cry8E和 CrySF蛋白均具有杀暗黑鳃金龟幼虫的活性。

Claims

权利要求书
I . 一种苏云金芽孢杆菌菌株 BaclUus thuringie is )，其在中国微生物菌种保藏管理委员 会普通微生物中心的保藏编号为 CGMCC NO.1242。
2. 权利要求 1的菌株在杀灭鞘翅目害虫中的应用。
3. —种从权利要求 1所述的苏云金芽抱杆菌菌株分离克隆的 C A§E基因， 其特征是该基 因具有 SEQ ID NO 1所示的核苷酸序列。
4. 一种 Cry8Eal蛋白，其特征是该蛋白是由权利要求 3的基因所编码，且具有 SEQ ID NO 2所示的氨基酸序列。
5. 一种蛋白，其特征在于其氨基酸序列与权利要求 4所示的 CrySEa l蛋白的氨基酸序列 同源性不低于 83. 5%。
6. 一种从权利要求 1所述的苏云金芽孢杆菌菌株分离克隆的 c ^F基因， 其特征是该基 因具有 SEQ ID NO 3所示的核苷酸序列。
7. 一种 Oy8Fal蛋白，其特征是该蛋白是 ώ权利要求 5的基因所编码，且具有 SEQ ID NO 4所示的氨基酸序列。
8. 一种蛋白，其特征在于其氨基酸序列与权利要求 7所示的 Cry8Fal蛋白的氨基酸序列 同源性不低于 83. 5%。
9. 一种增强对鞘翅目害虫毒性的方法，其特征是该方法协同组合使用权利要求 3的 cry8E. 基因和权利要求 5的 基因。
10.—种转基因微生物， 其特征在于微生物内转化有权利要求 3所述的 基因。
I I .一种转基因微生物， 其特征在于微生物内转化有权利要求 6所述的 基因。
12.—种转基因微生物， 其特征在于微生物内转化有权利要求 3所述的 y8E基因和权利 要求 6所述的 cr^F基因。
13.—种转基因植物， 其特征在于植物内转化有权利要求 3所述的 rySE基因。
14.一种转基因植物， 其特征在于植物内转化有权利要求 6所述的
Figure imgf000012_0001
基因。
15.—种转基因植物， 其特征在于植物内转化有权利要求 3所述的 ^£基因和权利要求 6所述的 c ^F基因。
16.—种生物制剂， 其特征在于含有权利要求 4所述的 C:ry8Ea l蛋白。
1 7.—种生物制剂， 其特征在于含有权利要求 7所述的 CrySFal蛋白。
18.—种生物制剂， 其特征在于含有权利要求 4所述的 Cry8Eal蛋白和权利要求 7所述的 Cry8Fa l蛋白。
PCT/CN2005/001133 2004-11-16 2005-07-27 Souche de bacillus thuringiensis et genes a haute activite larvicide sur les coleopteres Ceased WO2006053473A1 (fr)

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