WO2010118633A1 - 杀虫晶体蛋白基因Cry4Cb1及其编码蛋白和应用 - Google Patents

杀虫晶体蛋白基因Cry4Cb1及其编码蛋白和应用 Download PDF

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WO2010118633A1
WO2010118633A1 PCT/CN2010/000485 CN2010000485W WO2010118633A1 WO 2010118633 A1 WO2010118633 A1 WO 2010118633A1 CN 2010000485 W CN2010000485 W CN 2010000485W WO 2010118633 A1 WO2010118633 A1 WO 2010118633A1
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protein
gene
bacillus thuringiensis
pests
cry4cb1
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French (fr)
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郑爱萍
李平
朱军
王玲霞
王世全
邓其明
李双成
刘怀年
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四川农业大学
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a novel Bt protein and its encoding gene and application. Background technique
  • Bacillus thuringimsis is a Gram-positive bacterium, which is widely distributed. It forms a companion crystal composed of protein with insecticidal activity, also known as insecticidal crystal protein. (Insectididal crystal proteins, ICPs for short), ICPs are encoded by the cr_y gene, which is highly toxic to sensitive insects and not to higher animals and humans. In recent decades, Bt has been widely used in control A variety of pests such as Lepidoptera, Diptera, Coleoptera. In addition, Bt also has a controlling effect on a variety of pests such as Hymenoptera, Homoptera, Orthoptera, and Corydalis, and plant pathogenic nematodes, mites, and protozoa. At present, Bt has become a powerful substitute for chemically synthesized pesticides in the control of farmland pests, forest pests and sanitary pests. Bt is also an important genetic source for transgenic insect-resistant engineering plants.
  • Cyt protein has cytosolic properties, synergistic effect on certain Cry proteins and delays insect resistance ( Wu, D., Johnson, JJ, and Federici, BA 1994. Synergism of mosquitocidal toxicity between CytA and CrylVD Proteins using inclusion Sproduced from cloned genes of Bacillus thuringiensis. Mol. Microbiol. 13 :965-972; Wirth, MC Georghiou, GP, and Federeci, BA 1997. CytA enables CrylV endotoxins of Bacillus thuringiensis to overcome high levels of CrylV resistance in the mosquito, Culex Quinquefasciatus . Proc. Natl. Acad. Sci. 94: 10536-10540 )
  • Bacillus thuringiensis has been found for more than 100 years, and it has been widely used in the control of crop and horticultural plant pests, forest pests and sanitary pests, and has also achieved good results.
  • Bacillus thuringiensis due to the large-scale and repeated use of Bacillus thuringiensis, many insect populations have successively developed resistance to insecticidal crystal proteins to varying degrees.
  • the use of Bt insecticidal crystal protein-based insecticides has been used for more than 50 years. Initially, insect resistance to Bt has not been detected.
  • resistance problems have continued to exist in laboratories and It has been confirmed in field trials (M C G aU ghey, WH 1985.
  • a first object of the present invention is to provide a new BT virulence protein resource for the above deficiencies. .
  • a second object of the invention is to provide a gene encoding the protein.
  • the invention is a novel strain Bacillus thuringiensis HS 18-1 isolated from the soil of Chengdu Plain in Sichuan province.
  • the virulence test on HS 18-1 showed that HS18-1 has extremely high virulence against coleopteran pests, lepidopteran pests, and dipteran pests.
  • a pair of specific primers was designed according to the conserved sequence of Cry4 gene, and the genomic DNA was amplified. The results showed that the gene existed in the strain, and the full-length gene primer was further designed, and the Cr_y ⁇ gene was cloned.
  • the nucleotide sequence of the strain was SEQ ID No. As shown by l, the full length of the sequence SEQ ID No. 1 was 3474 bp, and the analysis showed that the GC content was 35.90%, encoding a protein consisting of 1157 amino acids. The amino acid sequence thereof was determined as shown in SEQ ID No. 2. The whole sequence was predicted by the bacterial sigma7.0 promoter program on the softberry website. The sequence containing the RNA polymerase activation site upstream of the gene coding region was named as cr ⁇ Cb. The present invention further analyzed the amino acid of the Cry4Cbl protein. Composition (see Table 1).
  • the Bt protein of the present invention further comprises a protein derived from Cry4Cbl having the amino acid sequence of SEQ ID No. 2 substituted, substituted and/or increased by one or several amino acids, having the same activity as the Cry4Cbl protein.
  • a gene of the invention includes a nucleic acid sequence encoding the protein.
  • codons suitable for expression of a particular species can be used, as desired, in view of the degeneracy of the codons and the preferences of the codons of the different species.
  • genes and proteins of the present invention can be cloned or isolated from strain HS18-1 or obtained by DNA or peptide synthesis.
  • the gene of the present invention can be operably linked to an expression vector to obtain a recombinant expression vector capable of expressing the protein of the present invention, and the expression vector can be further transformed by a transgenic method such as Agrobacterium-mediated method, gene gun method, pollen tube pathway method or the like.
  • the vector is introduced into a host, and a transformant having a transgenic Cr ⁇ C gene, such as a crop or a fruit tree, is obtained to have an insect-resistant activity.
  • Figure 1 shows the cr CW full-length gene clone, in which M, marker; l, cry4Cbl gene.
  • Figure 2 shows the restriction endonuclease map of the recombinant plasmid pET-4Cb, wherein 1 recombinant plasmid pET-4Cb; 2, double digestion with pDE-30a with Nde l+EcoR I; 3, Nde l+EcoR I double digestion pET -4Cb; 4, inserted DNA; Ml, M2 are Marker.
  • Figure 3 shows the SDS-PAGE assay for expression of Cry4Cbl in co/z' BL21 (DE3), where M is the protein marker; 1. Negative control (E. con BL21(DE3)(pET-30a)); Cleavage supernatant; 3. Cry4Cbl inclusion body.
  • Bacillus thuringiensis new strain, which was released on October 21, 2008 in the General Microbiology Center of China Microbial Culture Collection Management Committee (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Dayi Road, Chaoyang District, Beijing 100101, China) The deposit is classified as Bacillus thuringiensis, and the accession number is CGMCC No.2817.
  • the total DNA of strain HS 18-1 was extracted using a genomic DNA purification kit (purchased from Thermos).
  • the primer sequences were designed as follows:
  • cry4F was designed and synthesized based on the sequence of the open reading frame of the cry4Cbl gene: 5'-GCGCATATG(Ni/eI) ATGTCTAATCGTTATCAACGGTACCC-3'; cry4R: 5' - CGGAATTC (EcoR I) TCACTCGTTCATACAAATCAACTCGA-3', at the 5th, primer Nde I and EcoR I restriction sites, respectively.
  • the BtMC28 plasmid was used as a template for amplification.
  • the amplified product was double digested with NI and oR I.
  • the digested product was ligated with the double-digested vector pET-30a(+) to transform E. coli ⁇ 5 ⁇ competent state.
  • the cells were extracted and subjected to plasmid electrophoresis to verify that the size of the insert was in accordance with the intended purpose (Fig. 2), and then transferred to the recipient strain.co/i.BL21 (DE3).
  • the recombinant plasmid was named pET-4Cb, and the recombinant containing the recombinant plasmid was named co/z'.BL21 (4Cb).
  • SDS-PAGE analysis showed that the expression product of the cr ⁇ Cb7 gene was precipitated after sonication of the cells (Fig.
  • the Bt protein of the present invention can be used to prepare Bt insecticides, which can be transformed into cotton, corn, rice Crops such as vegetables and vegetables have the corresponding insect-resistant activities, thus reducing the use of pesticides and reducing environmental pollution. It has important economic value and application prospects.

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Description

杀虫晶体蛋白基因 Cry4Cb1及其编码蛋白和应用
技术领域
本发明涉及生物技术领域， 具体涉及一种新的 Bt蛋白及其编码 基因和应用。 背景技术
在人类生产过程中，虫害是造成农业生产损失及影响人类健康的 重要因素， 据 FAO统计， 全世界农业生产每年因虫害造成的经济损 失高达 14%, 病害损失达 12%, 草害损失达 11%。 损失额高达 1260 亿美元， 相当于中国农业总产值的一半， 英国的 4倍多。 另外， 蚊媒 病在预防医学中占有重要位置，其中登革热和黄热病等蚊媒病传播力 强、 流行面广、 发病率高、 危害性大。 据 WHO统计， 全世界每年感 染登革热人数多达 8000万， 我国的海南省在 1980和 1986年曾经暴 发过两次登革热， 发病分别达到 437469例和 113589例。 登革热和黄 热病主要由埃及伊蚊传播。
为了减少这些损失， 多年来， 对农作物害虫及蚊虫普遍釆用化学 防治手段进行防治， 但由于化学农药的长期、 大量使用， 造成了对环 境的污染， 农副产品中农药残留量增加， 给人类的生存和健康带来了 危害。 此外， 化学农药在杀灭害虫的同时, 也杀伤了天敌及其它有益 物， 破坏了生态平衡。 与化学防治相比, 生物防治具有安全、 有效、 持久的特点。 并且避免了化学防治带来的一系列问题。 因此， 生物防 治技术成了人们研究的热点。 在生物杀虫剂中， 苏云金芽孢杆菌是目 前世界上用途最广、 产量最大的一类微生物杀虫剂。
苏云金芽孢杆菌 Bacillus thuringimsis, 简称 Bt )是一种革兰氏 阳性细菌， 它的分布极为广泛， 在芽孢形成的同时可形成具有杀虫活 性的由蛋白质组成的伴胞晶体，又名杀虫晶体蛋白（ Insectididal crystal proteins, 简称 ICPs ), ICPs是由 cr_y基因编码的， 对敏感昆虫有强烈 毒性， 而对高等动物和人无毒性。 近几十年来， Bt 已广泛应用于控 制多种鳞翅目、 双翅目、 鞘翅目等害虫。 此外， Bt还对膜翅目、 同 翅目、 直翅目、 食毛目等多种害虫及植物病原线虫、 螨类、 原生动物 有控害作用。 目前在农田害虫、 森林害虫及卫生害虫的防治中 Bt已' 成为化学合成农药的有力替代品， Bt还是转基因抗虫工程植物重要 的基因来源。
自 1981年 Schnepf从菌株 HD-lDipel中克隆了第一个能表达杀 虫活性的基因以来 ( Adang MJ et al, Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta, Gene, 1985 , 36 (3): 289 - 300. ), 人们已经分离克隆了 390多种编码杀虫晶体蛋 白的基因， 根据编码的氨基酸序列同源性它们被分别确定为不同的 群、 亚群、 类和亚类 (Crickmore N， Zeigler D R, Feitelson J, et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev,1998,62:807-813; htt ://www.biols . susx.ac.uk/Home/Neil_Crickmore/Bt/)。 一般而言 , Cryl,Cry2和 Cry9等毒蛋白对鳞翅目害虫有效；其中研究的最多的是 Cryl和 Cry9类蛋白，它们编码的杀虫晶体蛋白分子量为 130-140kD, 许多基因目前已被广泛应用于植物的鳞翅目害虫的防治 (Kozie, M. G, Beland, G. L., Bowman, C, et al. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology, 1993， 11 : 194-200; Perlak, FJ., Deaton, R. W., Armstrong, T. A. , et al. Insect resistant cotton plants, bio/technology, 1990; 8:939-943; Van Frankenhuyzen, K.,Gringorten, L., and Gauhier, D.1997. Cry9Cal toxin, a Bacillus thuringiensis insecticidal crystal protein with high activity against the spruce bud worm (Choristoneura fnniferana). Appl. Environ, Microbviol.63:4132-4134; 王飞， 2001 , 苏 云金芽孢杆菌特异菌株生物学特性及 cr ;P新基因的研究， 硕士论文， 南开大学）。 苏云金芽胞杆菌以色列亚种（ thuringiensis subsp. israelensis, 简称 Bti)产生的毒素蛋白对蚊虫具有很好杀虫活性，被广 泛运用于蚊虫的防治 （ Goldberg L J, and Margalit J， 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentU, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti, and Culex pipiens. Mosqito News, 37: 355-358; )。 同时， Cyt蛋白具有溶细胞性， 对某些 Cry蛋白具有增效作用及延缓昆虫的抗性（ Wu, D.， Johnson, J. J., and Federici, B. A.1994. Synergism of mosquitocidal toxicity between CytA and CrylVD Proteins using inclusion sproduced from cloned genes of Bacillus thuringiensis. Mol. Microbiol.13 :965-972; Wirth, M. C. Georghiou, G. P., and Federeci, B. A. 1997. CytA enables CrylV endotoxins of Bacillus thuringiensis to overcome high levels of CrylV resistance in the mosquito, Culex quinquefasciatus . Proc. Natl. Acad. Sci. 94: 10536-10540 )
自本世纪初发现苏云金芽胞杆菌至今己有 100多年的历史，在农 作物和园艺植物害虫、森林害虫以及卫生害虫的防治方面得到广泛的 应用， 也起到良好的效果。 但是， 由于大规模和反复使用苏云金芽胞 杆菌， 许多昆虫种群己相继在不同程度上对杀虫晶体蛋白产生了抗 性。 以 Bt杀虫晶体蛋白为基础的杀虫剂的使用巳有 50多年的历史， 最初一直没有检测到昆虫对 Bt的抗性，但是，上世纪 80年中期开始， 抗性问题不断在实验室及田间试验中得到证实（M CGaUghey,W. H. 1985. Insect resistance to the biological insecticide Bacillus thuringiensis. Science. 229: 193-195 ), 原因主要是持续使用单品种及亚致剂量的 Bt以及 Bt转 基因抗虫植物的应用造成昆虫种群长期受到杀虫剂的选择压力。 1985 年， McGaughey报道仓库谷物害虫印度谷螟 （ Plodia interpunctella ) 在 Dipel (Bt subsp.kurstaik HD-1的商品制剂）的选择压力下， 繁殖 15 代后， 抗性增加 97倍； 在高剂量选择压力下， 抗性可增加 250倍。 1990年， 在夏威夷首次证实大田中的小菜蛾对 Bt杀虫剂产生了明显 的抗性（Tabashnik， B .E ., Finson, N ., Groeters, F.R., et al. 1994. Reversal of resistance to Bacillus thuringiensisin Plutella xylostella. Proc.Natl.Acad.Sci.USA.91 :4120-4124 ), 上世纪 90年代以来， 在我国 应用 Bt杀虫剂时间较长的深圳、 广州、 上海等地， 发现 Bt杀虫剂对 小菜蛾防治效果明显下降， 意味着抗性巳经形成 （冯夏， 1996. 广东 小菜蛾对苏云金杆菌的抗性研究.昆虫学报, 39 (3):238-244； Hofte, H., Van Rie, J" Jansens, S" Van Houtven, A" Vanderbmggen, H" and Vaeck, M.， 1988. Monoclonal antibody analysis and insecticidal spectrum of three types of lepidopteran-specific insecticidal crystal proteins of Bacillus thuringiensis. A^\. Environ. Microbiol. 54: 2010-2017 )。目前发 现在实验室及田间至少有十几种昆虫对 B t及其杀虫晶体蛋白产生了 抗性， 用选择压力数学模型预测到， 在 Bt转基因抗虫植物选择压力 的条件下， 昆虫将会产生抗性（ Schnepf， E.， Crickmore, N.， Van Pie, J., et al. 1998. Bacillus thuringiensis and its pesticidal Crystal proteins.Microbiol. Mol. Biol. Rev.65 (3):77 5-806 )。 另外， 有研究证明 Bti在大田的使用中尚未发现抗性问题（ Regis L， et al, 2000. The use of bacterial larvicides in mosquito and black fly control programsin Brazil.Mem. Instituto Oswaldo Cruz, 95: 207-210. )，但是抆虫对其抗性 问题不断在实验室中得到证实， 这种情况也可能会在大田中出现 ( Georghiou G P5 and Wirth M C， 1997. Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Applied and Environmental Microbiology, 63 : 1095-1101. )o
为避免抗性昆虫所造成的损失，寻找新的高毒力基因资源是解决 这个问题的有效途径， 这对我国的生物防治有着十分重要的意义。 发明内容
本发明的第一个目的在于针对上述不足提供一种新的 BT毒力蛋 白资源。 .
本发明的第二个目的在于提供编码所述蛋白的基因。
本发明的目的还在于提供上述蛋白及基因的应用。
本发明从四川省成都平原土壤中分离得到的苏云金芽孢杆菌 ( Bacillus thuringiensis )新菌株 HS 18- 1。 通过对 HS 18-1的毒力测试 表明， HS18-1 对鞘翅目害虫、 鳞翅目害虫、 双翅目害虫等等， 均具 有极高的毒力。
根据 Cry4 基因保守序列设计 1 对特异引物， 扩增其基因组 DNA, 结果表明该菌株存在 基因， 进一步设计其全长基因引 物， 克隆得到 Cr_y α基因， 其核苷酸序列如序列表 SEQ ID No. l所 示，序列 SEQ ID No.l的全长为 3474bp，分析表明， GC含量为 35.90%, 编码 1157个氨基酸组成的蛋白。 经测定， 其氨基酸序列如 SEQ ID No.2所示。在 softberry网站釆用 bacterial sigma7.0 promoter程序对全 序列进行预测表明， 在基因编码区上游含有 RNA聚合酶活化位点的 序列， 将该基因命名为 cr^Cb 本发明进一步分析了 Cry4Cbl蛋白 的氨基酸组成 （见表 1 )。
Cry4Cbl蛋白的氨基酸组成
Figure imgf000006_0001
应当理解， 本领域技术人员可根据本发明公开的氨基酸序列， 在 不影响其活性的前提下， 取代、 缺失和 /或增加一个或几个氨基酸， 得到所述蛋白的突变序列。 例如在非活性区段， 将第 20位的 Ser替 换为 Met。 因此， 本发明 Bt蛋白还包括 SEQ ID No.2所示氨基酸序 列经取代、 替换和 /或增加一个或几个氨基酸， 具有 Cry4Cbl蛋白同 等活性的由 Cry4Cbl 衍生得到的蛋白质。 本发明基因包括编码所述 蛋白的核酸序列。
此外， 应理解， 考虑到密码子的简并性以及不同物种密码子的偏 爱性， 本领域技术人员可以根据需要使用适合特定物种表达的密码 子。
本发明的基因和蛋白质可以从菌株 HS18-1中克隆或分离得到， 或者通过 DNA或肽合成的方法得到。
可将本发明基因与表达载体可操作地连接，得到能够表达本发明 蛋白的重组表达载体， 进而可以通过诸如农杆菌介导法、 基因枪法、 花粉管通道法等转基因方法， 将所述表达载体导入宿主， 得到转 Cr^C 基因的转化体， 例如农作物或者果树等植物， 使其具备抗虫 活性。
此外， 还可以通过发酵本发明菌株 HS18-1 , 得到含有 Cry4Cbl 蛋白的发酵液， 将其制备成杀虫剂， 用于农作物害虫的防治。 本领域 技术人员还可以将上述基因转化细菌或真菌，通过大规模发酵生产本 发明 Bt蛋白。
本领域技术人员还可以根据本发明公开的基因， 将其转化棉花、 玉米、 水稻、 蔬菜等农作物， 使其具备相应的抗虫活性。 从而降低农 药的使用量， 减少环境污染， 具有重要的经济价值和应用前景。 附图说明
图 1显示的是 cr CW全长基因克隆，其中 M, marker; l, cry4Cbl 基因。 图 2显示的是重组质粒 pET-4Cb的酶切鉴定图谱，其中 1重组质 粒 pET-4Cb; 2,用 Nde l+EcoR I双酶切 pET-30a; 3， Nde l+EcoR I双酶 切 pET-4Cb; 4,插入的 DNA; Ml、 M2为 Marker。
图 3显示的是在 co/z' BL21(DE3)中表达 Cry4Cbl的 SDS-PAGE 检测 ， 其 中 M 为 蛋 白 marker; 1. 阴 性对照 （ E. con BL21(DE3)(pET-30a) )； 2.裂解上清； 3. Cry4Cbl包涵体。 具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的 限制。 在不背离本发明精神和实质的情况下， 对本发明方法、 步骤或 条件所作的修改或替换， 均属于本发明的范围。
若来特别指明，实施例中所用的技术手段为本领域技术人员所熟 知的常规手段。 实施例 1 w w基因的克隆
本发明从四川省成都平原土壤中分离得到的苏云金芽孢杆菌
( Bacillus thuringiensis )新菌株， 该菌株已于 2008年 10月 21 曰在 中国微生物菌种保藏管理委员会普通微生物中心（地址： 北京巿朝阳 区大屯路甲 3号， 中国科学院微生物研究所， 邮编 100101 )保藏， 分类命名为苏云金芽孢杆菌 ( Bacillus thuringiensis )， 保藏号为 CGMCC No.2817。
本例通过如下方法克隆得到 Cry4Cbl基因的全长序列。
采用基因组 DNA纯化试剂盒 (购自赛百盛公司）提取菌株 HS 18-1 的总 DNA。 设计引物序列如下：
P1 : 5' ATGTCTAATCGTTATCAACGGTACCC 3 '
P2: 5 ' TCACTCGTTCATACAAATCAACTCGA 3 '
PCR反应体系：
10 X buffer 2.5μ1
MgCl2(25mM) 1.5μ1
Taq酶 0·2μ1 dNTPs(2.5mM) 2μ1
2μ1
模板 5μ1
最终反应体积 25μ1
热循环反应： 94°C预变性 5min; 94°C变性 lmin, 52°C退火， 72 °C延伸 2min, 30个循环； 72°C延伸 5min; 4°C停止反应。 扩增反应 产物在 1 %琼脂糖凝胶上电泳 ,置凝胶成像系统中观察 PCR扩增结果。 结果如图 1所示， 通过扩增得到了约为 3.5kb的序列， 将该序列进行 测序， 其核苷酸序列如 SEQ ID No. l所示， 与目的序列一致。
实施例 2 Cry4Cbl基因的表达及杀虫活性测定 根据 cry4Cbl基因开放阅读框两端序列，设计并合成一对特异性 引物 cry4F: 5'-GCGCATATG(Ni/eI) ATGTCTAATCGTTATCAACGGTACCC-3'; cry4R: 5'-CGGAATTC (EcoR I) TCACTCGTTCATACAAATCAACTCGA-3',分别 在 5，端引物 Nde I和 EcoR I酶切位点。以 BtMC28质粒为模板进行扩 增， 扩增的产物采用 N I和 oR I进行双酶切， 酶切产物与同样进 行双酶切后的载体 pET-30a(+)连接， 转化 E. coli ΌΗ5α感受态细胞， 提取其质粒酶切电泳验证了插入片断大小符合预期目的后 （图 2 ), 再转入受体菌 .co/i.BL21(DE3)。 将重组质粒命名为 pET-4Cb, 含重 组质粒的重组子命名为 co/z'.BL21(4Cb)。 SDS-PAGE 分析表明 cr ^Cb7基因的表达产物在菌体超声破碎后的沉淀中（图 3 ), 分子量 约为 130kDa左右， 与预测的蛋白分子量相符。 c ^C 基因表达产 物分别对甜菜夜蛾，棉铃虫及伊蚊的生测结果表明： 表达产物对这三 种虫都具有较好的杀虫活性。 对棉铃虫杀虫活性最高， LC50为 10.06 g/mL; 对伊蚊的 ^为 18.41 g/mL; 对甜菜夜蛾杀虫活性最 低， C50为 30.29 g/mL。 蛋白对鳞翅目杀虫活性的的测定方法参见 ( Song FP， Zhang J， Gu AX, et al., 2003. Identification of cryll- ype genes from Bacillus thuringiensis strains and characterization of a novel cryll- pe gene. Appl. Environ. Microbiol 69: 5207-5211), 蛋白对双翅 目杀虫活性的的测定方法参见（ Ibarra JE， del Rincon MC, Sergio Ο ύζ: et al., 2003. Diversity of Bacillus thuringienisis Strains from Latin America with Insecticidal Activity against Different Mosquito Species. Appl Environ Microbiol 69: 5269-5274). 工业实用性 本发明 Bt蛋白可以用于制备 Bt杀虫剂，所述基因可以转化棉花、 玉米、 水稻、 蔬菜等农作物， 使其具备相应的抗虫活性， 从而降低农 药的使用量， 减少环境污染， 具有重要的经济价值和应用前景。

Claims

1、 一种 Bt蛋白 Cry4Cbl, 其是：
1) SEQIDNo.2所示的氨基酸序列组成的蛋白质； 或
2) SEQIDNo.2所示的氨基酸序列经取代、 缺失和 /或增加一个 或几个氨基酸且具权有同等活性的由 1)所述蛋白质衍生的蛋白质。
2、 编码杈利要求 1所述蛋白的基因。
3、 如杈利要求 2所述的基因， 其核苷酸序列如 SEQIDNo.l所 示。
4、 含有权利要求 2或 3所述基因的表达载体。
5、 由杈利要求 4所述表达载体转化的宿主细胞。
6、 如杈利要求 5所述的宿主细胞， 其为植物宿主细胞。
7、 含有权利要求 1所述蛋白的杀虫剂。 书
8、 权利要求 2或 3所述基因或权利要求 4所述表达载体在制备 转基因植物中的应用。
9、 杈利要求 2或 3所述基因或杈利要求 4所述表达载体在提高 植物抗虫性中的应用。 ·
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