WO2010078708A1 - 苏云金芽胞杆菌杀线虫晶体蛋白基因cry1518-35 - Google Patents

苏云金芽胞杆菌杀线虫晶体蛋白基因cry1518-35 Download PDF

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WO2010078708A1
WO2010078708A1 PCT/CN2009/001302 CN2009001302W WO2010078708A1 WO 2010078708 A1 WO2010078708 A1 WO 2010078708A1 CN 2009001302 W CN2009001302 W CN 2009001302W WO 2010078708 A1 WO2010078708 A1 WO 2010078708A1
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bacillus thuringiensis
crystal protein
gene
nematicidal
protein
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PCT/CN2009/001302
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孙明
郭素霞
王鹏霞
彭东海
阮丽芳
喻子牛
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华中农业大学
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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

Definitions

  • Bacillus thuringiensis nematicidal crystal protein gene C ⁇ 5 S 5 FIELD OF THE INVENTION The present invention is in the field of biological control and biotechnology. The isolation and cloning of the insecticidal crystal protein gene of Bacillus thuringiensis is related to biopesticide genetic engineering and transgenic plants. BACKGROUND OF THE INVENTION
  • Bacillus thuringiensis thuringiensis (Bt) is a type of Gram-positive bacterium widely present in soil. During its growth and development, it can form endogenous spores, which are particularly toxic to insects due to their formation of spores.
  • ICPs Insecticidal Crystal Proteins
  • Bacillus cereus which secretes enterotoxin
  • Bacillus anthracis Bacillus anthracis
  • the crystal protein produced by Bacillus thuringiensis can be used for 10 species of more than 500 species of insects such as Lepidoptera, Diptera, Coleoptera, Hymenoptera, and Homoptera, as well as protozoa, linear animals, and flat animals.
  • Certain harmful species in the door also have specific biological activities (Schnepf HE, Crickmore N, Rie JV, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean D H.
  • Bacillus thuringiensis wild strains generally contain multiple genes encoding insecticidal crystal proteins. Self
  • the gene is a pesticidal gene derived from Bacillus thuringiensis encoding a parasporal crystal protein or a gene having obvious sequence similarity to a known c ⁇ y gene
  • the yi gene is a companion crystal protein gene encoding cytolytic activity or A gene that has significant sequence similarity to a known Cyt protein.
  • the homology of the amino acid sequences deduced from the full-length genes is divided into four grades. The boundary between the stages is 95%, 78% and 45% homology. As of December 2008, B.
  • thuringiensis insecticidal crystal protein gene has accumulated 55 classes of 436 genes and 2 classes of 27 genes (Crickmore N, Zeigler DR, Feitelson J, Schnepf E, van Rie J, Lereclus D, Baum J , Dean DH. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62 :807-813 ; http: ⁇ www.lifesci.sussex.ac.uk/home Neil_Crickmore/Bt/ Index.html).
  • Root knot nematode Plant parasitic nematodes, mainly parasitic on the roots, tubers, bulbs or bulbs of infected plants, which absorb nutrients from the host, causing growth and development, deformity, dwarfing and even death of the host plants, which bring significant agricultural production every year. loss. They can be parasitic on more than 10 plants, including soybeans, peanuts, tobacco, cucumbers, peony, etc., while plants such as Solanaceae, Cucurbitaceae, Cruciferae, etc. are particularly seriously affected (Liu Weizhi. Plant pathogens) Nematode Beijing: China Agricultural Press, 2000).
  • Root-knot nematodes Traditional methods for controlling root-knot nematodes mainly include crop rotation, fallow cultivation and chemical control. However, in many developing countries, people must rely on perennial crops and crop continuous cropping. Rotation and fallow are difficult to control root-knot nematodes; and chemical control methods can cause great pollution to the environment. Therefore, the prevention and control of root-knot nematodes by biological methods has gradually become one of the research hotspots in this field. Regarding the research on the control of plant parasitic nematodes by Bacillus thuringiensis, the earliest report was that in 1972, Prasad et al.
  • Ivanova reports that certain Bacillus thuringiensis preparations can be used to control root-knot nematodes on cucumbers and tomatoes; while Ensard has also achieved significant effects in controlling banana perforating nematodes with Bacillus thuringiensis preparations (Ivanova T S. Efficiency of biopreparation in control) Of gall nematode in protected soil. Agrokhimiya, 1996, 3 : 101 - 106.; Ensard J. Effects of three microbial broth cultures on growth and populations of free living and plant-parasitic nematodes on banana. European Journal of Plant Pathology, ⁇ 99 451-463).
  • Bacillus thuringiensis YBT-1518 strain is a flagellate strain isolated and preserved in our laboratory. It can form rice-like companion crystals during the formation of its spores. Bioassay shows that its crystal protein is related to Caenorhabditis. Elegans and Wokou; I. Meloidgyne hapla has high activity.
  • the present invention resides in the isolation of novel nematicidal crystal protein genes for the construction of microbial insecticides and transgenic plants resistant to nematodes.
  • the present invention isolates and clones a novel nematicidal crystal protein gene from Bacillus thuringiensis YBT-1518 It was found to be highly toxic to Northern root-knot nematodes, revealing the application of the crystal protein in the field of controlling root-knot nematodes.
  • the present invention is implemented as follows:
  • the above gene sequence provided by the present invention is a novel nematicidal crystal protein gene, which can be applied to transform microorganisms and plants to express Cryl518-35, so that the receptor organism exhibits a poisoning activity against nematodes,
  • the biological control of parasitic nematodes has a wide range of applications.
  • SEQ ID NO: 1 is the nucleotide sequence of the B. thuringiensis nematicidal crystal protein gene isolated and cloned according to the present invention, and the sequence listing SEQ ID NO: 2 is its coding sequence;
  • Figure 1 is a physical map of a BAC clone in which the nematicidal crystal protein gene ⁇ 7 ⁇ 57 ⁇ -35 is present in the present invention
  • Figure 2 is a construction diagram and physical map of the cloning vector pUC18-75 ⁇ -35 in the embodiment of the present invention
  • Figure 3 is a diagram showing the construction of the overexpression vector ⁇ 0225 and its physical map in the embodiment of the present invention
  • Figure 4 is a SDS-PAGE electrophoresis analysis of the nematicidal crystal protein gene expressed and purified in JM109 in the present invention
  • the present invention uses Bacillus thuringiensis YBT-1518 as a source strain of the nematicidal crystal protein gene c 5 -35, and the source of the strain is found in the literature: Yu, Z., P. Bai, W. Ye, F. Zhang, L. Ruan , Z. Yu, and M. Sun. A Novel Negative Regulatory Factor for Nematicidal Cry Protein Gene Expression in Bacillus thuringiensis. J. Microbiol. Biotechnol. 18(6): 1033-1039.
  • the strain has no flagella, can form typical long-grain crystals, and is highly toxic to root-knot nematodes and C. elegans.
  • Bacillus thuringiensis strain YBT-1518 was activated overnight, transferred to 5 mL of fresh LB medium at 1/100 (vA inoculum size (LB medium formula: tryptone 1%, yeast extract 0.5%, chlorinated) Sodium 0.5%, pH 7.0), cultured at 28 ° C, 200 rpm to mid-logarithmic growth phase.
  • LB medium formula: tryptone 1%, yeast extract 0.5%, chlorinated
  • the plasmid prepared in the previous step was incompletely digested by adding appropriate amount of wdin, and the 5-12 kb fragment was recovered and ligated into the vector PHT304 to transform Escherichia coli DH5a, and the ampicillin resistant plate (containing 100 g/mL ampicillin, 100 g) was applied. /mL IPTG and 80 g/mL of X-gal), the resistant plates were placed in a 37 ° C incubator for 14 h, and the white spots were picked from fresh LB plates (containing 100 g/mL of ampicillin), ie A plasmid library of strain YBT-1518 was randomly selected and 1000 clones were randomly selected and stored in a -80 ° C refrigerator.
  • the average insert size is 8 kb, and 1000 clones can cover approximately 2 to 3 times the entire plasmid genome (assuming a total plasmid genome size of 300 kb and a copy number of 3 for each plasmid).
  • upstream primer P1 primer sequence: 5, -CGCGGATCCATGAATAATATTAATAAGAAG-3
  • downstream primer ⁇ 2 based on the specific sequence of the gene and introduced restriction sites at both ends ⁇ mffl and / / (primer sequence: 5,-CCCAAGCTTCTAAGATATATAAGCATTTAAAG-3,), and PCR amplification using the recombinant plasmid PBMB0229 as a template to obtain the target gene.
  • the PCR reaction system and procedure are as follows:
  • the 25 ⁇ L reaction system contains: 2.5 ⁇ L lOxPCR reaction buffer, 1 dNTP (2.5 mM each), 0.5 specific upstream primer PI (20 mM), 0.5 specific downstream primer ⁇ 2 (20 mM), 0 ⁇ 2 ⁇ template (plasmid ⁇ 0229) , 0.25 Ex Taq enzyme, add sterile deionized water to 25 ⁇ .
  • the PCR reaction parameters and procedures were: 94 ° C, 5 min, 1 cycle; 94 ° C, 20 s, 52 ° C, 20 s, 72 ° C, 1.5 min, 30 cycles; 72 ° C, 10 min, 1 cycle.
  • the amplified target fragment was purified by PCR product recovery kit (purchased from Omega Biotech Co., Ltd.) and then ligated to the T vector pMD18-T Simple Vector (purchased from TaKaRa) by T/A cloning to obtain cryI518-containing 35 full-length sequence of recombinant plasmid p ⁇ JC ⁇ S-cryl518-35 (see Figure 2).
  • the recombinant plasmid was then transformed into E. coli (£. co/) DH5a, and ampicillin resistant plates (Amp R , final concentration 100 g/mL) were coated.
  • the resistant plate was cultured in a 37 ° C incubator for 12-16 h.
  • Cry6Aa2 The most similar protein to this peptide is the Cry6Aa2 protein, which has an amino acid sequence identity of 29.7% and a similarity of 40.4%, according to the naming principle of the crystal protein gene (see Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean DH. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.
  • Example 2 Expression and purification of nematicidal crystal protein gene in Escherichia coli JM109 and determination of its biological activity
  • nematicidal crystal protein gene 5 -3 ⁇ 5 in Escherichia coli JM109 is a large number of expression of Cryl518-35 protein.
  • Applicants converted the recombinant expression plasmid pEMB0225 carrying its coding sequence to E. coli JM109 (purchased) From Tiangen, the recombinant strain JM109/pEMB0225 was obtained.
  • the recombinant strain was inoculated in 5 mL of LB liquid medium (additional final concentration of ⁇ /mL ampicillin) and activated overnight. Transfer to a 50 mL LB liquid medium at 1: 100 (v/v) and incubate at 37 ° C to OD 6 . .
  • 1.0 mmol/L of isopropyl-BD-thiogalactoside i.e., IPTG, purchased from Sigma Co.
  • IPTG isopropyl-BD-thiogalactoside
  • the above-mentioned 50 mL recombinant JM109/pEMB0225 3h induction culture was centrifuged at 12000 rpm for 30 s to collect the cells, and the cells were disrupted by ultrasonic (technical parameters: power 400 W, crushed for 30 s, intermittent 30 s), centrifuged at 12000 rpm for 15 min, and the supernatant was taken. .
  • the supernatant was then purified on a Ni-IDA affinity chromatography column (His nickel column) (purchased from Novagen) to purify the specific protein Cryl518-35 (the specific purification procedure was carried out according to the kit operating instructions).
  • the final purified product was subjected to SDS-PAGE electrophoresis. The results are shown in Fig. 4.
  • the purified protein was aligned with the molecular weight standard of the protein, and the estimated molecular weight was 35 kDa, which was basically consistent with the expected crystal protein Cryl518-35 molecular weight of 35.2 kDa.
  • Nematicidal crystal protein of the present invention Cryl518-35 was successfully expressed in E. coli JM109.
  • the second-instar larvae of Meloidogyne incognita (Me/oWogv /wp/a) were used as target nematodes to detect the biological activity of Cry protein on nematodes. Take tomato roots infected with northern root-knot nematodes and rinse them off with tap water. The root knot nematode egg mass was removed from the root, sterilized twice with 0.5% NaClO, and then incubated at 25 °C for 3 to 5 days. The hatched nematode was used as a target for bioassay.
  • Bioassay was performed on 96-well plates, and 40 second-instar larvae were inhaled per well (specific methods: Yu Ziquan, Wang Ganlan, Liu Bin, Zou Xue, Yu Ziniu, Sun Ming. 2007.
  • Bacillus thuringiensis companion crystal protein pair Establishment of a bioassay method for plant parasitic nematodes and screening of highly virulent strains. Journal of Agricultural Biotechnology 15, 867-871). The whole experiment was set to 5 ⁇ 7 concentration gradients, and each concentration was set to 3 replicates. 20 g/mL BSA was used as a negative control, and the purified Ciy6Aa2 protein was used as a positive control. After 5 days, the mortality was counted, and the mortality was corrected and converted into a probability value. The protein concentration was converted into a logarithmic value, and the regression equation between the two was calculated to calculate LC 5Q . The results are shown in Table 1.
  • Table 1 Activity of nematicidal crystal proteins Cryl518-35 and Cry6Aa2 against northern root-knot nematodes Crystal protein regression equation R 2 value semi-lethal concentration ( ⁇ g/mL)
  • the nematicidal crystal protein Cryl518-35 obtained by the present invention shows high insecticidal activity against the northern root-knot nematode, semi-lethal concentration It was 7.43 g/mL and was comparable to the nematicidal activity (7.55 g/mL) of the known nematicidal crystal protein Cry6Aa2. These results demonstrate that the nematicidal crystal protein Oyl518-35 obtained by the present invention has high activity against northern root-knot nematodes.
  • the crystal protein Cryl518-35 discovered by the invention has high virulence to the root-knot nematode, and provides a new genetic resource for the biological control of the northern root-knot nematode.
  • the protein, Cryl518-35 has shown broad application prospects in the field of microbial insecticides and transgenic plants for controlling plant root-knot nematodes.

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Description

苏云金芽胞杆菌杀线虫晶体蛋白基因 C ^5 S 5 技术领域 本发明属于生物防治和生物技术领域。 涉及苏云金芽胞杆菌杀虫晶体 蛋白基因的分离与克隆， 与生物农药基因工程以及转基因植物有关。 背景技术 苏云金芽胞杆菌 thuringiensis, Bt)是一类广泛存在于土壤中的 革兰氏阳性细菌， 在其生长发育过程中， 能够形成内生芽胞， 因其伴随芽 胞形成而产生对昆虫具有特异毒性的杀虫晶体蛋白 （ Insecticidal Crystal Proteins, ICPs) , 而有别于分泌肠毒素的蜡状芽胞杆菌 (Bacillus cereus ) 和 引起炭疽病的炭疽芽胞杆菌 (Sac /^ m2 /zraC^)。 研究表明， 苏云金芽胞杆 菌产生的晶体蛋白可以对鳞翅目、 双翅目、 鞘翅目、 膜翅目、 同翅目等昆 虫纲 10个目 500多种昆虫以及原生动物、 线形动物门、 扁形动物门中某些 有害种类也有特异的生物活性 （Schnepf H E, Crickmore N， Rie J V， Lereclus D, Baum J, Feitelson J, Zeigler D R, Dean D H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev, 62: 775-806)。 Aroian的 实验室用实验充分证明了苏云金芽胞杆菌对线虫的特异活性 （Wei* J.Z., Hale* K.， Carta L.， Platzer E.， Wong C, Fang S.C., and Aroian R.V. (2003). Bacillus thuringiensis Crystal proteins that target nematodes. Proc. Natl. Acad. Sci. 100: 2760-2765
苏云金芽胞杆菌野生菌株一般含有多个编码杀虫晶体蛋白的基因。 自
确 认 本 1981年 Schnepf和 Whiteley从库斯塔克亚种 （subsp. kurstaki) 菌株 HD- 1 中克隆得到第一个杀虫晶体蛋白基因以来， 陆续发现并鉴定了许多新的杀 虫晶体蛋白基因。 此后， 新的杀虫晶体蛋白基因的克隆一直是苏云金芽胞 杆菌研究领域中的热点。 因此在 1995 年的无脊椎病理学年会上成立了由 Crickmore等学者组成的杀虫晶体蛋白基因命名委员会， 并于 1996年正式 提出了苏云金芽胞杆菌杀虫晶体蛋白按氨基酸序列同源性进行分类的新的 分类系统， 规定了命名规则和分类原则。 其规定： 基因是来自苏云金芽 胞杆菌编码伴胞晶体蛋白的杀虫基因或者与已知 c ^y基因有明显的序列相 似性的基因， yi基因是编码具有溶细胞活性的伴胞晶体蛋白基因或与已知 编码 Cyt蛋白有明显的序列相似性的基因。 根据全长基因推导的氨基酸序 列的同源性分为四个等级。 级与级之间的界限为同源性 95%、 78%和 45%。 截至 2008年 12月，苏云金芽胞杆菌杀虫晶体蛋白基因已累计达到 55类 436 种 基因和 2类 27个 基因 ( Crickmore N, Zeigler DR, Feitelson J， Schnepf E, van Rie J, Lereclus D, Baum J， Dean DH. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62 :807-813 ； http：〃 www.lifesci.sussex.ac.uk/home Neil_Crickmore/Bt/index.html)。
根结线虫 （
Figure imgf000003_0001
的植物寄生线虫，主要寄生于被感 染植物的块根、 块茎、 鳞茎或球茎上， 从寄主体内吸取营养， 造成寄主植 物的生长发育不良、 畸形、 矮化甚至死亡， 每年都给农业生产带来重大损 失。 它们可以寄生于 1 10 多种植物， 包括大豆、 花生、 烟草、 黄瓜、 牡丹 等， 而茄科、 葫芦科、 十字花科等植物的受害尤其严重 （刘维志. 植物病原 线虫学北京： 中国农业出版社， 2000)。 由于其生活史短， 分布面广， 目前 已经成为农业生产上最重要的病原生物之一 （Widmer T L， Abawi G S. Mechanism of suppression of Meloidogyne hapla and its damage by a green manure of Sudan grass. Plant Disease, 2000, 85(5): 562-568.)。 同时又由于根 结线虫的侵入往往会使真菌和细菌更易侵染植物， 是诱发植物病害的重要 原因之一 （汪来发， 杨宝君， 李传道. 根结线虫生物防治研究进展. 南京林 业大学学报（自然科学版 2002, 26(1 ): 64-68.)。
传统的防治根结线虫的方法主要包括轮作、 '休耕和化学防治。 但是在 许多的发展中国家， 人们必须依靠多年生作物和作物连作， 轮作和休耕都 很难用于防治根结线虫； 而化学防治的方法又会给环境带来很大污染。 因 此， 通过生物的方法防治根结线虫逐渐成为该领域的研究热点之一。 关于 苏云金芽胞杆菌对植物寄生线虫防治的研究， 最早的报道为 1972年 Prasad 等首次发现该菌的 β-外毒素即苏云金素对根结线虫的卵和幼虫有较高的毒 杀作用 （ Prasad S S V, Tilak K V B R， Gollakota K G. Role of Bacillus thuringiensis var. thuringiensis on the larval survivability and egg hatching of Meloidogyne spp., the causative agent of root-knot disease. J. Invertebr. Pathol. , 1972, 20: 377-378.)。 随后的研究又发现苏云金素可以明显降低线虫对植物 的侵染 ( Ignoffo C M， Dropkin V H. Deleterious effects of thermostable toxin of Bacillus toxin of Bacillus thuringiensis on species of soil-inhabiting my eel iophagus , and plant-parasitic nematodes. J. Kans. Entomol. Soc , 1977, 50: 394-398.; Devidas P, Cibulski R J， Rehberger L. Evaluation of Bacillus thuringiensis exotoxin for nematode control. Nematologica, 1988, 34(3): 249-301.)。 上个世纪 80年代 Bone等人的研究首次证实了苏云金杆菌晶体 蛋白对线虫的杀虫活性 （ Bone L W, Bottjer K P， Gill S S. Trichostrongylus colubriformis: Egg lethality due to Bacillus thuringiensis crystal toxin. Exp. Parasitol , 1985， 60: 3 14-322.)。之后， 美国 Mycogen公司的大量研究工作也 进一步证实了苏云金芽胞杆菌伴胞晶体对线虫的作用 （ Bradfish G A (1992). Process for controlling lepidopteran pests. EP 19920920639.)。 此外他们还克隆 了多个具有线虫活性的毒素蛋白的编码基因并申请了相关专利， 而我们所 能看到的关于苏云金芽胞杆菌对线虫的防治的报道也多见于这些专利中的 描述。
目前，已经有越来越多的抗线虫的苏云金杆菌制剂在田间应用。 Sharma 等用苏云金亚禾中 (subsp. i/7wr "<^era )禾卩以色列亚种 (subsp. ^rae/era^)菌齐 lj 处理大麦根际土壤对南方根结线虫 Meloidogyne ^ ^ 'to)起到了一定的 防效； 此外， 他们还发现， 用以色列亚种菌株 Bti-H14的毒素蛋白处理大豆 和玉米种子，能够使大豆胞囊线虫的寄生率显著降低 （ Sharma R D.
Figure imgf000005_0001
thuringiensis: a biocontrol agent of Meloidogyne incognica on barely. Nematologia Brasilera, 1994, 18: 79-84.)。 Ivanova则报道某种苏云金芽胞杆 菌制剂能够用于防治黄瓜和番茄上的根结线虫； 而 Ensard则用苏云金杆菌 制剂在防治香蕉穿孔线虫方面也取得了显著效果 （ Ivanova T S. Efficiency of biopreparation in control of gall nematode in protected soil. Agrokhimiya, 1996, 3 : 101 - 106.; Ensard J. Effects of three microbial broth cultures on growth and populations of free living and plant-parasitic nematodes on banana. European Journal of Plant Pathology, \ 99 451-463 )。 然而， 随着 Bt 杀虫剂应用的推广和使用强度的不断增加， 目标害虫的抗性现象陆续被科 学家们所报道。研究人员从目标害虫对 Bt杀虫剂的抗性机制的研究中发现， 昆虫对 Bt杀虫剂的抗性的产生与受体的识别与结合密切相关。 因此， 新的 杀虫晶体蛋白基因的克隆和应用已经成为预防与控制目标害虫对 Bt杀虫剂 产生抗性的关键途径， 是各种防治策略的核心内容。 而目前杀线虫晶体蛋 白基因资源十分有限， 因此， 寻找与克隆新的杀线虫晶体蛋白基因已经成 为苏云金芽胞杆菌研究领域中最活跃的部分之一。 苏云金芽胞杆菌 YBT- 1518菌株是由我室分离并保存的无鞭毛菌株， 它能够在其芽胞形成过 程中形成米粒状的伴胞晶体， 生物测定表明， 它的晶体蛋白对秀丽小杆线 虫 ( Caenorhabditis elegans )禾口； I匕方丰艮结线虫 ( Meloidgyne hapla )具有较高 的活性。 本发明在于分离新的杀线虫晶体蛋白基因， 用于构建抗线虫的微 生物杀虫剂和转基因植物。 发明内容 本发明从苏云金芽胞杆菌 YBT- 15 18中分离并克隆了一种新型的杀线虫 晶体蛋白基因
Figure imgf000006_0001
， 发现它对北方根结线虫具有高毒力， 揭示了该晶 体蛋白在防治根结线虫领域的应用。
本发明是这样实现的：
申请人从苏云金芽胞杆菌菌株 YBT- 15 18中分离到一个新的晶体蛋白基 因 cr_ 5 S-35。 经过测序发现本发明的晶体蛋白基因 θ ·5 -35其编码区由 861个碱基组成， 具有序列表 SEQ ID NO: 1所示的核苷酸序列。经过序列分 析发现本发明的 cr_ 5 -35基因编码的蛋白 Cry l 5 18-35是由 286个氨基酸残 基组成， 具有序列表 SEQ ID NO : 1所示的氨基酸序列， 预计分子量大小为 34 kDa。通过原核表达和室内生物测定证实本发明的基因 c ^ ^-35在微生 物中表达的 Cryl518-35蛋白， 能够对北方根结线虫有毒杀作用，这就预示着 该基因可以用于构建抗线虫的转基因植物。
本发明提供的上述基因序列是一种新的杀线虫晶体蛋白基因， 该基因可 以应用于转化微生物和植物，使之表达出 Cryl518-35，使受体生物表现出对 线虫的毒杀活性， 在寄生线虫的生物防治方面具有广泛的应用价值。
更详细的技术方案参考实施例部分的描述。 附图说明 序列表 SEQ ID NO: 1是本发明分离克隆的苏云金芽胞杆菌杀线虫晶体蛋白 基因的核苷酸序列， 序列表 SEQ ID NO: 2是其编码序列；
图 1 : 是本发明实施例中杀线虫晶体蛋白基因 ^7·57<§-35所在的 BAC克隆子 的物理图谱；
图 2: 是本发明实施例中克隆载体 pUC18- 75 §-35的构建图及其物理图 谱；
图 3 : 是本发明实施例中超量表达载体 ρΕΜΒ0225的构建图及其物理图谱； 图 4:是本发明实施例中杀线虫晶体蛋白基因 在 JM109中表达纯化 的 SDS-PAGE电泳分析；
M: 蛋白质分子量标准；
1： 纯化后的 Cryl518-35杀线虫晶体蛋白。 具体实施方案 以下叙述是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。 有关 DNA的标准操作方 法和所使用的药品均参考 《分子克隆实验指南》 所描述的内容 （参见萨姆 布鲁克和拉塞尔， 2001， 分子克隆实验指南， 第三版， 金冬雁等(译)， 科学 出版社， 北京） 。 本发明中所涉及的其他各种实验操作， 均为本领域的常 规技术， 文中没有特别说明的部分， 本领域的普通技术人员可以参照本发 明申请日之前的各种常用工具书、 科技文献或相关的说明书、 手册等加以 实施。 实施例 1 苏云金芽胞杆菌 YBT-1518中杀线虫晶体蛋白基因 的 克隆
本发明以苏云金芽胞杆菌 YBT-1518作为杀线虫晶体蛋白基因 c 5 -35的来源菌株，该菌株的来源见文献： Yu, Z.， P. Bai, W. Ye, F. Zhang, L. Ruan, Z. Yu, and M. Sun. A Novel Negative Regulatory Factor for Nematicidal Cry Protein Gene Expression in Bacillus thuringiensis. J. Microbiol. Biotechnol. 18(6): 1033-1039。 该菌株无鞭毛， 能形成典型的长米粒状晶体， 对根结线虫和秀丽小杆线虫具有高毒力。
1. 苏云金芽胞杆菌 YBT-1518总质粒的抽提
将苏云金芽胞杆菌菌株 YBT-1518过夜活化， 按 l/100(vA 的接种量转 接至 5 mL新鲜的 LB培养基中 （LB培养基配方： 胰蛋白胨 1%， 酵母提 取物 0.5%，氯化钠 0.5%， pH7.0), 28°C， 200 rpm 培养至对数生长中期。 STE(10 mM Tris-HCl, 1 mM EDTA pH 8.0)洗涤菌体 1次； 加入 90 μL溶液 1(50 mM 蔗糖， 25 mMTris.HCl(pH8.0)， 10 mM EDTA)悬浮菌体， 再加 100 mg/mL的溶菌酶 (溶液 I配制) ΙΟ μί, 冰浴 2 h以上； 加入 200 新鲜配制 的溶液 11(0.2 M NaOH, 1% SDS)，轻缓混匀后置冰上冰浴 5〜10 min;加 150 μL溶液 III(5 M KAc 60 mL， 冰乙酸 11.5 mL， 定容至 100 mL )， 混匀后置 冰上放 5 min; 以 12,000 rpm离心 5 min, 吸取上清液到一个新的离心管， 苯酚 /氯仿 /异戊醇溶液 (25:24: 1， v:V:V)抽提 2次；上清液中加入 2倍体积的无 水乙醇或等体积的异丙醇， 于室温静置 5〜10 min; 12,000 离心 5 min， 弃 上清， 加入 70%乙醇， 离心洗涤沉淀 1次； 真空干燥沉淀， 用 30 μί含有 20 g/mL RNase的 TE溶液（1 mM EDTA, 10 mM Tris-HCl, pH 8.0)溶解沉 淀。
2. 苏云金芽胞杆菌 YBT-1518总质粒文库的构建
将上步制备的质粒加入适量 wdin进行不完全酶切， 回收 5-12 kb片段连 接于载体 PHT304转化大肠杆菌 DH5a，并涂布氨苄青霉素抗性平板 (含有 100 g/mL的氨苄青霉素， 100 g/mL IPTG和 80 g/mL的 X-gal)， 抗性平板置于 37°C培养箱中培养 14 h后，挑取白斑于新鲜的 LB平板 (含有 lOO g/mL的氨苄 青霉素)，即得菌株 YBT-1518的质粒文库，随机挑取 1000个克隆保存于 -80°C 冰箱， 从中随机挑取 12个克隆抽取质粒之后进行/ ^dlll酶切和 0.8%琼脂糖 电泳检测得该文库的平均插入片段大小为 8kb， 1000个克隆大约可以覆盖整 个质粒基因组的 2〜3倍 (假设质粒基因组总大小为 300 kb, 每个质粒的拷贝 数为 3个)。
3. 苏云金芽胞杆菌 YBT-1518中杀线虫晶体蛋白基因 cr 5/S-35的克隆 利用基因 cr_y 的特异片段为探针，从上述构建好的总质粒文库中筛选到 了一个阳性克隆子 EMB0229, 对该克隆子的测序 （由 Irwitrogen公司完成） 表明该重组子中所含有一个大小为 10kb左右的质粒 pBMB0229(见图 1)。对该 质粒进行序列分析发现该质粒上除了编码有基因 C ^4«2外， 在这个基因的 下游还存在一个同 高度相似的基因。 申请人依据该基因的特异序列 并在两端引入酶切位点 ^mffl和//^ΛΠ设计上游引物 P1 (引物序列为： 5，-CGCGGATCCATGAATAATATTAATAAGAAG-3，）和下游引物 Ρ2(引物 序列为： 5，-CCCAAGCTTCTAAGATATATAAGCATTTAAAG-3，）， 并以重 组质粒 PBMB0229为模板进行 PCR扩增以获得目标基因， PCR反应的体系和 程序如下：
25 μL反应体系包含： 2.5 μL lOxPCR反应缓冲液， 1 dNTP (各 2.5 mM) ， 0.5 特异性上游引物 PI (20 mM) , 0.5 特异性下游引物 Ρ2 (20 mM) ， 0·2 μί模板 （质粒 ρΒΜΒ0229) ， 0.25 Ex Taq酶， 加灭菌 去离子水至 25 μί。 PCR反应参数和程序为： 94°C， 5 min, 1个循环； 94°C， 20 s, 52 °C, 20 s, 72 °C, 1.5 min, 30个循环； 72°C， 10 min, 1个循环。
将扩增到的目标片段通过 PCR产物回收试剂盒（购自 Omega生物技术 公司）纯化回收后通过 T/A克隆连接到 T载体 pMD18-T Simple Vector上（购 自 TaKaRa 公司） ， 得到含有 cryI518-35 全长序列的重组质粒 p\JC\S-cryl518-35 (；见图 2).。之后将该重组质粒转化大肠杆菌 (£. co/)DH5a, 并涂布氨苄青霉素抗性平板 (AmpR， 终浓度为 100 g/mL)。将抗性平板置于 37°C培养箱中培养 12-16h，待转化子长到一定大小以后，通过质粒快检（大 肠杆菌质粒快检方法参考： 张桂敏等， 一种简便快速筛选重组子的方法， 湖北大学学报（自然科学版 )，2005, 27 (3): 280-281.) 对转化子进行筛选。 将 筛选到的转化子用 5 mL的液体 LB培养基活化培养， 然后抽提质粒进行酶 切验证，酶切片段大小与预期大小一致。最后将筛选到的阳性转化子用 5mL 的液体 LB培养基活化培养， 取 lmL的过夜培养物送样测序（由 Irwitrogen 公司完成）。测序结果显示该基因具有如序列表 SEQ ID NO: l中所示的核苷 酸序列， 申请人将该基因命名为 c^ 57S-35 (基因命名以斜体小写表示）。 通过软件分析预测此段编码序列可编码一个如序列表 SEQ ID ΝΟ: 1中所示 的由 286个氨基酸组成的多肽片段， 预测其分子量为 34kDa, 申请人将其 命名为 Cryl 518-35 (蛋白命名以正体大写表示）。
4. 苏云金芽胞杆菌 YBT-1518中杀线虫晶体蛋白基因 c^ J/S- 的序列 分析
对 Cr_yA5/«^ 5的进一步序列分析表明， 该基因由 861个核苷酸组成， 编码 一个由 286个氨基酸组成的多肽， 其理论分子量为 34-kDa， 在 GenBank上进 行的 BlastP分析发现， 同该多肽最相似的蛋白质为 Cry6Aa2蛋白， 其氨基酸 序列的一致性为 29.7 %，相似性为 40.4% , 依据晶体蛋白基因的命名原则 (参 见文献 ( Crickmore N, Zeigler DR, Feitelson J, Schnepf E, van Rie J, Lereclus D, Baum J, Dean DH. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:807-813), 该基因将被归为 I类新型 cr_y基因。 实施例 2 杀线虫晶体蛋白基因 在大肠杆菌 JM109中的表达纯化 及生物活性测定
1. 杀线虫晶体蛋白基因 cr_yA57<5-35在大肠杆菌中超量表达载体的构建 将实施例 1中获得的含有基因 cr_ 5/S-35的重组质粒 pUC 18-cr> /5 S-^5 经过 ^ HI和/^ 双酶切， 同时将质粒载体 pQE30进行相同的双酶切， 然 后将上述载体和外源的酶切产物通过 0.8%的琼脂糖凝胶分离后， 切下目标 片段并通过 DNA凝胶回收试剂盒 （购自 Omega生物技术公司） 纯化回收。 之后将两者通过 T4 DNA连接酶（购自 TaKaRa生物技术公司）连接， 从而获 得了含有基因 c ^5 35全长 ORF的重组表达质粒 pEMB0225 (见图 3 ) ， 将 该重组表达质粒转化大肠杆菌 c0/ DH5a (购自 Tiangen公司） ， 并抽提质 粒分别进行 ^ HI和 / /^///双酶切验证，酶切片段大小与预期大小一致。进 一步的测序结果表明 c ^57S-35基因被正确地插入了到了表达载体 pQE30 中。
2. 杀线虫晶体蛋白基因 5 -3·5在大肠杆菌 JM109中的表达和纯化 为了大量表达 Cryl518-35蛋白， 申请人将上述携带有其编码序列的重 组表达质粒 pEMB0225转化到大肠杆菌 JM109 (购自 Tiangen公司） 中， 得到了重组菌 JM109/ pEMB0225。 将该重组菌株接种于 5 mL LB液体培养 基中 (另外附加终浓度为 ΙΟΟ μΒ/mL氨苄青霉素)， 过夜活化。 以 1 : 100 (v/v) 转接至 50 mL LB液体培养基中， 置于 37°C摇床培养至 OD6。。为 0.5-0.8左 右以后，加入 1.0 mmol/L 的异丙基 -B-D-硫代半乳糖苷（即 IPTG，购自 sigma 公司） 于 37°C诱导培养 3h。 将上述 50 mL重组菌 JM109/ pEMB0225的 3h 诱导培养物经过 12000 rpm离心 30s收集菌体， 利用超声波（技术参数： 功 率 400 W， 破碎 30s， 间歇 30s) 将细胞破碎后 12000 rpm离心 15min取上 清。 然后将上清液过 Ni-IDA亲和层析柱 （His镍柱） （购自 Novagen公司） 提纯该特异蛋白 Cryl518-35 (具体的纯化步骤按照试剂盒操作说明书进 行）。 对最终的纯化产物进行 SDS-PAGE电泳检测， 结果如图 4所示， 提纯 的蛋白与蛋白分子量标准比对， 估算分子量为 35 kDa, 与预计的晶体蛋白 Cryl518-35分子量大小 35.2 kDa基本吻合， 证明本发明的杀线虫晶体蛋白 Cryl518-35在大肠杆菌 JM109中得到了成功的表达。
3. 晶体蛋白 Cryl518-35对北方根结线虫生物活性的测定
以北方根结线虫 (Me/oWogv /wp/a)的二龄幼虫作为靶标线虫检测 Cry 蛋白对线虫的生物活性。 取被北方根结线虫感染的番茄根， 用自来水冲洗 干净。从根部取下根结线虫卵块，用 0.5 % NaClO消毒 2次，然后 25°C 孵 化 3〜5d， 孵化出的线虫即作为生物测定的靶标。 生物测定在 96孔板上进 行，每孔吸入 40头 2龄幼虫（具体方法参考文献：余子全， 王乾兰， 刘斌， 邹 雪， 喻子牛， 孙明. 2007. 苏云金芽胞杆菌伴胞晶体蛋白对植物寄生线虫生 物测定方法的建立和高毒力菌株的筛选. 农业生物技术学报 15， 867-871 )。 整个实验设 5〜7个浓度梯度， 每个浓度设 3个重复， 用 20 g/mL BSA作 为阴性对照， 纯化的 Ciy6Aa2蛋白作为阳性对照。 5天后统计死亡率， 将 死亡率校正后换算成几率值， 蛋白浓度换算成对数值， 求出二者之间回归 方程计算 LC5Q， 结果见表 1。
表 1 : 杀线虫晶体蛋白 Cryl518-35 和 Cry6Aa2对北方根结线虫的活性检测 晶体蛋白 回归方程 R2值 半致死浓度 （^g/mL)
Cry6Aa2 y=1 .0297x+4.0961 0.9896 7.55
Cryl 518-35 y=1 .1067x+4.0359 0.9658 7.43 从上表中可以看出， 本发明得到的杀线虫晶体蛋白 Cryl518-35对北方 根结线虫显示出了很高的杀虫活性， 半致死浓度为 7.43 g/mL， 而且与己知 的杀线虫晶体蛋白 Cry6Aa2的杀线虫活性（7.55 g/mL)相当。 这些结果证 明本发明得到的杀线虫晶体蛋白 Oyl518-35对北方根结线虫具有高活性。
本发明发现的晶体蛋白 Cryl518-35对北方根结线虫具有高毒力， 为北方 根结线虫的生物防治提供了一种新的基因资源。 作为一种新的杀线虫晶体 蛋白， Cryl518-35在防治植物根结线虫的微生物杀虫剂和转基因植物领域显 示出了广泛的应用前景。

Claims

权利要求:
1、 一个来自于苏云金芽胞杆菌的杀线虫晶体蛋白基因 c ^5^-35， 它的 核苷酸序列如序列表 SEQIDNO: 1所示。
2、 一个来苏云金芽胞杆菌的杀线虫晶体蛋白基因 它编码的 蛋白具有如序列表 SEQIDNO: 2所示的氨基酸序列。
3、权利要求 2所述的蛋白在制备防治植物寄生线虫的微生物制剂中的应 用。
4、权利要求 2所述的蛋白在防治植物寄生线虫的转基因微生物和转基因 植物中的应用。
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