Nematicidal Activity of Supernatant from Bacillus thuringiensis Fermentation
-
摘要: 室内条件下，分别测定了苏云金杆菌Bt 00-50-5菌株液体和固体发酵物对南方根结线虫的毒力效果。结果显示，牛肉膏蛋白胨液体发酵法在72 h所得上清液的毒性最高，南方根结线虫死亡率达到99.0%，而固体麸皮发酵物对线虫的最高毒性为发酵8 h，南方根结线虫死亡率为89.0%。液体发酵上清液经葡聚糖凝胶G-75柱层析分离，柱下物在280 nm处的吸收值呈现出4个峰Ⅰ、Ⅱ、Ⅲ和Ⅳ，其值0.601，1.475，1.641和0.392，其中峰Ⅱ、Ⅲ、Ⅳ处收集的蛋白对南方根结线虫有毒杀作用，分子量最小的Ⅳ蛋白杀线虫活性最高。上清液天然聚丙烯酰胺凝胶电泳分离杀虫蛋白结果显示6条清晰蛋白带，其中37 kDa蛋白含量最多。
-
关键词:
- 苏云金杆菌00-50-5 /
- 发酵上清液 /
- 南方根结线虫 /
- 天然聚丙烯酰胺凝胶电泳
Abstract: Nematocidal activities of fermentation products of Bacillus thuringiensis 00-50-5 culture using a liquid or solid medium in the lab was investigated. The results showed that the supernatant collected from the 72-h-fermentation on abeef-extract broth showed a 99.0% lethality on Meloidogyne incongnita; while that from the 8-h-fermentation on a bran-based medium, 89.0%.The supernatant collected from the liquid fermentation was subsequently subjected to a Sephadex G-75 column chromatography separation. Four peaks with the absorbance of 0.601, 1.475, 1.641 and 0.392 at 280nm were present. The bioassay on them indicated that the proteins found at Peak Ⅱ, Ⅲ, and Ⅳ were toxic to M. incognita, and among them, the one from Peak Ⅳ with the smallest molecular weight exerted the greatest nematocidal effect onthe parasite. The native-PAGE profile on the supernatant showed 6 visible protein bands including a 37 kDa protein, which was the highest in amount. -
图 1 液体发酵不同时间取样离心后上清液对南方根结线虫的毒性
注：图中不同字母表示在0.05水平差异显著。下同。
Figure 1. Nematocidal toxicity of centrifuged supernatants from liquid broths of B. thuringiensis culture after varied fermentation durations
图 2 固体发酵不同时间取样离心后上清液对南方根结线虫的毒性
Figure 2. Nematocidal toxicity of supernatants from solid substrate medium of B. thuringiensis culture after varied fermentation durations
图 3 液体发酵不同时间取样的上清液在280 nm处的吸收值
Figure 3. Absorbance at 280 nm of supernatant from liquid broths of B. thuringiensis culture after varied fermentation durations
图 4 固体发酵不同时间取样的上清液在280 nm处的吸收值
Figure 4. Absorbance at 280 nm of supernatant from solid substrate medium of B. thuringiensis culture after varied fermentation durations
图 5 溶解Bt 00-50-5发酵产物离心上清液干粉蛋白的柱层析
注：±表示微毒，死亡率在5%~15%；+表示有毒，死亡率在20%~35%；+++表示毒力较高，死亡率大于75%。
Figure 5. PAGE profile of dissolvedprotein from Cfs powder after liquid B. thuringiensis 00-50-5 fermentation and supernatant centrifugation
-
[1] SCHNEPF E, CRIEKMORE N, VAN R J, et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiology and Morlecular Biology Reviews, 1998, 62(3):775-806. https://www.ncbi.nlm.nih.gov/pubmed/9729609 [2] FRANKENHUYZEN K V, TONON A. Activity of Bacillus thuringiensis cyt1Ba crystal protein against hymenopteran forest pests[J]. Journal of Invertebrate Pathology, 2013, (113):160-162. http://www.ncbi.nlm.nih.gov/pubmed/23545068 [3] FRANKENHUYZEN K V. Insecticidal activity of Bacillus thuringiensis crystal proteins[J]. Journal of Invertebrate Pathology, 2009, 101(1):1-16. doi: 10.1016/j.jip.2009.02.009 [4] PAYNE J, NARVA K E, UYEDA K A, et al. Polynucleotides encoding a 130 kDa pesticidal protein from Bacillus thuringiensis isolate PS201T6. U.S.6, 150, 165.[P]. 2000-11-21. [5] GOMEZ I, PARDO-LOPEZ L, MUNOZ C, et al. Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis[J].Peptides, 2007, 28(1):169-173. doi: 10.1016/j.peptides.2006.06.013 [6] PAYNE J M, HICKLE L A. Bacillus thuringiensis isolates active against Phthiraptera pests[P]. U.S. 5, 237, 746, 1993-12-28. [7] HOFTE H, WHITELEY H R. Insecticidal crystal proteins of Bacillus thuringiensis[J]. Microbiology and Molecular Biology Reviews, 1989, 53:242-255. [8] MOHAMMED S H, SEADY M A E, ENAN M R, et al. Biocontrol efficiency of Bacillus thuringiensis toxins against root-knot nematode, Meloidogyne incognita [J]. Journal of Cell and Molecular Biology, 2008, 7(1):57-66. http://www.researchgate.net/profile/Nasser_Eldin_Ibrahim/publication/221875386_Biocontrol_efficiency_of_Bacillus_thuringiensis_toxins_against_root-knot_nematode_Meloidogyne_incognita/links/0912f505783cd6b801000000.pdf?origin=publication_detail [9] DEVIDAS P, REHBERGER L A. The effects of exotoxin (Thuringiensin) from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans[J]. Plant Soil, 1992, 145:115-120. doi: 10.1007/BF00009547 [10] WEI J Z, HALE K, CARTA L, et al. Bacillus thuringiensis crystal proteins that target nematodes[J]. National Academy of Sciences, 2003, 100 (5):2760-2765. doi: 10.1073/pnas.0538072100 [11] 龙海波, 孙艳芳, 白成, 等.海南省象耳豆根结线虫的鉴定研究[J].热带作物学报. 2015, 36(2):371-376. http://www.cnki.com.cn/Article/CJFDTOTAL-RDZX201502025.htm [12] CANDAS M, LOSEVA O, OPPERT B. Insect resistance to Bacillus thuringiensis:Alterations in the indianmeal moth larval gut proteome[J]. Molecular and Cellular Proteomics, 2003, 2(1):19-28. doi: 10.1074/mcp.M200069-MCP200 [13] BAI C, VICK B A, YI S H. Characterization of a new Bacillus thuringiensis isolate highly active against Cochylis hospes[J]. Current Microbiology, 2002, 44:280-285. doi: 10.1007/s00284-001-0003-y [14] BAI C, BARKER J F, SHU X Y. A new Bacillus thuringiensis isolate with an unusually high activity against the Banded sunflower moth[C]//Proceedings of the 23rd Sunflower Research Workshop. National Sunflower Association, 2001:63-69. [15] BAI C, DEGHEELE D, JANSENS S, et al. Activity of insecticidal crystal proteins and strains of Bacillus thuringiensis against Spodoptera exempta[J]. Journal of Invertebrate Pathology, 1993, 62:211-215. doi: 10.1006/jipa.1993.1101 [16] BAI C, VICK B A. Fermentation of a new Bacillus thuringiensis strain[J]. HELIA, 2001, 24(35):47-54. https://www.researchgate.net/publication/266331623_FERMENTATION_OF_A_NEW_Bacillus_thuringiensis_STRAIN [17] 崔云龙.苏云金芽孢杆菌液体培养物上清液中杀虫活性物质的研究[J].微生物学报, 1993, 33(1):62-68. http://www.cnki.com.cn/Article/CJFDTOTAL-WSXB199301010.htm [18] PANG A S D, GRINGORTEN J L, BAI C. Activation and fragmentation of Bacillus thuringiesis δ-endotoxin by high concentrations of proteolytic enzymes[J]. Canadian Journal of Microbiology, 1999, 45:816-825. doi: 10.1139/w99-086 -
下载:
点击查看大图
计量
- 文章访问数: 1376
- HTML全文浏览量: 154
- PDF下载量: 174
- 被引次数: 0
