||Pseudomonas taiwanensis TKU015殺蟲蛋白之基因選殖與特性研究
||Molecular Cloning and Characterization of an Insecticidal Protein from Pseudomonas taiwanensis TKU015
||Graduate Institute of Life Sciences
Pseudomonas taiwanensis TKU015為台灣篩選出的土壤菌， 經由檢查其殺蟲蛋白之胺基酸序列發現和Pseudomonas entomphila L48之殺蟲蛋白具有極高的相似性，故設計引子選殖出其殺蟲蛋白基因作進一步的研究。用聚合酶連鎖反應擴增之片段有兩段，為先前已被選殖出來，大小為2 kb的活性片段，及運用Genome Walker TM擴增原序列基因C端片段所得全長3 kb片段 。將殺蟲蛋白基因接合到pET-32 載體上，再轉形到大腸桿菌BL21宿主表達， 在37度下以IPTG誘導搖瓶過夜，以表現目標蛋白。利用HisTag將目標蛋白進一步純化出來，再去除多餘的鹽類，最後將純化的重組蛋白對果蠅幼蟲做生物活性測試，並比較探討不同蛋白質長度片段之殺蟲活性的差異。
||The availability of effective and cheap insecticides heralded an agricultural revolution. The insecticides not only help farmers increase yields, but also lower food prices. However, use of these insecticides is detrimental since some of these persist in the environment and accumulate in living organisms, causing various fatal diseases and are also toxic to nontarget species. In time new resistant strains of insects emerge, requiring increased doses of insecticides and introduction of new insecticides. Recently, bioinsecticides are being used as an alternative to the insecticides. Bioinsecticides are certain types of pesticides derived from natural materials including animals, plants, and bacteria. The toxic action of bioinsecticides is often specific to a single group or species of insects, does not hazardous to non-target species. They are also degraded in sunlight. The most widely used bioinsecticides are microbial pesticides.
Pseudomonas taiwanensis TKU015 was isolated from Taiwan soil and was found that the amino acid sequence of one of its insecticidal proteins was similar to Pseudomonas entomphila L48, so the primers was designed to clone this insecticidal protein gene in further studies. By using polymerase chain reaction method, the 2-kb DNA fragments encoding insecticidal toxin was obtained as described previously. By using Genome WalkerTM kit, the 3-kb fragments encoding full-length insecticidal toxin was obtained. The insecticidal toxin genes were subcloned into the pET-32 Xa/Lic vector and then transformed into E. coli BL21. The recombinant insecticidal toxins were expressed by the transformed E. coli BL21 by induction with IPTG at 37 degrees overnight. The target proteins fused with His-tag were purified and de-salted. Finally, the purified insecticidal proteins were analyzed its insecticidal effect on Drosophila larvae.
中文摘要 ············································································· I
英文摘要 ············································································· III
目錄 ··················································································· V
圖目錄 ················································································ VIII
表目錄 ················································································ X
第一章 序論 1
第二章 文獻回顧 2
2.0 殺蟲劑之簡介 2
2.1 無機殺蟲劑 2
2.2 有機殺蟲劑 2
2.3 生物型殺蟲劑 4
2.3.1 植物類殺蟲劑 4
2.3.2 生化活性殺蟲劑 4
2.3.3 微生物殺蟲劑 5
2.4 假單孢菌 7
2.4.1 假單孢菌簡介 7
2.4.2 假單孢菌殺蟲性質 8
第三章 材料與方法 16
3.1 實驗菌株、載體、果蠅 16
3.2 分子生物實驗套組 17
3.3 培養基 18
3.4 試藥 19
3.4.1 PCR試藥 19
3.4.2 DNA電泳試藥 19
3.4.3 抗生素試藥 20
3.4.4 轉形試藥 20
3.4.5 聚丙烯醯胺膠體電泳試藥 20
3.4.6 快速蛋白質液相層析純化試藥 21
3.5 實驗儀器 22
3.6 Pseudomonas taiwanensis TKU015生長曲線及菌數 25
3.7 應用 GenomeWalkerTM 尋找殺蟲蛋白基因 25
3.10 重組殺蟲蛋白之純化 27
3.11 快速蛋白質液相層析純化 27
3.13 聚丙烯醯胺膠體電泳 28
3.14 重組蛋白生物毒殺活性試驗 28
第四章 結果與討論 37
4.1 Pseudomonas taiwanensis TKU015生長曲線及菌數探討 37
4.1.1 Pseudomonas taiwanensis TKU015生物毒性測試 37
4.2 應用GenomeWalkerTM找出Pseudomonas taiwanensis TKU015殺蟲蛋白序列全長 37
4.2.2 殺蟲蛋白功能性區域預測 38
4.3 Pseudomonas taiwanensis TKU015殺蟲蛋白基因選殖 39
4.3.1 殺蟲蛋白基因接合在pET32 Xa/LIC載體上 39
4.3.2 基因轉形 39
4.4 生物活性測試 40
第五章 結論 51
第六章 參考文獻 52
圖2.1 DDT的結構式 ································································ 10
圖2.2 毒魚藤素結構式 ····························································· 11
圖2.3 除蟲菊結構式 ································································ 12
圖2.4 蘇力菌電顯圖 ································································ 13
圖2.5 Pseudomonas entomophila 毒殺機制範例 ······························· 14
圖3.1 Pseudomonas taiwanensis TKU015殺蟲蛋白基因選殖實驗流程圖 24
圖3.2 GenomeWalkerTM 流程圖 ··················································· 34
圖3.3 pET32a載體 ··································································· 35
圖3.4果蠅生長流程 ································································· 36
圖4.1(a) Pseudomonas taiwanensis TKU015 在LB 液態培養基之生長曲線 ··························································································· 42
圖4.1 (b) Pseudomonas taiwanensis TKU015在LB液態培養基之菌數變化 ··························································································· 42
圖4.2培養120 h之Pseudomonas taiwanensis TKU015之生物活性試驗 43
圖4.3 運用GenomeWalkerTM 基因特異性引子GSP1、GSP2找出 Pseudomonas taiwanensis TKU015 殺蟲蛋白序列全長 ······················· 44
圖4.4 Pseudomonas taiwanensis TKU015 3-kb 殺蟲蛋白全長 ·············· 45
圖4.5殺蟲蛋白之結構區域 ························································ 46
圖4.6 以Genome WalkerTM所得全長片段設計p470及p471引子，對
Pseudomonas taiwanensis TKU015 進行聚合酶連鎖反應所得3-kb殺蟲蛋白產物之電泳圖 ········································································· 47
圖4.7 3-kb殺蟲蛋白各純化步驟之SDS-PAGE電泳分析圖 ················ 48
圖4.8 2-kb殺蟲蛋白各純化步驟之SDS-PAGE電泳分析圖 ················ 49
圖4.9轉殖殺蟲蛋白基因至大腸桿菌及純化蛋白之毒殺活性比較 ········ 50
表2.1 Pseudomonas spp.毒殺昆蟲比較整理表 ·································· 15
表3.1 實驗使用之引子與PCR產物 ············································· 30
表3.2 GenomeWalkerTM PCR配方 ················································ 31
表3.3 GenomeWalker TM PCR流程 ················································ 31
表3.4 PCR配方······································································· 32
表3.5 PCR流程······································································· 32
表3.6 FPLC buffer配方 ····························································· 33
表4.1培養時間對Pseudomonas taiwanensis TKU015毒殺活性之影響 ··· 41
林詠迪。2009。 Pseudomonas sp.TKU015 殺蟲蛋白之基因選殖與純化。碩士論文。淡江大學生命科學系研究所。
高穗生、謝奉家、 林宗俊、 曾瑞堂。2004。 兼具殺蟲與抗菌作用之線蟲共生細菌－光桿菌。植物保護學會會刊 46:163-172。
Akhurst, R. and G. B. Dunphy. 1993. Parasites and pathogens of insects. N. Beckage, S. Thompson, and B. Federici. ed. Academic Press, New York, NY.
Anadón, A., M. R. Martínez-Larrañaga, and M. A. Martínez. 2009. Review use and abuse of pyrethrins and synthetic pyrethroids in veterinary medicine. Vet. J. 182:7–20.
Anthony, M. A. and B. H. Harvey. 1936. Toxicological study of derris. J. Ind. Eng. Chem. 28:815–821.
Beard, J. 2006. DDT and human health . Sci. Total. Environ.355: 78 – 89.
Carlinia, C. R. and M. F. Grossi-de-Sa´. 2002. Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. Toxicon. 40:1515–1539.
Chattopadhyay, A., N. B. Bhatnagar, and R. Bhatnagar. 2004. Bacterial insecticidal toxins. Crit. Rev. Microbiol. 30:33–54.
Clyne, P. J., C. G. Warr, M. R. Freeman, D. Lessing, J. Kim, and J. R. Carlson. 1999. A novel family of divergent seventransmembrane proteins: candidate odorant receptors in Drsophila. Neuron. 22:327–338.
EPA. Biopesticides. U. S. Environmental Protection Agency. 2002. Available at: http://www.epa.gov/agriculture/tbio.html#Advantages.
Forst, S., B. Dowds, N. Boemare, and E. Stackebrandt. 1997. Xenorhabdus spp and Photorhabdus spp. Bugs that kill bugs. Annu. Rev. Microbiol. 51:47–72.
Kriege, J., E. Grosse-Wilde, T. Gohl, and H. Breer. 2005. Candidate pheromone receptors of the silkmoth Bombyx mori. Europ. J. Neurosci. 21:2167–2176.
Li, D., Q. Sun , M. Huang , J. Zhang, S. Bai, L. Zheng, J. Zhao, D. Qiu, L. Li, Z. Yang, M. You, G. Liu, Y. Zhang, C. Zhang, and S. Li . 2007. Agrobacterium–mediated genetic transformation of Elymus breviaristatus with Pseudomonas pseudoalcaligenes insecticidal protein gene. Plant. Cell. Tissue. Organ. Cult. 89:159–168.
Liu, S. G., W. Zhu, Z. R. Yang, S. R. Ge, and X. Y. He. 1995. Isolation and identiﬁcation of a pathogen of grasshoffers. Acta. Microbiol. Sin. 35:86–90.
Min, H., L. Daxu, X. Wenliang, G. Jianhua, Z. Jian, Z. Jie, Y. Zhirong, and S. Qun. 2008. Transformation of core Pseudomonas pseudoalcaligene insecticidal protein gene and its insecticidal expression in tobacco. Acta. Microbiol. Sin. 48:1198-202.
Pai, H. H., W. C. Chen, and C. F. Peng. 2004. Cockroaches as potential vectors of nosocomial infections. Infect. Control Hosp. Epidemiol. 25: 979–984.
Padmanabhan, V. , G. Prabakaran, K. P. Paily , and K. Balaraman. 2005. Toxicity of a mosquitocidal metabolite of Pseudomonas fluorescens on larvae & pupae of the house fly, Musca domestica . Indian. J. Med. Res. 121: 116-119.
Peng, R., A. Xiong, X. Li, H. Fuan, and Q. Yao. 2003. Aδ-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity. Appl Microbiol Biotechnol. 63: 300–306.
Petell, J. K., R. O. Fatig III, G. L. Orr, B. W. Schafer, J. A. Strickland, K. Sukhapinda, A. T. Woodsworth, and L. Guo. 1999. Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins purification and characterization of toxin A and toxin B. J. Biol. Chem. 274:9836-9842.
Péchy-Tarr, M., D. J. bruck, M. Maurhofer, E. Fischer, C. Vogen, M. D. Henkels, K. M. Donahue, J. Grunder, J. E. Loper, and C. Keel. 2008. Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens. Environ. Microbiol. 10:2368–2386.
Prabakaran, G., K. P. Paily, V. Padmanabhan, S. L. Hoti, and K. Balaraman. 2003. Isolation of a Pseudomonas fluorescens metabolite exotoxin active against both larvae and pupae of vector mosquitoes. Pest. Manag. Sci. 59:21-24.
Sadanandane, C., C. M. Reddy, G. Prabakaran, and K. Balaraman. 2003. Field evaluation of a formulation of Pseudomonas fluorescens against Culex quinquefasciatus larvae and pupae. Acta. Trop. 87:341-343.
Samiyappana, R., R. R. Commarea, R. Nandakumara, A. Kandana, S. Sureshb, M. Bharathib, and T. Raguchandera. 2003. Pseudomonas ﬂuorescens based bio-formulation for the management of sheath blight disease and leaffolder insect in rice. Crop. Prot. 21:671–677.
Shihua, D. and Y. Zhirong. 2001. N-Terminal analysis and antibody preparation of insecticidal protein from Pseudomonas pseudoalcaligenes. Acta. Microbiol. Sin. 41:642–645.
Vallet-Gely, I., B. Lemaitre, and F. Boccard. 2008. Bacterial strategies to overcome insect defences. Nat. Rev. Microbiol. 6:302-313.
Vodovar, N., M. Vinals, P. Liehl, A. Basset, J. Degrouard, P. Spellman, F. Boccard, and B. Lemaitre .2005. Drosophila host defense after oral infection by an entomopathogenic Pseudomonas species. PNAS. 102 :11414 –11419
Vosshall, B. L., H. Amrein, S. P. Morozov, A. Rzhetsky, and R. Axel. 1999. A spatial map of olfactory receptor expression in the Drosophila antenna. Cell. 96:725–736.
Waterfield, N., A. Dowling, S. Sharma, P. J. Daborn, U. Potter, and R. H. Ffrench-Constant. 2001. Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli. Appl. Environ. Microbiol. 67:5017-5024.
Witzgall, P., P. Kirsch, and A. Cork. 2010. Sex pheromones and their impact on pest management. J. Chem. Ecol. 36:80–100.
Zhang, J., J. Zhao, D. Li, S. Liu, L. Li, Q. Sun, M. Huang, and Z. Yang. 2009. Cloning of the gene encoding an insecticidal protein in Pseudomonas pseudoalcaligenes. Ann. Microbiol. 59:45-50.
Zhao, J., X. Luo, D. H. Chen, and Z. R. Yang. 2004. Study on the locusts energy metabolize ability inhibited by the insecticidal protein puriﬁed from Pseudomonas pseudoalcaligenes. Acta. Microbiol. Sin. 44:365–368