殺蟲劑具有提高農業生產量、降低食品價格並具有成本低廉、效果迅速的優點。但隨者時間改變昆蟲會逐漸產生抗藥性，此外，殺蟲劑對周遭環境及非目標生物也具有毒害，會危害人體健康。近期，生物性殺蟲劑逐漸成為新式殺蟲劑研究的方向。生物性殺蟲劑乃採用天然物質原料，例如動物、植物、微生物等。它擁有對週遭環境污染性小及針對目標害蟲具專一性的特點。現又以微生物所生產的毒殺蛋白殺蟲劑為市場大宗。
 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.

目錄
頁次
誌謝
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
3.3.1果蠅培養基	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
3.8大腸桿菌表現載體構築及轉形	26
3.9重組蛋白質之表現	27
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
3.12去鹽	28
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
4.2.1殺蟲蛋白基因序列之比較	38
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


圖目錄
頁次
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
IX
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


表目錄
頁次
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

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