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

張興慈(Hsin-Tzu Chang)

指導教授 ： 王三郎

共同指導教授 ： 劉嚞睿(Je-Ruei Liu)

淡江大學/理學院/生命科學研究所碩士班/碩士(2010年)

https://doi.org/10.6846/TKU.2010.00097

摘要

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。

關鍵字

Pseudomonas taiwanensis ； 基因選殖 ； 殺蟲蛋白 ； 殺蟲劑

並列摘要

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.

並列關鍵字

Pseudomonas taiwanensis ； insecticidal protein ； gene cloning

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