含melanin黑色素基因组载体之建构及其在萤光性假单胞菌之表现(精)

含melanin黑色素基因組載體之建構及其在螢光性假單胞菌之表現

Construction of a Mel Operon Containing Vector and Its Expression in Pseudomonas Fluorescens

中文摘要

化學性農藥(Chemical pesticids)的大量使用易造成生態環境污染、藥物殘留、農作物生產率降低甚而導致食品安全危機等問題，因此應用現代生物技術(Modern biotechnology)開發以微生物所生產的選擇性高、對人畜及環境危害低的安全性生物農藥(Biological pesticides)已是一必然趨勢。螢光性假單胞菌(Pseudomonas fluorescens) 屬非病原性根圈性微生物(Non-pathogenic rhizobacteria)，它可以有效抑制多種病原菌的活動和繁殖。經研究證實，這類微生物的病原拮抗機制主要源自所分泌的抗生物質，其中更以具砒硌(Pyrrol)環分子結構的pyrrolnitrin最具代表性。Pyrrolnitrin為光敏感物質(Photosensitive substance)，其pyrrol ring上與亞氨基(Imino group)的結合的單鍵，易因紫外光的激發而斷裂導致抑菌活性的喪失，因此如何有效保護其分子結構的完整性以發揮抑菌效果，便成為發展此類型生物殺菌劑(Biological fungicides)的關鍵所繫。Melanin黑色素為化粧品上泛用的天然紫外光保護劑(Natural UV protectants)，其生成機制及相關基因組(Operon)已漸被瞭解。本論文即是針對melanin黑色素的光保護特性，擬利用基因重組技術，將源自鏈黴菌(Streptomycents)的melanin黑色產生產性相關基因（mel基因組）轉殖到P. fluorescens進行基因表現，以保護所分泌的抗生物質，進而改良此類型生物殺菌劑的缺失。

在此之前本實驗室曾經針對pyrrolnitrin，利用melanin黑色素進行光保護效果評估，結果證實了其保護抗生物質免於被光分解失活的效力。根據此一事實，我們首先從耕作土

壤中，利用選擇性分離方法以及365nm紫外光長波光照的原理，選別了16株P. fluorescens，分別予以安定化及低溫保存。針對這些菌株我們進一步檢驗其內生性質體(Cryptic plasmid)的存在，做為後續篩選重組DNA宿主的根據。考慮菌種高耐藥性及轉殖質體DNA的不穩定等因素，我們最後選擇了編號PFC009的菌株做為接受外來質體的宿主。

在菌種鑑定方面，我們利用電子顯微鏡觀察、革蘭氏染色以及生理生化特殊分析等方法確認菌株所屬。我們同時利用高效能液相層析(HPLC)方法確認該菌株具有pyrrolnitrin的生產性。在含melanin黑色素基因表現質體DNA的建構方面，我們曾嘗試過多種策略，其中以利用1.帶有mel基因組和Ampicillin耐藥性基因的大腸桿菌(E. coli)表現質體pIF413 (3.9kb)，以及2.帶有Kanamycin耐藥性基因的廣宿廣性載體(Broad host range vects)pKT230(11.9kb)，兩個質體所建構的新質體pMP-101能以電擊法(Electroporation)被分別轉殖到目標宿主(Target hose) P. fluorescens PFC009和仲介宿主(Intermediate hose) E. coi JM109菌體內進行mel基因組的表現，因而確認其為穿梭性載體(Shuttlevector)。P. fluorescens PFC009在添加兩價銅離子、酪氨酸以及IPTG的固態培養基上，於接菌24小時後即可觀察到其melanin黑色素的生成。經重複轉接測試結果，含質體pMP-101的P. fluorescens PFC009，在不含選擇性標記(Selective markers)抗生素的培養基中仍可穩定表現melanin黑色素基因，因此推測其基因表現的安定性係源自質體pKT230上的轉位子(transposon)Tn903；藉由轉位作用(transpositon)可將質體pPM-101上的mel基因組併合到宿主細胞的染色體DNA上，為了確認轉位作用的發生與否，我們進行質體pMP-101的抽取及利用限制酵素XhoI和EcoRI剪出pIF413上面mel基因組上的部份片斷(1.4kb)作為探針(Probe)，進行南方點墨法(Southern blot)以確認mel基因組的存在位置，結果證明mel基因組在菌體內雖然未發生的自然併合(Integration)現象，但是可以穩定地存在宿主細胞內。此外我們利用SDS-PAGE電泳分析及西方點墨法(Western blot)證實酪氨酸酵素(Tyrosinase)在宿主細胞的生成，同時以酵素

活性測定並予以定量。我們以紫外線照射法比較PFC009野生株和melanin生產性的PFC009轉形株對紫外線的抵抗，證明在相同劑量的紫外線照射下轉形株比野生株有更高的存活率。為尋找可用來包覆種子的擔體，我們也測試了0.5% water agar作為油菜種子包覆擔體的利用性，由於agar本身也是一種營養物質，造成以它作為擔體的實驗組有發黴並且油菜的發芽率和生活狀況都較差，因此我們應該另外尋找具有黏性但是不會引起發黴的擔體，幫助PFC009包覆在油菜種子表面，以進一步探討PFC009的病害防治效果。

英文摘要

Chemical pesticides have been intensively applied in agricultural practice for years. The rapid developments of environmental pollution, residual contamination, reduction of crop productivity, and the damage to human and animal health are significant problem to the aware of. Therefore, it is quite urgent to develop safer and selective biological pesticides by using modern biotechnology. Pseudomonas fluorescens are aggressive colonizers of plant rhizophere and some of them have been used to control soil borne fungal diseases. The major antifungal compound for these bacteria has been proved to be the pyrrolnitrin, a pyrrol-type antibiotic with a wide range of antifugal spectrum. Pyrrolnitrin is a photosensitive substance, which can be readily decomposed by the UV irradiation due to the existence of such a chemically unstable moiety as pyrrol ring in its molecule. The cleavage of single bonds which link to the imino group in the pyrrol moeity of pyrrolnitrin will eliminate its antagonistic activity toards phytopathogens. Melanins are a large diverse family of naturally occurring biopolymers that can be used as UV-protective additives for cosmetic products. The genes involved in melanin biosynthersis in the bacterium Streptomyces antibioticus have recently been

clarified. In order to develop a more UV-resistant and longer-lasting biofungicide, we consider it is neceessary to prevent the pyrrolnitrin from UV damage. We postulated that transferred a functionable melanin-synthesizing gene operon (mel operon) to the pyrrolnitrin-producing Pseudomonas fluorescens could solve the UV problem in improving the shortcomings of Pseudomonas-type biofungicides currently being used.

Previously, we have demonstrated the protective effects of melanin pigment to pyrrolnitrin and some UV-sensitive antibiotics in vitro. In this study, we have focused in:

1.Selecting a soil borne pyrrolnitrin-producing Pseudomonas fluorescens PFC009, for the use of recombinant DNA host (selective isolation method, 365nm UV inspection, cryptic plasmid examination, antibiotic identification/quantitation and strain identification);

2.Constructing a mel gene containing shuttle vector pMP-101 between Pseudomonas fluorescens PFC009 and Escherichia coli JM109 (pIF413 and pKT230 ligation);

3.Transfoming the constructed plasmid pMP-101 to the target host Pseudomonas fluorescens PFC009 (electroporation);

4.Evaluating the performance of mel gene expression in Pseudomonas fluorescensn PFC009 (quantitative analysis of tyrosinase activity, Southern blotting

and Western blotting);

5.Establishing an in vitro and in vivo biological evaluation system for the comparison of the transformant and the parent strain on the crop protective ability against UV radiation.