- 學位論文
轉殖蟲生真菌黑色素生合成基因以增加其逆境之抗性
Transformation of entomopathogenic fungi with melanin biosynthesis genes to enhance anti-stress capability
摘要
於田間應用昆蟲寄生菌來防治害蟲常遇到如高溫、乾燥、高UV、輻射等逆境，降低成活率，因而減損其防治效率，據以往之文獻報導，黑色素對於生物不同物種之抗逆境或致病能力有極為密切之關係，故為克服此種瓶頸，乃嘗試應用基因重組技術將其他會產生黑色素真菌之黑色素生合成基因（polyketide synthase、scytalone dehydratase、1,3,8-trihydroxynaphthalene reductase）轉殖到昆蟲寄生菌（黑殭菌Metarhizium anisopliae var. anisopliae、白殭菌Beauveria bassiana、擬青黴菌Paecilomyces javanicus），再探討其耐逆境及侵染昆蟲寄主之能力。本試驗首先應用簡併式引子對（degenerate primers）利用聚合酶鏈鎖反應（PCR，polymerase chain reaction）之技術，以磚格孢菌（Alternaria alternata）之genomic DNA作為模板增幅出polyketide synthase（長約700 bp），scytalone dehydratase（長約250 bp），1,3,8-trihydroxynaphthalene reductase（長約750 bp）基因之核酸片段，再以DIG標識當為探針，篩檢由研究室所建構之磚格孢菌（A. alternata）基因體之Fosmid library，並對polyketide synthase與1,3,8-trihydroxynaphthalene reductase呈正反應之選殖株將其挑出，增殖並萃取genomic DNA，利用散彈槍方式建構DNA library（shotgun DNA library），進行定序組合，最後將長41279 bp之fosmid選殖株完全解序，此DNA序列可轉錄至少六個基因：polyketide synthase、1,3,8-trihydroxynaphthalene reductase與4個未知功能的基因，但所預期之基因scytalone dehydratase並不包括在內，此將進一步以限制酶剪切、南方氏電泳分析，並期望可得到預期之結果，此基因預期應座落於polyketide synthase與1,3,8-trihydroxynaphthalene reductase之間；此外也應用RACE，將polyketide synthase、scytalone dehydratase、1,3,8-trihydroxynaphthalene reductase等基因之全長度解序，其開放讀架（open reading frame）之全長度分別為6483 bp、519 bp、801 bp。並建構pCAM-GF-GT-Scy（以GFP作為selection marker，內建有scytalone dehydratase 之full length cDNA）、pCAM-GH-GT-Tri（以hygromycinr作為selection marker，及1,3,8-trihydroxynaphthalene reductase之full length cDNA）Ti-plasmid 轉型載體（binary vector），以電穿孔儀將其送入農桿菌（Agrobacterium tumefaciens EHA105 strain）中，將利用農桿菌轉型法將目標基因轉入昆蟲寄生菌：黑殭菌、白殭菌、擬青黴菌，選出轉型株，並以南方氏、北方氏點墨雜合，配合綠色螢光融合蛋白（Green fluorescence fusion protein, GFP）檢測，目前已成功將此等黑色素生合成基因轉入昆蟲寄生菌染色體，初步觀察黑殭菌之轉型株，以螢光顯微鏡可以觀察到孢子與菌絲表現綠色螢光，並且於光學顯微鏡下可看到與野生型不同之黑色菌絲。後續將進行轉型株之基因型與表現型之確認以及生物活性檢測。
並列摘要
Application of entomopathogenic fungi to control insect pests in the field was usually not effective as anticipated due to the stressed and harsh environmental conditions such as high temperature, desication and high sunlight UV radiation. To circumvent the obstacles encountered, we attempt to transform the prevalent entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae var. anisopliae, and Paecilomyces javanicus with melanin biosynthesis genes: polyketide synthase, scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase, melanin proved characterized with antistress and corelated with virulent capacity of some pathogenic fungi. Part of polyketide synthase, scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase gene fragments with length of 700, 250, 750 bp, respectively were amplified by polymerase chain reaction（PCR）using degenerate primers from Alternaria alternata, and being labeled with DIG as probes to screen the Fosmid library. The library was constructed based on the genomic DNA extracted from the mycelium of the dematiaceous A. alternata , which has been documented possessing three melanin biosynthesis genes clustered in a 30 Kb DNA contig. The Fosmid clones exhibiting positive signal against polyketide synthase and 1,3,8-trihydroxynaphthalene reductase were selected to construct shotgun library for sequencing and assembling. The assembled 41279 bp contig blastingX with nr NCBI revealed the encoded polyketide synthase, 1,3,8-trihydroxynaphthalene reductase plus four hypothetical proteins genes, flanked with function unknown DNA sequences. Unexpectedly, the scytalone dehydratase gene was not discovered within the contigs. However, all the full open reading frame of polyketide synthase, scytalone dehydratase, 1,3,8-trihydroxynaphthalene reductase genes were cloned and their full-length open reading frames accessed by rapid amplification of cDNA ends（RACE）. To further ascertain the existance of scytalone dehydratase gene, the specific Fosmid clones DNA will be restricted by an array of enzymes and verified by Southern blotting. We also constructed Ti-plasmid binary vectors, pCAM-GF-GT-Scy pCAM-GH-GT-Tri, which harboured GFP and scytalone dehydratase, hygromycinr and 1,3,8-trihydroxynaphthalene reductase gene insertions respectively, which has been cotransfered into the targeted entomopathogenic fungi B. beauveria, M. anisopliae var. anisopliae and P. javanicus by Agrobacterium tumefaciens Ti-plasmid mediated transformation using electroporation device. Preliminarly light and fluorescent microscopy showed the successful transformation as evident by the green fluorescent conidia and mycelium, or the darkened mycelium, which otherwise have not been observed in wild types. Experiments with respect to the phenotype、genotype and bioassay are undergoing and expected to come up with promising outcome.