曾顯雄臺灣大學：植物病理與微生物學研究所鐘珮哲Chung, Pei-ChePei-CheChung2007-11-272018-06-292007-11-272018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/58077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。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.中文摘要...................................................................................................................1 英文摘要.................................................................................................................3 前言................................................................................................................5 壹、前人研究..............................................................................................7 一、黑色素之分佈位置........................................................................................7 二、真菌黑色素之功能................................................................................8 （一）保護並阻止輻射之傷害......................................................................8 （二）抵抗酵素的分解...........................................................................9 （三）抵抗極端的溫度................................................................................9 （四）與金屬離子黏附............................................................................10 （五）微生物黑色素之氧化還原能力........................................................10 （六）真菌黑色素是致病因子..................................................................11 （七）真菌黑色素是否可抵禦乾燥環境........................................................13 （八）對於殺真菌劑具有抗性................................................................14 三、真菌黑色素之生合成...........................................................................14 （一）DOPA melanin....................................................................................... 15 （二）GDHB melanin.......................................................................................16 （三）Catechol melanin.................................................................................. 17 （四）DHN melanin........................................................................................... 18 四、Agrobacterium mediated transformation............................................20 五、蟲生真菌.........................................................................................................25 貳、材料與方法..............................................................................................27 一、DNA level.......................................................................................................27 （一）基因體 DNA萃取............................................................................................ 27 （二）黑色素形成相關基因選殖................................................................................28 （三）建構Fosmid library.........................................................................................33 （四）DIG標定之探針..............................................................................................38 （五）南方氏雜合................................................................................................38 （六）Fosmid colony hybridization..........................................................................42 （七）Colony hybridization..................................................................................43 （八）挑選正反應訊號之選殖株建構shotgun library及解序...............................45 二、RNA level.............................................................................................................46 （一）RNA 萃取........................................................................................................46 （二）反轉錄聚合酵素鏈鎖反應RT-PCR................................................................48 （三）RACE（Rapid Amplification of cDNA Ends）.............................................49 三、Agrobacterium mediated transformation.................................................53 （一）電穿孔competent cell之製備與電穿孔之流程..............................54 （二）Construction of binary vector-pCAM-GH-GT-Tri...................................55 （三）Construction of binary vector-pCAM-GF-GT-Scy...................................59 （四）Using electroporation method to transform Agrobacterium strain.............62 （五）蟲生真菌之來源與培養...................................................................................62 （六）Genetic transformation........................................................................62 參、結果.........................................................................................................64 一、DNA level..................................................................................................64 （一） 基因體DNA萃取...........................................................................................64 （二） 黑色素形成相關基因選殖.........................................................................64 （三）建構Fosmid library........................................................................65 （四）DIG標定之探針.........................................................................65 （五）南方氏雜合..............................................................................65 （六）Fosmid colony 南方氏雜合........................................................................66 （七）Colony hybridization..................................................................................66 （八）挑選正反應訊號之選殖株建構shotgun library................................66 二、RNA level...........................................................................................67 （一）RNA extraction...........................................................................................67 （二）反轉錄聚合酵素鏈鎖反應RT-PCR...............................................................67 （三）RACE（Rapid Amplification of cDNA Ends）............................................67 三、Agrobacterium mediated transformation....................................................68 （一）Construction of binary vector-pCAM-GH-GT-Tri....................................68 （二）Construction of binary vector-pCAM-GF-GT-Scy.....................................69 （三）Using electroporation method to transform Agrobacterium strain........70 （四）Genetic transformation...................................................................................70 肆、討論..............................................................................................................72 一、Alternaria alternata生合成黑色素之相關基因.................................................72 二、Fosmid library 正反應選殖株解序結果與黑色素生合成基因分析................74 三、黑色素合成相關基因的cDNA全長.................................................................75 四、以Agrobacterium tumefaciens作為轉型之工具..............................................75 五、以農桿菌作為真菌轉型之媒介....................................................................76 六、未來展望................................................................................................79 伍、圖.............................................................................................................80 陸、參考文獻.................................................................................................103 柒、附錄................................................................................................................117 捌、附圖.........................................................................................................123 圖序 圖一、電泳磚格孢菌（Alternaria alternata）之基因體核酸，以確認其長度。…………………………………………………………………………81 圖二、以polyketide synthase、scytalone dehydratase、1,3,8-trihydroxynaphthalene reductase基因保守性序列，設計簡併式引子對，進行聚合酶聯鎖反應增幅所得預期之長度核酸產物電泳圖。…………………………………………82 圖三、建構磚格孢菌（Alternaria alternata）Fosmid library回收40 Kb DNA片段之濃度測試電泳圖。……………………………………………………..83 圖四、磚格孢菌（Alternaria alternata）基因體DNA以限制酶PstI、KpnI、EcoRI、HindIII進行酵解，並以polyketide synthase作為探針，進行南方氏雜合之壓片結果。…………………………………………………………………84 圖五、磚格孢菌（Alternaria alternata）基因體DNA以限制酶EcoRI、HindIII進行酵解，以scytalone dehydratase作為探針，進行南方氏雜合之壓片結果。…………………………………………………………………………..85 圖六、磚格孢菌（Alternaria alternata）基因體DNA以限制酶PstI、KpnI、EcoRI、HindIII進行酵解，以1,3,8-trihydroxynaphthalene reductase作為探針，進行南方氏雜合之結果。…………………………………………………..86 圖七、Fosmid sub-clones of Alternaria alternata selected after Southern hybridization……………………………………………………………...87 圖八、以DIG標示polyketide synthase、1,3,8-trihydroxynaphthalene reductase之核酸探針，以南方氏雜合法進行兩次篩檢所建構之磚格孢菌(Alternaria alternata)之Fosmid library。……………………………………………….88 圖九、建構shotgun library並解序之後，得到aaf01018E1的全長度序列41279bp………………………………………………………………………89 圖十、磚格孢菌(Alternaria alternata)之polyketide synthase基因比對Prodom資料庫所界定之功能性區域(functional motif)。………………………….90 圖十一、Alternaria alternata之RNA萃取與scytalone dehydratase RACE之結果…………………………………………………………………………..91 圖十二、1,3,8-trihydroxynaphthalene reductase進行RACE之結果…………….92 圖十三、經由RACE所獲知之scytalone dehydratase之全長度711bp之基因。………………………………………………………………………..93 圖十四、經由RACE所獲知之1,3,8-trihydroxynaphthalenereductase之全長度1029bp之基因。………………………………………………………….94 圖十五、所建構之pCAM-GH-GT-Tri(binary vector)利用限制酶剪接以確認黏合產物是否正確之電泳圖。………………………………………………..95 圖十六、所建構用以攜帶Hygr、1,3,8-trihydroxynaphthalene reductase基因之pCAM-GH-GT-Tri binary vector。……………………………………..96 圖十七、所建構之pCAM-GF-GT-Scy (binary vector)利用限制酶剪接以確認黏合產物是否正確之電泳圖。………………………………………………….97 圖十八、所建構用以攜帶GFP、scytalone dehydratase基因之pCAM-GF-GT-Scy binary vector。…………………………………………………………….98 圖十九、黑殭菌（Metarhizium anisopliae var. anisopliae）以scytalone dehydratase與1,3,8-trihydroxynaphthalene reductase基因轉型之表現型特徵光學鏡檢。…………………………………………………………………………99 圖二十、螢光顯微鏡鏡檢轉入scytalone dehydratase與1,3,8-trihydroxynaphthalene reductase兩基因之黑殭菌轉型株與野生株。……………………………………………………………100 圖二十一、黑殭菌與擬青黴菌野生株與轉型株之表現型。…………………….101 圖二十二、比較五種不同真菌催化黑色素生合成前趨物之polyketide synthase之功能性區域。…………………………………………………………1023106288 bytesapplication/pdfen-US黑色素黑色素生合成相關基因農桿菌轉殖法蟲生真菌抗逆境生物防治melaninmelanin synthesis related genesAgrobacterium-mediated-transformationentomopathogenic fungiantistressbiocontrolotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58077/1/ntu-94-R92633002-1.pdf