曾顯雄臺灣大學:植物病理與微生物學研究所王淞民Wang, Song-MinSong-MinWang2010-05-112018-06-292010-05-112018-06-292009U0001-2807200917171300http://ntur.lib.ntu.edu.tw//handle/246246/181985摘要 木黴菌（Trichoderma spp.），為ㄧ毀滅性超寄生菌(destructive mycoparasite)，自1930年代即已被研發成生物防治劑，而應用於作物或苗圃、苗木地上部或土媒真菌性病害之防治，但常因逆境而使其於田間之防治效果不如預期。黑色素（melanin）廣泛存在各種生物體內，具有保護減低生物受到UV傷害、並能提高微生物對高溫、乾燥之耐性。為了提昇木黴菌之抗逆境之能力與生物防治效能，將磚格孢菌（Alternaria alternata）之黑色素生合成基因polyketide synthase（PKS）、 scytalone dehydratase（SCD）及1,3,8- trihydroxynaphthalene reductase（THN），構築於Ti plasmid轉型載體pCAMBIA 1300中。載體pCAM-GPD-GFP-PKS以GFP為selection marker，內建有PKS之full length gDNA，載體pCAM-GPD-HYG-Tri-Scy以hygromycinr為selection marker，內建SCD與THN之full length cDNA。應用農桿菌之轉型系統（Agrobacterium tumefaciens mediated transformation, ATMT），將此基因轉入木黴菌T. harzianum與T. reesei，使轉型株能表現該基因並產生黑色素，更進而測試其對逆境之耐受性，以及生物防治之能力。T. harzianum以原生質體作為轉殖材料，共獲得7株轉型株， Southern blot顯示其中3株具有兩個黑色素生合成PKS基因，以及各一個SCD與THN基因。T. reesei分別以原生質體與分生孢子做為轉殖材料，共獲得5株各具一個PKS、SCD及THN基因之轉殖株，以及5株僅帶有SCD與THN兩基因之轉型株。生理活性檢測顯示出T. harzianum轉型株對UV-B耐受性是野生株之兩倍，於35℃下，轉型株比野生株生長快速，於水活性為0.945，轉型株活力亦較佳，而對於病原菌Colletotrichum gloeosporioides、Phellinus noxius、Phytophthora parasitica等，轉型株皆具有更高之侵染能力。當連續照射UV-B 10分鐘後，培養24小時內，轉型株T. reesei分生孢子發芽率比野生株高將近10倍。在水活性0.929，轉型株發芽率為35.68%，而野生株僅13%，兩者相差2.7倍。但轉型株、野生株對P. noxius、P. parasitica之侵染能力相近。Abstractrichoderma, a destructive mycoparasite, has been studied and being used to biocontrol plant fungal disease for more than 70 years. Nevertheless, the control efficacy in fields sometime was lower than anticipation, mainly due to the biotic or abiotic stress encountered. Previously melanin was proved unequivocally with the capacity to enhance immense organisms to counteract the stressed conditions or increase virulence toward animal or plant hosts. Attempt to circumvent the obsticals encountered in biocontrol, the melanin biosynthesis genes encoding polyketide synthase（PKS）, scytalone dehydratase（SCD）, and 1, 3, 8-trihydroxynaphthalene reductase (THN) cloned from Alternaria alternata were engineered into the Trichoderma harzianum and T. reesei by Agrobacterium tumefaciens mediated transformation（ATMT）. To carry out transformantion, two shuttle vectors were constructed, using pCAMBIA 1300 binary vector as backbone, one vector（pCAM-GPD-GFP-PKS）inserted with PKS and green fluorescence protein（GFP）, while the other（pCAM-GPD-HYG-Tri-Scy）harbors SCD, THN and hygromycin-B phosphotransferase（hygromycinr）；both vectors using common Aspergillus GPD promoter and Trpc terminator to drive the transcription. Totally, seven T. harzianum transformants derived from protoplast were obtain, and five T. reesei transformants from protoplasts or conidia were secured. Transformants T. harzianum 3-3 and T. reesei 3-1 possess one copy of PKS, SCD and THN gene, respectively, whereas transformants T. harzianum 11-1 harbors 2 coppies of PKS and one copy of SCD and THN. The T. harzianum transformant exhibited two-fold tolerance toward UV-B irradiation than wild type, and also showed higher growth rate at 35℃, and at lower water activity（aw）at 0.945. Additionally, the transformant also possessed higher virulence toward fungal pathogens Colletotrichum gloeosporioides, Phellinus noxius, and Phytophthora parasitica compared with wild type. While the T. reesei transformant after irradiation with UV-B at the dosage of 3744 mJ/cm2 for 10 min, exhibited 10 times more germination rate than wild type, so as to the germination rate at lower water activity（aw 0.929）, with 35.68% versus 13% between the two strains. However, there are no significant difference in regarding the virulence between the transformant and wild type strains.目次文摘要………………………………………………………………………………1文摘要………………………………………………………………………………2言……………………………………………………………………………………4、前人研究………………………………………………………………………….6、木黴菌（Trichoderma spp.）……………………………………………………6、生物防治機制………………………………………………………………….....7（一）超寄生………………………………………………………………………..7（二）細胞壁分解酵素……………………………………………………………..7（三）抗生物質……………………………………………………………………9（四）營養競爭……………………………………………………………………9（五）刺激植物產生抗性與防禦機制……………………………………………10、影響木黴菌防治能力之環境因子………………………………………………11、黑 色 素(melanin)………………………………………………………………12一）黑色素保護並防止紫外線過量之傷害……………………………………13二）黑色素影響病原菌之致病力………………………………………………13三）抵抗乾燥、高溫環境………………………………………………………14四）黑色素對細胞壁之影響……………………………………………………14、農桿菌轉型（Agrobacterium-mediated transformation）…………………….14（一）Agrobacterium tumefaciens致病機制……………………………………..14（二）農桿菌轉型機制……………………………………………………………15 2-1. 植物細胞表面接受器與農桿菌之附著……………………………...16-2. T-complex之胞內運送………………………………………………..17-3. T-complex進入細胞核………………………………………………..17-4. T-complex與染色體接合、蛋白水解及T-DNA插入………………10三）真菌轉型……………………………………………………………………20、材料與方法……………………………………………………………………..23、轉型木黴菌之選擇…………………………………………………………..23 Ⅰ、菌種來源與對峙培養測試…………………………………………………23、DNA level……………………………………………………………………….23、菌體培養……………………………………………………………………..23、基因體DNA萃取…………………………………………………………....23、DNA電泳……………………………………………………………………24、黑色素生合成相關基因的primer設計與PCR條件設定………………....25、pGEM®-T Vector（Promega）for T-A cloning……………………………..27、抽取質體DNA之方法……………………………………………………….28、純化PCR產物與電泳膠中之DNA…………………………………………..30、南方氏雜合.....................................................................................................30、RNA level……………………………………………………………………....33、菌體培養…………………………………………………………………….33、RNA萃取…………………………………………………………………….34、電泳分析…………………………………………………………………….34、DNase處理…………………………………………………………………..34、One-step RT-PCR……………………………………………………………34、Agrobacterium mediated transformation……………………………………….35一）電穿孔competent cell 之製備與電穿孔操作流程………………………..36 Ⅰ、電穿孔competent cell製備…………………………………………………...36 Ⅱ、電穿孔流程…………………………………………………………………..36二）構築binary vector pCAM-GPD-GFP-PKS…………………………………..36三）構築binary vector pCAM-GPD-HYG-Tri-Scy………………………………38四）電穿孔傳送建構binary vector至A. tumefaciens EHA105…………………...41五）超寄生菌之培養…………………………………………………………......41六）黑色素生合成基因之轉殖…………………………………………………..42 Ⅰ、原生質體製備………………………………………………………………..42、Agrobacterium-mediated transdormation…………………………………….43、轉型株之檢測………………………………………………………………..43、轉型株生理特性檢測…………………………………………………………..44一）不同培養溫度對轉殖株菌落生長之影響………………………………..44二）不同培養溫度對轉殖株孢子發芽之影響………………………………..44三）水活性之耐受性……………………………………………………………..44 Ⅰ、發芽率……………………………………………………………………….44Ⅱ、菌落生長……………………………………………………………………..45四）UV之耐受性……………………………………………………………….45五）轉型株之超寄生能力……………………………………………………..45、結果……………………………………………………………………………..47、對峙培養………………………………………………………………………..47、DNA level……………………………………………………………………….47、Agrobacterium mediated transformation………………………………………..48一）Construction binary vector pCAM-GPD-GFP-PKS………………………….48二）Construction binary vector pCAM-GPD-HYG-Tri-Scy……………………..48三）基因轉型……………………………………………………………………..49 3-1. Trichoderma harzianum轉型與轉型株之檢測……………………………...49 3-2.Trichoderma reesei轉型與轉型株之檢測………………………………........51四）轉型株之生理活性測試……………………………………………………..53 Ⅰ、Trichoderma harzianum………………………………………………………53-1-1.溫度對分生孢子發芽之影響……………………………………………..53 4-1-2.溫度對菌落生長之影響………………………………………………......53 4-1-3.水分活性對分生孢子發芽與菌落生長之影響....………………………..53-1-4.UV之耐受性………………………………………………………………54-1-5.超寄生能力……………………………………………………………......54 Ⅱ、Trichoderma reesei…………………………………………………………...55-2-1.溫度對菌落生長之影響…………………………………………………..55 4-2-2.溫度與發芽率之關係……………………………………………………..55 4-2-3.水活性與孢子發芽之關係……………………………………………..56-2-4.紫外線耐受性測試………………………………………………………..56 4-2-5.超寄生能力………………………………………………………………..56、討 論……………………………………………………………………………58、圖表……………………………………………………………………………..63、參考文獻……………………………………………………………………….101錄一 實驗試劑配方……………………………………………………………..112錄二論文附圖……………………………………………………………………116application/pdf8377333 bytesapplication/pdfen-US黑色素生合成基因超寄生菌農桿菌轉型Melanin biosynthesis geneTrichodermaATMThttp://ntur.lib.ntu.edu.tw/bitstream/246246/181985/1/ntu-98-R95633015-1.pdf