基轉超寄生菌Trichoderma spp.黑色素生合成基因以提昇其逆境抗性與致病力

Abstract

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。

Abstract Trichoderma, 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.