利用轉殖菸草探討Pyramiding蛋白質酶抑制劑對廣泛昆蟲及 病源之抗性

Abstract

甘薯的sporamin（胰蛋白酶抑制劑）基因已證實具有抗蟲效果，而芋頭中的cystatin（胱胺酸蛋白酶抑制劑）基因亦已證實具有抗線蟲以及抗黴菌等功效，本論文之主要目的是結合兩個基因轉殖於作物中，以創造一個具有抗蟲、抗黴菌及抗細菌之多重抗性 (mutiple resistance）的生技作物 (biotechnological crop）。 利用本實驗室所研發的合成啟動子（synthetic promoter), pMSPOA 驅動上述兩個蛋白酶抑制劑基因，然後轉殖於菸草中，經過hygromycin抗生素抗性篩選得到轉殖株後，利用南方轉印、北方轉印以及蛋白質活性分析確認兩個轉基因在菸草中之有效表達後，進行抗玉米穗蟲（Helicoverpa armigera Hubner）、軟腐病菌（Erwinia carotovora）及青枯病菌（Ralstonia solanacearum）之抗性試驗，實驗結果證實此轉基因對玉米穗蟲具有致死與延滯生長發育之效果，LT50（lethal 致死時間）分別在3.7天至5.8天之間，明顯較對照組減少。對於抗細菌及抗黴菌亦有明顯效果，細菌感染後之繁殖量，僅有對照組之25%，因此，此研究證實兩種蛋白脢抑制劑的共同效應確實可賦予作物具有抗蟲、抗細菌及抗黴菌的多重抗性功效。

Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. The major aim of this thesis was to develop a trait which confers dual broad spectrum resistance against insects and phytopathogens. On the research front, a variety of conventional and more novel methods have been employed to introduce multiple genes into plants, but all techniques suffer from certain drawbacks. This study was undertaken to determine the effect of two protease inhibitor genes under the control of two wound inducible synthetic promoter, pMPSOA, in tobacco W38. To achieve this purpose, Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco plants containing both the genes were selected for resistance to hygromycin, and confirmed by PCR. Furthermore, Northern blot analysis revealed that stable expression of transgenes. Progeny analysis of T0 plants confirmed the inheritance of transgene to the next generation. The polyphagous moth Helicoverpa armigera (Hubner) is one of the world’s most important agricultural pests. Larval of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Furthermore, second instar H. armigera larvae reared on transgenic lines showed reduced LT50 values of respectively, as compared to larvae collected from the wild type. Transgenic tobacco overexpressing the stacked genes exhibited also strong resistance against phytopathogens such as Erwinia carotovora, Ralstonia solanacearum, Pythium aphanidermatum, Alternaria alternata. Maceration of leaf tissue was analyzed from transgenic and wild type tissue revealed that bacterial populations were significantly reduced compared to wild type. Microscopic observation in the wild type and transgenic line showed that transgenic line exhibited limited the growth of hyphae and leaf remains green. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to obtain broad-spectrum resistance against insects and phytopathogens.