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|標題:||Cloning of Cucumis metuliferus Cm4 gene and its effects on transgenic Nicotiana benthamiana
|關鍵字:||Cucumis metuliferus;PRSV;Cm4;CRK;resistance;刺角瓜;木瓜輪點病毒;Cm4;鈣依賴相似蛋白激酶;抗病性||引用:||林育宗。2012。刺角瓜抗木瓜輪點病基因之選殖與功能分析。國立中興大學農藝 學系博士論文。台中。 柳建安。2004。植物基因轉殖與分子檢測技術。教育部顧問室植物生物技術教學 資源中心。台中。 Abbasi, F., H. Onodera, S. Toki, H. Tanaka and S. Komatsu. 2004. OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath. Plant Mol. Bio. 55: 541-552. Baebler, S., K. Witek, M. Petek, K. Stare, M. Tusek-Znidaric, M. Pompe-Novak, et al. 2014. Salicylic acid is an indispensable component of the Ny-1 resistance-gene-mediated response against Potato virus Y infection in potato. J. Exp. Bot. 65: 1095-1109. Baures, I., T. Candresse, A. Leveau, A. Bendahmane and B. Sturbois. 2008. The Rx gene confers resistance to a range of potexviruses in transgenic Nicotiana plants. Mol. Plant Microbe. Interact. 21: 1154-1164. Bendahmane, A., K. Kanyuka and D.C. Baulcombe. 1999. The Rx gene from potato controls separate virus resistance and cell death responses. 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刺角瓜 (Cucumis metuliferus) Cm4基因透過genome block walking和快速擴增cDNA末端法，解序出基因組和cDNA全長以及Cm4基因之啟動子序列。Cm4基因組全長共5054 bp，cDNA全長共1722 bp，可編碼573個胺基酸並轉譯出帶有絲/蘇氨酸蛋白激酶domain的鈣依賴相似蛋白激酶 (Ca2+-dependent protein kinase-related kinase)。刺角瓜中只帶有一套Cm4基因，而作為轉殖材料的菸草 (Nicotiana benthamiana) 及番茄 (Solanum lycopersicum) 中沒有和Cm4序列相似的基因。當刺角瓜感染木瓜輪點病毒 (Papaya ringspot virus, PRSV) 以及機械傷害時，Cm4基因會在系統葉誘導表現，推測Cm4基因和刺角瓜的系統防禦相關。為了解Cm4基因在異源植物表現是否會影響到植物的抗病性，構築Cm4基因組表現以及cDNA過表現載體之後進行菸草和番茄轉殖，並以聚合酶連鎖反應檢測轉殖株。從菸草轉殖Cm4基因組表現的子代發現有子葉較大的情形，但觀察四週大轉殖株則與野生型的型態則沒有差異。檢測菸草轉殖株的抗病性，將不同轉殖品系各15株，分別接種馬鈴薯Y病毒 (Potato virus Y, PVY)、番椒葉脈斑駁病毒 (Pepper veinal mottle virus, PVMV)、辣椒葉脈斑駁病毒 (Chilli veinal mottle virus, ChiVMV)、蕪菁嵌紋病毒 (Turnip mosaic virus, TuMV)，發現所有表現Cm4基因之轉殖株皆感病。Cm4基因表現不能使菸草具有抗病性，但接種病毒會誘導Cm4基因表現，Cm4基因可能參與系統防禦訊號的傳遞，然而Cm4基因是否會影響刺角瓜的抗病性，則須在日後檢測Cm4靜默之刺角瓜轉殖株才可得知。
A partial cDNA-AFLP fragment of Cm4 was initially identified in Cucumis metuliferus. Subsequently, the full length of Cm4 genomic and cDNA fragments were cloned using genome block walking and rapid amplification of cDNA end (RACE). The C. metuliferus genomic and cDNA fragments of Cm4 gene contain 5054 and 1722 bp in which translated 573 amino acids and coded for Ca2+-dependent protein kinase-related kinase (CRK) containing serine/threonine protein kinase domain. Southern blot showed that C. metuliferus had one copy of Cm4 but there was no homolog existing in Nicotiana benthamiana and Solanum lycopersicum. The expression of Cm4 was induced by Papaya ringspot virus (PRSV) inoculation and machine infection on systemic leaves indicating its role in systemic defense response to wounding and pathogen infections. To investigate the effect of Cm4 with regard to virus resistance, the full length of Cm4 genomic and cDNA fragments were constructed in expression vectors and genetic-engineered into N. benthamiana and S. lycopersicum by Agrobacterium-mediated transformation. Transgenic plants harboring Cm4 gene were obtained. Some tobacco transgenic plants expressing Cm4 genomic fragment showed enlarged cotyledons but no difference was observed in 4-weeks-old of these plants. All the transgenic tobacco showed susceptible symptom after inoculated with Potato virus Y (PVY), Pepper veinal mottle virus (PVMV), Chilli veinal mottle virus (ChiVMV) and Turnip mosaic virus (TuMV). This study suggested that C. metuliferus Cm4 genomic and cDNA fragments might not participate in potyvirus resistance in tobacco. However, its resistant role against PRSV in C. metuliferus need to be verify only when C. metuliferus transgenic plants obtained in the future.
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