Cloning of Cucumis metuliferus Cm4 gene and its effects on transgenic Nicotiana benthamiana

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.目次 內容 頁次 中文摘要 ............................................................i 英文摘要 ............................................................ii 目錄 .................................................................iii 表目錄 .................................................................v 圖目錄 .............................................................vi 壹、 前人研究 .....................................................1 一、 植物的抗病機制 ............................................1 二、 逆境下植物的鈣離子累積與訊號傳遞 ........................2 三、 CDPK/SnRK家族之介紹 .....................................4 四、 CDPK/SnRK家族對逆境影響之研究 .............................5 貳、 材料與方法 一、 試驗材料 ....................................................9 二、 植物DNA萃取 ................................................9 三、 植物RNA萃取 ................................................9 四、 Genome walking .......................................10 五、 快速擴增cDNA末端法 ..................................12 六、 載體構築及農桿菌培養 ..................................13 七、 刺角瓜基因轉殖 .............................................15 八、 番茄基因轉殖 .............................................16 九、 菸草基因轉殖 ............................................16 十、 聚合酶連鎖反應 .......................................17 十一、 反轉錄聚合酶連鎖反應 ................................17 十二、 膠體電泳分析 ......................................17 十三、 探針製備 ..............................................18 十四、 南方墨點法分析 .........................................18 十五、 北方墨點法分析 ...........................................19 十六、 刺角瓜及菸草之病毒接種與性狀分析 .................20 參、 結果 一、Cm4基因之選殖 ......................................21 二、Cm4基因結構及功能預測 ..................................22 三、Cm4基因套數之檢測 ...................................23 四、PRSV感染刺角瓜對Cm4基因表現量之影響 ..................23 五、刺角瓜、番茄及菸草轉殖Cm4基因及轉殖株檢測 ................24 六、轉殖株外表型及抗感病分析 ..............................25 七、Cm9基因之選殖 .............................................26 八、Cm9基因套數之檢測 .....................................28 九、刺角瓜靜默及表現Cm9之基因轉殖 ............................28 肆、討論 一、Cm4與CRK蛋白之相似性 .................................29 二、Cm4參與系統性防禦之反應 ...........................30 三、菸草中表現Cm4基因對抗病性之影響 ......................32 伍、參考文獻 .................................................3