Effects of overexpressing a Cucumis metuliferus Cm1 gene on abiotic stress tolerance in tobacco

非生物逆境如乾旱及鹽害等皆會影響植物生長及生理反應，嚴重時會導致植株生長受阻、葉片萎凋與黃化等現象，植物有許多對抗非生物逆境的機制包括脯胺酸累積與逆境相關蛋白的表現等，先前的研究發現蛋白抑制子(proteinase inhibitor)參與植物耐逆境途徑，能減少逆境相關蛋白的降解以提升耐性。本試驗從刺角瓜(Cucumis metuliferus)中選殖出編碼Serine蛋白酶抑制子Cm1基因，利用農桿菌轉殖技術轉至圓葉菸草(Nicotiana benthamiana)中，研究在鹽害及乾旱逆境下Cm1對圓葉菸草的影響。以聚合酶連鎖反應檢測Cm1的轉殖株，進一步用南方墨點法分析T2及T3世代，篩選出轉殖株同質體，分別得到帶有兩重複Cm1基因的轉殖品系Y1-13及帶有單一重複的轉殖品系Y17-12。進一步用北方墨點法確認轉殖株中Cm1基因表現情形，並藉由西方墨點法檢測轉殖株中Cm1蛋白的表現。在鹽害及乾旱逆境下，觀察轉殖株的種子發芽率及生理反應，結果顯示Cm1過表現能提升種子於鹽害及乾旱逆境下的發芽率，但Cm1對於植株處理鹽害及甲基茉莉酸後並無提升葉圓片對逆境的耐性。另一方面發現轉殖株葉圓片處理乾旱逆境後葉綠素會明顯下降，而Cm1轉殖品系Y1-13相較於其他品系有較高的葉綠素，顯示Cm1對於植株在乾旱逆境下能抑制葉圓片葉綠素的降解。Abiotic stresses such as drought and salt stresses affect plant growth and physiological responses , including growth inhibition, leaves wilting, and yellowing. There are many mechanisms of plants to abiotic stresses such as the accumulation of proline and other related proteins. Previous studies have shown that the proteinase inhibitor may take part in the response to abiotic stresses by reducing degradation of stress -related proteins to improve stress-tolerence. In this study, a gene, Cm1, coding Serine protease inhibitor was isolated from Cucumis metuliferus. To investigate the effect of overexpression Cm1 gene on abiotic stresses in Nicotiana benthamiana by Agrobacterium-mediated transformation. Cm1 was detected from transgenic plants by using polymerase chain reaction. Two independent Cm1 homogeneous lines were obtained including Y17-12 ( one copy of Cm1 gene) and Y1-13 (two copies of Cm1 gene). The expression of Cm1 gene was verified by Northern blot analysis. The expression of Cm1 protein in transgenic line was detected by western blot analysis. Further more, Cm1 increased seed germination under salt and drought-stresses , but did not affect the tolerance of adult transgenic plants under salt and MJ treatments. On the other hand, the chlorophyll content of leaf discs decreased significantly under drought stress, while a Cm1 transgenic line, Y1-13, has higher chlorophyll comparing to the test lines. This indicated that Cm1 might retard chlorophyll degredation in plants under drought stress.目錄 內容 頁次 中文摘要............................6 貳、 材料與方法 一、 試驗材料....................................................8 二、 農桿菌載體構築及培養........................................8 三、 菸草基因轉殖................................................8 四、 植物DNA的萃取 ............................................9 五、 植物RNA的萃取.............................................9 六、 聚合酶連鎖反應.............................................10 七、 膠體電泳分析...............................................10 八、 探針製備...................................................11 九、 南方墨點法分析.............................................11 十、 北方墨點法分析.............................................12 十一、 西方墨點法分析.........................................13 十二、 幼苗鹽害逆境處理.......................................14 十三、 逆境發芽試驗...........................................14 十四、 葉圓片非生物逆境處理...................................14 十五、 植株性狀調查...........................................15 (一)株高...................................................15 (二)鮮重...................................................15 (三)葉片乾重...............................................15 (四)脯胺酸測定. ............................................15 (五)總蛋白量測定...........................................15 (六)葉綠素測定.............................................15 (七)脂質過氧化程度測定.................................16 十六、統計分析..................................................16 參、 結果 一、 菸草轉基因分析及套數檢測..................................17 二、 菸草轉殖株同質體篩選......................................17 三、 菸草轉殖株Cm1基因表現分析................................18 四、 轉殖株Cm1蛋白表現分析....................................18 五、 鹽害逆境試驗..............................................18 (一)鹽害逆境發芽試驗. ......................................18 (二)鹽害逆境植株試驗.......................................19 六、 乾旱逆境試驗..............................................20 (一)乾旱逆境發芽試驗.......................................20 (二)乾旱逆境葉圓片試驗.....................................20 七、甲基茉莉酸對植株影響.......................................22 肆、討論 一、轉殖株基因表現分析.........................................23 二、非生物逆境下對種子發芽之影響...............................23 三、非生物逆境下對植株生長之影響...............................24 四、Cm1基因參與非生物逆境.....................................26 伍、參考文獻........................................................2