台農67號甘藷傷害誘導cDNA之選殖與特性測定

Abstract

在自然環境裡，因為植物不具自由活動的能力，所以在面對眾多攝食者或微生物的侵害時，其本身自我防禦機制則扮演相當重要的抵抗或保護角色。為了釐清植物防禦系統的作用機制及探討此機制可能參與的角色與功能為何，本篇論文之目的在於研究參與植物防禦機制的基因。 本實驗選用台農67號甘藷為試驗材料，對甘藷葉片施以機械傷害，利用mRNA differential display方式選殖出在mRNA層次具有差異性的cDNA的片段共45個，其中3個cDNA片段與已知的逆境誘導基因的序列有高度相似性。以Northern blotting方式研究這3個基因在傷害處理與逆境賀爾蒙作用下之表現模式。實驗結果發現與peroxidase 相似的DD27在機械傷害、可誘發防禦機制的誘導物polygalacturonic acid (PGA)、chitosan、和植物賀爾蒙 methyl jasmonate、salicylic acid、ethephon處理下mRNA表現量均增加，而在abscisic acid (ABA)處理下mRNA 表現量沒有明顯改變。更進一步，鈣離子抑制劑ethylene glycol tetraacetic acid (EGTA)和LiCl處理時並不會延遲或影響傷害後DD27 mRNA表現量的增加。此外，和metallothionein-like protein相似的DD03在機械性傷害、與MeJA、ethephon、ABA、SA處理下其mRNA表現量均沒有明顯的變化。然而，序列相似於membrane Protein的DD37 在機械性傷害處理後mRNA表現量在傷害後10分鐘內立刻減少。若給予鈣離子抑制劑處理則發現EGTA（鈣離子螫合劑）可延遲DD37 mRNA表現量下降的現象，但LiCl（抑制胞器內鈣離子釋出）並沒有影響。另一方面，PGA、 chitosan、MeJA、ethephon、ABA和SA處理下DD37 mRNA表現量均會受到影響而下降，但不同的處理條件其mRNA表現情況具有時問點與表現量上的差異。由上述結果可知植物面對外來逆境時，細胞內各基因的表現是具有相當大的差異性的。

In the nature, plants are immobile and require special mechanism to survive after injured by chewing insects or microorganism. So plant's self-defensive system is very important. The purpose of this study is to isolate the wound-induced mRNA from sweet potato (Ipomoea batatas, cv Tainung 67), and further to analyze the defense mechanism of sweet potato. Genes, which were induced 2 or 12 hours after wounded, were screened using mRNA differential display. Among the 45 cDNA clones recovered in this screen, three genes are similar to the reported stress-induced genes. These three genes expressed differently in wounding. The gene for DD27, a peroxidase, was induced when wounding, polygalacturonic acid (PGA), chitosan, methyl jasmonate(MeJA), salicylic acid(SA), or ethephon was applied. The accunulation of DD27 mRNA was not affected by treating abscisic acid (ABA). When plant was wounded, the expression of DD27 gene was not reduced by Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) and compounds affecting the release of intracellular Ca2+ from vacuoles. However, the expression of DDO3, a metallothionein-like protein, was not significantly affected by wounding or treated with MeJA, ethephon, ABA, SA. Furthermore, the gene expression of DD37, a membrane protein, abolished at 10 mm after wounding. The application of Ca2+ chelator EGTA can delay the decrease of DD37 mRNA due to wounding. Nevertheless, DD37 transcipation level was reduced by PGA, chitosan, MeJA, ethephon, ABA and SA. These results indicate that the gene expression of the wounded plants is complicated and delicate.