阿拉伯芥轉植株中百合ASR蛋白參與葡萄糖與乙烯之訊息傳遞及百合ASR蛋白 轉殖煙草之選殖

Abstract

LLA23為鐵砲百合（Lilium longiflorum Thunb. cv. Snow Queen）花粉專一性蛋白，在花粉發育成熟後期的乾燥時期會大量累積。LLA23屬於ABA-, stress-, and ripening-induced （ASR）蛋白質的一員，且35S::LLA23轉殖阿拉伯芥具有耐旱、耐鹽及對ABA不敏感的特性。利用阿拉伯芥T4代轉殖株進行糖逆境下的訊息傳遞研究。以中低濃度(6.9~167mM)的葡萄糖及甘露醇處理，野生型與轉殖株的發芽率差異不明顯，但濃度越高有越降低的趨勢。以高濃度葡萄糖(333 mM)及甘露醇(mannitol)處理會降低種子的發芽率，但35S::LLA23轉殖株發芽率較野生型高。較高的萌芽率顯示，35S::LLA23轉植株對於葡萄糖有較高的耐受性，也表示百合ASR蛋白質可能影響轉殖株中葡萄糖的訊息傳遞。觀察小苗生長狀況，在高糖下野生型小苗生長受抑制，但35S::LLA23小苗生長不受抑制。顯示後萌發時期轉殖株也較野生型有較高的耐受性。利用RT- PCR 分析，在高糖逆境下，部分逆境相關基因的表現量在35S::LLA23轉殖株中與野生型相較有提高或降低的現象，表示百合ASR蛋白質可能調控與逆境相關的基因。以乙烯前驅物1-aminocyclo- propane-1-carboxylic acid（ACC）處理則發現，35S::LLA23轉殖株對於頂端鉤過度彎曲、胚軸短胖以及根部延長受抑制等現象的反應較不敏感，推測百合ASR蛋白有可能參與乙烯的訊息傳遞路徑。為了測試經濟作物轉殖百合ASR基因一樣具有耐旱的特性，我們將同一構築以膿桿菌轉殖方式送入菸草。利用PCR分析及抗體免疫偵測，已獲得六棵表現此百合ASR蛋白質的T0代轉植株，後續將篩選同型結合子轉殖株以進行耐旱與耐鹽能力的測試和分析。

A pollen-specific protein, LLA23, accumulates at the later stage of pollen maturation in lily(Lilium longiflorum Thunb. cv. Snow Queen) anthers. LLA23 is a member of ABA-, stress-, and ripening-induced (ASR) proteins. 35S::LLA23 lines in Arabodopsis exhibit drought and salt tolerance and show insensitivity to ABA. T4 transgenics were used to analyze sugar signal transduction. When treated with low to intermediate (6.9~167mM) concentrations of glucose or mannitol, difference in germination rate is insignificant between wild-type (Col-0) and transgenic plants, but the germination rate decreases as the glucose concentration increases. High level of glucose (333mM) or mannitol inhibits seed germination of both wild- type and transgenics. However, the germination rate of 35S::LLA23 plants is higher than wild-type. 35S::LLA23 exhibits reduced insensitivity to glucose, suggesting the involvement of LLA23 in the pathway of glucose signal transduction. The seedling growth of wild type plants is arrested under high concentration of glucose while 35S::LLA23 seedlings keep growing. It suggests that 35S::LLA23 seedlings display resistance to glucose at the stage of post-germination. RT-PCR analysis shows different patterns of stress-related genes in transgenic plants under the treatment of 333 mM glucose. It suggests that LLA23 may act as a regulator controlling the expression of these stress-related genes. When treated with ethylene precursor, 1-aminocyclo-propane-1-carboxylic acid (ACC), 35S::LLA23 plants show reduced sensitivity in exaggerated curvature of the apical hook, shortening and radial swelling of the hypocotyl and an inhibition of root elongation. Therefore, it suggests that LLA23 may also involve in ethylene signal transduction. To investigate the possible drought tolerance of economic plants, we have transformed the LLA23 gene into tobacco by Agrobacterium. Six T0 transgenic lines expressing the LLA23 protein are confirmed by PCR and immunoblot analyses. The homozygote plants will be further selected for drought resistance analysis.