文心蘭萳茜品系甘露糖結合型凝集素啟動子的調控機制之分析

Abstract

儲藏性蛋白會大量存在於植物的儲藏器官如種子、塊莖、球莖和根莖等。它們可幫助植物在逆境期間存活，或是在種植期間提供營養給新生植物。而儲藏性蛋白也表現了生物活性幫助抵抗害蟲、病原體或非生物性逆境。因此儲藏性蛋白可能扮演儲藏和防衛兩種角色。在植物塊莖已知和防衛相關的蛋白有幾丁質酶和凝集素（lectin）。其中許多文獻報導指出，凝集素具有抗蟲、抗真菌、抗微生物等的防禦功能。大部分目前已知的植物凝集素可依據他們的結構和演化相關的蛋白質分為七種家族。在文心蘭假球莖中大量存在之mannose-binding lectin 可歸類於monocot mannose-binding lectins 的家族中。由於本實驗室過去的研究發現，文心蘭的假球莖內含有大量的mannose-binding lectin，但對於其生理功能仍不清楚。因此期望藉由研究mannose-binding lectin 基因之啟動子，進一步了解其調控機制及特性。首先利用genome walking 方法，進行文心蘭mannose-inding lectin（MBL）基因啟動子的選殖，利用實驗室已知的MBL cDNA 序列，設計具專一性的引子，利用文心蘭Genome Walker 基因庫為模板及由Universal Genome Walker Kit 所提供的引子進行二次PCR 釣取MBL 基因上游5’端啟動子的DNA 序列，約2Kb，命名為pOMBL1，我們將此啟動子以PlantCARE 網站進行啟動子cis-acting elements sites 之分析，得知啟動子片段中除了TATA-box 及CAAT-box 外，尚包含2 個ABRE、2 個WUN-motif、3 個TGA-box、3 個W-box、4 個MYB 及6 個MYC protein binding sequence。之後將pOMBL1 構築於雙向載體pBI101 中，用於洋蔥表皮基因槍法轉殖及阿拉伯芥轉殖，也將pOMBL1 構築於雙向載體pCABIA1381z 及pCAMBIA1302 中，用於水稻和菸草轉殖。為了確定pOMBL1 是否可誘導下由基因表現，我將pOMBL1::GUS 載體以基因槍法轉殖至洋蔥表皮細胞中，經組織染色已確定所釣取之pOMBL1 可啟動下游GUS 報導基因的表現。接著，藉由農桿菌進行阿拉伯芥基因轉殖，結果發現GUS 基因在阿拉伯芥不同組織部位之表現量有所差異，其根部和莖部的表現量皆大於葉部。且當阿拉伯芥2、4、7、14、30 天之T3 轉殖株以不同誘導試劑處理並進行組織染色，可得知此啟動子皆可受IAA、ABA、JA 和SA 的誘導，但以前三者之誘導能力較強。若選取三週大之阿拉伯芥轉殖株，以不同誘導試劑處理或病原真菌感染後進行RT-PCR 反應，則顯示此啟動子在ABA100μM 處理或病原真菌感染下會明顯地受到誘導。 另外在水稻轉殖方面，將已轉殖pOMBLl::GUS 之水稻癒合組織以不同誘導試劑處理並進行組織染色，得知此啟動子明顯受ABA 和JA 的誘導。若將水稻不同大小之幼苗進行wounding 處理及組織染色，也顯示此啟動子可被受傷誘導。再者將已轉殖pOMBLl::GFP之水稻癒合組織以不同誘導試劑處理或病原真菌感染後進行RT-PCR 反應，結果顯示此啟動子在ABA、JA 和SA 處理或病原真菌感染下會明顯地受到誘導，卻可能受IAA 抑制。最後在菸草轉殖方面，利用已轉殖pOMBLl::GFP 之菸草葉片進行癒合組織的誘導，並利用共軛焦螢光顯微鏡（Confocal microscope）進行觀察，已經確定pOMBLl::GFP 有插入至菸草genome 中。接著，將已轉殖pOMBLl::GFP 之菸草癒合組織以不同誘導試劑處理或病原真菌感染後進行RT-PCR 反應，結果顯示此啟動子在ABA 和SA 處理或病原真菌感染下會明顯地受到誘導，此結果與阿拉伯芥相似。然而，為了確定MBL 基因在文心蘭假球莖中是否會受到不同賀爾蒙處理而誘導其大量表現，我選取同一時期之文心蘭以ABA、SA、IAA 和JA 處理後進行北方墨漬分析，探討其表現量差異，結果顯示MBL 基因明顯地受到ABA、JA 和SA 誘導，而可能受IAA 抑制，此與轉殖水稻分析的結果相符合。 由轉殖阿拉伯芥、菸草與水稻經分析的結果具有些許的差異，我們認為可能MBL 基因啟動子在雙子葉及單子葉中的調控機制有所不同而導致。另外，MBL 基因啟動子無論在阿拉伯芥、水稻或菸草中皆可受病原真菌感染而誘導，我們推測或許MBL 基因在文心蘭中對抗病原真菌入侵時扮演某些角色。

The monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins, which until now have been isolated from species of the Amaryllidaceae, Alliaceae, Araceae, Bromeliaceae, Lilliaceae, and Orchidaceae. Monocot mannose-binding lectins have been found in most vegetative tissues such as leaves, flowers, ovaries, bulbs, tubers, rhizomes, and roots. Previous study showed that monocot mannose-binding lectins are believed to play a role in the plant’s defense against sucking insects, nematodes, other invertebrates and even fungi. A mannose-binding lectin (MBL) has been isolated from Oncidium Gower Ramsey. It is an abundant storage protein in Oncidium pseudobulb. In order to understand the role of the promoter region of MBL in the regulating of gene expression, the 2Kb 5’-flanking region of MBL was further cloned and sequenced by PCR-based genomic walking method. A number of putative regulatory motifs were identified, including two ABRE, two WUN-motifs, three W-boxes, three TGA-boxes, four MYBs and six MYC protein binding sequences. The promoter of MBL was fused to the GUS and GFP sequences, then the resulting constructs were used to transform Arabidopsis, rice and tobacco. In Arabidopsis transgenic plants, the histochemical analysis revealed the expression of MBL promoter::GUS was detected at high level in shoot apical meristems, hypocotyls and roots at different stages of development. In RT-PCR analysis, we found that the GUS expression in roots and stems were higher than that in leaves. Besides, we found that the GUS activity was induced by IAA, JA and SA, but especially by ABA and Blumeria gramini f.sp. hordei. In rice transgenic calli and plants, the histochemical and RT-PCR analysis indicated that the GUS activity was prominantly induced by JA, ABA, SA and Blumeria gramini f.sp. hordei, but suppressed by IAA. This expression pattern was similar to that of the MBL gene in Oncidium. In addition, histochemical analysis of GUS activity in rice seedlings revealed that the GUS activity was also induced by mechanical wounding treatment. Otherwise, In tobacco calli, the RT-PCR analysis revealed that the GFP activity was induced by ABA, SA and Blumeria gramini f.sp. hordei. This result in tobacco was similar to that in Arabidopsis. In summary, the data suggest that the regulation of the MBL gene may be not the same in monocots and dicots. Moreover, it also suggested that the MBL genes in Oncidium maybe play a role in the plant’s defense against fungi.