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標題: Functional characterization of a NCED gene from Phaius tankervilliae
作者: 林立
Li Lin
關鍵字: 鶴頂蘭;PtNCED1
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蘭花的種子細微,具有構造簡單但無胚乳的球型胚。許多地生蘭的成熟種子在無菌播種時發芽率低,前人研究發現,蘭花種子進入成熟階段時,內生性ABA含量居高不下,因此推測成熟種子的低發芽率可能與ABA累積有關。先前李勇毅博士自鶴頂蘭中分離出一個基因,推測為9-cis-epoxycarotenoid dioxygenase (NCED),將之命名為PtNCED1。NCED功能是將9-cis xanthophylls催化成ABA的前驅物xanthoxin。南方墨點分析確認了PtNCED1在鶴頂蘭中為單一基因。因為該基因在種子早期直到成熟階段皆持續表現,推測此未下降的PtNCED1轉錄會造成ABA累積,可能導致胚胎發育停滯,並阻礙種子萌發。為了證明PtNCED1具有NCED酵素活性,以及確定其在阿拉伯芥中的同功基因,向ABRC購得AtNCED缺陷株。由於AtNCED6和 AtNCED9在種子發育過程中高度表現,因而選用其同型合子的突變株作為互補實驗的對象,並以35S啟動子大量表現PtNCED1。然而發現互補後的轉殖株無法正常生長,甚至不產果莢,因此實驗無法進一步分析。PtNCED1/mGFP5蛋白主要分佈於葉綠體,與前人文獻所述相符。最後,利用菸草的暫時性轉殖,發現大量表現PtNCED1會增加ABA的累積,證明PtNCED1確實參與ABA的生合成。

Orchid seeds are characterized by their tiny size, with a globular stage embryo and without endosperm. Seed germination in vitro of several terrestrial orchids is difficult while the seed matured. Previous studies have shown that the endogenous ABA level remained high in the mature seeds, suggest a cause for their low germination rate. Isolated from Phaius tankervilliae by Dr. Lee, a gene putatively encode 9-cis-epoxycarotenoid dioxygenase (NCED) that catalyze conversion of 9-cis xanthophylls to xanthoxin, an ABA precursor, was named as PtNCED1. Southern hybridization confirmed its single gene status in Phaius tankervilliae. As PtNCED1 was expressed in early seeds and persistent till seeds maturation, it is speculated that the un-descending PtNCED1 transcripts may cause an accumulation of ABA, so to arrest the embryo development and block the seed germination in Phaius. To demonstrate that the PtNCED1 encode enzyme with NCED activity, and to identify its orthologous gene in Arabidopsis, 11 T-DNA knock-out lines of AtNCED1, 2, 5, 6, or 9 were obtained from Arabidopsis Biological Resource Center (ABRC). As AtNCED6 and -9 were highly expressed in the developing seeds, their homozygotic T-DNA insertion lines were chosen for functional complementation test and the PtNCED1 were expressed under controlled by 35S promoter. However, the complemented transgenic plants grown weakly and barely produced seeds, therefore could not be further analyzed. Coincided with other species, PtNCED1/mGFP5 was localized mainly in chloroplast. Finally, a significantly increased ABA content was detected in tobacco leaves that transiently overexpressed PtNCED1, demonstrating that PtNCED1 is involved in ABA biosynthesis.
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