植物從營養時期進入開花的階段在其生活史上是一個很重要的發育轉變,而文心蘭假球莖在此階段的轉變機制仍然不明。於是我們建構文心蘭subtractive EST library[假球莖EST扣減葉子EST]來找尋文心蘭開花相關基因。在EST library中peroxidase, mannose binding lectin,sodium/dicarboxylate cotransporter,及stress-resistant gene有較高的表現量,其中以peroxidase的表現頻率佔了EST library 12.1% 最令人感到興趣。我們在文心蘭cDNA library中得到9個全長class III peroxidase[POX]基因,並從比對生物資訊的資料庫中發現到這群POX是第一次在單子葉植物中被找到,而且具有與auxin結合的胺基酸片段(binding domain)。 為了進一步了解這群POX的生理功能,完成POXs在不同生長階段及不同組織之Northern blot分析,發現到這些POXs在未抽花梗的假球莖中會大量表現,在抽3cm花梗時表現量又降低,所以這些POXs可能會參與開花起始時期的生理代謝過程。藉由兩種POX fusion protein(GFP及GST)的轉殖分析,發現這群POX protein主要是表現在細胞間隙中。與IAA進行in vitro assay時也發現這群POX protein會將IAA降解。將其中七個POXs轉殖到阿拉伯芥中進行生理分析,發現到阿拉伯芥提早開花的功能。利用GC-MS檢測轉殖株中auxin的含量,發現這些轉植株中auxin的含量比野生型低。基於上述實驗結果,推測出這群POX可能在文心蘭抽出花芽前大量表現,使文心蘭假球莖內的IAA被降解,因此造成假球莖內auxin/cytokinin的荷爾蒙比例失去平衡,進而誘導花芽的產生。
Oncidium gower ramsey is an important orchid for cut flower industry in Taiwan. Its pseudobulb is a crucial organ for inflorescence initiation and flower development. In order to investigate the genes expression involved in pseudobulb development and in flowering initiation process, a subtractive cDNA library was generated. cDNA produced from mature pseudobulb was subtracted by cDNA of leaf tissue, then constructed into pGEM-T to generate an EST libraries. More than one thousand ESTs were sequenced and analyzed. Interestingly,the class III peroxidase (POX) genes occupied 12.1.% of total ESTs. They seems to play an important role for flowering. In order to study the physiological function, we isolated nine class III peroxidase cDNA genes from cDNA library. These gene members are show 43-98% identity each other. We constructed fusion protein of POX to GFP, for bombarding to Onion cell.We found that POX was localized in the intercellular space . We also constructed POX fusion protein vector with GST, and found that it reacted with IAA. Transgenic ectopic express of POXs genes in Arabidopsis with POXs gene caused prominently early flowering within two weeks. Using GC-MS to detect IAA concentration in these transgenic plants, IAA concentration was lower than WT. In Northern blotting analysis, we found that these genes were highly expressed in Oncidium pesudobulb before flower initiation, but expression of these genes were decreased in Oncidium pesudobulb with 3cm flower bud. By RT-PCR analysis, POX13 was specifically expressed in Oncidium pesudobulb and also enhanced in stress condition such as chilling (40C)、ABA(100uM) or H2O2(100uM). These results imply that class III peroxidase in Oncidium may regulate flowering process in Oncidium pseudobulb.
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