南西文心蘭啟動子pOgCHRC之功能特性研究

Abstract

切花文心蘭 ( Oncidesa ) 為台灣切花最大宗之外銷花卉，其為目前相當重要之經濟作物。然近年切花文心蘭因品系缺乏變化，使出口總值趨於平緩。為突破此一瓶頸，本實驗室利用RNA干擾技術 ( RNA interference ) 進行基因轉殖，創造出新白花品系——蜜雪文心蘭( Oncidesa Honey Snow MF )。此基因轉殖載體之啟動子pOgCHRC ( chromoplast-specific carotenoid-associated protein ) ，為文心蘭花部組織專一表達之啟動子。研究顯示，白花文心蘭具有受光照影響花色之特性。經由光照試驗結果顯示，白花文心蘭於較高強度光照之環境下其花色偏黃之比例增加。為了解影響花色變化之因素，本論文針對參與在此RNA干擾現象內之相關因子做進一步之分析，包括：RNA干擾之目標基因OgPSY、類胡蘿蔔素生合成路徑之基因、驅動RNAi效果之啟動子pOgCHRC。結果顯示，啟動子pOgCHRC於此花色變化現象中扮演關鍵之角色。進一步以阿拉伯芥轉殖分析此啟動子之特性，發現此啟動子pOgCHRC於-1390至-1034之區域，可能存在一負調控因子，將此段序列做序列分析，顯示其上具有一段Box 4之調控因子 ( ATTAAT )，Box 4為一段與光調控有關之保守序列。而在其序列-1033至-712之區段，具有一段CGT-motif ( CCTGAACGC ) 及一段GT1-motif ( GGTTAAT )，此區段會增強啟動子pOgCHRC之活性，而高強度光照會抑制啟動子之活性。研究結果顯示pOgCHRC啟動子於-1033至-1具有最強之表達能力，因此此片段可提供未來提升文心蘭轉殖株花色品質之理想啟動子。

In Taiwan, Oncidesa is the top cut-flower exported to Japan. In our previous study, a new transgenic white florets “Oncidesa Honey Snow MF” was generated by silencing phytoene synthase (OgPSY). The RNAi construct is driven by a tissue-specific promoter: pOgCHRC (The promoter of chromoplast-specific carotenoid-associated protein), which is specifically expressed in floral tissues. However, some of the Oncidesa MF showed yellow or pale yellow flower. The ratio of white and yellow flowers seems to be influenced by temperature or light intensity. To realize the effects on flower colour, we analyzed the carotenoid biosynthesis gene and OgCHRC. We demonstrated that the activity of pOgCHRC is crucial for the formation of flower color. To study the regulatory mechanism of pOgCHRC, the different deleted promoter regions were cloned unto a GUS reporter gene and transformed into Arabidopsis thaliana. We identified that the promoter region from -1390 to -1034 repressed the activity of pOgCHRC. It contains a cis-regulating element: Box 4, which is predicted to be involved in light response. In the promoter region from -1033 to -712, the CGT-motif and the GT1-motif ware found, which may enhance the activity of pOgCHRC. The result shows that the promoter region from -1033 to -1 has the highest activity. In the future, the truncated promoter can be used to promote the RNA interference efficiency in Oncidesa transformants.