蘭花花器表達基因之啟動子活性分析

Promoter Activity Analysis of Flower Organ-Expressed Genes in Orchids

宋品慧(Pin-Hui Sung)

指導教授 ： 杜宜殷 ； 黃鵬林

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02123

摘要

為取得文心蘭苯基苯乙烯酮合成酶（chalcone synthase）基因之啟動子，以蝴蝶蘭苯基苯乙烯酮合成酶pOCHS01 cDNA為探針，篩選文心蘭基因組庫，經五次篩選後，共得到19個選殖系。依其限制酶圖譜可分成三群，分別選取選殖系λOgCHS1、λOgCHS55、λOgCHS133進行次選殖核酸定序，得到基因及其啟動子序列。三個文心蘭苯基苯乙烯酮合成酶基因分別命名為Og-CHS1、Og-CHS2及Og-CHS3，三個基因均具有兩個顯子和一個隱子，顯子與隱子交界處均遵守GT-AG原則，分別解碼出392、390、391個胺基酸，預測之分子量分別為43、42.7、42.7 KDa。文心蘭三個苯基苯乙烯酮合成酶之胺基酸序列與蝴蝶蘭 cDNA所演繹之胺基酸序列相似度分別為90.3%、90.3% 及84.2%，與其他作物之苯基苯乙烯酮合成酶基因演繹之胺基酸序列相比，同源性介於84.2~91.3%間。進一步分析蛋白質特性，結果顯示此三個文心蘭苯基苯乙烯酮合成酶皆具有保守性高的酵素活性區，並可能受到磷酸化修飾。依啟動子序列比對分析結果顯示，文心蘭苯基苯乙烯酮合成酶基因啟動子具多種response elements，除了可能接受光調控外，還可能受到乙烯、離層酸 (ABA)、激勃素 (GA)、高溫逆境、創傷、elicitor及水楊酸等因子，共同調控啟動子之作用。以文心蘭基因體DNA進行南方氏雜交分析，顯示文心蘭苯基苯乙烯酮合成酶基因為多拷貝基因。在基因表現分析方面，根據北方雜交分析結果，苯基苯乙烯酮合成酶基因於文心蘭之根、假球莖、葉、花苞、花等不同部位均有表現，尤以根部活性較高。在0、1、3、5、7、9天之不同採後天數的花朵，均有基因表現，由0天開始些微下降，第5天達高峰，之後遞減。在不同去花藥蓋處理天數中，表現趨勢相反，隨處理天數之增加而逐漸減少，直至第5天後，才又開始逐漸累積，而於第9天時，達到高峰。
為了進一步尋找可於蘭花花器大量表現、或具有組織器官表現特異性之啟動子，因此分別構築文心蘭苯基苯乙烯酮合成酶基因（Og-CHS）、蝴蝶蘭乙醯輔酶A氧化酶基因 (Pt-ACCA) 、蝴蝶蘭ACC合成酶基因（Pt-ACS）及蝴蝶蘭ACC氧化酶基因（Pt-ACO）之啟動子片段於含有報導基因GUS之載體中。以蝴蝶蘭及文心蘭花瓣為材料，進行啟動子活性暫時性表現分析，試驗結果顯示，Pt-ACO1啟動子之活性，最高可達對照組CaMV 35S啟動子活性的1.76倍。經由農桿菌媒介法，將上述之質體，分別進行菸草及阿拉伯芥之穩定性轉殖，以轉殖株之子代進行GUS活性組織化學染色分析。菸草轉殖株之初步分析結果顯示，Og-CHS的三個基因啟動子皆可表現於葉及根冠；Pt-ACCA啟動子於營養發育階段未偵測到基因表現；Pt-ACO1啟動子於根、莖及葉皆有活性表現，其中以莖及葉表現量較大；Pt-ACS1啟動子則於根部有部分活性。以阿拉伯芥轉殖株進行GUS活性分析，結果顯示Pt-ACS1啟動子於莖、葉柄與花器有GUS活性表現；Pt-ACO1啟動子GUS活性分佈主要於花器及果實表現最強，根、莖及葉亦有GUS活性表現；Pt-ACCA啟動子可於花藥大量表現根、莖有微弱GUS活性表現；Og-CHS1啟動子GUS活性於根之生長點有較強表現，莖及葉脈部分表現，花器及果梗皆有表現；Og-CHS2啟動子於根之生長點、莖及葉缺刻尖端有高活性GUS表現，柱頭及花梗亦有GUS活性分佈；Og-CHS3啟動子於根及莖有部分活性，主要於葉柄及未開放之小花苞有高活性表現，且可隨著不同發育時期，於不同組織部位產生特異性表現。

關鍵字

ACC氧化酶 ； ACC合成酶啟動子活性分析 ； 花器表達 ； chalcone合成酶

並列摘要

To obtain chalcone synthase genes from Oncidium, Phalaenopsis cDNA was used as probe to screen a genomic library of Oncidium. Approximately 1.5 × 106 plaque-forming units were screened and 19 independent clones were picked up. Based on restriction map analysis, these genomic clones were classified into three groups. Genes located in three genomic clones λOgCHS1, λOgCHS55 and λOgCHS133 were sequenced and named Og-CHS1, Og-CHS2 and Og-CHS3, respectively. All three genes contained two exons and one intron with consensus GT-AG dinucleotides situated at their boundaries. Compared with Phalaenopsis cDNA, the sequence homology of deduced amino acid for these 4 chalcone synthase genes was 84.2 ~ 91.3%. Several putative phosphorylation sites found in Og-CHS amino acid sequences indicated that post-translational modification might affect Og-CHS activity. Analyzing upstream promoter sequence of chalcone synthase genes, several predicted responsive elements related to light, ethylene, auxin, GA, temperature and wounding were found. Southern blot analysis indicated chalcone synthase genes from Oncidium were a multigene family. The mRNA encoding Og-CHS was found primarily in the roots. It also found in pseudobulbs, leaves, flower buds and flowers. Og-CHS mRNA was accumulated in Oncidium petals from the cutting day, reached to the maxium in the fifth day and then decreased progressively. The expression pattern changed significantly after pollinia cap dislodgment. Transient expression analysis of different promoters of flower organ-expressed genes by particle bombardment demonstrated that the promoter activity for Pt-ACO::GUS increased by 1.76-fold compared to that of CaMV35S::GUS. GUS gene was expressed in transgenic tobacco Og-CHS::GUS plants in leaves and crown whereas no GUS activity was detected in Pt-ACCA::GUS plants at vegetative stage. GUS staining was largely observed in roots, and also in stem and leaves for Pt-ACO::GUS. Pt-ACS1::GUS was expressed in leaves, and Pt-ACS2::GUS was expressed in stem and roots. Transgenic Arabidopsis plants exhibited differential expression of GUS at various developmental stages.

並列關鍵字

Flower organ-expressed genes ； promoter activity analysis ； chalcone synthase ； ACC synthase ； ACC Oxidase

參考文獻

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