蝴蝶蘭激勃素生合成相關基因之選殖與分析

Abstract

摘要 為瞭解蝴蝶蘭激勃素 (gibberellin, GA) 生合成路徑相關酵素GA 20-oxidase 及GA 2-oxidase 之基因特性，以阿拉伯芥同源基因為探針，篩 選蝴蝶蘭互補DNA 庫，選取pPhGA20ox-1-50 cDNA 進行定序分析，結果 顯示此cDNA 長1478bp，可轉譯372 個胺基酸，對應之基因命名為 PhGA20ox1，預測之分子量為41.9 kD，等電點 (isoelectric point) 為5.96。 PhGA20ox1 胺基酸序列中含有推測與GA 及共基質 (cosubstrate) 2-oxoglutarate 與二價鐵離子結合之GA 20-oxidase 保守序列LPWKET、 NYYPXCXXP 及三個組胺酸 (histidine)。南方氏雜交分析結果顯示 PhGA20ox1 在蝴蝶蘭基因組中為低拷貝基因，北方雜交分析則顯示此基因 在蝴蝶蘭植株根、花莖、葉、花朵、花苞、花梗中均有表現，且根部與葉 部表現量較高；此基因可受到過氧化氫誘導表現，以50 mM 過氧化氫誘導 時基因表現量最高。此外，UV 照射則無法誘導PhGA20ox1 基因表現。 GA 2-oxidase 選殖系pPhGA2ox-3-79 cDNA 長689 bp，可轉譯229 個 胺基酸，與其他物種之GA 2-oxidase 胺基酸序列相比，缺少N 端與C 端序 列。為取得基因全長，以pPhGA2ox-3-79 為探針篩選蝴蝶蘭基因組庫，選 取選殖系λphg1-1 進行限制酶圖譜及南方氏雜交分析、次選殖與定序分 析。λphg1-1 所含之GA 2-oxidase 基因命名為PhGA2ox1，取得之序列長 3841 bp，開放解讀框架 (open reading frame) 為954 bp，可轉譯317 個胺 基酸，預測之分子量為35.3 kD，等電點為5.9。基因結構具有兩個顯子 (exon) 及一個隱子 (intron)，隱子長1488 bp，第一與第二個顯子長度分別 為705 及249 bp，胺基酸序列含有推測與二價鐵離子結合之組胺酸及天門 冬胺酸 (aspartic acid)。PhGA2ox1 以蝴蝶蘭各部位進行北方雜交分析均偵 測不到PhGA2ox1 基因表現，此基因也不受UV 照射誘導，但處理50 mM 過氧化氫可誘導基因較高量之表現。

Abstract In order to understand the characteristics of gibberellin biosynthesis genes encoding GA 20-oxidase and GA 2-oxidase in Phalaenopsis, homologous DNA fragments from Arabidopsis thaliana were used as probes to screen Phalaenopsis cDNA library. Nucleotide sequence analysis revealed that pPhGA20ox-1-50 cDNA, designatedd as PhGA20ox1, was 1478 bp in length, encodes a polypeptide of 372 amino acid residues with a calculated molecular weight of 41.9 kD and isoelectric point 5.96. The deduced amino acid sequence of PhGA20ox1 contains conserved domain LPWKET, NYYPXCXXP and three histidines, which are considered to be involved in the binding of substrate GA, cosubstrate 2-oxoglutarate and Fe2+, respectively. PhGA20ox1 mRNA was expressed in roots, flower stem, leaves, flowers, buds and flower petiole of Phalaenopsis, especially in roots and leaves. Gene expression of PhGA20ox1 was induced by H2O2 with a highest transcript level induction at 50 mM. Moreover, expression of PhGA20ox1 did not respond to UV irradiation. One of the GA 2-oxidase cDNA, pPhGA2ox-3-79, has also been cloned from Phalaenopsis. This cDNA was 689 bp in length, and the deduced polypeptide of pPhGA2ox-3-79 cDNA lacked the N- and C- terminus as compared to other GA 2-oxidase amino acid sequences. To obtain the corresponding gene, pPhGA2ox-3-79 cDNA was used as the probe to screen genomic library of Phalaenopsis. Genomic clone λphg1-1 containing PhGA2ox1 was sequenced and characterized. The sequence analysis revealed that PhGA2ox1 was 3841 bp in length with a 954 bp open reading frame and encoding a 317 amino acid polypeptide. This gene contains two exons and one intron of 1488 bp. The deduced amino acids contained the histidine and aspartic acid residues which were found to be involved in the binding of Fe2+. No PhGA2ox1 transcripts were detected in Nothern analysis of different organs in Phalaenopsis, and PhGA2ox1 expression was not induced by UV. However, the PhGA2ox1 was expressed in Phalaenopsis upon exposure to 50 mM of H2O2.