蝴蝶蘭 MYB cDNAs 之選殖與分析

Abstract

植物 MYB 家族基因扮演轉錄因子的角色，其功能包括調控次級代謝、細胞形態發生、花朵香氣及顏色等。本試驗選殖蝴蝶蘭 MYB cDNA 並分析其特性和功能，期望未來應用於蝴蝶蘭等花卉之育種改良。利用阿拉伯芥 Production of Anthocyanin pigment 1 (PAP1) cDNA為探針，以溶斑雜交法篩選蝴蝶蘭 cDNA 庫一百五十萬個溶斑形成單位 (plaque forming unit, pfu)。篩選出三個可演繹出完整胺基酸序列之 MYB cDNA，命名為 PtMYB1、PtMYB2 和 PtMYB3。將其胺基酸序列與資料庫序列比對後，皆屬於 R2R3-MYB 轉錄因子，含有為兩個重覆 MYB 蛋白功能性組區，每一個功能性組區皆含有三個 α - helices。為了解 PtMYB 於蝴蝶蘭基因組分佈情形，南方氏雜交分析得知三個蝴蝶蘭 PtMYB 皆為低拷貝基因。分析蝴蝶蘭不同器官 PtMYB 之表現情形，結果 PtMYB1 和 PtMYB2 於花器表現量最高，PtMYB3 於根部表現量最高。而於不同荷爾蒙誘導處理條件下，PtMYB1 可受多種荷爾蒙誘導；PtMYB2 於水楊酸處理下，表現量最高；而 PtMYB3 於水楊酸和離層酸處理下，mRNA 累積量最多。於阿拉伯芥原生質體和洋蔥表皮細胞進行細胞內定位分析，結果顯示 PtMYB1 和 PtMYB2 皆表達於細胞核。

Functions of MYB gene family in plants are diverse including regulation of secondary metabolism, cellular morphogenesis, flower color, and fragrance etc. Plaque hybridization was employed to screen the cDNA library of Phalaenopsis for MYB cDNA using Arabidopsis thaliana PAP1 cDNA as probe. The MYB cDNAs were isolated, characterized and named PtMYB1, PtMYB2 and PtMYB3. All of them belong to R2R3-MYB subfamily containing two repeats and each repeat has a helix-turn-helix structure. Southern blot analysis indicated that all of three PtMYB were low copy genes. Northern blot analysis revealed that their expressions were tissue-specific. PtMYB1 and PtMYB2 had higher mRNA expression level in floral organs whereas PtMYB3 had higher mRNA expression level in roots. While PtMYB1 was found to be induced by several hormones, PtMYB2 and PtMYB3 were induced mainly by salicylic acid and abscisic acid. Subcellular targeting analysis in Arabidopsis protoplasts and onion epidermal cells indicated that PtMYB1 and PtMYB2 were localized in the nucleus.