蝴蝶蘭 MYB 基因之選殖與蛋白質定位分析

為了解蝴蝶蘭轉錄因子 MYB 基因之結構以及其功能，本研究進行蝴蝶蘭 MYB 基因之選殖。利用蝴蝶蘭轉錄因子 PtMYB1、PtMYB2 與 PtMYB3之 cDNA 作為探針，篩選蝴蝶蘭基因組庫，共獲得 21 個基因組選殖系，其中含有 PtMYB1 之基因組選殖系為 λPtMYB70，而含有 PtMYB2 之基因組選殖系為 λPtMYB261。經次選殖並定序分析後，獲得 PtMYB1 與PtMYB2 之完整基因，皆具有三個顯子與兩個隱子，且隱子邊界序列皆符合GT-AG 規則，此兩基因之解碼區位於第一與第二顯子內的長度皆分別為133 bp 與 130 bp；而啟動子序列經比對可能受到逆境、光照、激勃素、茉莉酸、水楊酸或機械式創傷等因子調控。蛋白質定位的部分，將 PtMYB3 與綠色螢光蛋白 (green fluorescent protien) 構築成融合載體，並於阿拉伯芥原生質體與洋蔥表皮細胞進行暫時性表達。結果顯示均與核染劑 DAPI 完全符合，確認 PtMYB3 蛋白質專一表達於細胞核中。

關鍵字

轉錄因子； MYB ；基因組選殖系；基因結構；啟動子

並列摘要

To understand the gene structure and function of the transcription factor MYB from Phalaenopsis, cDNA of PtMYB1, PtMYB2 and PtMYB3 were used as probes, to screen the genomic library of Phalaenopsis. The genomic clone λPtMYB70 and λPtMYB261 contain PtMYB1 gene and PtMYB2 gene, respectively. After subcloning and sequencing, PtMYB1 and PtMYB2 gene both possess three exons and two introns, whose junction sequences all follow the GT-AG rule. The exon 1 and exon 2 are 133 bp and 130 bp, respectively. According to the analysis of the promoter regions of PtMYB1 and PtMYB2, some cis-acting element related to stress, light, gibberellic acid, methyl jasmonate, salicylic acid, and wounding were found. In order to understand the protein localization, PtMYB3 cDNA fused with green fluorescent protein gene was transiently expressed in Arabidopsis protoplasts and onion epidermal cells. The results of transient assay showed that PtMYB3 specifically expressed in nucleus.