苦瓜、香蕉與蝴蝶蘭 EIN2同源基因之選殖與分析

更年性 (climacteric ) 果實及花卉於貯運途中易受乙烯影響，目前已知EIN2為乙烯訊息傳遞路徑中的正向調控因子，於多數植物中為單一拷貝，針對EIN2若應用RNA干擾技術 (RNA interference) 進行基因默化，將可阻擋乙烯訊息傳遞。故本研究進行苦瓜、香蕉以及蝴蝶蘭EIN2同源基因之選殖，首先以阿拉伯芥 C端保守區基因片段為探針篩選得到苦瓜cDNA，長度為2,828 bp，可轉譯 964 bp，命名為McEIN2，轉譯之胺基酸和胡瓜最相似，相似性約88%，再以苦瓜EIN2 cDNA為探針，篩選得到香蕉EIN2 cDNA，長1,837 bp，可轉譯545個胺基酸，命名為MhEIN2，其轉譯之胺基酸序列，與水稻最相似，同源性為 62.4%，兩者皆只含第6個顯子後半段及第7個顯子 EIN2 cDNA。以文心蘭EIN2 DNA之第2~4顯子序列和香蕉EIN2 cDNA作為篩選蝴蝶蘭 EIN2基因組庫探針，次選殖系λPtEIN2-7和λPtEIN2-103分別含有第2~4顯子和第6個顯子，推測第1隱子長度超過8 Kb，第4至第5個隱子長度超過 14 Kb，第6個隱子長度超過8 Kb，轉譯之胺基酸相似性和玉米相似，為53.4%。EIN2 C端為為重要傳遞訊息之區域，分別在苦瓜、香蕉與蝴蝶蘭EIN2 C端找到此四個此保守性區域。南方氏雜交分析中苦瓜和蝴蝶蘭 EIN2結果顯示皆為單一拷貝；利用Real-time PCR檢測香蕉EIN2 於不同部位均有表現，以根部表現量最低，胚株最高，花瓣和花藥無明顯差異；於果實發育不同時期之果皮和果肉，MhEIN2隨著發育階段之第3期達到高峰，而後緩降。

關鍵字

乙烯不敏感；阿拉伯芥； EIN2 ；核蛋白；顯子

並列摘要

Fruit ripening and petal senescence of climacteric crops are seriously affected by ethylene during the transportation. In many crops, EIN2 is a single copy gene and acts as a positive regulator. Therefore, EIN2 is a good candidate gene to be silenced by RNA interference for interruption of ethylene signal transduction. First of all, 3' end fragment of AtEIN2 gene was used as probe to screen cDNA library of bitter gourd and the McEIN2 cDNA is 2,828 bp long and encodes 964 amino acids. Second, McEIN2 cDNA was used as probe to screen cDNA library of banana and the MhEIN2 cDNA is 1,837 bp in length which encodes 545 amino acids. Both McEIN2 and MhEIN2 cDNAs are corresponding to 3' end part of exon 6 and exon 7. Amino acid sequence of McEIN2 shares 88% similarity with that of cucumber EIN2 and the similarity between MhEIN2 and ZmEIN2 from corn is 62.4%. Next, exon 2 to exon 4 of Oncidium EIN2 gene was used as probe to screen the genomic library of Phalaenopsis. λPtEIN2-7 and λPtEIN2-103 contain exon 2 to exon 4 and exon6, respectively. All the predicted lengths of intron 1, 4, 5, and 6 are longer than 6 kb. The coding sequence of exon 6 in PtEIN2 shares the highest similarity in amino acid sequence to ZmEIN2. Four conserved boxes which act as glucose sensor could be found in deduced amino acid sequences of McEIN2, MhEIN2, and PtEIN2 C-terminus. The Southern blot analysis indicated that the EIN2 genes from bitter gourd and Phalaenopsis belong to single copy gene. The abundance of MhEIN2 mRNA was found to be highest in ovule and lowest in root. During ripening of banana fruit, the expression of MhEIN2 increased consistent with the ripening stages, reached to the maximum at stage 3, and decline after stage 3.