苦瓜EIN3同源cDNA之選殖與分析

Cloning and Analysis of Ethylene Insensitive 3 Homologous cDNA from Momordica charantia L.

為了瞭解苦瓜果實後熟的乙烯訊息傳導機制，以進一步運用生物技術調控果實後熟，本研究進行EIN3同源cDNA之選殖與分析。首先根據已知之EIN3同源蛋白胺基酸序列演化保留區，設計專一性寡核苷酸引子。接著以苦瓜基因組DNA為模板，進行聚合酶連鎖反應。所得到之基因片段作為探針，篩選苦瓜cDNA庫後，得到選殖系pMEIL56，對應的基因命名為McEIL1，演繹的蛋白具有616個胺基酸，預測的分子量為70.3kDa，等電點為5.57。McEIL1蛋白具有EIN3及EIL（EIN3-like）之共同結構特徵，包括N端的酸性胺基酸區、五個演化保留區、一個proline-rich區、兩個核內定位訊號（nuclearlocalizationsignal, NLS）區、及必需之離胺酸。序列分析比對結果顯示，McEIL1的胺基酸序列與胡瓜CmEIL2相似度最高為87.9%，與阿拉伯芥EIN3序列具有51.8%的同源性。由南方氏雜交分析可知，McEIL1在苦瓜基因組中屬於單一拷貝基因。 In order to understand the mechanism of ethylene signal transduction mechanism in fruit ripening of bitter gourd (Momordica charantia) and to regulate ripening by means of biotechnology, EIN3 (ETHYLENE INSENSITIVE 3) homologous cDNAs were isolated and characterized from bitter gourd. Primers designed on the basis of amino acid conserved regions of EIN3 from different plant species and genomic DNA from bitter gourd were used to perform polymerase chain reaction (PCR). The PCR products were used as probes to screen cDNA library from bitter gourd. The gene of cDNA in clone pMEIL56 was desiginated as McEIL1 encoding a 616-amino acid polypeptide with predicted molecular weight of 70.3 kDa and isoelectric point of 5.57. All deduced polypeptides of identified cDNA clones contained complete structural features found in the Arabidopsis EIN3 protein, including an amino terminal acidic domain, five basic conserved domains, a proline-rich region, two nuclear localization signal, and the lysine residue essential for its function. Amino acid sequence homology between McEIL1 from bitter gourd and CmEIL2 from cucumber is 87.9%, and that between McEIL1 and Arabidopsis EIN3 is 51.8%. Southern blot analysis indicated that the McEIL1 gene from bitter groud is a single-copy gene.