苦瓜EDR1及CAN1同源基因之選殖與啟動子活性分析

Abstract

為取得苦瓜McEDR1基因及其啟動子，選取苦瓜基因組選殖系McEDR97、McEDR111進行定序分析，苦瓜McEDR1基因解碼區(coding region)長10 kb，由13個顯子與12個隱子所組成，可轉譯出一1,000個胺基酸組成的蛋白質，預測之分子量為109.12 kDa，等電點為5.51，並含有一個絲胺酸/蘇胺酸蛋白激酶組區（Serine/Threonine protein kinase domain）。苦瓜McEDR1之胺基酸序列與其他物種相比同源性約46%~51%，歧異主要發生在非激酶組區域。同時得到McEDR1啟動子序列3.1 kb，經cis-acting element保守性序列比對分析顯示，此啟動子可能受到離層酸、乙烯、創傷調控基因表現。利用McEDR1 cDNA全長為探針，對苦瓜全RNA進行北方雜交分析，結果顯示苦瓜McEDR1基因的表現在根中表現較多，而果實中則較少。而苦瓜McEDR1啟動子活性暫時性表達分析結果顯示，苦瓜McEDR1啟動子活性於雄花花瓣中表現量較高；於菸草轉殖植株中則在根部有明顯的表現，阿拉伯芥T1轉殖株在細胞分裂旺盛的生長點，如根毛、側根、芽體及葉基部等處可偵測到GUS活性。 苦瓜McCAN1基因全長4 kb，由9個顯子與8個隱子所銜接，可演繹出344個胺基酸組成的蛋白質，分子量為38 kDa，等電點為9.23，並在C-terminal具有Staphylococcal nuclease (SNase)保守性區域。McCAN1演繹胺基酸序列，與真核生物及原核生物的CAN1同源基因進行比對，結果顯示此基因在真核生物中的保守性約50%~70%，與原核生物相比則只有20%左右，歧異主要發生在N-terminal。另分析苦瓜McCAN11基因之演繹胺基酸轉譯起始密碼上游3.5 kb，根據啟動子序列分析結果，推測McCAN11基因的表現可能受到離層酸、乙烯與創傷等逆境所誘導。利用McCAN1 cDNA全長為探針，對苦瓜全RNA進行北方雜交分析，可知苦瓜McCAN1基因在根部具有最高的表現，莖、雄花與雌花表現次之，於葉及果實表現最少。啟動子活性分析方面，暫時性表現結果顯示，McCAN1::GUS於蝴蝶蘭花瓣，苦瓜雌、雄花花瓣均可偵測GUS活性；菸草之McCAN1::GUS轉殖植株之葉片及根部可偵測到GUS活性，另McCAN1::GUS於阿拉伯芥轉殖株之側根、簇生葉、花序、芽及果柄等處皆有表現。

Two genes from bitter gourd were chosen for further analysis. In order to obtain the promoter and coding sequences encoding Enhanced Disease Resistance 1 (EDR1) homolog from bitter gourd, two genomic clones McEDR97 and McEDR111 isolated from bitter gourd using cDNA as probe were sequenced and characterized. McEDR1 gene snaps about 10 kilobases, including 13 exons and 12 introns. The open reading frame of McEDR1 encodes a 1,000 amino acid with a predicted molecular weight of 109.12 KDa, and isoelectricpoint were 5.51. McEDR1 contained a conserved serine/threonine protein kinase domain in C-terminus. The amino acid sequence of McEDR1 shows 46-51% homology to other EDR1 othologoue whose most dynamics located in N-terminal non-kinase domain. According to the results of sequence analysis, 3.1 kilobases upstream from the translation start site in McEDR1 were 5’untranslate regions and promoter region, and conserved cis-acting elements responsive to abscisic acid, ethylene , and wounding were found in the promoter region. Northern blot analysis indicates that the expression of bitter gourd McEDR1 gene in all organs especially in the stem and female flowers. On the other hand, Arabidopsis transformed with McEDR1::GUS expressed GUS especially in young leaves. The other gene McCAN1 was homologous to Ca2+-dependent nuclease. McCAN1 sequence obtained 4 kb length and consisted 9 exons and 8 introns of 344 amino acid in McCAN1. Conserved cis-acting elements responsive to ABA, ethylene, and wounding were found in the promoter region of McCAN1. McCAN1 gene was predicted to translated 344 amino acid protein with a molecular mass of 38 KDa. McCAN1 contained a Staphylococcal nuclease homologue (SNase) domain in C-terminus. The amino acid sequence of bitter gourd McCAN1 shows 50-70% homology to other eukaryote organism, and about 20% homology to prokaryote, most dynamics located in N-terminus. Northern blot analysis indicates that the expression of bitter gourd McCAN1 gene in all organs especially in the root, and leaves were much less. According to the results of GUS histochemistry staining in promoter activity analysis, McCAN1::GUS was expressed in inflorescence and roo in Arabidopsis and root, leaf in tobacco transformants.