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cDNA Cloning and Sequence Analysis of 5-Hydroxyconiferaldehyde O-Methyltransferase from Eucalyptus camaldulensis
赤桉5-羥化針葉樹型乙醛O-甲基轉移酶(AldOMT)之互補核酸(cDNA)選殖及序列分析
摘要
從赤桉樹幹刮取發育中的次生木質部組織，並萃取總量RNA，再利用反轉錄之合酶連鎖反應(RT-PCR)，擴大複製出一條1101bp編碼製造5-羥化針葉樹型乙醛O-甲基轉移酶(5-hy droxyconiferaladehy de O-methyltransferase; AldOMT)之互補核酸片段(REOMT-11)，隨後將其選殖到PCRⅡ TOPO載體。經過核酸定序，REOMT-11含有1098bp的開放閱讀框(open reading frame)，可轉譯成366個胺基酸的一條多勝酶，其分子量的估測值爲39,900。接著再利用合成酶連鎖反應，擴大複製出5’端上游與3’端下游的核酸片段，而獲得全長度赤桉AldOMT cDNA之選殖，並定名爲EucOMT1。經胺基酸序列比對，選殖的EucOMT1明顯歸屬於caffeate O-methyltransferase(COMT)類型，而與caffeoy1-CoA O-methyltransferase(CCoAOMT)之類型相距甚遠。進一步的分析，發現EucOMT1具有4個可能連接Sadenosy1-L-methionine(SAM)的區段。
並列摘要
Total RNA of developing secondary xylem scraped from the woody stem of Eucalyptus camaldulensis was extracted and used as initial material for cloning. A 1101-bp cDNA fragment (REOMT-11) encoding 5-hydroxy coniferaldehyde O-methyltransferase (AldOMT) was am plified using reverse-transcription polymerase chain reaction (RT-PCR), and cloned into a PCRII TOPO vector. Nucleotide sequencing revealed that the REOMT-11 clone contained an open reading frame of 1098-bp cDNA, which encoded a polypeptide of 366 amino acid residues with a predicted molecular weight of 39,900. A full-length AldOMT cDNA clone (EucOMT1) was obtained by supplementing 2 other fragments, which were amplified from upstream and down stream regions of the REOMT-11 clone. In comparisons of deduced amino acid sequences, EucOMT1 was classified into the type of caffeate O-methyltransferase (COMT) which used to be defined as catalyzing the methylation of caffeic acid to ferulic acid, and away from the caffeoyl-CoA O-methyltransferase (CCoAOMT) class. In further analyses, 4 possible S-a denosyl-L-methionine (SAM) binding domains were found in the deduced amino acid sequence of the EucOMT1 clone.