- 期刊
- OpenAccess
cDNA Cloning and Molecular Characterization of 4-Coumarate: Coenzyme A Ligase in Eucalyptus camaldulensis
赤桉4-香豆酸：輔酶A接合酶(4CL)之互補核酸(cDNA)選殖與分子定性
摘要
對於植物酚基丙酸鹽類之生合成而言，4-香豆酸：輔配A接合酶(4-coumarate: coenzyme A ligases; 4CLs)是一群非常重要的酶，包括生合成木材三項主要組成分之一的木質素。為了能夠逐步進入遺傳調控桉樹木質素含量之領域，我們於此報告從赤桉(Eucalyptus camaldulensis)的次生發育木質部選殖2個4CL的互補核酸片段，分別定名為Euc4CLE3與Euc4CL1。其中，Euc4CLE3片段含有823 bp核酸，屬於開放閱讀框(open reading frame; ORF)的部分序列，可編碼轉譯成274個胺基酸。另一條1953 bp的Euc4CL1，經定序含有全長之cDNA，屬於ORF的1632 bp可轉譯成一條544個胺基酸的多胜肽，其分子量的估測值為59,470。經過胺基酸序列的群團分析，Euc4CLE3與Euc4CL1分屬兩個不同群的4CLs。進一步將Euc4CL1與歸屬同群其他植物的4CL進行蛋白質並列比對，結果顯示Euc4CL1是4CLs家族的成員。重組酵素分析辨別Euc4CL1對於木質素先驅物具有不同酵素活性與喜好度，隱約示意Euc4CL1參與木質素化。南方雜交確認Euc4CL1基因存在於赤桉染色體基因組。北方與西方雜交顯示Euc4CL1基因在次生發育木質部組織具有mRNA與蛋白質的清晰表達，進而闡釋Euc4CL1與木質素生合成以及木質部發育之緊密關聯性。
並列摘要
The 4-coumarate: coenzyme A ligases (4CLs) are a group of enzymes very important to the biosynthesis of plant phenylpropanoids including lignin, which is 1 of 3 major wood components. To gradually approach the genetic manipulation of the lignin content in Eucalyptus trees, we report on the cloning of two 4CL cDNAs designated Euc4CLE3 and Euc4CL1 (GenBank accession no., DQ147001) from secondary developing xylem tissues of E. camaldulensis Blakely. The Euc4CLE3 fragment containing 823-bp cDNA was a partial sequence of the open reading frame (ORF) encoding 274 amino acid residues. The other 1953-bp clone from nucleic acid sequencing revealed a full length of 4CL cDNA containing an entire ORF of 1632-bp cDNA, which encoded a polypeptide of 544 amino acid residues with a calculated molecular weight of 59,470. In comparisons of amino acid sequences, the Euc4CLE3 and Euc4CL1 clones were classified into distinct classes of 4CLs. Alignment of the deduced Euc4CL1 protein with other plant 4CLs in class Ⅰ showed that Euc4CL1 was a member of the 4CL family. Recombinant protein analyses indicated that the enzymatic activity and substrate preference of Euc4CL1 differed from those of lignin precursors, implying its involvement in lignification. Southern blot analysis confirmed the existence of native Euc4CL1 in the chromosomal genome of E. camaldulensis. Northern and western hybridizations verified that the Euc4CL1 gene was clearly expressed at the mRNA and protein levels in secondary developing xylem tissues, further verifying that Euc4CL1 is tightly associated with lignin biosynthesis and xylem development.