絲瓜簇葉病植物菌質體tuf基因之選殖及序列分析

Cloning and Nucleotide Sequence Analysis of a tuf Gene from Loofah Witches' Broom Phytoplasma

利用絲瓜簇葉病植物菌質體DNA為模板及兩個引作PCR反應，產生一段具1651個鹽基對的DNA片段。經核甘酸序列分析發現此DNA片段含有兩個譯讀框架，其中一個為合成部分之延長因子EF-G（fus基因）的譯讀框架，另一個為合成全長之延長因子EF-Tu（tuf基因）的譯讀框架。tuf基因含有1911個核甘酸，能合成一個具有396個氨基酸，分子量為43.8千道爾頓的蛋白質。此基因之核甘酸序列的G+C百分比為33.42，合乎膜柔綱之生物的特性。與其他細菌比較，在氨基酸序列上有高達53.9到61.8%的相似性，而與古生菌的相似性則低於26%。基因組上，只存有一個tuf基因版本。在親緣關係方面，由tufDNA序列分析所構成的親緣樹或是利用168 rDNA序列分析構成的親緣樹，顯示絲瓜簇葉病植物菌質體與其他菌種的親緣關係是相當一致的。

Using two primers and the enriched genomic DNA of a loofah witches' broom (LfWB) phytoplasma as the template, a 1651-bp DNA fragment was amplified in a PCR reaction. The DNA fragment contained two open reading frames (ORFs), one coding for part of the elongation factor EF-G (jus gene) and the other for the full-length EF-Tu (tuf gene). The tuf gene contained 1191 nucleotides encoding a 396-amino acid protein with a predicted molecular mass of 43.8 kDa. The sequence displayed a common characteristic of low G+C content of mollicutes and had a G+C content of 33.42%. It had a high degree of amino acid sequence homology, from 53.9 to 61.8%, with those of other bacteria except two archaea (lower than 26%). There was only one tuf gene locus on the genome. The phylogenetic relatedness of LfWB phytoplasma to other prokaryotes revealed by tuf DNA sequences was consistent with the result reported by 16S rDNA sequences.