絲瓜簇葉病植物菌質體tuf基因的選擇及序列特性分析

Abstract

本文以絲瓜簇葉病植物菌質體為材料，篩選其保留性高的tuf基因。我們的選殖策略為依據已發表的兩種動物菌質體幼Mycoplasmagenlitalium及M. galliseptium的tuf基因序列，設計了兩個PCR反應的引子(Tul、Tu2)，以絲瓜簇葉病植物菌質體為模版DNA，合成了一段長約400bp的DNA片段，作為篩選tuf基因的探針；並構築於λ ZAP的絲瓜簇葉病植物菌質體基因庫中，篩選到HI選殖株，HI之嵌入月段長約3.7kb，經序列分析的結果，此選殖株包含了一個完整的tuf基因及部分長度的fus基因。tuf基因全長1185bP，可以堆衍出395個胺基酸的蛋白質，並推測其分子量大小為47.3kDa。利用DNASTAR套裝軟體，分析絲瓜簇葉病植物菌質體與其他14種菌體之tuf基因DNA序列，發現絲瓜簇葉病植物菌質體與其他4種植物菌質體具60％以上的相似性而與4種動物菌質體具50~56％的相似性。同時也由此選殖株序列預測絲瓜簇葉病植物植物菌質體tuf mRNA的上游具有Shine-Dalgarno序列，而於其下游可形成一RNA髮夾構造(hairpin structure)。藉由研究絲瓜簇葉病植物菌質體tuf基因的結果，可輔助我們瞭解植物菌質體在Mollicutes的分類地位及與其他細菌的親緣關係，並發現以tuf基因之DNA序列與其他菌種所作之親緣關係圖與以16S DNA序列分析所得之結果相似。

Using the enriched genomic DNA of a phytoplasma associated with loofah witches , broom (LfWB phytoplasma) as the template and a pair of oligonucleotide primers designed according to the tuf gene sequences of Mycoplasma genitalium and 功紹 oplasma gallisePtium Mycoplasma genitalium, a four hundred-bp DNA fragment named Tu400 was amplified in a PCR reaction . A clone, HI containing a 3.7-kbp insert DNA fragment was isolated from a LfWB phytoplasma λ zap genomic library using the Tu400 as probe. Comparing with the DNA sequences in Genbank, the insert DNA contained two open reading frames (ORFs) encoding part of elongation factor EF-G (fus gene) and the full-length EF-Tu (tuf gene). The tuf gene contained 1185 nucleotides and encoded a 395-amino acid protein of 47.3 kDa. This gene had >60% similarity with four tuf sequences of phytoplasmas isolated from periwinkle, and 50-56% with four animal mycoplasmas. There was a Shine-Dalgrno sequence in the upstream region of the gene. The region downstream tuf forms a putative hairpin structure which signals the end of the transcription unit. The phylogenetic relatedness of LfWB phytoplasma to other prokaryotes constructed by using tuf DNA sequences displayed a similar result reported by using the 16S rDNA.