花生簇葉病菌質體RNA聚合酵素SIGMA FACTOR基因之選殖與分析(II)

Abstract

在進行花生簇葉病病原菌質體RNA 聚合酵素Sigma Factor 基因選殖時，以E. coli 、B. subtilis 、Lactococcus lactis 、Staphylococcus aureus 、Clostridium acetobutylicum 及Mycoplasma genitalium 等細菌之rpoD、sigA 基因進行比對，根 據其高保守性區域設計出一組PCR 引子，以花生簇葉病病原菌質體DNA 為模板 進行PCR 反應，得到一600 bp 之PCR 產物，並以此作為核酸探針，篩選花生簇 葉病病原菌質體之基因庫，得到一含1.8 kb 嵌入片段之選殖株重組質體pPSF3。 對pPSF3 之嵌入片段進行核甘酸序列分析可得到一個完整的ORF (open reading frame)，且在轉譯起始密碼AUG 上游有互補於植物菌質體16S rRNA 之3’端核 酸之序列，即Shine-Dalgarno 序列之存在，此為可能之核糖體結合位置(ribosomal binding site)。此ORF 之核甘酸序列經轉譯為胺基酸序列後，發現其中有啟動子 -10 及-35 辨識結合區域、中心酵素結合區域、及rpoD box，且基因大小及基因 結構均與其他細菌的sigma factor 基因相似，故以此推斷其為sigma factor 基因。 此基因之密碼利用性上並無以UGA 作為tryptophan 遺傳密碼之現象，且有與 Mollicutes 綱細菌在基因體特性上相同之"AT pressure"現象。而在南方氏雜合反 應的結果，則可發現在花生簇葉病植物菌質體中，可能有三個近似於其他真細菌 基因之rpoD 之sigma factor 基因。

In an effort to elucidate the molecular biology of phytoplasmas, a gene encoding the primary sigma factor gene of the phytoplasma associated with peanut witches’ broom (PNWB) was cloned and sequnced using PCR-based strategy. A 600-bp rpoD gene fragment was PCR-amplified and used as a nucleic acid probe for screening λ ZAP II genomic library of PNWB phytoplasma. The complete nucleotide sequence of the 1.8 kb insert DNA of pPSF3, one of the in vivo excised recombinants carrying a complete open reading frame (ORF), was determined. The ORF encodes a polypeptide contains -10 recognition and binding region, -35 recognition and binding region, core binding region and rpoD box. The gene organization and the nucleotide sequence in conserved region of the ORF are similar to other rpoD homologous sigma factor genes. According to the nucleic acid and amino acid sequence analyses and the results of Northern hybridization analyses, this gene was identified as a putative sigma factor gene. Based on the results of Southern hybridization analyses with the 600-bp fragment as a probe, it is suggested that three rpoD homologous sigma factor genes may exist in PNWB-phytoplasma.