Molecular Cloning of Sigma Factor Gene of Phytoplasma Associated with Peanut Witches' Broom

花生簇葉病菌質體sigma factor基因之選殖與分析

陳紹寬(Shau-Kwaun Chen) ； 朱佩文(Pei-Wen Chu) ； 何國傑(Kuo-Chieh Ho) ； 陳武揚(Wuu-Yang Chen) ； 林長平(Chan-Pin Lin)

《植物病理學會刊》 17卷4期 (2008/12) Pp. 279-287

https://doi.org/10.6649/PPB.200812_17(4).0003

摘要

本實驗從事花生簇葉病菌質體sigma factor基因之選殖研究，實驗中以六種細菌之rpoD，sigA基因高保守陸區域設計出一組PCR引子，以花生簇葉病菌質體DNA爲模板進行PCR反應而獲得一600bP大小之PCR產物，並以此作爲核酸探針進行花生簇葉病菌質體基因庫之篩選，得到含1.8kb嵌入片段之選殖株重組質體pPSF3。對pPSF3之嵌入片段進行核苷酸序列分析可得到一個完整的ORF(open reading frame)，且在其轉譯起始密碼AUG上游有互補於植物菌質體16S rRNA之3端核酸序列即Shine-Dalgarno序列之存在，此爲可能之核糖體結合位置(ribosomal binding site)此ORF之核苷酸序列經推衍爲胺基酸序列後，發現其中有啟動予-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的sigma factor基因相似，故據此推斷其爲sigma factor基因。在南方氏雜合反應的結果可發現在花生簇葉病菌質體中可能有三個類似於其他真細菌中rpoD基因之sigma factor基因。

關鍵字

Mollicutes綱 ； rpoD基因 ； RNA聚合酶 ； 聚合酶連鎖反應選殖策略

並列摘要

To elucidate the molecular biology of phytoplasmas, a gene encoding the primary sigma factor of the phytoplasma associated with peanut witches' broom (PnWB) was cloned and sequenced. One complete open reading frame (ORF) was determined on the insert of the selected recombinants. 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 ace similar to other prokaryotic rpoD homologous sigma factor genes. According to the nucleic acid and amino acid sequence analyses, along with the results of Northern hybridization analyses, this gene was identified as a putative sigma factor gene. Based on the results of Southern hybridization analyses, it suggests that three rpoD homologous sigma factor genes may exist in PnWB phytoplasma.

並列關鍵字

Mollicutes ； rpoD gene ； RNA polymerase ； PCR-based cloning