花生簇葉病病原菌質體rpoC基因之選殖與分析

Cloning and Analysis of rpoC Gene of Phytoplasma Associated with Peanut Witches' Broom

林啓揚(C. Y. Lin) ； 陳武揚(W. Y. Chen) ； 林長平(C. P. Lin)

《植物病理學會刊》 15卷3期 (2006/09) Pp. 129-138

https://doi.org/10.6649/PPB.200609_15(3).0001

摘要

本研究在於進行花生簇葉病菌質體rpoC基因之選殖與基因序列之分析。基因篩選的策略為先行比對革蘭氏陽性細菌之rpoC基因的核酸及胺基酸序列，找出其保守性區段後設計出簡併式(degenerate) PCR引子對Frpoc1/Rrpoc1再以感染花生簇葉病之日日春植株全DNA為模版增出一l.8kb大小含有rpoC基因部分序列之PCR產物以作為探針，並利用在一般原核生物中位於rpoC上游之rpoB與位於下游之rpsL基因之序列，以chromosomal walking的方式選殖出rpoC兩端之基因片段，如此共完成包含rpoB3΄端、rpoC及rpsL5΄端之4,817bp序列之選殖與定序。在核酸序列中從nt 201-4,340具有一完整之ORF1，並可推衍轉譯1,379個胺基酸序列，其胺基酸序列經比對後發現與其他細菌之rpoC基因之胺基酸序列具有相似性。將此一rpoC可能基因之胺基酸序列與E.coli K12之RpoC, Saccharomyces cerevisiae之RPO21,及Drosophila melanogaster之RNA Polymerase Ⅱ 215 KD subunit RPⅡ215之胺基酸序列做比對，在八個具保守性的區域中，發現在C，D，G等區域具有較高之相似度。另外由胺基酸序列行預測功能上之推估比較，發現所選殖出的花生簇葉病菌質體rpoC基因在功能上並沒有退化演化的跡象，此可能乃因每一個功能區在基因轉錄的運作上均有其必要性。經南方氏雜配反應(Southern hybridization)顯示rpoC基因在花生簇葉病菌質體應是single copy或low copy number。此外，由北方雜配反應與RT-PCR之給果亦證實所選殖之花生簇葉病菌質體，rpoC有轉錄成mRNA。

關鍵字

花生簇葉病 ； 植物菌質體 ； rpoC基因

並列摘要

To clone the rpoC gene that encodes the β' subunit of the RNA polymerase in phytoplasma associated with peanut witches' broom (PnWB), a pair of degenerate PCR primers Frpoc1/Rrpoc1 was designed based on the conserved regions of the rpoC genes of various prokaryotes. A 1.8-kb PCR fragment was amplified specifically from the PnWB phytoplasma-infected periwinkle plants using primer pair Frpoc1/Rrpoc1. Sequence analysis revealed that the PCR product has high sequence homology to other reported rpoC genes. To obtain the full length of the rpoC gene, a chromosomal walking strategy was conducted. Based on the conserved sequences flanking the rpoC gene, four PCR primers were designed. A total of 4,817-bp DNA fragment containing the full length of rpoC gene, partial rpoB gene (3' end), and partial rpsL (5' end) gene was amplified and sequenced. An open reading frame between nt 201 and nt 4,340 was identified as ORF1. Alignment of amino acid sequence of ORF1 with those of E. coli K12 RpoC, Saccharomyces cerevisiae RPO21, and Drosophi1a melanogaster RNA polymerase Ⅱ 215 KD subunit RPⅡ215 revealed that regions C, D, and G shared great similarities. Southern hybridization analysis indicated that rpoC is a one-copy or low-copy number gene in the PnWB phytoplasma genome. Northern hybridization and RT-PCR analyses both showed that mRNA of rpoC was transcribed in PnWB phytoplasma.

並列關鍵字

peanut witches' broom ； phytoplasma ； rpoC gene