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花生簇葉病菌質體polC基因之選殖與分析

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

摘要


本研究在於從事花生簇葉病菌質體polC基因之選殖,採用之選殖策略為先行收集其他革蘭氏陽性細菌polC基因之核酸及胺基酸序列,進行比對分析,根據其高保守性區域設計引子對F3-2/R6-2,再以感染花生簇葉病之日日春全DNA為模板進行PCR反應,藉此獲得一729bp大小之PCR產物,再將其選殖至pCR(上標 ®)Ⅱ-TOPO質體中,得到一重組質體pF3-2-15,針對pF3-2-15之嵌入片段進行核酸定序,並根據此序列設計專一性引子對F4/R6,經用PCR DIG標識法針對pF3-2-15之嵌入片段增幅出493bp之DIG標識片段,以作為polC基因篩選用核酸探針,從事花生簇葉病菌質體重組質體之篩選,如此遂獲得一選殖株重組質體pH15-40。重組質體pH15-40之嵌入片段長度為5221bP,其中包含一個完整ORF及一個不完整的ORF,分別稱之為ORF1 (4479bp)與ORF2 (502bp)。將由ORF1核酸序列推衍所得之胺基酸序列進行比對分析後,發現該序列與其他細菌的DNA聚合酶Ⅲ基因polC相似。此外,由ORF1所推衍出之胺基酸序列在一些重要的胺基酸位點上的保守性也很高,而且在ORF1所推衍之胺基酸序列中,在第400個至第562個胺基酸之問可發現exo domain,而在第1233個胺基酸至第1335個胺基酸之間可發現pol domain,因此推測ORF1應為花生簇葉病菌質體的polC基因。由ORF2之核酸序列推衍所得之胺基酸序列進行比對分析後,發現該序列與其他細菌的NADH oxidase。基因nox相似。以罹病日日春全RNA進行反轉錄聚合酶連鎖反應的結果顯示polC基因有轉錄情形發生,且選殖株pH15-40的嵌入片段包含polC基因全長。

關鍵字

DNA聚合酶 花生簇葉病 polC

並列摘要


To clone and analyze the polC gene of peanut witches' broom-phytoplasma (PNWB-phytoplasma), a pair of degenerate oligonucleotide PCR primer F3-2/ R6-2 were designed according to the conserved sequences of the polC gene of various Gram-positive bacteria. The total DNA from a periwinkle infected with PNWB-phytoplasma was prepared for PCR reaction to amplify a 729-bp PCR product. The PCR product was then cloned into the vector pCR(superscript ®)Ⅱ-TOPO to obtain a recombinant plasmid pF3-2-15. A 493-bp DIG-labeled PCR product was amplified by using the primers F4/R6 designed according to the sequence of the insert DNA of pF3-2-15 and was used as a nucleic acid probe for the screening of recombinant plasmids of PNWB-phytoplasma DNA. The recombinant plasmid pHl5-40 thus obtained comprises an insert DNA of 5221 nucleotides and encompasses a complete ORF of 4479 nucleotides and an incomplete ORF of 502 nucleotides, referred to as ORF1 and ORF2, respectively. The alignment of the primary structures of ORF1 and the DNA polymerase Ⅲs of other Gram-positive bacteria reveals strong conservation of long segments of sequences and perfect conservation of amino acid residues in the exo domain (400(superscript th)-562(superscript nd) amino acid) and the pol domain (1233(superscript rd) 1335(superscript th) amino acid). The analyses of nucleic acid sequence and amino acid sequence suggest that ORF1 is the putative polC gene of PNWB-phytoplasma. Moreover, the sequence of ORF2 is homologous to the NADH oxidase gene, nox, of other organisms. The results of RT-PCR reveal that the polC gene was transcribed when the total RNA from PNWB-phytoplasma infected periwinkle was applied as the template and the recombinant plasmid pHl5-40 encompasses the entire polC gene of PNWB-phytoplasma.

被引用紀錄


蘇意婷(2010)。植物菌質體罹病日日春花器葉片化、綠化之病徵發展及花形、花色決定基因之變化與植物菌質體菌量之關係〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2010.02423
呂沛穎(2009)。花生簇葉病菌質體 rpsT, serS 與 hflB 基因之選殖與分析〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2009.02268
黃婷榆(2009)。花生簇葉病菌質體dnaB1和dnaG基因之選殖與分析〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2009.02254
蔡涵雅(2007)。花生簇葉病菌質體group II intron之選殖與分析〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2007.02978

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