花生簇葉病菌質體dnaB1和dnaG基因之選殖與分析

Abstract

本研究在於從事花生簇葉病菌質體 dnaB1 和 dnaG 基因之選殖與基因序列之分析。首先利用本實驗室所研發之花生簇葉病菌質體專一性 PCR 引子對 281-5a/ 281-5b，以受花生簇葉病菌質體感染之日日春全 DNA 為模板進行 PCR 反應，經選殖 PCR 產物後，獲得選殖株 281-5。經定序分析後獲得 dnaB1 基因 5’ 端核酸序列。該 dnaB1 基因為指導複製解螺旋酶 (replicative DNA helicase) DnaB 之合成，且 dnaB1 基因在植物菌質體之基因體為 dnaB1-dnaG 之基因排序方式，其中 dnaG 基因所指導之引發酶 (DNA primase) DnaG 亦參與於 DNA 複製之過程中，因此以選殖株 281-5 之選殖片段核酸序列為基礎，同時進行 dnaB1 和 dnaG 基因之選殖。實驗中所採用之選殖策略為收集 GenBank 中已發表之各植物菌質體之 dnaG 基因核酸序列，進行比對分析後，並依據 dnaG 基因 5’ 端和 3’ 端高保守性區域分別設計簡併式 (degenerate) 反向引子 dnaBr2 及 RG2，並利用選殖株 281-5 之選殖片段序列設計正向引子 dnaB-1-R-1，並以引子對 281-5a/ dnaBr2 和 dnaB-1-R-1/ RG2 分別進行 PCR 反應，以增幅花生簇葉病菌質體之核酸片段，經選殖 PCR 產物後，分別獲得選殖株 B52 和 BG。選殖株 281-5, B52 和 BG 之選殖片段於重疊處之核酸序列相同度為 100%，且選殖株 BG 之選殖片段，於 NCBI 基因資料庫進行比對分析後，發現其 3’ 端序列與本研究室先前發表之選殖株 H13 選殖片段 (GenBank AY270153) 之 5’ 端核酸序列完全相同，因此利用選殖株 H13 之選殖片段核酸序列，設計反向引子 RHp1，並以引子對 dnaB-1-R-1/ RHp1 進行 PCR 反應，經選殖 PCR 產物後，獲得選殖株 BH。以電腦軟體分析本研究中所獲得之各選殖株選殖片段序列，經將其接續後獲得 3,593 bp 之核酸序列，經序列比對分析後得知，該核酸片段依序包含一 hypothetical protein gene 之 3’ 端序列, dnaB1及 dnaG 基因全序列，以及另一 hypothetical protein gene 之 5’ 端序列。以核酸探針 FB3-p 進行南方氏雜配反應 (Southern hybridization) 以分析 dnaB1 基因套組數時， 可獲得兩個雜配訊號，因此推測 dnaB1 基因在花生簇葉病菌質體基因體中具有兩個套組。此外，由 RT-PCR 反應的結果發現在花生簇葉病菌質體中 dnaB1 和 dnaG 基因可分別進行 mRNA 之轉錄。

A PCR-based strategy was conducted to obtain dnaB1 and dnaG genes of phytoplasma associated with peanut witches’ broom (PnWB) in this study. A PCR fragment was amplified specifically from the PnWB phytoplasma-affected periwinkle plants using primer pair 281-5a/ 281-5b designed in previous study. The PCR product was cloned and the DNA fragment of clone 281-5 was sequenced and revealed to have homology to the sequences of published dnaB genes. In phytoplasma genome, dnaB gene can be arranged in the order of dnaB-dnaG to encode the DnaB and DnaG proteins for DNA replication, respectively. To clone the dnaB1 and the downstream dnaG gene of PnWB phytoplasma, two degenerate oligonucleotide PCR primers dnaBr2 and RG2 in reverse direction were designed according to the 5’ and 3’ end conserved sequences of the dnaG gene of various phytoplasmas, and primer dnaB-1-R-1 in forward direction was also designed according to the sequence of the cloned fragment of 281-5. PCR fragments were then amplified specifically from the PnWB phytoplasma DNA using primer pairs 281-5a/ dnaBr2 and dnaB-1-R-1/ RG2. The PCR products were cloned and clones B52 and BG were obtained, respectively. Sequence analysis of the cloned fragment of BG revealed that the 3’ end sequence has high homology to that of clone H13 (GenBank AY270153) published by us previously. A primer RHp1 in reverse direction was then designed according to the sequence of the cloned fragment of H13. PCR fragment was amplified from the PnWB phytoplasma DNA using primer pair dnaB-1-R-1/ RHp1 to obtain clone BH. Sequences of the cloned fragments of 281-5, B52, BG and BH were analyzed and combined as a 3,593 bp sequence fragment. Sequence analysis showed that the fragment contain the 3’ end sequence of a hypothetical protein gene, dnaB1 gene, dnaG gene, and the 5’ end sequence of another hypothetical protein gene in order. Southern hybridization analysis indicated that there were two copies of dnaB1 gene in PnWB phytoplasma genome. RT-PCR analysis showed that mRNAs of dnaB1 and dnaG were transcribed independently in PnWB phytoplasma.