花生簇葉病菌質體dnaB1和dnaG基因之選殖與分析

Cloning and analysis of dnaB1 and dllaG genes of peanut witches'-broom phytoplasma

"以專一性PCR引子對281-5a/281-5b針對受花生簇葉病菌質體感染之日日春全DNA進行PCR反應，經選殖（cloning）該PCR產物片段並定序後獲得dnoB1基因5'端之核酸序列。依據該片段之核酸序列設計正向引子dnaB-1-R- 1'並收集GenBank中登錄之各植物菌質體之dnaG基因核酸序列，進行比對分析後，於該基因近5'端和3'端處之高保守性區域分別設計反向之degenerate引子dnaBr2及RG2，並以引子對281-5a/dnaBr2和dnaB-1-R-1/RG2進行PCR反應，經選殖該等PCR產物後分別獲得選殖株B52和BG。B52及BG之選殖片段經序列比對分析後發現B52之3'端序列與BG之5'端序列重壘，且BG之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，因此依據H13之選殖片段序列設計反向引子RHp1，並以引子對dnaB - 1-R- 1/RHP1進行PCR反應而獲得選殖株BH。經將本研究中所獲得之選殖片段序列重疊接續後共獲得3,593 bp之核酸序列，其中包含兩個hypothetical protein gene部分序列和dnoB1及dnaG基因全長序列。由南方氏雜配反應（Southern hybridization）結果推測danB1基因在花生簇葉病菌質體之基因體中真有兩個套組。此外，由RT-PCR反應的結果發現在花生簇葉病菌質體中danB1和dnaG基因可分別進行mRNA之轉錄。"

"A PCR-based strategy was conducted to obtain dnaBl and dnaG genes of peanut witches' broom (PnWB) phytoplasma in this study. A PnWB phytoplasma specific PCR primer pair 281-5a/ 281-5b was used to amplify the total DNA ofPnWB-infected periwinkle to obtain clone 281-5, and the cloned fragment was sequenced and revealed to have homology to the sequences of published dnaB genes. To clone the dnaBJ and the downstream dnaG gene of PnWB phytoplasma, two degenerate primers dnaBr2 and R02 in reverse direction were designed according to the 5-' and 3-' 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. The PCR primer pairs 281-5a/ dnaBr2 and dnaB-1-R-1/ RG2 were used to amplify the inserts of clones B52 and BG, respectively. Sequence analysis of the cloned fragments of B52 and BG revealed that the 3' end sequence of the cloned fragment of BG has high homology to that of clone H13 (GenBank AY270153). A primer RHp1 in reverse direction was then designed according to the sequence of the cloned fragment of H13, and the PCR prime r pair dnaB-1-R-1/ RHp1 was then used to amplify the insert of the clone BH. Sequences of the cloned fragments of 281-5, B52, BO and BH were analyzed and combined as a 3,593 bp sequence fragment. Sequence analysis showed that the fragment contain two partial hypothetical protein genes , dnaBl gene and dnaG gene . 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 separately in PnWB phytoplasma."