以逢機定序方式選殖花生簇葉病菌質體之質體及插入序列

Cloning and Sequencing of Extrachromosomal DNA and Insertion Sequence of Phytoplasma Associated with Peanut Witches' Broom Using Random Sequencing

魏慧珍(H. C. Wei) ； 林長平(C. P. Lin)

《植物病理學會刊》 13卷2期 (2004/06) Pp. 143-154

https://doi.org/10.6649/PPB.200406_13(2).0006

摘要

以逢機定序的策略對花生簇葉病菌質體的全DNA(total DNA)進行分析，以罹病日日春植株之全DNA及健康植株之全DNA為核酸探針，配合差異性雜配反應(differential hybridization)對花生簇葉病菌質體構築在lambda Zap*II及 pBluescriptII SK (-) phagemid 上之基因庫行篩選，共選殖了108個選殖株，逐一共完成了其中八個選殖株之核苷酸定序及分析比對，經由比對結果顯示，其中，選殖株C2及選殖株H13中所具有的putative ORFs，與NCBI基因庫中之已有的資料具較高之相似性。選殖株C2中之一個open reading frame(ORF)與病毒的replication protein(Rep) gene 具有相似性。因植物菌質體之質體(extrachromosomal DNA) 曾被報導與病毒的Rep gene 具有相似性，因此以選殖株C2已知序列設計反向引子對，對罹病日日春全DNA進行反向聚合酵素連鎖反應(inverse polymerase chain reaction, IPCR)實經電泳分析潔果只出現單一條帶，顯示選殖株C2之嵌入DNA為一環狀(circular)質體。以嵌入片段中之Rep 基因。另外，選殖株H13中之一個ORF兩個側也發現反向重複序列(inverted repeat sequence)，因此推斷此選殖株H13所嵌入之DNA片段中具有花生簇葉病菌質體基因體中之插入序列。本研空中不論以該插入序列或以其間之序列為核酸探針進行南方氏雜配反應，結果皆為單一訊號，顯示此花生簇葉病菌中所發現之插入序列只具有單一套組(single copy)或者是低套組(low copy)。

關鍵字

質體 ； 插入序列 ； 花生簇葉病 ； 植物菌質體

並列摘要

A random (“shotgun”) sequencing strategy was conducted to investigate the genome of the phytoplasma associated with peanut witches’ broom (PNWB) in this study. Total DNA of periwinkle infected with PNWB phytoplasma and that of healthy periwinkle were used as probes for differential screening of lambda Zap*II genomic libriay and pBluescriptII SK (-) recombinant plasmids of PNWB-Phytoplasma. Eight out of 108 random clones thus selected were completely sequenced and anylyzed. The insert DNA of recombinant plasmid C2 consists of 4226 nucleotides and encompasses an ORF homologous to the Rep genes found in the extrachromosomal DNA of other organisms. A pair of oligo-nucleotide primers C2R1/C2F1 for inverse polymerase chain reaction (IPCR) were designed according to the nucleicacid sequences of the recombinant plasmid C2, and a 265 bp fragment was amplified only with the total DNA prepared from diseased periwinkles infected with PNWB phytoplasma as the template for IPCR reaction. The result indicates that the 4.2 kbp insert DNA of the recombinant plasmid C2 is a circular form DNA (plasmid). The results of Southern hybridization analysis using Rep gene fragment of clone C2 as a probe suggested that multiple copies of Rep gene may exist in PNWB-Phytoplasma. Recombinant clone H13 encompasses a complete ORF of 3880 nucleotides homologous to the transposase gene of other organisms. An eight- nucleotide inverted repeats between nucleotides 1711- 1718 and 2735-2742 near the ORF was found and a conserved DDE motif in this ORF was identified. According to the insertion sequence, it suggests that only one copy of transposase and insertion sequence may exist in PNWB-phytoplasma.

並列關鍵字

extrachromosomal DNA ； nsertion sequence ； eanut witches' broom ； hytoplasma