花生簇葉病病原菌質體gyrB和gyrA基因之選殖與分析

Cloning of gyrB and gyrA Genes of Phytoplasma Associated with Peanut Witches' Broom

莊景光( J. G Chuang) ； 林長平( C. P Lin)

《植物病理學會刊》 9卷4期 (2000/12) Pp. 157-166

https://doi.org/10.6649/PPB.200012_9(4).0005

摘要

Gyrase酵素是由gyrB基因分別轉譯出的gyrA蛋白（A次單位元）及gyrB蛋白（B次單位元）以A2B2型式所組成之四連體蛋白。本研究在進行簇葉病病原菌質體gyrB及gyrA基因之選殖時，首先根據Acholeplasma laidlowii及Mycoplasma spp.等革蘭氏陽性細菌之gyrB基因序列之高保守性區域設計出PCR引子對GBF2/GBR3，再以感染花生簇葉病病原菌質體的日日春植物全DNA為模板進行PCR反應，獲得一約1.4kb大小之PCR產物。將此PCR產物選殖定序後，再以此序列設計出PCR引子對GBF3/GBR5，以PCR增幅出篩選gyrB基因的0.8kb核酸探針，再由感染花生簇葉病病原菌質體的日日春植物全DNA之基因庫篩選含gyrB基因片段之選殖株，我們獲得一選株重組質體pPGB 1-4，定序此一5308bp的EcoRI DNA嵌入片段，發現其包含三個完整open reading frame (ORFs 1-3)及一段與其他細菌gyrA基因5’端有同質性的DNA片段。而藉由pPGB 1-4上己知序列及比對其他細菌之gyrA基因核酸序列，再另行設計出PCR引子對GAF1/GAR2及GAF3/GAR3，則可自罹病日日春全DNA中增幅出位於gyrA基因中且大小為0.5kb之gyrA篩選用探針，並成功的選殖出另一重組質體pPGA 2-3，其EcoRI嵌入片段經核酸序列解奓得3840bp。此序列與pPGB 1-4的嵌入片段序列(5308 bp)共同進行核酸序列分析，再獲得另一完整的ORF(ORF4)序列。由ORF3及ORF4推衍所得之胺基酸序列進行比對分析後，發現其基因大小及基因結構與其他細菌的gyrB及gyrA基因具最高相似度。藉此推斷，ORF3及ORF4應分別為花生簇葉病病原菌質體之gyrB及gyrA基因。由南方氏雜配反應的結果可發現在花生簇葉病病原菌質體中應僅具有單一套(single copy)之gyrB基因及gyrA基因。而由RT-PCR反應的結果可發現，在花生簇葉病病原菌質體中gyrB及gyrA基因應是以gyrB-gyrA共同轉錄(cotranscription)的方式進行轉錄。

關鍵字

gyrB基因 ； gyrA基因 ； 花生簇葉病 ； 植物病原菌質體

並列摘要

DNA gyrase is a type II topoisomerase that is a tetrameric molecule composed of two A and two B subunits, which are encoded by the gyrA and gyrB genes, respectively. In order to clone the gyrB gene of phytoplasma associated with peanut witches' broom (PNWB), a pair of oligo- nucleotide PCR primers GBF2/GBR3 were designed according to the conserved amino acid sequences of the gyrB gene of various prokaryotes including Acholeplasma laidlawii, and Mycoplasma spp. Total DNA from diseased periwinkle infected with phytoplasma associated with peanut witches' broom (PNWB) was prepared for PCR reaction to amplify a 1458 bp-PCR fragment of the gyrB gene of the phytoplasma. A 768 bp-PCR product was further amplified by using the internal primer pair GBF3/GBR5 synthesized according to the sequence of the 1458 bp- PCR fragment. The 768-bp DNA fragment was DIG-labeled and used as a probe for screening of the PNWB-phytoplasma genomic library. A positive clone, pGDB 1-4, was selected and the sequence of the insert DNA was determined. The sequence of the insert DNA (5308 bp) contained three complete open reading frames (ORFs 1-3), one of which showed sequence homology to the gyrB genes of many bacteria, and a partial sequence homology to the gyrA genes of many bacteria. The specific primer pair GAF3/GAR3 was then designed based on the sequences of the partially sequenced gyrA gene and the conserved domains of the gyrA gene product. The primer pair was used to amplify a 462 bp gyrA gene fragment from the PNWB-phytoplasma DNA and used as a probe to screen for the gyrA gene-containing clone from the PNWB-phytoplasma genomic library. Clone pPGA 2-3 was found and the sequence of the insert was determined. The complete sequence (3840 bp) of the insert was linked to the 5308-bp sequence previously determined and an ORF(ORF 4) was found to be homologous to the gyrA genes of several bacteria. Based on the homology data, we concluded that ORF 3 and ORF 4 represent the gyrB and gyrA genes of the PNWB-phytoplasma, respectively. Southern hybridization analysis indicated that PNWB- phytoplasma has only one copy of gyrB and gyrA in the genome; and the results of RT-PCR analyses indicated that gyrB and gyrA may be cotranscribed as a polycistronic mRNA in PNWB- phytoplasma.

並列關鍵字

gyrB gene ； gyrA gene ； peanut witches' broom ； phytoplasma