Cloning and Sequence Analyses of recA Gene of Phytoplasma Associated with Peanut Witches' Broom

花生簇葉病菌質體recA基因之選殖與分析

朱俞蓉(Yu-Rong Chu) ； 陳武揚(Wuu-Yang Chen) ； 林長平(Chan-Pin Lin)

《植物病理學會刊》 15卷4期 (2006/12) Pp. 211-218

https://doi.org/10.6649/PPB.200612_15(4).0001

摘要

本實驗將Escherichia coli, Bacillus subtilis, Acholeplasma laidlawii, Mycoplasma genitalium, Mycoplasma pulmonis及Mycoplasma mycoides subsp. Mycoides等細菌之recA基因進行序列比對，根據其保守性較高之區域設計出PCR引子對recA-1f/recA-1r，以花生簇葉病病原菌質體DNA為模板進行booster PCR反應，得到一323 bp之PCR產物，此PCR產物之序列中含有一EcoRⅠ切位，將此PCR片段或將其EcoRⅠ酵解後所獲之76bp片段以DIG進行標識以作為核酸探針，並用於篩選花生簇葉病病原菌質體之λ ZAPⅡ EcoRⅠ基因庫，得到選殖株重組質體Prec1（含2.3比之嵌入片段）及pRBC9（含2.5kb之嵌入片段）。對此323bp之PCR產物、pREC1及pREC9之嵌入片段進行核苦酸序列分析，將其重疊之序列聯結後可得到一個完整的ORF(open reading frame)，且在轉譯起始密碼AUG上游有互補於植物菌質體165rRNA之3'端核酸序列之Shine-Dalgarno序列之存在，其可能為核糖體結合位置(ribosomal binding site)。此ORF之核苷酸序列經推衍為肢基酸序列後，發現其基因大小及基因結構均與其他的細菌之recA基因相似，故由此推斷其為植物菌質體之recA可能基因。其基因在密碼利用性上並無以UGA作為tryptophan轉譯密碼之現象，且該基因序列有與其他Mollicutes綱細菌在基因體特性上相同之AT-rich現象。而在南方氏雜配反應的結果則可發現在花生簇葉病植物菌質體之基因體中應僅具有單一套組(single copy)之recA基因。

關鍵字

花生簇葉病菌質體 ； recA基因

並列摘要

The recA gene, which plays an important role in DNA repair of eubacteria, was cloned and analyzed from phytoplasma associated with peanut witches' broom (PnWB) in this study. In order to clone the recA gene, a pair of degenerate PCR primers, recA-1f and recA-1r, was designed according to the sequences of recA gene of Escherichia coli, Bacillus subtilis, Acholeplasma laidlawii, Mycoplasma genitalium, Mycoplasma pulmonis, and Mycoplasma mycoides subsp. mycoides. Total DNA prepared from the diseased periwinkle plant infected with PnWB phytoplasma was then used as the DNA template for booster PCR to amplify the partial sequences of recA gene of PnWB phytoplasma with primer pair recA-1f/ recA-1r. An amplified 323 bp-PCR product harboring an EcoRⅠ site was cloned in vector pCR2.1 and the recombinant plasmid pTA1 was obtained. The cloned insert of pTA1 and the 76bp fragment of the EcoRⅠ restricted 323 bp-PCR product was then used as a nucleic acid probe for screening the λ ZAPⅡ EcoRⅠ genomic library of PnWB phytoplasma. Two in vivo excised recombinants, pREC1 (with a 2.3 kb insert) and pREC9 (with a 2.5 kb insert) were obtained and sequenced. The sequences of the 323 bp-PCR product overlap with the sequences of the cloned inserts of pREC 1 and pREC9 that flanking to each other at the EcoRⅠ cutting sequences of the 323 bp-PCR product. The cloned insert of pREC1 contained the upstream sequences and the 5' region of recA gene, and the cloned insert of pREC9 contained the 3' region and the downstream sequences of the gene. A complete open reading frame (ORF) was thus constructed and identified based on the sequences of the 323 bp-PCR product, and the DNA inserts of pREC1 and pREC9. The ShineDalgano sequence complementary to the 3'-end sequence of 16S rRNA of phytoplasmas was found 12-16 nucleotides upstream of AUG start codon of the ORF. The gene organization, the nucleotide sequence, and the deduced amino acid sequence in the conserved regions of the ORF are similar to those of the other recA genes of eubacteria. Therefore, this gene was identified as a putative recA gene.

並列關鍵字

Peanut witches' broom phytoplasma ； recA gene