Sequence Analysis of Genes Coding for the Molecular Chaperones GrpE, DnaK, and DnaJ from Phytoplasma Associated with Peanut Witches' Broom

花生簇葉病菌質體molecular chaperones GrpE，DnaK和DnaJ基因之選殖與分析

朱佩文(Pei-Wen Chu) ； 陳紹寬(Shau-Kwaun Chen) ； 陳武揚(Wuu-Yang Chen) ； 林長平(Chan-Pin Lin)

《植物病理學會刊》 16卷4期 (2007/12) Pp. 215-224

https://doi.org/10.6649/PPB.200712_16(4).0005

摘要

本實驗從事花生簇葉病菌質體dnaK和dnaJ基因之選殖研究。實驗中根據八種細菌之dnaK基因高保守性區域設計出PCR引子KF1，KF2，KR1，以花生簇葉病菌質體DNA為模板，先以KF1/KR1為引子對進行PCR反應，再繼續以KF2/KR1為引子對其PCR產物進行半巢式(semi-nested)PCR，如此可增幅出一大小為750 bp之PCR產物，並以其全長或將其經EcoRI酵解後之200 bp片段作為核酸探針，針對以內限制酶EcoRI構築的花生簇葉病菌質體基因庫進行篩選，分別得到含2.7 kb嵌入片段之選殖株重組質體PbSK1-1及合有2.3 kb嵌入片段之選殖株重組質體pBSK3-1。對上述之750 bp片段、Pbk1-1及pBSK3-1之嵌入片段進行核苷酸序列分析，可發現共有四個完整的ORFs (open reading frames)。將各ORFs核苷酸序列推衍為胺基酸序列，分別和aster yellows witches' broom phytoplasma (AYWB phytoplasma)等物種之序列進行比對，發現各ORFs、分別與hrcA，grpE，dnaK及dnaJ等基因之序列最為相似，且在序列分析中均可找到相同之基因結構與主要之功能區域(domains)。基於此等基因之排列與序列特性，本研究指出molecular chaperon Hsp70應存在於花生簇葉病菌質體中。

關鍵字

Mollicutes綱 ； dnaK操縱子 ； 半巢式聚合酵素連鎖反應

並列摘要

A PCR-based strategy was used for cloning dnaK and dnaJ genes of phytoplasma associated with peanut witches' broom (PnWB). Three nucleotide primers, KF1, KF2, KR1 were designed based on the conserved regions of eight bacterial dnaK genes. A 750-bp dnaK gene fragment was amplified using primer pair KF1/KR1, and then KF2/KR2 in semi-nested PCR. The 750 bp fragment and it's EcoRI-digested 200 bp fragment were used as probes for screening a genomic library of PnWB phytoplasma. Two recombinant plasmids, pBK1-1 (2.7 Kb) and pBSK3-1 (2.3 Kb), were obtained and sequenced. Based on the sequence analyses of the 750 bp dnaK fragment and the inserts of pBK1-1 and pBSK3-1, four open reading frames were identified to be arranged in the order of hrcA, grpE, dnaK and dnaJ. Chromosomal arrangement of these genes in PnWB phytoplasma is identical to those of aster yellows witches' broom phytoplasma, onion yellows phytoplasma and other bacteria phylogenetically related to phytoplasmas. This implies that the primitive bacterial molecular chaperone Hsp70 machine may exist in PnWB phytoplasma and its possible function when exposed to stress could be similar to those of other prokaryotes.

並列關鍵字

Mollicutes ； dnaK operon ； semi-nested PCR