Cloning of rpoC Gene Encoding RNA Polymerase β’ Subunit of Phytoplasma Associated with Peanut Witches’ Broom

• Main Authors: Chi-Yang Lin, 林啟揚
• Other Authors: Chan-Pin Lin
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/34956870565112706552

碩士 === 國立臺灣大學 === 植物病理學研究所 === 91 === The active holoenzyme of bacterial RNA polymerase consists of a core (α2ββ’) and a sigma (σ) factor.β’ is the largest subunit in the enzyme complex and responsible for binding the DNA template to catalyze RNA synthesis. To clone the gene encoding theβ’ subunit of RNA polymerase in phytoplasma associated with peanut witches’ broom (PNWB-phytoplasma), a pair of degenerate PCR primers, FrpoC1 and RrpoC1, were designed based on the conserved region of the rpoC genes characterized in various prokaryotes bacteria. A 1.8 kb PCR fragment was amplified specifically from the diseased periwinkle infected with phytoplasma associated with peanut witches’ broom. Sequence analysis revealed that this PCR product shared high similarity to part of the other reported rpoC gene. To obtain the full length rpoC gene, a PCR chromosomal walking strategy was conducted. Primers for the genes positionally conserved in the upstream and downstream of the rpoC gene were designed. A total of 4.8 kb region which contained the full length of rpoC gene, and partial other upstream rpoB and downstream rpsL genes was obtained. An Open reading frame from nt 201 to nt 4,340 was identified and named ORF1. Amino acid alignment of ORF1 with Saccharomyces cerevisiae RPO21, Drosophila melanogaster RNA polymerase II 215KD subunit RPII215, E. coli K12 rpoC, revealed C, D, and G regions shared greater similarities among the eight reported conserved regions. Southern hybridization analysis suggested that rpoC is one copy or low copy in the PNWB-phytoplasma genome. According to the results of northern hybridization and RT-PCR analyse, rpoC and rpsL maybe organize as a single transcription unit; however, rpoB is possibly not cotrnscribed with rpoC.