Cloning and Analysis of rpsT, serS and hflB Genes of Peanut Witches''-Broom Phytoplasma
Date Issued
2009
Date
2009
Author(s)
Lu, Pei-Ying
Abstract
To investigate serS gene in peanut witches’-broom (PnWB) phytoplasma, subgenomic libraries of PnWB phytoplasma were constructed in this study. Clone lib_speI_4-19 was obtained from the PnWB phytoplasma SpeI-restriction subgenomic library by colony hybridization using PCR- amplified probe WS2-5 based on the partial sequence of serS gene of PnWB phytoplasma obtained in our previous study. To obtain the 5’ end sequence of serS, another probe WS8-6 was amplified based on the sequence of cloned fragment of lib_speI_4-19. Clone lib_HindIII_2-36 was then selected from the PnWB phytoplasma HindIII-restriction subgenomic library. Sequences of the cloned fragments of lib_speI_4-19 and lib_HindIII_2-36 were integrated and analyzed to reveal that the sequences may cotain four putative open reading frames (ORFs 1-4). The deduced amino acid sequences of ORF1, ORF2 and ORF3 showed homology with those of rpsT gene, serS gene and hflB gene, respectively. On the other hand, amino acid sequence of the ORF4 probably encodes a hypothetical protein with no significant homology to any known sequences. To clone complete rpsT gene, a degenerate PCR primer was designed according to the 5’ conserved region of phytoplasma rpsT gene, a clone c_wsf6mr7_rpsT with full length of rpsT gene was then obtained using PCR-based cloning strategy. The 3,902 bp genomic DNA sequence of PnWB phytoplasma was determined from the sequences of the cloned fragments of lib_speI_4-19, lib_HindIII_2-36 and c_wsf6mr7_rpsT using SeqMan sequence analysis program. The fragment contains full length of rpsT gene, serS gene, hflB gene and an incomplete ORF4 in order. The rpsT gene encodes a 30S subunit ribosomal protein S20. The serS gene encodes a seryl-tRNA synthetase with three highly conservative motifs. Both proteins were involved in translation and related to protein biosynthesis. The hflB gene encodes an ATP-dependent Zn metalloendoprotease of the AAA family (ATPases associated with diverse celluar activities) with two functional domains responsible for protein degradation. PCR fragments that spanned the rpsT-serS and serS-hflB intergenic region were amplified separately in reverse transcription-PCR (RT-PCR) using the total RNA prepared from PnWB-affected periwinkle as template, and the sequences of PCR products were identical to the corresponding sequences of the 3,902 bp PnWB phytoplasma genomic fragment. RT-PCR experiments indicate that the three genes are expressed as a single transcript, demonstrating that they constitute an operon.
Subjects
peanut witches’-broom phytoplasma
subgenomic library
rpsT gene,
serS gene
hflB gene
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