Cloning and Analysis of dnaB1 and dnaG Genes of Peanut Witches’-Broom Phytoplasma
Date Issued
2009
Date
2009
Author(s)
Huang, Ting-Yu
Abstract
A PCR-based strategy was conducted to obtain dnaB1 and dnaG genes of phytoplasma associated with peanut witches’ broom (PnWB) in this study. A PCR fragment was amplified specifically from the PnWB phytoplasma-affected periwinkle plants using primer pair 281-5a/ 281-5b designed in previous study. The PCR product was cloned and the DNA fragment of clone 281-5 was sequenced and revealed to have homology to the sequences of published dnaB genes. In phytoplasma genome, dnaB gene can be arranged in the order of dnaB-dnaG to encode the DnaB and DnaG proteins for DNA replication, respectively. To clone the dnaB1 and the downstream dnaG gene of PnWB phytoplasma, two degenerate oligonucleotide PCR primers dnaBr2 and RG2 in reverse direction were designed according to the 5’ and 3’ end conserved sequences of the dnaG gene of various phytoplasmas, and primer dnaB-1-R-1 in forward direction was also designed according to the sequence of the cloned fragment of 281-5. PCR fragments were then amplified specifically from the PnWB phytoplasma DNA using primer pairs 281-5a/ dnaBr2 and dnaB-1-R-1/ RG2. The PCR products were cloned and clones B52 and BG were obtained, respectively. Sequence analysis of the cloned fragment of BG revealed that the 3’ end sequence has high homology to that of clone H13 (GenBank AY270153) published by us previously. A primer RHp1 in reverse direction was then designed according to the sequence of the cloned fragment of H13. PCR fragment was amplified from the PnWB phytoplasma DNA using primer pair dnaB-1-R-1/ RHp1 to obtain clone BH. Sequences of the cloned fragments of 281-5, B52, BG and BH were analyzed and combined as a 3,593 bp sequence fragment. Sequence analysis showed that the fragment contain the 3’ end sequence of a hypothetical protein gene, dnaB1 gene, dnaG gene, and the 5’ end sequence of another hypothetical protein gene in order. Southern hybridization analysis indicated that there were two copies of dnaB1 gene in PnWB phytoplasma genome. RT-PCR analysis showed that mRNAs of dnaB1 and dnaG were transcribed independently in PnWB phytoplasma.
Subjects
dnaB1 gene
dnaG gene
gene cloning
Southern hybridization
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