Cloning and Analysis of Group II Intron in Peanut Witches' Broom Phytoplasma
| Autor: | Han-Ya Tsai, 蔡涵雅 |
|---|---|
| Rok vydání: | 2007 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 95 A random ("shotgun") sequencing strategy was conducted to investigate the genome libraries of peanut witches'' broom (PnWB) phytoplasma in this study. After screening the PnWB-phytoplasma libraries by using R1-1 probe amplified from the conserved region of partial matR gene in PnWB phytoplasma, a total of seven clones of matR gene containing group II intron sequences of peanut witches'' broom (PnWB) phytoplasma were obtained and analyzed. Except clone EI42 that lacked 5’ sequence of group II intron, the group II intron sequences of other six clones contained both 5’ and 3’ boundary sequences. Except clone EV33, the conserved sequences of IBS2 (intron binding site 2), IBS1, EBS2 (exon binding site 2) and EBS1 were identified in these six clones. This may be inferred that these six group II introns may transpose in the genome of PnWB phytoplasma mainly through retrohoming mechanism. After sequence alignment, it was revealed the matR genes in these seven group II introns may comprise the conserved RVT (reverse transcriptase), X (maturase) and En (endonuclease) domains. Premature translation stop codon, translational frameshift, and sequence deletion of matR genes were identified in various clones. Based on the sequence analyses of these seven clones, they were implied to be the various versions of group II intron located in different parts of PnWB genome. Besides, the amino acid sequences of matR gene in EV9 and HIII57 were distinguishable to those of the others, and these group II introns were therefore classified to two classes in PnWB phytoplasm based on the sequence heterogeneity. In Northern hybridization analyses by using R1-1 probe, several RNA transcripts of matR gene were revealed. The hybridization signal of 2.4-2.5 kb in size was the expected length of group II intron and was inferred to be the group II intron RNA excised from the pre-RNA. The longer 5.0 kb Northern hybridization signal fragment was inferred to the unspliced pre-RNA and the two shorter fragments about 1.5 kb in size were inferred to be the deficient group II intron RNAs. A 2400 bp-PCR fragment containing almost the complete sequence of group II intron was amplified in reverse transcription-PCR (RT-PCR) with the total RNA prepared from PnWB-affected periwinkle as template and the sequence was revealed to be identical to that of EV33 clone. This implied that the group II intron in clone EV33 may carry out gene expression. Though there is one advance stop codon found in the D domain in matR gene of EV33, it was revealed that the IEP may still be a functional protein lacking En domain to assist group II intron RNA movement. The results indicate that group II introns have high rates of intron gain and loss and thus create the diversity of group II introns in PnWB-phytoplasma genome. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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