Cloning and Analysis of Plasmids of Periwinkle Leaf Yellowing Phytoplasma
Date Issued
2010
Date
2010
Author(s)
Chen, Ling-Ling
Abstract
A new phytoplasma disease named as periwinkle leaf yellowing (PLY) was first observed in a herbal flower nursery in Dayuan Township (Taoyuan county, Taiwan) in August 2005. Analysis of 16S rDNA, 16S-23S rDNA ISR, and partial 23S rDNA sequence revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows (AY) group (16SrI group). The periwinkle plants infected with PLY phytoplasma isolate 2005, PLY phytoplasma isolate 2008 and PLY phytoplasm isolate 2009 were designated as 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkle, respectively. For cloning the plasmids from abovementioned isolates of PLY phytoplasma, phytoplasmal DNAs extracted from 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkles were used as templates for PCR and inverse PCR amplification. Two plasmids, p05PLY-1 and p05PLY-2, were cloned from PLY phytoplasma isolate 2005. A plasmid p08PLY-1 was cloned from PLY phytoplasma isolate 2008. Two plasmids, p09PLY-1 and p09PLY-2, were cloned from PLY phytoplasma isolate 2009. Sequence analyses of these plasmids revealed that they had highest identity with the sequences of plasmid pOYW of onion yellows (OY) phytoplasma wild strain, and all of the five plasmids contain rep gene and ssb gene. Southern blot analysis using total DNA extracted from 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkle digested with seven restriction endonucleases for electrophoresis, and rep partial gene sequence as probe was performed to confirm the existence of these five plasmids. Hybridization bands of expected sizes were detected in total DNA extracted from 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkle. Bands of unexpected sizes were also detected and were presumed to be the sequences of other plasmids that may exist in the PLY phytoplasma cell. The phylogenetic tree based on the analysis of the amino acid sequences of Rep proteins of phytoplasma plasmids, geminiviruses and various bacterial plasmids was constructed. The results indicated that the Rep proteins of the five plasmids of PLY phytoplasma were closely related to those of bacterial plasmids. Absolute quantification using real-time PCR was performed to estimate the copy number of plasmids in PLY phytoplasma cell. The real-time PCR primer pair repRT-f3/repRT-r3 that can amplify the rep gene sequences of plasmids was applied to determine the copy number of plasmids containing rep gene. On the other hand, the real-time PCR primer pair p05RT-f3/p05RT-r3 that can amplify the sequences only existed in plasmid p05PLY-1 and p05PLY-2 was used to determine the copy number of these two plasmids specifically. The copy number of plasmids containing the p05RT-f3/p05RT-r3 amplified sequence was thus revealed to be less than that of plasmids containing rep gene. The result suggested that plasmids other than p05PLY-1 and p05PLY-2 did exist in the PLY phytoplasma isolate 2005.
Subjects
periwinkle leaf yellowing phytoplasma
phylogenetic analyses
real-time PCR
rep gene
ssb gene
Southern blot analyses
Type
thesis
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