Objective: To clone plasmid from chinaberry witches'-broom phytoplasma and analyse its molecular characterization.
Methods: Fragments of one plasmid (pCWBFq) in chinaberry witches'-broom phytoplasma-Fuqing strain (CWBFq) were amplified with primer pairs which were designed according to plasmid sequences published on NCBI. Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares. Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe.
Results: One complete plasmid was sequenced from CWBFq. pCWBFq comprised 4446 bp and had a nucleotide content of 73.5% A + T and encoded six proteins. Protein P2, P3, P4 and P5 of pCWBFq contained 3, 2, 1 and 2 tranmembrane domains respectively, and their predicted signal peptide values were 0.989, 0.505, 0.918 and 0.914 respectively. Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9.6% -85.6% , however, the homology of different SSB proteins was between 74.0% - 89.4%. Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq. In addition, The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain (PaWBNy), periwinkle virescence phytoplasma-Hainan stanin (PeVHn), chinaberry witches'-broom phytoplasma-Fuzhou strain (CWBFz) and mulberry dwarf phytoplasma - Puyang strain (MDPy), whereas, no hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain (JWBBj), cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain (BiWBNc).
Conclusion: The plasmid encoded a replication associated protein (RepA) and a single-stranded DNA binding protein (SSB), which were for the replication of plasmid. Four putative proteins encoded by the plasmid were predicted to contain one or more hydrophobic transmembrane domains, respectively, and presumably to be localized to the membrane. The alignment and homology analysis as well as phylogenetic analysis to the DNA and encoded protein amino acid sequences of the whole plasmids and single ORFs on the known phytoplasmal plasmids showed that the different homologous sequences have distinct variation, among which the repA gene with the largest diversity appeared in all the known plasmids while ssb with less variation were only found in 16SrI plasmids. CWBFq, PaWBNy, PeVHn, CWBFz and MDPy possessed distinct plasmids in terms of number and size, whereas there was no plasmid detected in JWBBj, CLYXc and BiWBNc, perhaps as a result of low homology among repA genes in plasmids of JWBBj, CLYXc and BiWBNc.