关键词: 植原体, 质粒, 序列分析, 系统进化分析

Abstract: On the basis of the known phytoplasmal plasmid sequences, two pairs of specific primers were designed and used for amplication by PCR. Two amplified DNA fragments with different sizes covering the whole plasmid were obtained through two round of PCR amplification. After being sequenced and spliced,four phytoplasmal full-length circular plasmid DNA sequences, including the plasmids of mulberry dwarf(pMDPy) in Puyang,Henan Province, periwinkle(Catharanthus roseus)virescence (pPEVHn) in Hainan Province, paulownia witches’-broom (pPaWBG33D) in Yanzhou, Shandong Province and chinaberry witches’-broom phytoplasma strains (pCWBFq-2) in Fuqing, Fujian Province were achieved, with the sizes of 3 833, 3 943, 3 843 and 3 913 bp, respectively. They are predicted to encode five proteins,open reading frame ORF1 encoding replication associated protein(RepA),ORF5 encoding binding protein(SSB)and ORF2-4 encoding given unknown function proteins. pMDPy is the first plasmid determined from mulberry dwarf phytoplasma.Sequence analysis of non-coding regions (NCR) of the four kinds of plasmids revealed that the NCR sequences between ORF1 and ORF2 existed promoters and ribosome binding sites.The four phytoplasmal plasmid sequences were compared with the phytoplasmal plasmid sequences found in GenBank through multiple alignments and phylogenetic analysis; the results showed that these four plasmids and other 16Sr I group plasmid could be grouped into a large branch,which is consistent with the phylogenetic tree created from 16S rDNA comparisons. This research would not only provide further insight into the different phytoplasmal plasmid structure and function, host specificity and plasmid variation and evolution within different phytoplasma species or strains,but also offer new theoretical basis for a multiple genes and index-based system for phytoplasmal classification.

Key words: phytoplasma, complete circular plasmid, sequence analysis, phylogenetic analysis