长春花小叶病植原体质粒DNA克隆及其分子特征

Complete Sequence of a Full-length DNA and Molecular Characterization of one Plasmid from Periwinkle Little Leaf Phytoplasma

为了测定长春花小叶病植原体质粒及分析其分子特征,扩增长春花小叶病植原体质粒片段,然后进行反向扩增,拼接得到质粒全序列,且预测质粒编码蛋白的跨膜区、亚细胞定位等。结果表明,获得的质粒全长为4 102 bp,A+T含量为75.18%,编码5个蛋白,且将该质粒命名为pPLLHn-1。其中ORF的2、3、4编码蛋白分别含有2、1、2个跨膜区,ORF3的信号肽(Singnal peptide,SP)的信号值为0.948,ORF3编码的氨基酸序列与洋葱黄化植原体质粒EcOYW1参与质粒拷贝数控制的蛋白(Copy number control protein)具有97%的同源性。此结果说明长春花小叶病植原体质粒pPLLHn-1编码的5个蛋白中,除与质粒复制有关的RepA和SSB外,另外3个均为跨膜蛋白,根据分泌蛋白的特征分析初步推断ORF3蛋白为分泌蛋白。

DNA fragments of the plasmid（pPLLHn-1）in Periwinkle little leaf phytoplasma Hainan strain（PLL-Hn）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 among the pPLLHn-1 plasmid sequences were completed by using bioinformatic softwares.The plasmid comprised 4 102 bp and had a nucleotide content of 75.18% A＋T and encoded five ORFs.ORF2,ORF3 and ORF4 of pPLLHn-1 contained 2,1 and 2 tranmembrane domains respectively,and signal peptide values of ORF3 was 0.948.Homologous comparison showed that Cop homology between ORF3 and the Cop of EcOYW1 was 97%.Two ORFs of the pPLLHn-1 plasmid encoded a replication associated protein（RepA）and a single-stranded DNA binding protein（SSB）,respectively.Other three putative proteins encoded by the plasmid were predicted to contain one or more transmembrane domains respectively.According to the characteristics of secreted proteins,ORF3 protein was concluded a secreted protein.