植物菌質體之優勢免疫膜蛋白基因 imp, idpA 與 amp 之選殖與分析

Abstract

Previous studies have shown that an immunodominant membrane protein is a major portion of the total cellular membrane proteins in most phytoplasmas. Genes encoding immunodominant membrane proteins have been identified and sequenced from several taxonomic groups of phytoplasmas and have been classified into three distinct types: type 1 immunodominant membrane protein Imp, type 2 immunodominant membrane protein IdpA, and type 3 immunodominant membrane protein Amp. No amino acid similarity was revealed among different type of immunodominant membrane proteins. The sequence identity of immunodominant membrane protein genes between phytoplamas was also evidenced to be lower than those of their upstream or downstream genes or non-coding regions. In this study, cloning and sequencing of these three types immunodominant membrane protein genes in Taiwan were performed for three groups of phytoplasmas including the pear decline phytoplasma (PDTWII phytoplasma) and peanut witches'-broom (PnWB) phytoplasma in 16SrII group which have imp genes, poinsettia branch-inducing (PoiBI) phytoplasma in 16SrIII group which has idpA gene, and periwinkle leaf yellowing (PLY) phytoplasma and paulownia witches'-broom (PaWB-Taiwan) phytoplasma in 16SrI group which have amp genes. Sequence analysis of these cloned immunodominant membrane protein gene fragments revealed that the sequence identities were very low, especially among the sequences of hydrophilic domains. Each phytoplasmal immunodominant membrane protein gene has their own distinct sequence even the phytoplasmas belong to the same group. These distinct immunodominant membrane protein gene sequences can be used for the differentiation of groups or subgroups of various phytoplasmas. Specific PCR primer pairs ampf5/Amp1R and Amp1F/ampr4 were designed based on distinct regions of PLY phytoplasma and PaWB-Taiwan phytoplasma amp gene sequences for their identification and classification. RT-PCR analysis showed that the imp, amp genes of PLY phytoplasma and the imp gene of PnWB phytoplasma were transcribed in PLY phytoplasma and PnWB phytoplasma.