Cloning and Analysis of Phytoplasmal Immunodominant Membrane Protein Genes imp, idpA and amp

在植物菌質體優勢免疫膜蛋白相關之研究中顯示，其為植物菌質體細胞膜之主要蛋白。優勢免疫膜蛋白基因已陸續由多種植物菌質體中被選殖出來，這些基因所編碼之膜蛋白依據其嵌入於細胞膜上之構型，計可區分為不同的三個類型，分別為第一型優勢免疫膜蛋白 Imp (immunodominant membrane protein, Imp)、第二型優勢免疫膜蛋白 IdpA (immunodominant membrane protein A, IdpA) 以及第三型優勢免疫膜蛋白 Amp (antigenic membrane protein, Amp)。此三型優勢免疫膜蛋白之間在胺基酸序列上不具相似性。此外，在植物菌質體中之優勢免疫膜蛋白基因之序列相同度，亦被證實是低於其上下游基因或是 non-coding regions。本研究利用聚合酵素連鎖反應並配合基因選殖技術，由本實驗室現有之植物菌質體中，選殖獲得花生簇葉病菌質體之第一型 imp 優勢免疫膜蛋白基因之全長序列及台灣梨衰弱病第二群菌質體之第一型 imp 優勢免疫膜蛋白基因之部分序列 、聖誕紅叢枝誘導性植物菌質體之第二型 idpA 及第一型 imp 優勢免疫膜蛋白基因之全長序列以及日日春葉片黃化病及台灣泡桐簇葉病菌質體之第三型 amp 及第一型 imp 優勢免疫膜蛋白基因之全長序列。依序列比對分析之結果，顯示同一型各優勢免疫膜蛋白基因間之序列相同度甚低，尤其在 hydrophilic domain 間之序列相同度更低於其他 domain 間之序列相同度。儘管為同一 16Sr group 中之植物菌質體，其彼此間之優勢免疫膜蛋白基因序列亦具有差異性之區域，因此可據以區分不同 16Sr group 或 subgroup 之植物菌質體。針對台灣目前僅有之同屬 16SrI group 之日日春葉片黃化病菌植體及台灣泡桐簇葉病菌質體，就其第三型優勢免疫膜蛋白基因 amp 序列間具差異性之區域，所設計出之專一性 PCR 引子對 ampf5/Amp1R 及 Amp1F/ampr4，以 PCR 反應可成功區分此二親緣關係相近之植物菌質體。此外，由反轉錄聚合酵素連鎖反應 (RT-PCR) 之結果顯示，日日春葉片黃化病菌質體第一型 imp 及第三型 amp 優勢免疫膜蛋白基因以及花生簇葉病菌質體第一型 imp 優勢免疫膜蛋白基因均可轉錄出 mRNA。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