植物菌質體之優勢免疫膜蛋白基因imp，idpA及amp之選殖與應用

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

程諭揚(Y. Y. Cheng) ； 林長平(C. P. Lin)

《植物病理學會刊》 19卷1期 (2010/03) Pp. 9-18

https://doi.org/10.6649/PPB.201003_19(1).0002

摘要

植物菌質體之研究顯示優勢免疫膜蛋白爲植物菌質體細胞膜之主要蛋白。優勢免疫膜蛋白依據其嵌入於細胞膜上之構型，可區分爲三個類型，分別爲第一型優勢免疫膜蛋白Imp (immunodominant membrane protein)、第二型優勢免疫膜蛋白IdpA(immunodominant membrane protein A)以及第三型優勢免疫膜蛋白Amp(antigenic membrane protein)。本研究利用聚合酵素連鎖反應(PCR)並配合基因選殖技術，獲得花生簇葉病菌質體imp基因之全長序列、臺灣梨衰弱病第二群菌質體imp基因之部分序列、聖誕紅叢技誘導性植物菌質體imp基因及idpA基因之全長序列、日日春葉片黃化病菌質體imp基因及amp基因之全長序列、台灣泡桐簇葉病菌質體之imp基因及amp基因之全長序列。比對序列發現屬於同一16Sr group中之植物菌質體，彼此間同一型的優勢免疫膜蛋白基因序列具有差異性之區域，可據以區分不同16Sr group或subgroup之植物菌質體。針對台灣目前僅有同屬於16Srl群之日日春葉片黃化病菌質體及台灣泡桐簇葉病菌質體，根據第三型優勢免疫膜蛋白基因amp序列間具差異性之區域，設計出對兩菌質體專一性的PCR引子對ampf5/Amp1R及Amp1F/ampr4，PCR可成功區分此二親緣關係相近之植物菌質體。此外，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imp及amp基因以及花生簇葉病菌質體第一型imp基因均可於植物菌質體中轉錄出mRNA。

關鍵字

優勢免疫膜蛋白 ； 聚合酵素連鎖反應 ； imp基因 ； idpA基因 ； amp基因 ； 反轉錄聚合酵素連鎖反應

並列摘要

Previous studies have shown that immunodominant membrane protein constitute a major portion of cellular membrane proteins in most phytoplasmas. Genes encoding immunodominant membrane proteins have been identified and sequenced from several taxonomic groups of phytoplasmas. Based on their topologies in the membrane, the phytoplasma immunodominant membrane proteins can be 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 types of immunodominant membrane proteins. The sequence identity of immunodominant membrane protein genes between phytoplamas was lower than those of their upstream or downstream genes or non-coding regions. In this study, cloning and sequencing of these three types of immunodominant membrane protein genes were performed for three groups of phytoplasmas, including imp genes from the pear decline phytoplasma (PDTWII phytoplasma) and peanut witches'-broom (PnWB) phytoplasma of 16SrII group. idpA gene from poinsettia branch-inducing (PoiBI) phytoplasma of 16SrIII group, and amp genes from periwinkle leaf yellowing (PLY) phytoplasma and paulownia witches'-broom (PaWB-Taiwan) phytoplasma of 16Srl group. Nucleotide sequences of imp, idpA, or amp genes were different among phytoplasmas of the same group. The distinct immunodominant membrane protein gene sequences can be used for differentiating members within the same groups or subgroups of various phytoplasmas. Based on the distinct regions of amp gene PLY phytoplasma and PaWB-Taiwan phytoplasma. PCR primer pairs ampf5/Amp1R and Amp1F/ampr4 specific for identification and classification of the two phytoplasmas were successfully developed. RT-PCR showed that both the imp, and amp genes of PLY phytoplasma and the imp gene of PnWB phytoplasma were transcribed in PLY phytoplasma and PnWB phytoplasma, respectively.

並列關鍵字

immunodominant membrane protein ； polymerase chain reaction ； imp gene ； idpA gene ； amp gene ； RT-PCR