日日春葉片黃化病植物菌質體之質體選殖與分析

Abstract

日日春葉片黃化病植物菌質體 (periwinkle leaf yellowing phytoplasma, PLY phytoplasma) 為本研究室於 2005 年於台灣桃園縣大園鄉發現並命名之新種植物菌質體，其 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR) 及部分 23S rDNA 序列經比對分析後，顯示其親緣關係與第一群 (16SrI group) 植物菌質體最為接近。本研究將感染 2005, 2008 及 2009 年三個不同年份之日日春葉片黃化病植物菌質體分離株之日日春病株分別命名為 05PLY日日春、 08PLY 日日春及 09PLY 日日春，並針對三個不同年份之日日春病株全 DNA，利用 PCR 及 inverse PCR 進行日日春葉片黃化病植物菌質體之質體序列選殖與增幅。結果顯示於 05PLY 日日春選殖到兩個日日春葉片黃化病植物菌質體之質體 p05PLY-1 及 p05PLY-2；於 08PLY 日日春選殖到日日春葉片黃化病植物菌質體之質體 p08PLY-1；於 09PLY 日日春選殖到兩個日日春葉片黃化病植物菌質體之質體 p09PLY-1 及 p09PLY-2 。經過序列分析，發現五個日日春葉片黃化病植物菌質體之質體皆與洋蔥黃萎病植物菌質體野生種之質體 pOYW 有最高相同度，且五個質體序列上皆帶有與複製相關的 rep 基因 (replication protein gene) 及 ssb 基因 (single-strand binding protein gene)。本研究以 rep 基因片段序列作為核酸探針，搭配七種限制酵素針對日日春病株全 DNA進行南方氏雜配反應，確認日日春葉片黃化病植物菌質體質體之大小及存在情形。圖譜結果顯示，在 05PLY 日日春、08PLY 日日春及 09PLY 日日春中，皆有與預期相符之片段訊號產生，初步推測本研究所發現之五個質體確實存在於日日春葉片黃化病植物菌質體細胞內。此外，圖譜亦顯示與預期不符之片段訊號產生，推測在受日日春葉片黃化病植物菌質體感染之日日春病株中，尚有其他未被選殖到的質體存在。本研究將植物菌質體之質體、geminiviruses 及細菌質體之 Rep 蛋白胺基酸序列進行譜系分析，確認植物菌質體質體之 Rep 蛋白之親緣關係，結果顯示本研究所發現之五個日日春葉片黃化病植物菌質體質體之 Rep蛋白為類似於細菌質體之 Rep 蛋白。本研究以兩組 real-time PCR 引子對，利用同步聚合酵素連鎖反應 (real-time PCR)，針對罹病植株全 DNA 進行其體內日日春葉片黃化病植物菌質體細胞內質體之套組數測定。當以引子對repRT-f3/ repRT-r3 進行 real-time PCR 時，可測定帶有 rep 基因之質體套組數；相對地，當以引子對 p05RT-f3/p05RT-r3進行 real-time PCR 時，可增幅帶有非 rep 片段之質體，因該增幅片段僅在 p05PLY-1 及 p05PLY-2 質體出現，故可測定此二質體之套組數。在本研究中針對 05PLY 植株全 DNA以引子對 p05RT-f3/p05RT-r3 測得之質體套組數小於帶有 rep 基因之質體套組數，因此推測在 05PLY 日日春中，日日春葉片黃化病植物菌質體細胞內除已選殖之 p05PLY-1 及 p05PLY-2 二質體外，應仍有其他帶有 rep 基因之質體存在。

A new phytoplasma disease named as periwinkle leaf yellowing (PLY) was first observed in a herbal flower nursery in Dayuan Township (Taoyuan county, Taiwan) in August 2005. Analysis of 16S rDNA, 16S-23S rDNA ISR, and partial 23S rDNA sequence revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows (AY) group (16SrI group). The periwinkle plants infected with PLY phytoplasma isolate 2005, PLY phytoplasma isolate 2008 and PLY phytoplasm isolate 2009 were designated as 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkle, respectively. For cloning the plasmids from abovementioned isolates of PLY phytoplasma, phytoplasmal DNAs extracted from 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkles were used as templates for PCR and inverse PCR amplification. Two plasmids, p05PLY-1 and p05PLY-2, were cloned from PLY phytoplasma isolate 2005. A plasmid p08PLY-1 was cloned from PLY phytoplasma isolate 2008. Two plasmids, p09PLY-1 and p09PLY-2, were cloned from PLY phytoplasma isolate 2009. Sequence analyses of these plasmids revealed that they had highest identity with the sequences of plasmid pOYW of onion yellows (OY) phytoplasma wild strain, and all of the five plasmids contain rep gene and ssb gene. Southern blot analysis using total DNA extracted from 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkle digested with seven restriction endonucleases for electrophoresis, and rep partial gene sequence as probe was performed to confirm the existence of these five plasmids. Hybridization bands of expected sizes were detected in total DNA extracted from 05PLY periwinkle, 08PLY periwinkle and 09PLY periwinkle. Bands of unexpected sizes were also detected and were presumed to be the sequences of other plasmids that may exist in the PLY phytoplasma cell. The phylogenetic tree based on the analysis of the amino acid sequences of Rep proteins of phytoplasma plasmids, geminiviruses and various bacterial plasmids was constructed. The results indicated that the Rep proteins of the five plasmids of PLY phytoplasma were closely related to those of bacterial plasmids. Absolute quantification using real-time PCR was performed to estimate the copy number of plasmids in PLY phytoplasma cell. The real-time PCR primer pair repRT-f3/repRT-r3 that can amplify the rep gene sequences of plasmids was applied to determine the copy number of plasmids containing rep gene. On the other hand, the real-time PCR primer pair p05RT-f3/p05RT-r3 that can amplify the sequences only existed in plasmid p05PLY-1 and p05PLY-2 was used to determine the copy number of these two plasmids specifically. The copy number of plasmids containing the p05RT-f3/p05RT-r3 amplified sequence was thus revealed to be less than that of plasmids containing rep gene. The result suggested that plasmids other than p05PLY-1 and p05PLY-2 did exist in the PLY phytoplasma isolate 2005.