利用real-time PCR進行花生簇葉病菌質體質體pPNWB套組數之測定與台灣梨衰弱病菌質體之檢測

Determination of the copy number of plasmid pPNWB of peanut witches’ broom phytoplasma and multiplex detection of pear decline phytoplasma using real-time PCR

李芷芸(Chih-Yun Lee)

指導教授 ： 林長平

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02650

摘要

在分類上屬於第二群植物菌質體之花生簇葉病菌質體 (peanut witches’ broom phytoplasma, PnWB phytoplasma)，在本研究室先前針對其基因體的研究中發現其具有一環型質體 (plasmid) pPNWB (Wei, 2004)，欲了解該質體於PnWB phytoplasma細胞內之套組數 (copy number)，遂利用菌質體內已知的單一套組數基因 (single copy) rpoB，作為參考序列以同步聚合酵素連鎖反應 (real-time PCR) 進行PnWB phytoplasma胞內質體pPNWB套組數之分析，結果發現pPNWB之套組數為一變動值。配合單一套組基因rpoB之定量分析，發現PnWB phytoplasma於嫁接一至六周後在日日春植株內持續增殖，此時質體pPNWB也會於植物菌質體族群在植株中增加之際在PnWB phytoplasma細胞內增加其套組數。利用同樣之定量方式進行PnWB phytoplasma 16S rRNA gene (16S rDNA) 套組數之測定，依本研究real-time PCR之結果，推斷在PnWB phytoplasma應具2個套組之16S rDNA，此一結果與目前已分析之第一群、第三群與第十群植物菌質體均具有2個16S rDNA套組之結果相符。其套組數之確定，將有助於藉由分析植物菌質體16S rDNA序列，定量植物菌質體於寄主植物內細胞之數量，本研究即將此結果應用至第二群台灣梨衰弱病菌質體 (PDTWII phytoplasma) 菌體數量之分析。台灣梨樹衰弱病 (pear decline-Taiwan, PDTW)，最早在1994年六月台灣中部東勢、和平兩地梨樹栽培區發現，本實驗室研究之初先證實該病害之病原為分類地位屬於第十群 (group 16SrX) 之PDTW phytoplasma，隨後並證實其可藉由黔梨木蝨 (Cacopsylla qianli ) 與中國梨木蝨 (C. chinensis) 傳播，本實驗室隨後於2005年在中國梨木蝨蟲體內發現第二群 (group 16SrII) 之PDTWII phytoplasma，並於2006年發現此兩群植物菌質體可複合感染 (coinfection) 梨樹，為檢測並追蹤此二群植物菌質體在罹病梨樹及媒介昆蟲內消長的情形，在本研究中針對二者之16S rDNA序列設計出多重同步聚合酵素連鎖反應 (multiple real-time PCR) 引子對，配合以real-time PCR為基礎之定量分析，發現以往以PCR檢測，確認有第十群PDTW phytoplasma感染之梨樹病株檢體DNA內，亦可偵測到第二群之PDTWII phytoplasma之存在，確認其為複合感染，顯示田間罹病植株受兩群植物菌質體複合感染之情形相當普遍。經靈敏度測試則發現multiplex real-time PCR優於multiplex PCR，而real-time PCR亦優於PCR，此一定量技術之建立將有利於將來追蹤兩群台灣梨衰弱病菌質體於梨樹及媒介昆蟲體內之細胞數量及二者間族群消長情形之觀察。

關鍵字

16S核醣體RNA基因 ； 多重同步聚合酵素連鎖反應 ； 台灣梨衰弱病菌質體 ； 質體pPNWB ； 同步聚合酵素連鎖反應

並列摘要

A plasmid, named pPNWB, was isolated from peanut witches’ broom phytoplasma (PnWB phytoplasma) in our lab previously. To estimate the copy number of pPNWB in PnWB phytoplasma cell, absolute quantification was implemented by real-time PCR with previously reported single copy gene, rpoB, in PnWB phytoplasma as a reference sequence. Variation of the copy number of pPNWB was found in different PnWB phytoplasma-infected periwinkle plants in this study. The copy number of pPNWB in PnWB phytoplasma cell increased as the number of PnWB phytoplasma cell increased in periwinkle plant after grafting was noticed. Same real-time PCR-based absolute quantification strategy was also applied to determine the copy number of 16S rRNA gene (16S rDNA) in PnWB phytoplasma. The PnWB phytoplasma, a 16SrII phytoplasma, was revealed to harbor two copies of 16S rRNA gene in it’s genome. The result is the same as those of other phytoplasma members of 16SrI, 16SrIII and 16SrX group. PDTWII phytoplasma, one of the causative agent of PDTW disease, is also the member of group 16SrII and may also carry two copies of 16S rDNA in it’s genome. The copy number of 16S rDNA, which is relatively easier to be cloned from phytoplasma than other genes, can be used as a reference sequence to quantify the number of phytoplasm cell in plant tissue. Pear decline in Taiwan (PDTW) was first reported in 1994 in orchards of central Taiwan. PDTW phytoplasma can be transmitted by two pear psyllas, Cacopsylla qianli and C. chinensis. PDTWII phytoplasma was first identified in pear psyllas in 2005. In 2006, the co-infection of PDTW phytoplasma and PDTWII phytoplasma was also evidenced in pear trees. To detect these two phytoplasmas in pear plants and insect vectors, the specific primer pairs for multiplex real-time PCR and real-time PCR were designed and applied effectively in this study. Many DNA samples prepared from PDTW phytoplasma-infected pears confirmed by PCR previously were thus reexamined using real-time PCR. PDTWII phytoplasma was detected in many of these samples. The results indicated that the co-infection of PDTW and PDTWII phytoplasmas in pear trees and inscet vectors may be very common in fields. The multiplex real-time PCR and real-time PCR-based detection also showed higher sensitivity than those of multiplex PCR and conventional PCR. The real-time PCR-based quantification of PDTW and PDTWII phytoplasmas established in this study will be applied to monitor the fluctuations of these two phytoplasmas’ populations in pear trees and insect vectors.

並列關鍵字

16S rDNA, multiplex real-time PCR, pear decline in Taiwn, phytoplasma, plasmid pPNWB, real-time PCR

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