以人工重組感染性選殖株探討木瓜輪點病毒基因變異 對致病性之影響

Abstract

木瓜為台灣重要果樹，感染木瓜之病害中以木瓜輪點病毒(Papaya ring spot virus, PRSV)引起的木瓜輪點病(Papaya ring spot)最具破壞性。此病毒屬於馬鈴薯Y病毒科(Potyviridae)，馬鈴薯Y病毒屬(Potyvirus)，使寄主葉片產生嵌紋、變形甚至絲狀化、果實表面出現水浸狀輪斑等病徵。PRSV主要分為P型及W型，P型可感染番木瓜科及葫蘆科，W型只能感染葫蘆科。PRSV-P型目前依照葉片上呈現之病徵又分為嚴重嵌紋(severe mottling, SM)、嚴重嵌紋壞疽(severe mottling with necrosis, SMN)與畸形(deformation, DF)三個主要系統。三系統間具有病理性及分子性之差異，顯示PRSV田間演化情況複雜，目前田間病害以SMN與DF兩系統為主導優勢，兩系統基因序列相似度極高病徵卻截然不同，值得探入探討造成病徵不同之決定因子(symptom determinants)。SMN系統與DF系統全長序列相似度為96.5%，各基因核苷酸序列相似度約為96-99%，靠近5’端的序列相似度較低，如P3基因相似度95.8-96.7%，P1基因低於95%，5’UTR則在93.0-94.2%，本論文將以實驗室SMN與DF兩系統具感染力之全長cDNA選殖株，選擇兩系統間差異性較大之P1基因序列進行重組置換。以精確phusion PCR增幅DF系統之5’端至P1的基因片段，後接SMN的片段，構築成5’-DF(P1)-SMN的重組cDNA選殖株 ; 也以同樣的方法構築5’端至P1為SMN片段，後接DF片段另一重組5’-SMN(P1)-DF選殖株。以胞外轉錄(in vitro transcription)將兩感染性選殖株合成完整的病毒基因體RNA transcripts，機械接種於最感病的木瓜寄主台農二號，發現5’-DF(P1)-SMN重組選殖株的病徵表現以DF為主，5’-SMN(P1)-DF重組選殖株亦然，顯示P1基因對病徵有影響，但不是唯一的病徵決定因子。

Papaya ring spot caused by Papaya ring spot virus (PRSV) , one of the most destructive diseases in papaya. PRSV belonging to the genus Potyvirus, family Potyviridae. PRSV-infected papaya trees show mosaic, distorted and shoestring-like symptoms on the leaves, and sunken ring spots on the fruits. PRSV categorized into 2 types, PRSV-P type and PRSV-W type. The P type infects both papayas and cucurbits whereas the W type only infects cucurbits. Based on the incited symptoms, the PRSV-P type further devided into SM (severe mottling), SMN (severe mottling with necrosis) and DF (deformation) strains. Between these 3 strains have apparently differences on pathological and molecular. This study investigate the genomic variations and find the key genomic areas associated with pathogenicity among different PRSV strains through the research with artificially recombinant infectious clones infecting papaya hosts. Based on the previously constructed infectious clones of PRSV SMN and DF strains, two new recombinant infectious named 5’-DF(P1)-SMN and 5’-SMN(P1)-DF clones were further made. The 5’-DF(P1)-SMN clone has a head of DF (5’UTR and P1 fragment of DF) followed by the SMN fragment, and the 5’-SMN(P1)-DF clone has a head of SMN (5’UTR and P1 fragment of SMN) followed by the DF fragment. The recombinant genomic RNA trascripts were synthesized through in vitro transcription, and they were used to individually inoculate the TN2 papayas. The results showed that symptom express of both two recombinant clones similar to PRSV-DF. It revealed that P1 gene contribute part of pathogenicity, but not the only one gene affect symptom express.