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Serological Properties of Nucleocapsid Proteins of Five Tospoviruses and Production of N Gene Transgenic Plants with Serogroup-specific Resistance
|摘要:||番茄斑萎病毒屬(Tospovirus)之四主要血清型代表性病毒分別為蕃茄斑萎病毒（tomato spotted wilt virus, TSWV）、花生輪點病毒 (groundnut ringspot virus, GRSV)、鳳仙花壞疽斑點病毒 (impatiens necrotic spot virus, INSV) 及西瓜銀斑病毒（watermelon silver mottle virus,WSMV）。晚近一種於台灣中部地區花生上分離的番茄斑萎病毒屬病毒被稱為花生黃化扇斑病毒 (peanut chlorotic fan-spot virus, PCFV) ，先前的實驗結果顯示PCFV與TSWV、INSV及WSMV無血清關係，唯缺與第二血清型之血清反應。本實驗將第二血清型中的GRSV接種至奎藜 (Chenopodium quinoa) 後，以簡易之方法將其29 kDa 之核鞘蛋白純化，並將此蛋白免疫紐西蘭白兔製備其抗血清，所得之抗血清經免疫擴散反應（immunodiffusion），間接酵素連結反應法 (indirect ELISA)及西方轉漬法（Western blotting），結果顯示PCFV與第一、二、三、四血清型均無血清關係，各番茄斑萎病毒NPs之抗血清只與其本身對映之抗原有血清反應，但TSWV與GRSV間有相當強烈之血清反應。
本實驗室已有具專一抗性之WSMV 核鞘蛋白基因 (N gene) 之轉殖煙草，本研究進一步構築GRSV、TSWV與INSV之N 基因轉殖煙草，以期能得到高專一抗性可作為生物檢定四種主要血清型病毒之轉殖煙草。本實驗利用農桿菌（Agrobacterium tumefaciens）轉移法將TSWV、GRSV、INSV個別之核鞘蛋白基因轉殖至煙草中，經康黴素（kanamycin）篩選後所得到之轉基因株系，接著進行接種實驗，結果得到七個只抗TSWV，但不抗GRSV、INSV及WSMV的轉殖菸草株系；五個只對GRSV有抗性，但不抗TSWV、INSV及WSMV的株系；八個只抗INSV，但不抗TSWV、GRSV及WSMV的株系。實驗結果顯示這些抗病株系均對本身血清型之病毒具備專一抗性，將來可以利用作為區分此四種主要番茄斑萎病毒血清型之鑑別性指示植物。以聚合脢連鎖反應（polymerase chain reaction, PCR）偵測分析則證實TSWV、GRSV及INSV的N基因存在轉殖植物中。以西方轉漬法偵測轉基因煙草中核鞘蛋白的表現，結果顯示具高度抗性的株系中只有I-30株系微弱表現核鞘蛋白，剩餘之株系皆不表現核鞘蛋白，推論此種高度抗性為RNA誘導之基因消沈現象（RNA-mediated gene silencing）。
Tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV), impatiens necrotic spot virus (INSV), and watermelon silver mottle virus (WSMV) are typical representatives of serogroup I, II, III, and IV, respectively, in the genus Tospovirus. Peanut chlorotic fan-spot virus (PCFV) was found in central Taiwan recently. Previous studies indicated that PCFV is serologically unrelated to TSWV, INSV, and WSMV. However, the serological relationships between PCFV and the serogroup II viruses have not been established yet. In this study, the NP of GRSV was purified from GRSV- infected Chenopodium quinoa by a simple procedure. The specific antiserum to the purified NP was produced from rabbit and used to clarify its serological relationship with PCFV. In immunodiffusion tests, indirect ELISA, and western blotting, the antisera to each NP of TSWV, GRSV, INSV, WSMV, and PCFV only reacted with their homologous antigens and no cross reactions with the heterologous antigens were observed except that there were reactions between TSWV and GRSV. The results indicated that the NP of PCFV is serologically unrelated to those of TSWV, GRSV, INSV, and WSMV. In this study, we attempted to establish a simple and fast method by using transgenic tobacco with specific resistance to differentiate TSWV, GRSV, INSV, WSMV of the four major serogroups. The WSMV N gene has been successfully transferred into tobacco in previous study. In this investigation, N gene of each TSWV, GRSV and INSV was transferred into tobacco to generate specific resistance to the corresponding viruses. After selection on the kanamycin medium, the verified transgenic tobacco lines were subjected to mechanical inoculations with TSWV, GRSV, INSV, and WSMV, separately, to evaluate their resistance to infection of each virus under greenhouse conditions. Seven TSWV N gene transgenic lines were found highly resistant to TSWV, but not to GRSV, INSV, and WSMV. Similarly, five GRSV N gene transgenic lines and eight INSV N gene transgenic lines were found highly resistant only to the homologous virus but not resistant to the heterologous viruses. The previous generated WSMV N gene transgenic tobacco line was also proved only resistant to WSMV, but susceptible to TSWV, GRSV, and INSV. It appears that these highly resistant lines can be used to differentiate tospoviruses of TSWV, GRSV, INSV, and WSMV in the four major serogroups just by mechanical inoculations. The presence of each N gene in transgenic tobacco lines was confirmed by PCR amplification. The expression of the transgenes was detected by Western blotting. The NPs of the plants of the highly resistant lines were not detectable except the line I-30, suggesting that the resistance in most of the highly resistant lines were due to RNA-mediated gene silencing. WSMV is the major limiting factor for growing watermelon and other cucurbits in Taiwan. In this study, effects to generate the transgenic watermelon expressing the WSMV N gene were also attempted. Seven months after selection on kanamycin medium, the putative transgenic watermelon shoots were obtained. Whether the WSMV N gene was intergrated, and expressioned in the putative transgenic watermelon and whether they are resistant to WSMV infection remain to be further investigated.
|Appears in Collections:||植物病理學系|
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