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研究生:林翠芊
研究生(外文):Lin, Choy-Chieng
論文名稱:竹嵌紋病毒及其衛星核酸於寄主植物之系統性分佈
論文名稱(外文):The Systemic Spread of Bamboo Mosaic Virus and its Associated Satellite RNA in Plants
指導教授:林納生
指導教授(外文):Lin, Na-Sheng
學位類別:碩士
校院名稱:文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:63
中文關鍵詞:竹嵌紋病毒衛星核酸
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竹嵌紋病毒含有單一正股的 RNA 基因體,屬於 Potexvirus 病毒群,其威染之竹類植物,以麻竹和綠竹為主。竹嵌紋病毒衛星核酸是 Potexvirus 病毒群中唯一發現的衛星核酸,其中兩個衛星核酸分離株 BSF4 和 BSL6,其核甘酸序列的差異只有6.9%,但生物特性卻迥然不同。衛星核酸 BSF4 對協助病毒 BaMV-S 的複製和所引起的病徵。不會產生明顯的影響,且能在菸草植物 (Nicotiana benthamiana) 中有效的進行系統性移動;反之,衛星核酸 BSL6 會干擾協助病毒 BaMV-S 的複製和延遲病徵之表現,並且不能在菸草植物進行有效的系統性移動。
為了要探討竹嵌紋病毒和衛星核酸在寄主植物體內的系統性分佈,故以免疫染色法,了解病毒顆粒或鞘蛋白於植物體內累積之細胞型態和濃度差異。將感染BaMV-O 分離株(不含衛星核酸)之綠竹 (Bambusa oldhamii) 和感染 BaMV-V 分離株(含衛星核酸)之泰山竹 (B. vulgaris) 的未展開卷心葉及其幼嫩莖部,先以石蠟包埋切片後,再以抗 BaMV-O 病毒顆粒的血清行免疫金染色,繼之以銀加層擴大反應 (silver enhancement) 置於光學顯微鏡下觀察。無論是 BaMV-O 或 BaMV-V 感染的未展開卷心葉,其呈免疫正反應之處即銀粒子累積之部位,均位於不連續的鑲嵌片段區域內。病毒或鞘蛋白之累積,以葉肉細胞 (mesophyll cell, MC)、表皮細胞(epidermal cell, EC) 鞘細胞砲延伸纖維 (bundle sheath extension fiber, EF)、維管束兩旁空腔 (fusoid cavities, FC) 和鞘細胞 (bundle sheath, BS) 出現頻率最高,維管束導管 (metaxylem, MX)、木質部簿壁細胞 (xylem parenchyma, XP)、木質部纖維 (xylem fiber, XF)、篩管 (sieve element, SE) 和伴細胞 (companion cell, CC) 也可偵測到,但頻率不高。葉鞘和莖之切片,其正反應部位多位於維管束組織周圍的薄壁細抱 (PC)、鞘細胞延伸纖維 (EF) 和表皮標 (EC),但葉鞘維管束韌皮部 (phleom,Ph) 和早成木質部 (protoxylem, PX) 以及莖之維管束早成木質部 (PX) 亦發現有反應。對照組的切片以免疫前血清 (preimmune serum) 染色,無論是卷心葉或幼嫩莖的細胞皆無反應。本實驗以抗病毒顆粒血清偵測竹嵌紋病毒或其鞘蛋白於竹葉和莖的系統性分佈,其結果異於一般對病毒分佈的了解,因為木質部 (MX)、鞘細胞延伸纖維 (EF)、早成木質部 (PX)、維管束兩旁空腔 (FC) 和木質部纖維 (XF),在生理上皆為死細胞,但卻有大量的病毒或鞘蛋白的累積。
為了要了解兩個衛星核酸分離株於菸草植物體中系統性移動的差異,分別以BaMV-S RNA、BaMV-S RNA 加 BSL6 生體外轉錄體和 BaMV-S RNA 加 BSF4生體外轉錄體等三組接種菸草,再以接種後不同天數分別採收接種葉或系統葉,以墨點雜交 (dot blot hybridization) 方式偵測基因體 RNA 和衛星核酸。接種 16 天後,無論是單獨接種 BaMV-S RNA 或共同接種衛星核酸之菸草植物,其系統葉皆可偵測到基因體 RNA,單獨接種 BaMV-S RNA 於菸草之系統葉皆無偵測到衛星核酸的反應,而共同接種衛星核發的菸草,10株有9株系統葉可偵測到 BSF4,但另10株中僅有 1 株可偵測到 BSL6。將此含有 BSL6 和 BSF4 反應之系統葉分別傳接至健康菸草,於接種20天後,此共同接種衛星核酸的菸草,10株的系統葉中,皆可偵測到 BSF4,若10株中僅有4株可偵測到BSL6,顯示 BSL6 在轉接的第二代菸草中移動的連度較轉接第一代之菸草為快。當第二代感染的系統葉,再轉接至第三代菸草時,接種 14 天後,發現共同接種衛星核酸的菸草系統葉,7株皆可偵測到衛星核酸 BSF4,而另15株中有10株可偵測到衛星核酸 BSL6。顯示衛星核酸 BSL6在菸草植物體中經轉接三代後,其系統性移動的速率增加。進而分別抽取所有上述不同轉接次數的系統葉 RNA,進行 RT-PCR (reverse transcription-polymerase chainreaction) 反應,再以衛星核酸各個專一性限制酉每進行切割反應。結果顯示,無論 BSF4或 BSL6,其三代菸草系統葉之衛星核酸子代,仍皆源自於接種之衛星核酸,並朱受到另一個衛星核酸的污染。

Bamboo mosaic virus (BaMV) has a genome consisting of single-stranded, positive-sense RNA. The virus is a member of the potexvirus group. Most susceptible bamboo species belong to the genera Bambusa and Dendrocalamus. Satellite BaMV (satBaMV) is the only one known to be associated with potexvirus group. Two satBaMVs, BSF4 and BSL6, with a 6.9 % difference in nucleotide sequence, exhibit distinct phenotypes in infected plants. BSF4 has little effect on BaMV RNA replication nor of symptom expression caused by BaMV. Moreover, BSF4 spreads in tobacco systemically and efficiently. In contrast, BSL6 not only reduces replication and delays systemic symptom development but also is inefficient for long distance transport in tobacco (Nicotiana benthamiana).
Immunogold-silver staining of the infected tissues was used for localization of BaMV capsid protein in systemically infected bamboo plants. The roll leaves and young stems of common bamboo (Bainbusa vulgaris) infected with BaMV-V (with satellite) and green bamboo {B. oldhamii) infected with BaMV-O (free of satellite) were embedded in paraffin, processed for immuno-staining, reacted with anti-BaMV-O virion serum and labelled with immunogolds followed by silver enhancement for visualization. The localization of silver aggregates was found to be limited in some areas showing a mosaic-like pattern. The virion or coat protein could be detected frequently in the leaf mesophyll, epidermal cells, bundle sheath extension fibers, fusoid cavities and bundle sheaths occasionally observed in the metaxylem, xylem parenchyma cells, xylem fibers, sieve elements and companion cells. However, silver aggregates in stem sections were detected in parenchyma cells surrounding vascular, protoxylem and epidermal cells. Moreover, sections of leaf sheath showed silver aggregates in epidermal cell, bundle sheath extension fibers, parenchyma cells, bundle sheaths, parenchyma cells of xylem, fiber of xylem, sieve elements and companion cells. Preimmune serum was used as control and no labelling was observed. The metaxylem, bundle sheath extension fibers, fusoid cavities, protoxylem and fiber of xylem are dead cells physiologically but labels were frequently observed in these structures.
To analyze the difference in systemic movement of the two isolates of satellite RNA, tobacco plants were infected with BaMV-S RNA, BaMV-S RNA with BSL6, BaMV-S RNA with BSF4, respectively. Inoculated and systemic leaves were harvested at different time intervals for the detection of viral RNA and satBaMV using dot blot hybridization. At 16 days post-inoculation (d.p.i.), viral RNA was detectable in systemic leaves of tobacco. Nine out of ten plants coinfected with BSF4 transcripts showed the presence with satBaMV whereas only 1 out of 10 plants coinfected with BSL6 was positive for satBaMV in the upper non-inoculated leaves. At second passage, all of the plants coinfected with BSF4 showed the presence of satellite at 20 d.p.i. wheresa 4 out of 10 plants coinfected with BSL6 were positive for satBaMV. After the third passage, 7 out of 7 and 10 out of 15 plants were positively detected with BSF4 and BSL6, respectively, in their coinfected systemic leaves at 14 d.p.i. . These results indicated that the third passage showed the fastest movement systemically for BSL6 satBaMV. To verify whether the satRNA in the systemic leaves is BSF4 or BSL6 in every passage, restriction enzymes were used to map the cDNA derived from reverse transcription-polymerase chain reaction of total RNAs from each samples. The results indicated that no evident changes in satRNA sequence was found during the whole process.

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