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研究生:邱慧琪
研究生(外文):Chiou, Hui-Chi
論文名稱:由構樹嵌紋病株分離之一種carlavirus特性之研究
論文名稱(外文):Characterization of a carlavirus isolated from common mulberry (Broussonetia papyrifera (L.) L´H’erit. ex Vent.) with mosaic symptom in Taiwan
指導教授:陳滄海陳滄海引用關係
指導教授(外文):Chen, Tsang-Hai
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:植物保護系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2008
畢業學年度:95
語文別:中文
論文頁數:77
中文關鍵詞:桑科構樹構樹嵌紋病毒
外文關鍵詞:MoraceaeBroussonetia papyriferaCommon mulberry mosaic viruscarlavirus
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構樹 (Broussonetia papyrifera (L.) L´H’erit. ex Vent.) 為桑科(Moraceae)落葉性喬木或灌木,葉可供作豬、牛、羊、鹿的飼料,樹皮可造紙,由於其耐煙塵,可作為工廠和礦山區的綠化用樹,亦常為平地或低海拔區域的先驅植物,目前台灣地區並無經濟栽培,皆為道路旁綠化或荒地之野生化植物,分佈零散。2005年於屏東地區田間發現構樹葉片呈現嚴重黃綠嵌紋病徵,病害發生率為42 %。經週年觀察,發現田間病徵會隨季節變化,而有由嚴重至輕微甚至消失之現象。田間病葉經由奎藜(Chenopodium quinoa)作單斑分離,可分離出一種絲狀病毒,病毒粒子大小約為600-650 nm。病毒熱不活化溫度為70℃,耐稀釋度為10-4,室溫(24℃)下活性可維持4天,冷凍(-80℃)則活性可保存10個月。電顯觀察奎藜單斑及黃化嵌紋構樹病組織之切片,可於細胞質中見到病毒粒子成束狀之聚集及細胞胞器之病變。機械接種16科64種供試植物,僅藜科的奎藜、紅藜(C. amaranticolor)、綠藜(C. murale)及豆科之豇豆(Vigna unguiculata)等4種植物會被感染,葉部呈現黃色局部斑點病徵。種子媒介傳播發病率為15.2 %。每100g新鮮奎藜病葉,經高低速交替離心及硫酸銫密度梯度離心純化後,可獲得純化病毒收量約為3.6 mg。經電泳分析估算病毒鞘蛋白分子量約為36 kDa。白兔經免疫注射,可製備岀力價1024之抗血清;瓊脂擴散反應可與單斑奎藜病葉、純化病毒及田間構樹嵌紋病葉呈現同源正反應,不會與Carlavirus屬之 Lily symptomless virus (LSV)及Lycoris virus T (LVT)發生沉澱反應。以針對Carlavirus屬病毒聚合酶基因之廣效性引子對進行RT-PCR,構樹嵌紋病葉及所接種之奎藜病葉皆可增幅出一286 bp核酸產物,經解序及與GenBank中已知25種59支分離株之carlaviruses序列比對後,發現其與Blueberry scorch virus及Lily symptomless virus之核苷酸序列相同度(percent identity) 最高僅達77%。綜合以上結果顯示,本研究之構樹病毒分離株可能為文獻所未曾記載之一種carlavirus,建議暫時先將其命名為構樹嵌紋病毒 (Common mulberry mosaic virus)。
Broussonetia papeyrifera (L.) L´H’erit. ex Vent. casually named as common mulberry in Moraceae family is a kind of deciduous arbor or shrub which leaves can be used as feeds for pigs, cattle, sheep and deer, and barks can be used for paper-making. It is a pioneer plant growing in plains or regions with lower altitude. At present, the commercial cultivation has never been implemented in Taiwan, there are only pioneer plants on roadsides or wastelands, so they are distributed widely but sporadically in Taiwan. In 2005, a mosaic symptom on common mulberry were found in Pingtung. The disease incidence was about 42% by field survey. Symptoms fluctuated seasonally. A filamentous virus of 600-650 nm in particle size, were successfully isolated by mechanical inoculation from common mulberry with mosaic symptom. The thermal inactivation point of the virus was 70℃, dilution end point was 10-4 and the longevity in vitro was 4 days at 24℃ and more than 10 month at -80℃. It had a very narrow host range restricted to three chenopodiaceous plant species and one leguminosae plant species among 64 tested plant species belonging to 16 families. Numerous bundles of virus particles scattered through the cytoplasm were found in local lesion of Chenopodium quinoa and mosaic leaf of common mulberry. The disease incidence was about 15.2% by seed transmission. A purified virus preparation was obtain with a yield of 3.6 mg / 100g from diseased leaf. A virion protein with molecular weight of 36 kDa was detected where purified virus was denatured with sodium dodecyl sulfate (SDS) and analyzed by electrophoresis in polyacrylamide gels (SDS-PAGE) and by Western blotting. An antiserum with a titre of 1024 was obtained by immunizing a white rabbit with the purified virions. In SDS-agar gel double diffusion test, the antiserum against the common mulberry virus isolate reacted strongly with its homologous antigens, but not to antigens of two other carlaviruses, i.e. Lily symptomless virus (LSV) and Lycoris virus T (LVT). A sequence of 286 nucleotides (nts) was found and the percent of nucleotide identities of the polymerase gene were not higher than 77% comparing to 59 strains of 25 carlavirus species. Based on the results mentioned above show that the virus isolated from of common mulberry is a carlavirus which is proposed to be as Common mulberry mosaic virus tentatively.
目錄

中文摘要................................................ I
Abstract............................................... III
謝誌.................................................... V
目錄.................................................... VII
圖表目錄 ................................................X
壹、前言 ................................................1
貳、前人研究............................................. 3
一、感染桑科植物之病毒種類及特性........................... 3
(一)、Mulberry latent virus ( MLV,桑潛隱病毒)............ 3
(二)、Maclura mosaic virus ( MacMV,桑橙嵌紋病毒 )......... 4
(三)、Fig leaf chlorosis virus ( FigLCV,無花果黃葉病毒 ).. 4
(四)、Mulberry ringspot virus (MRSV,桑輪點病毒).......... 4
二、Carlavirus 屬病毒之特性.............................. 5
參、材料與方法........................................... 8
一、田間病害發生調查...................................... 8
二、構樹嵌紋病周年性病徵變化調查........................... 8
三、病毒之來源與分離...................................... 8
四、病毒之周年性分離...................................... 8
五、寄主範圍測試......................................... 9
六、病毒之生體外物理性質測定............................... 9
(一)、熱不活化溫度(Thermal inactivation point)測定........ 9
(二)、耐稀釋度(Dilution end point)測定.................. 9
(三)、耐保存性(Longevity in vitro)測定.................. 10
七、電子顯微鏡之觀察...................................... 10
(一)、負染法(Negative staining)......................... 10
(二)、超薄切片法(Ultrathin section)..................... 10
八、種子媒介傳播試驗...................................... 11
九、病毒之純化........................................... 11
十、病毒鞘蛋白分子量測定.................................. 12
十一、抗體之製備......................................... 12
(一)、抗血清之製作....................................... 12
(二)、免疫球蛋白(Immunoglobulin G,IgG)之製備........... 13
1、DE23 cellulose分離柱之填裝............................ 13
2. 免疫球蛋白之純化...................................... 13
十二、血清學反應......................................... 14
(一)、抗血清力價測定.................................... 14
(二)、瓊脂雙向擴散反應(SDS- agar gel double diffusion test) ................................................14
(三)、西方轉漬反應(Western blotting).................... 14
十三、聚合酶及鞘蛋白基因之定序與分析 ........................16
(一)、反轉錄聚合酶連鎖反應(one step RT-PCR)............... 16
(二)、電泳膠體分析....................................... 17
(三)、聚合酶基因之選殖.................................... 17
(四)、選殖株篩選......................................... 18
(五)、聚合酶基因之定序與分析.............................. 18
肆、結果................................................ 19
一、田間病徵及病害發生調查 ................................19
二、構樹嵌紋病周年性病徵變化調查........................... 19
三、病毒之來源與分離...................................... 19
四、病毒之周年性分離...................................... 19
五、寄主範圍測試......................................... 19
六、病毒之物理性質測定.................................... 20
(一)、熱不活化溫度(Thermal inactivation point)測定........ 20
(二)、耐稀釋度(Dilution end point)測定................... 20
(三)、耐保存性(Longevity in vitro)測定................... 20
七、電子顯微鏡的觀察...................................... 20
(一)、負染法(Negative staining)........................ 20
(二)、超薄切片法(Ultrathin section)...................... 20
八、種子媒介傳播試驗..................................... 21
九、病毒之純化.......................................... 21
十、病毒鞘蛋白分子量測定.................................. 21
十一、血清學反應......................................... 22
(一)、抗血清力價測定..................................... 22
(二)、瓊脂雙向擴散反應(SDS- agar gel double diffusion test) ................................................22
(三)、西方轉漬反應(Western blotting).................... 22
十二、聚合酶及鞘蛋白基因之定序與分析........................ 23
(一)、反轉錄聚合酶連鎖反應(one step RT-PCR)............... 23
(二)、核苷酸序列之解讀................................... 24
(三)、核苷酸序列之分析................................... 24
伍、討論................................................ 25
陸、圖表................................................ 30
柒、參考文獻............................................. 58
捌、附錄................................................ 63
玖、作者簡介............................................. 66
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