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賴美津教授實驗室執行科技部天然氣水合物計畫，研究生翁杰愔自台灣西南海域變形前緣區取得活塞岩心底泥樣品分離純化出甲烷囊菌屬新種菌株Methanoculleus taiwanensis ... 資料載入處理中... 跳到主要內容 臺灣博碩士論文加值系統 ::: 網站導覽| 首頁| 關於本站| 聯絡我們| 國圖首頁| 常見問題| 操作說明 English |FB專頁 |Mobile 免費會員 登入| 註冊 功能切換導覽列 (167.99.71.17)您好！臺灣時間：2021/12/2715:08 字體大小：       ::: 詳目顯示 recordfocus 第1筆/ 共1筆  /1頁 論文基本資料 摘要 外文摘要 目次 參考文獻 電子全文 QRCode 本論文永久網址: 複製永久網址Twitter研究生:魏文心研究生(外文):Wen-HsinWei論文名稱:台灣西南海域深海底泥甲烷囊菌屬新種及甲烷古菌病毒之純化分類鑑定與病毒基因體解析論文名稱(外文):IsolationandcharacterizationofgenusMethanoculleusspeciesandtheirvirusesfromdeepseamarinesedimentoffshoreSWofTaiwan指導教授:賴美津指導教授(外文):Mei-ChinLai口試委員:湯森林、黃啟裕口試委員(外文):Sen-LinTang、Chi-YuHuang口試日期:2018-01-18學位類別:碩士校院名稱:國立中興大學系所名稱:生命科學系所學門:生命科學學門學類:生物學類論文種類:學術論文論文出版年:2018畢業學年度:106語文別:中文論文頁數:111中文關鍵詞:甲烷古菌、甲烷古菌病毒、甲烷囊菌屬外文關鍵詞:Methanogen、Methanogenvirus、Methanoculleus相關次數: 被引用:4點閱:135評分:下載:0書目收藏:0 三域生物都可能被病毒感染，許多細菌噬菌體與真核生物病毒都是顯學，但有關古菌病毒的描述卻僅佔少數。

好氧極端高鹽古菌因較易培養，其相關噬菌體型態病毒被報導與研究的較早。

近年來，泉太古生物門的高溫古菌多樣化病毒形態與病毒離開宿主後於高溫環境下的形態結構變化與適應演化，是目前相當被關注的主題研究。

然而在過去二十年間幾乎沒有關於甲烷古菌病毒的研究發表。

賴美津教授實驗室執行科技部天然氣水合物計畫，研究生翁杰愔自台灣西南海域變形前緣區取得活塞岩心底泥樣品分離純化出甲烷囊菌屬新種菌株MethanoculleustaiwanensisCYW4T，以穿透式與掃描式電子顯微鏡均觀察到病毒及CYW4T細胞於後對數期被病毒裂解現象。

本論文研究進一步測量CYW4T於不同生長期的細胞密度OD、甲烷產量與顯微觀察，確認病毒於後對數期與平穩期間裂解釋出，菌量下降，菌液澄清。

甲烷古菌病毒VMta(VirusofMethanoculleustaiwanensis)，病毒顆粒為球形，有二十面體衣殼蛋白(capsidprotein)，外部還有外套膜(envelope)包覆，直徑大小約為100nm。

VMta病毒具雙股DNA含70,262個鹼基對，G+C含量為54.5%，有101個開放讀序框架(ORFs)。

VMta基因體序列經BLASTX及HHpred比對，顯示可能利用穿刺蛋白將DNA送入宿主中，並以滾環型複製機制(rollingcyclereplication,RCR)複製DNA，經由終端酶將DNA包裝進空的衣殼蛋白，最後由裂解性酵素，例如內溶素(endolysin)破壞宿主而出。

本研究亦自台灣西南海域好景海脊取得岩心樣品純化出新甲烷菌株Methanoculleussp.CWC-02，經由親緣關係比對，菌株CWC-02與MethanoculleusmarisnigriJR1T有98.4%相似度，而兩株菌的平均核苷酸一致性(ANI)為86.81%；且基因組對基因組距離計算(GGDC)分析結果為32.50%，顯示菌株CWC-02應為Methanoculleus屬下之新“種”菌株。

菌株CWC-02能利用甲酸及H2/CO2及2-propanol、2-butanol等二級醇為基質行甲烷化作用，最適生長溫度為37℃。

菌株CWC-02生長至平穩期於TEM的觀察下，也發現有許多的VPL(virus-likeparticle)自細胞裂解而出，推測菌株CWC-02也被裂解病毒感染。

於天然氣水合物潛力區以甲烷為主的生態系中，碳源與營養的加速循環，對此生態系的影響與可能的甲烷天然氣累積值得進一步探討。

VirusesinfectallThreeDomainsoflifes,howevertheknowledgeofarchaealvirusesislimited.Lately,thediversevirusmorphologyandtheindependentvirusdevelopmentoutsidetheextremethermophilic/acidophilicarchaealhostattractedtheattentiononarchaealvirusinvestigations.Currently,over100archaealviruseshavebeendiscovered,mostrelatedtothermophilicCrenarchaeaandextremehalophilicEuryarchaea.However,almostnonemethanoarchaealviruswasreportedforthepasttwodecades.Fromthecoresedimentonthetopofgashydratehabitatinthedeformationfrontsiteoffshoresouth-westernTaiwan,MethanoculleustaiwanensisCYW4Twasenriched,isolatedandcharacterized.CelllysiswasobservedatstationaryphaseandalyticviruswhichcausedthelysisofstrainCYW4TwasobservedunderTEM.Thesphericalshapevirion,about100nm,withtheicosahedralcapsidsandenvelopewasnamedasVMta(VirusofMethanoculleustaiwanensis).VMtaisadsDNAviruswhichcontained70,262bpwithamol%G+Ccontentof54.5and101putativeopenreadingframes(ORFs).BaseontheVMtagenomeinformationandanalysisthroughBLASTXandHHpred,itwassuggestedthattheVMtamayinjectedtheDNAintothehostbythepunctureproteinandreplicatedtheirgenomesthroughtherolling-circlereplication(RCR)mechanism.TheviralDNAswerepackedintotheemptycapsidbytheterminaseenzymesandvironslefthostcellwiththeendolysindisruption.Methanoculleussp.CWC-02wereenrichedfromthemarinesedimentoftheGoodweatherridge.PhylogeneticanalysisrevealedthatstrainCWC-02wascloselyrelatedtoMethanoculleusmarisnigriJR1T(98.4%16SrRNAgenesequencesimilarity).GenomerelatednessbetweenstrainCWC-02andMethanoculleusmarisnigriJR1Twascomputedusingbothaveragenucleotideidentity(ANI)andgenome-to-genomedistancecalculator(GGDC)withvaluesof86.81%and32.50%,respectively.Accordingtogenomicdata,itispatentthestrainCWC-02maythenovelnewspecies.StrainCWC-02usedH2plusCO2,formate,2-propanoland2-butanolascatabolicsubstrates.Theoptimumgrowthtemperaturewas37℃.ThestationaryphasegrowthofstrainCWC-02cellsalsoobservedvirus-likeparticlesunderTEM.Theoccurrenceofmethanoarchaealvirusesatthemethane(gas)hydrateecosystemsindicatedthatvirusesmayplayaroleinaccelerationofthecarbonandnutritionalcycling. 摘要iAbstractii目錄iii表目錄vii圖目錄viii壹、前言1貳、前人研究4一、甲烷古菌(太古生物)(Methanogen)4二、自甲烷天然氣水合物潛力區純化的甲烷古菌發現甲烷古菌病毒5三、原核生物病毒6(一)病毒分類6(二)細菌病毒6(三)古菌病毒7(四)好氧極端高鹽古菌病毒81.雙股DNA(dsDNA)好氧極端高鹽古菌病毒82.單股DNA好氧極端高鹽古菌病毒9(五)高溫古菌病毒101.泉太古生物門病毒(Crenarchaealvirus)102.廣域太古生物門病毒(Euryarchaealvirus)13(六)甲烷古菌病毒13四、病毒感染作用及複製過程14(一)DNA病毒複製機制15(二)滾環型複製(Rolling-circlereplication,RCR)15參、材料與方法17第一部分-古菌病毒純化及特性分析17一、甲烷古菌培養與病毒純化17(一)台灣甲烷囊菌(MethanoculleustaiwanensisCYW4T)培養17(二)PEG沉澱17(三)蛋白質層析儀(Fastperformanceliquidchromatography,FPLC)18(四)病毒溶液濃縮19二、穿透式電子顯微鏡(Transmissionelectronmicroscopy,TEM)觀察19三、甲烷古菌病毒DNA萃取19四、核酸酶作用20五、蛋白質電泳20(一)VMta病毒樣品處理20(二)SDS-PAGE20(三)銀染色法(Silverstain)21六、脂質之萃取及分析22(一)甲烷古菌細胞膜與甲烷古菌病毒脂質萃取22七、病毒基因體定序與序列分析比對23(一)核酸與蛋白質序列分析23(二)HHpred分析24(三)CDD(Conserveddomaindatabase)比對分析24第二部分-甲烷古菌純化及特性分析25一、採樣時間地點與樣品甲烷古菌增殖進度25二、藥劑與培養基25(一)除氧操作系統(Hungatestation)25(二)MB/W與MM/W液態培養基配製25(三)還原劑及碳源配製26(四)抗生素溶液配製27(五)TGC(thioglycollate)培養基配製27三、甲烷古菌增殖培養與厭氧轉殖接種28(一)接菌28(二)氣相層析儀偵測樣品甲烷氣體28(三)抗生素添加28(四)連續稀釋法(serialdilution)29(五)Roll-tube29四、微生物形態觀察30(一)位相差顯微鏡觀察細胞30(二)穿透式電子顯微鏡(Transmissionelectronmicroscopy,TEM)觀察細胞30(三)場發射掃描式電子顯微鏡(Field-emissionscanningelectronmicroscope,FE-SEM)觀察細胞30五、生長與生理生化特性分析31(一)碳源利用測試31(二)抗生素抗性測試32(三)生長曲線測量32(四)溫度生長範圍測試33六、甲烷古菌染色體DNA萃取33七、核酸純度與定量分析34八、聚合酶連鎖反應(Polymerasechainreaction,PCR)34九、核酸電泳分析35十、PCR增幅樣品切膠純化回收35十一、質體轉型作用36(一)勝任細胞的製備36(二)質體轉形作用37(三)質體抽取與純化37十二、核酸定序與序列分析38(一)16SrRNA基因定序與序列分析38(二)染色體DNA定序與序列分析38肆、結果與討論40一、病毒VMta40(一)病毒VMta與台灣甲烷囊菌CYW4T的關係40(二)VMta病毒純化41(三)VMta為雙股DNA病毒41(四)VMta病毒結構分析41(五)VMta病毒脂質組成分析42(六)VMta病毒蛋白組成分析42(七)病毒基因體分析431.VMta病毒序列與蛋白功能預測432.病毒結構相關序列分析433.DNA複製相關序列分析444.病毒組裝與釋出宿主相關功能序列分析45(八)VMta病毒的生命週期45二、深海底泥甲烷古菌之增殖培養與純化47(一)自好景海脊(Goodweatherridge)純化的Methanoculleussp.CWC-02之細胞形態、系統演化分類與生理生化特性分析47(二)細胞型態47(三)系統演化分類分析48(四)生理生化與生長特性分析49伍、結論與展望51陸、表與圖52柒、參考文獻89捌、附錄99 朱高鈴。

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台灣西南海域興新興能源－天然氣水合物資源調查與評估：地球化學調查研究：台灣西南海域地質微生物(細菌與太古生物)多樣性調查與天然氣水合物形成與分解機制探討(4/4)。

中央地質調查所報告第100-25-B號，委辦計畫編號(100-5226904000-02-03)。

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