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研究生:蘇俊豪
研究生(外文):Jun-Hao Su
論文名稱:多齒新米蝦(Neocaridina denticulate)表現序列標籤系統的建立與應用
論文名稱(外文):Development and Application of the Expressed-Sequence-Tag System Derived from Neocaridina denticulate
指導教授:宋宏紅宋宏紅引用關係
指導教授(外文):Hung-Hung Sung
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:116
中文關鍵詞:多齒新米蝦鄰苯二甲酸酯類化合物生物監測表現序列標籤
外文關鍵詞:Neocaridina denticulatephthalate estersbiomonitorexpressed sequence tag
相關次數:
  • 被引用被引用:1
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環境荷爾蒙為可干擾生物體內分泌系統正常作用的外因性內分泌干擾物(Endocrine disrupting chemicals, EDCs),鄰苯二甲酸酯類化合物(Phthalate esters, PAEs) 為此類的物質之一。本研究選用已知會影響淡水長臂大蝦(Macrobrachium rosenbergii)的四種PAEs,包括鄰苯二甲酸二乙酯(diethyl phthalate, DEP)、鄰苯二甲酸二己酯(dihexyl phthalate, DHP)、鄰苯二甲酸二丙酯(dipropyl phthalate, DPrP)及鄰苯二甲酸二苯酯(diphenyl phthalate, DPP),在實驗室的條件下浸泡處理多齒新米蝦後,利用建立的表現序列標誌(Expressed sequence tags, ESTs)資料庫,進行基因表現的測定,除了探討PAEs對蝦子生理影響外,同時評估多齒新米蝦及其ESTs系統作為生物指標系統的可行性。
實驗蝦子分別以0.1、1.0及10.0 mg/L 三種劑量PAEs的池水浸泡一天後,針對選定的28種ESTs以RT-PCR測定並分析基因表現的變化。所有結果與不含PAEs池水浸泡的控制組蝦子作比較,發現13種ESTs表現會受DEP影響;16種ESTs表現會受DPrP影響;2種ESTs表現會受DHP影響;9種ESTs表現會受DPP影響。進一步將其中已知功能的ESTs作相關性分析,顯示DEP、DPrP及DPP三種PAEs對多齒新米蝦的生理影響效應相似,主要是防禦和代謝相關功能,影響的功能性基因包括防禦相關的QM protein(FL592852)、hemocyanin(FL640908)、heat shock 70 kD protein cognate(FL640930)、glutathione S-transferase class mu(FL640931)及dehydrogenase 1(FL640932);呼吸相關的mitochondrion(FL640918);轉譯相關的elongation factor 1-alpha(FL640913);代謝相關的cathepsin-L like cysteine protease mRNA(FL640915)、glutamata dehydrogenase(FL640917)、Trehalose-6-
phosphate synthase 1(FL640934);至於DHP對蝦子的生理影響較低。
接著,降低處理濃度為0.01 mg/L和0.05 mg/L以浸泡蝦子後,分析ESTs對四種PAEs的靈敏度。在統計水準p &;lt; 0.05下與控制組作比較,結果有7個ESTs的表現在0.01 mg/L時仍有明顯變化;在統計水準p &;lt; 0.1下,在0.01 mg/L時發生明顯變化的ESTs增加至10個。將ESTs之表現幅度設定為大於或小於控制組的50%時,28段ESTs中有14段ESTs推測可以作為監測的生物指標(Bioindicator)。
由本研究結果推測,由多齒新米蝦所建立的ESTs系統不但可以作為環境污染物(如環境荷爾蒙PAEs)對物種生理影響的研究工具,也可以作為潛在汙染之監測或環境復育的評估。
Endocrine disrupting chemicals (EDCs) which are xenobiotic chemicals such as phthalate esters (PAEs) can interfere with the endocrine functions of orginasms. In this study, the expressed sequence tag (ESTs) system developed from shrimp Neocaridina denticulate and four PAEs of aquatic pollutants, including diethyl phthalate (DEP), dihexyl phthalate (DHP), dipropyl phthalate (DPrP) and diphenyl phthalate (DPP), were used to study the effect of PAEs on physiological functions of N. denticulate and to assess the feasibility of the ESTs system to monitor the pollution of aquatic environment. After shrimps were separately immersed four PAEs with different concentrations (0 mg/L, 0.1 mg/L, 1.0 mg/L and 10.0 mg/L of pond water) for one day, the expression of 28 ESTs were detectd by semi-quantitative RT-PCT. This result showed that, comparing with the control group, 13, 16, 2 and 9 ESTs of them were significantly changed in DEP-, DPrP-, DHP- and DPP-treated groups, respectively; among them, the effect of DEP, DPrP and DPP on the functional known ESTs are similar, mainly including both the defence-related and metabolism-related ESTs, MQ protein (FL592852), hemocyanin (FL640908), heat shock 70 kD protein cognate (FL640930), glutathione S-transferase class mu (FL640931) and dehydrogenase 1 (FL640932), mitochondrion (FL640918), elongation factor 1-alpha (FL640913), cathepsin-L like cysteine protease mRNA (FL640915), glutamata dehydrogenase (FL640917), trehalose-6- phosphate synthase 1 (FL640934). As for DHP, its effect on shrimp was lower than othe three PAEs.
To determine the sensitivity of the tested ESTs to the four PAEs, shrimps were immersed in lower dosages (0.01 mg/L and 0.05 mg/L). There were 7 ESTs in 0.01-mg/L-treated groups that the expression significantly changed when was statistically analysed at p<0.05, but 10 ESTs at p<0.1. When the expressed-ratio of ESTs larger or lower than that of control 50 % is decided as the standard, it was suggested that 14 of 28 ESTs could be use as biological molecule indicators.
According to the results, the study indicated that the ESTs system developed from N. denticulate can be used as a tool in studying the effect of environment pollution but also in monitoring the hidden risk of aquatic pollutants.
中文摘要1
英文摘要3
壹、前言5
貳、文獻回顧9
一、外因性內分泌干擾物質9
二、鄰苯二甲酸酯類化合物12
1. PAE的特性與流布14
2. PAE對生物的影響16
3. PAE的管理18
三、生物檢測方式19
1. 指標生物21
2. 生物指標(Bioindicator)22
代謝合成產物體(metaboliomics)23
蛋白質體(proteomics)25
基因體(genomics)26
四、多齒新米蝦(Neocaridina denticulate)28
?礡B實驗目的與策略30
肆、材料方法31
一、養殖環境31
二、實驗用蝦31
三、實驗蝦的使用標準31
四、鄰苯二甲酸酯類化合物的製備32
五、多齒新米蝦浸泡處理33
六、表現序列標籤(Expressed sequence tags; ESTs)的表現34
1. 表現序列標籤(EST)資料庫的整合與專一性引子的確認34
2. 蝦組織Total RNA的萃取34
3. 反轉錄作用35
4. 聚合?○s鎖反應及半定量測定35
伍、結果38
一、實驗用多齒新米蝦的篩選38
二、整合與篩選的表現序列標籤系統39
三、鄰苯二甲酸酯類化合物對EST表現的影響39
四、EST對PAE的靈敏度42
陸、討論45
一、因PAE而受到影響的EST46
二、PAE結構對蝦子生理功能的影響48
三、四種PAE對於蝦子不同生理功能上扮演的角色50
1. PAE對蝦子防禦功能的影響50
2. PAE對蝦子代謝功能的影響52
3. PAE對蝦子基因表現相關功能的影響55
4. PAE影響蝦子生理作用之可能機制的探討55
四、表現序列標籤(EST)系統作為汙染監測的可能性57
柒、結論61
捌、參考文獻62
玖、圖表79
附錄110
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