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研究生:張家彥
研究生(外文):Jia-yan Zhang
論文名稱:繖花龍吐珠對嘉磷塞抗藥之特性研究-5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)基因及分子鑑定
論文名稱(外文):The characteristics of glyphosate-resistant Hedyotis corymbosa: 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)gene and molecular identification.
指導教授:袁秋英袁秋英引用關係
指導教授(外文):Chiou-Ing Yuan
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:生化科技研究所碩士班
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:2012
畢業學年度:100
語文別:中文
論文頁數:84
中文關鍵詞:嘉磷塞抗藥性繖花龍吐珠EPSPS
外文關鍵詞:glyphosateHedyotis corymbosa (L.) Lamresistance5-enolpyruvylshikimate 3-phosphate synthase (EPS
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繖花龍吐珠(Hedyotis corymbosa (L.) Lam),為茜草科耳草屬中的一年生草本植物,分佈於台灣全省低海拔地區,此植物對於除草劑嘉磷塞(glyphosate)具高度耐受性,本研究針對繖花龍吐珠測試其與嘉磷塞之劑量反應、shikimic acid含量及5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)酵素等生理反應,探討其抗藥特性。台中市霧峰區果園旁採集之繖花龍吐珠,經育苗至12-15葉齡,噴施嘉磷塞0.1-26.24 kg ai ha-1劑量,施藥後14日,以乾重為基礎,經由loglogistic程度估算感性(S)及抗性(R)繖花龍吐珠之ED50分別為0.41及5.04 kg ai ha-1。繖花龍吐珠幼葉shikimic acid 含量之測定顯示,隨施藥後天數及藥劑劑量增加,感性繖花龍吐珠shikimic acid含量亦逐漸增加,施藥後10日,shikimic acid大量累積約為抗性繖花龍吐珠shikimic acid含量的42.8倍。抗與感性繖花龍吐珠EPSPS基因選殖,經由設計專一性引子進行RT-PCR反應,可擴增約950 bp之核酸片段,再以 RACE(5''- and 3''-rapid amplification of cDNA ends)技術,完成抗及感性嘉磷塞繖花龍吐珠EPSPS cDNA之解序,抗及感性繖花龍吐珠EPSPS cDNA長度皆為1551 bp,僅有11個鹼基之差異,其中於抗及感性繖花龍吐珠第319鹼基分別為guanine(G)及cytosine(C),經序列轉譯後敏感植株胺基酸 107proline(P),於抗性植株為 106serine(S)。進而利用PCR-RFLP 及 AS-PCR 技術,於感性株可將EPSPS全長cDNA切割為332 bp及163 bp二核酸片段;或經由PCR測定可增幅抗性繖花龍吐株為350 bp專一性核酸條帶,建立抗嘉磷塞繖花龍吐珠之分子鑑定方法。此分子檢測方法可供抗除草劑雜草監測及管理之用。
Hedyotis corymbosa is a Rubiaceae plant that developing resistant to glyphosate in Taiwan since 2008s. In some areas, this resistant weed is becoming a serious problem for farmer. Hedyotis corymbosa seedlings at 12-15 leaves stage were foliar sprayed with glyphosate ranging from 0.1-26.24 kg ai ha-1. Dose-response was established based on dry weight of seedlings at 14 days after spray. The 50% effect dose (ED50) estimated from loglogistic equation was 0.41 and 5.04 kg ai ha-1 for susceptible and resistant type, respectively. Susceptible Hedyotis corymbosa showed obvious accumulation of shikimic acid starting one day after glyphosate spray. At 10 days after spray, shikimic acid of susceptible Hedyotis corymbosa was about 42.8 times higher than that of resistant type. We used RT-PCR and E. coli propagation to prepare the related target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of Hedyotis corymbosa. EPSPS cDNA were 1,551 bp for both resistant and susceptible Hedyotis corymbosa. Sequence data revealed a total 11 bp difference between the resistant and susceptible type. Sequence transcribed data showed that amino acids for resistant and susceptible type were 107serine and 107proline, respectively. EPSPS activity assay showed that inhibition (I50) of resistant type was 20 times higher than that of susceptible type. We designed two sets of primers with reference to specific base to that of resistant and susceptible Hedyotis corymbosa. These primers were used in PCR-RFLP or AS-PCR for molecular assay of Hedyotis corymbosa collected from central Taiwan. Ten of the 14 populations showed characteristic fragment around 350 bp of resistant Hedyotis corymbosa, and were clearly distinguishable with that of 4 susceptible types.
中文摘要 ----------------------------------------------------------------------------- I
英文摘要 ----------------------------------------------------------------------------- III
目錄------------------------------------------------------------------------------------ VI
縮寫對照 ---------------------------------------------------------------------------- IX
壹、 前言 ---------------------------------------------------------------------------- 1
貳、 前人研究
一、 繖花龍吐珠之簡介---------------------------------------------------------- 3
二、 嘉磷塞之特性介紹---------------------------------------------------------- 4
(一)、嘉磷塞理化及毒性--------------------------------------------------------- 4
(二)、嘉磷塞之作用機制及傷害徵狀------------------------------------------- 5
(三)、藥劑於植體之吸收、傳導及代謝方式---------------------------------- 6
(四)、嘉磷塞對植體shikimic acid含量之影響--------------------------------- 9
(五)、嘉磷塞標的酵素EPSPS之特性-------------------------------------------- 10
三、 嘉磷塞之抗藥性--------------------------------------------------------------- 12
(一)、 全球抗及耐嘉磷塞之雜草種類------------------------------------------- 12
(二)、 抗嘉磷塞植物之作用模式------------------------------------------------- 14
四、 抗嘉磷塞雜草之分子鑑定--------------------------------------------------- 17
參、 材料與方法 -------------------------------------------------------------------- 18
一、 材料 ----------------------------------------------------------------------------- 18
(一) 、 嘉磷塞對繖花龍吐珠藥劑劑量反應測定------------------------------- 18
(二)、 嘉磷塞對繖花龍吐珠之葉片Shikimic acid含量測定------------------ 19
(三)、 抗性及感性繖花龍吐珠之EPSPS基因選殖及序列比較-------------- 20
(四)、 EPSPS 蛋白表現、分析、純化------------------------------------------- 30
(五)、 抗及感性繖花龍吐珠EPSPS基因之南方氏墨點法分析-------------- 40
肆、 結果與討論---------------------------------------------------------------------- 49
一、 嘉磷塞對繖花龍吐珠之劑量反應------------------------------------------- 49
二、 繖花龍吐珠噴施嘉磷塞葉片內shikimic acid 含量測定---------------- 51
三、 抗及感性繖花龍吐珠EPSPS基因之選殖及序列比較-------------------- 52
四、 抗及感性繖花龍吐珠EPSPS融合蛋白之構築及表現------------------- 53
五、 含有抗或感性繖花龍吐珠EPSPS大腸桿菌之嘉磷塞耐受性測定--- 54
六、 抗及感性繖花龍吐珠EPSPS活性分析------------------------------------- 54
七、 抗及感性繖花龍吐珠EPSPS基因之南方墨點法分析-------------------- 55
八、 利用PCR-RFLP及AS-PCR技術,建立抗性繖花龍吐株之分子鑑定方法--------------------------------------------------------------------------------------------- 56
伍、 參考文獻 ------------------------------------------------------------------------ 58
陸、 表 --------------------------------------------------------------------------------- 68
柒、圖 --------------------------------------------------------------------------------- 69
捌、附錄 ------------------------------------------------------------------------------ 80

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