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題名 | 野茼蒿(Conyza sumatrensis)對嘉磷塞之抗藥性及5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)基因研究=Glyphosate-resistant Broadleaf Fleabane (Conyza Sumatrensis) : Dose Response and Variation Associated with Target Enzyme (EPSPS) |
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作者姓名(中文) | 袁秋英; 謝玉貞; 林李昌; 蔣慕琰; | 書刊名 | 植物保護學會會刊 |
卷期 | 48:3 民95.09 |
頁次 | 頁229-241 |
分類號 | 433.85 |
關鍵詞 | 嘉磷塞; 抗藥性; 野茼蒿; EPSP synthase基因; 分子特性; Glyphosate; Resistance; Conyza sumatrensis; EPSP synthase; Molecular characteristics; |
語文 | 中文(Chinese) |
中文摘要 | 嘉磷塞(glyphosate)為臺灣最常使用之非選擇性除草劑,於農地之使用約20年,近年各地野茼蒿(Conyza sumatrensis (Retz.) Walker)已呈現田間抗藥性之情況,構成防治管理上日趨嚴重之問題。本研究利用野茼蒿對嘉磷塞之劑量反應、shikimic acid含量變化及5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)基因選殖,探討野茼蒿對嘉磷塞之抗藥特性。由台中縣地區採集之52族群野茼蒿種子,經播種、育苗至6-8葉齡,噴施次致死量(0.3 kg ai ha-1)之嘉磷塞(41% 溶液),初步篩選代表性之感性(susceptible, S)及抗性(resistant, R)野茼蒿。針對感、抗性野茼蒿幼苗,再進行0.05-1.6 kg ai ha-1嘉磷塞之劑量反應,施藥後14日,以乾重為基礎,經由log-logistic 程式估算感、抗性野茼蒿之ED50分別為0.07及0.56 kg ai ha-1,ED50 R/S值為8。抗性野茼蒿於施藥後2-3日,shikimic acid含量略增加,但隨天數增加而逐漸降為1.9 μmol g-1。感性野茼蒿於施藥後7日,shikimic acid累積為18 μmol g-1,約為抗性野茼蒿shikimic acid含量的9.5倍。利用反轉錄聚合酶鏈鎖反應(reverse transcription-polymerase chain reaction, RT-PCR)選殖感及抗性野茼蒿EPSPS基因,各有3條cDNA (EPSPS-1、EPSPS-2、EPSPS-3),感及抗性野茼蒿EPSPS-1 開放讀碼區(open reading frame, ORF)完全相同,長度為1341 bp,感及抗性野茼蒿EPSPS-2及EPSPS-3的ORF皆為1338 bp,相似度亦皆為96%。抗性野茼蒿EPSPS2有11個胺基酸與感性野茼蒿EPSPS2不同,其中抗性野茼蒿的4 個高度保留胺基酸之變異分別為68glycine (Gly, G)→glutamic acid (Glu, E)、196arginine (Arg, R)→Gly、416Arg→ cysteine (Cys, C)及428serine (Ser, S)→Cys,此4處胺基酸的改變,可能為抗藥性的原因之一。 |
英文摘要 | Glyphosate -resistant and -susceptible types were obtained from preliminary screening of 52 populations of Broodleaf fleabane from orchards in Taichung country where suspected resistance have been reported. Broadleaf fleabane (Conyza sumatrensis (Retz.)Walker)) seedlings at 6-8 leaf stage were sprayed with glyphosate at rates ranging from 0.05-1.6 kg ai ha-1. Dose-response relationship was established based on dry weight of seedlings 14 days after spray. The median effective doses (ED50) estimated from log-logistic equation were 0.07 and 0.56 kg ai ha-1 for susceptible and resistant type, respectively. Susceptible broadleaf fleabane accumulated more shikimic acid than resistant type under 1 kg ai ha-1 of glyphosate. Seven days after spray, shikimic acid of susceptible broadleaf fleabane was 18 μmol g-1, about 9.5 times higher than that of resistant type. We explored the molecular characteristics of target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of broadleaf fleabane using RT-PCR, E. coli transformation and restriction enzymes to generate the related target enzyme. There are three EPSPS cDNAs for both resistant and susceptible broadleaf fleabane. Open reading frame for EPSPS1, EPSPS2 and EPSPS3 of resistant and susceptible type were 1341, 1338 and 1338 bp, respectively. Sequence data revealed 96% identity and 11 amino acids difference between EPSPS2 for the resistant and susceptible type. Resistant broadleaf fleabane was associated with 68glycine, 196arginine, 416arginine and 428serine, as compared with glutamic acid, glycine, cysteine and cysteine. GenBank data comparison suggested that variation of amino acid position at 68, 196, 416 and 428 is important in the change from susceptibility to resistance. |
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