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題名 | 美洲假蓬(Conyza Bonariensis)對嘉磷塞之抗藥性與5-enolpyruvylshikimate-3-phosphate Synthase (EPSPS)基因研究=Resistant Responses and Characteristics of 5-enolpyruvylshikimate-3-phosphate Synthase (EPSPS) in Hairy Fleabane (Conyza bonariensis) to Glyphosate |
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作者 | 林李昌; 葉茂生; 蔣慕琰; 袁秋英; Lin, Li-chang; Yeh, Mau-shing; Chiang, Mou-yen; Yuan, Chiou-ing; |
期刊 | 作物、環境與生物資訊 |
出版日期 | 20081200 |
卷期 | 5:4 2008.12[民97.12] |
頁次 | 頁268-280 |
分類號 | 433.85 |
語文 | chi |
關鍵詞 | 美洲假蓬; 嘉磷塞; 抗藥性; EPSP synthase; Conyza bonariensis; Glyphosate; Resistance; |
中文摘要 | 摘要 本研究利用美洲假蓬(Conyza bonariensis (L.) Cron) 對嘉磷塞(glyphosate)之劑量反 應、shikimic acid 含量變化及5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS)基因 選殖,探討美洲假蓬對嘉磷塞之抗藥特性。 由臺中縣採集美洲假蓬39 個族群的種子,經 播種、育苗至6-8 葉齡,噴施0.3 kg ai ha-1 嘉磷塞(41% 溶液)次致死劑量,初步篩選抗 性(resistant; R)及感性(susceptible; S)美洲假 蓬。再針對抗及感性美洲假蓬幼苗,進行 0.13-12.00 kg ai ha-1 嘉磷塞之劑量反應分 析,施藥後14 日以乾重為基礎的ED50 R/ ED50 S=14。美洲假蓬葉片shikimic acid 含 量之測定顯示, 施藥7 日後感性植株之 shikimic acid 含量為38 μmol g-1,是抗性植 株之5 倍以上。經由美洲假蓬EPSPS 之基因 選殖結果, 抗及感性美洲假蓬皆有3 個 EPSPS cDNA (EPSPS1 、EPSPS2 及 EPSPS3),EPSPS1 開放讀碼區(open reading frame;ORF)為1,341 bp,而EPSPS2 及 EPSPS3 的ORF 較EPSPS1 短,皆為1,338 bp,抗及感性美洲假蓬相對應的EPSPS1、 EPSPS2 及EPSPS3 蛋白質只有1、0 及1 個 胺基酸的差異, 然而抗及感性美洲假蓬 EPSPS1 分別與EPSPS2 及EPSPS3 比對,相 同度(identity)為85-86%,有多達34-36 個胺 基酸的差異;抗及感性美洲假蓬EPSPS2 與 EPSPS3 則相同度較高,只有6 個胺基酸的差 異。抗性美洲假蓬經純化的EPSPS1、EPSPS2 及EPSPS3 酵素活性,於嘉磷塞藥劑處理後, EPSPS1 於4 mM 劑量幾乎完全喪失活性; EPSPS2 及EPSPS3 於0.5-2.0 mM 劑量,活 性隨濃度增加而緩慢降低,EPSPS1、EPSPS2 及EPSPS3 活性的I50 值分別為0.23、1.42 及 1.36 mM,顯示EPSPS2 及EPSPS3 對嘉磷塞 的親和性較EPSPS1 低約6 倍。推測美洲假 蓬的高耐受性與EPSPS2、EPSPS3 蛋白質含 量的多寡有關,或是另有其他抗性機制。 |
英文摘要 | ABSTRACT Glyphosate-resistant (R) and –susceptible (S) types of hairy fleabane (Conyza bonariensis (L.) Cronq.) were obtained through preliminary screening on 39 populations from orchards in Taichung Hsien where suspected resistant varieties have been reported. Hairy fleabane seedlings at 6-8 leaf stage were foliar sprayed with glyphosate ranging from 0.13 to 12.00 kg ai ha-1. Dose-response relationship was established based on dry weight of seedlings 14 days after spraying. The ED50 estimated based on the loglogistic analysis model was 0.46 and 6.40 kg ai ha-1 for S and R types, respectively. Susceptible hairy fleabane accumulated more shikimic acid than R type under 1 kg ai ha-1 of glyphosate. At 7 days after spraying, shikimic acid of S hairy fleabane was 38 μmol g-1, about 5 times higher than that of R type. We explored the molecular characteristics of target enzyme 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS) of hairy fleabane using RT-PCR, E. coli transformation and restriction enzymes to generate the related target enzyme. There are three EPSPS cDNAs for both R and S hairy fleabane. The length of open reading frame for EPSPS1, EPSPS2 and EPSPS3 of R and S types were 1,341, 1,338 and 1,338 bp, respectively. Amino acid sequence data revealed 85-86% identity and 34-36 amino acids differences between EPSPS1 to EPSPS2 and/or EPSPS3 for the R and S types, respectively. We used RT-PCR and E. coli propagation to prepare EPSPS of hairy fleabane. EPSPS activity assay showed that inhibition (I50) of EPSPS2 and/or EPSP3 were 6 times higher than that of EPSPS1 in R type. The contents of high tolerant EPSPS2 or EPSPS3 protein are one of the possible explanations for glyphosate-resistance. |
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