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

林李昌(Li-Chang Lin) ； 葉茂生(Mau-Shing Yeh) ； 蔣慕琰(Mou-Yen Chiang) ； 袁秋英(Chiou-Ing Yuan)

《作物、環境與生物資訊》 5卷4期 (2008/12) Pp. 268-280

https://doi.org/10.30061/CEB.200812.0006

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1341bp，而EPSPS2及EPSPS3的ORF較EPSPS1短，皆為1338bp，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4mM劑量幾乎完全喪失活性；EPSPS2及EPSPS3於0.5-2.0mM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1.36mM，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。

關鍵字

美洲假蓬 ； 嘉磷塞 ； 抗藥性 ； EPSP synthase

並列摘要

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 1341, 1338 and 1338 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.

並列關鍵字

Conyza bonariensis ； Glyphosate ； Resistance ； EPSP synthase