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抗嘉磷塞雜草-繖花龍吐珠(Hedyotis corymbosa) 5-烯醇丙酮酸莽草酸-3-磷酸合酶之點突變及分子快速鑑定

A Molecular Characterization of Point-Mutations in the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Enzyme of Glyphosate-Resistant Biotypes of Hedyotis corymbosa

摘要


農地雜草對化學除草劑的抗藥性問題日漸嚴重,目前全球抗除草劑之雜草已有249 種,造成藥劑使用量的增加及農地生態多樣性的失衡等問題。本研究針對抗嘉磷塞 (glyphosate) 的繖花龍吐珠 (Hedyotis corymbosa (L.) Lam.),測試其對嘉磷塞的劑量反應、莽草酸 (shikimic acid)含量及關鍵酵素5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) 活性,探討其點突變(point mutation) 的抗藥特性。以12 ~ 15 葉齡的繖花龍吐珠植株,經噴施0.10 ~ 26.24 kg a.e./ha 不同濃度之嘉磷塞,施藥後14 日,以乾重為基礎,經由log-logistic 程式估算繖花龍吐珠感性生物型 (Sbiotype) 及抗性生物型 (R biotype) 之ED50 (50% 傷害程度) 分別為0.46 及4.58 kg a.e./ha,抗性增加約10 倍。感性繖花龍吐珠於施藥後莽草酸含量亦逐漸增加,施藥後10 日,莽草酸約為抗性生物型的36.5 倍。利用RT-PCR 增幅及比對抗及感性生物型EPSPS cDNA 長度皆為1,551 bp,僅有11 個鹼基之差異,其中於抗及感性生物型第532 鹼基分別為胸腺嘧啶 (thymidine) 及胞嘧啶 (cytosine),經序列轉譯後第178 個胺基酸分別為絲氨酸 (serine) 及脯氨酸 (proline),此處之改變可能為抗性生物型EPSPS 對嘉磷塞親和性降低之主要原因。再將抗性及感性生物型之EPSPScDNA,經轉殖於大腸桿菌 (Escherichia coli) 大量複製,純化的EPSPS,經1 mM 嘉磷塞藥劑反應,顯示抗性生物型EPSPS 活性為感性生物型的3 倍。於抗及感性生物型EPSPS cDNA 第532鹼基差異處,分別設計兩組專一性引子,利用單核苷酸多型性 (single nucleotide polymorphism, SNP) 及聚合酶鏈鎖反應─限制性片段長度多型性 (PCR-RFLP) 技術,進行11 個繖花龍吐珠抗及感生物型之分子鑑定,於其中4 個抗性植株可檢出約350 bp 之DNA 條帶,7 個感性植株可利用HaeIII 酵素檢出。此分子檢測方法針對現階段臺灣農地的繖花龍吐珠因目標酵素之變異,建立快速檢驗抗嘉磷塞的生物型,可應用於抗性生物型的分布與擴散之監測,亦做為調整用藥種類與管理方法之依據。

並列摘要


Weed resistance to herbicides has become a major concern for crop production worldwide. We investigated the resistance of Hedyotis corymbosa to glyphosate in Taiwan, focusing on dose-response, shikimic acid level, and the production of target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). For this, Hedyotis corymbosa seedlings at the 12 ~ 15 leaf stage were foliar sprayed with 0.10 to 26.24 kg a.e./ha glyphosate. Dose-response was established based on the dry weight of seedlings 14 days after glyphosate treatment. ED_(50) was estimated using a log-logistic equation, and was found to be 0.46 or 4.58 kg a.e./ha for susceptible and resistant biotypes, respectively. Susceptible H. corymbosa showed obvious accumulation of shikimic acid. Ten days following glyphosate treatment, shikimic acid content of susceptible H. corymbosa was 36.5 times higher than that of resistant H. corymbosa. Furthermore, after using real-time polymerase chain reaction (RT-PCR) to prepare the related target enzyme EPSPS of H. corymbosa and treating plants with 1 mM glyphosate, an EPSPS activity assay showed that the EPSPS content of the resistant biotype was 3 times higher than that of the susceptible biotype. We also generated EPSPS cDNA by performing RT-PCR with specific primers designed according to sequence data from GenBank. In so doing, we determined that EPSPS cDNA was 1,551 bp for both resistant and susceptible H. corymbosa. However, sequence data revealed nucleotides differences in 11 bp between resistant and susceptible biotypes. For example, the resistant biotype had a thymidine (T) at position 532, whereas the susceptible biotype had a cytosine (C) at that position. Moreover, the amino acid at position 178 is the major target site among glyphosate resistant grasses around the world, and transcribed sequence data revealed that amino acids at this position were serine178 and proline178 for resistant and susceptible biotypes, respectively. Finally, we designed two sets of primers with reference to 532 bases and HaeIII specific to EPSPS nucleotides of resistant and susceptible H. corymbosa. These primers were used in single nucleotide polymorphism (SNP) and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assays for detecting of H. corymbosa collected from southern Taiwan. We found that 4 out of 11 H. corymbosa populations possessed resistant biotypes and were clearly distinguishable from the 7 populations with susceptible biotypes.

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