請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/95773
標題: 鑑定來自兩個對殺線蟲劑感受性不同之水稻葉芽線蟲的乙醯膽鹼酯酶基因
Cloning three acetylcholinesterase genes from two Aphelenchoides besseyi with different nematicide-susceptibilities
作者: 徐榮愷
Jung-Kai Hsu
關鍵字: 水稻葉芽線蟲
Aphelenchoides besseyi
acetylcholinesterase inhibitors
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摘要: 水稻葉芽線蟲 (Aphelenchoides besseyi)之Fm與Rl兩品系依序分別分離自鳥巢蕨與水稻,為兩不同門 (Phylum)的寄主植物,原生環境有極大的差異,且寄主範圍、生殖模式亦不同。在前人研究中發現兩品系多個寄生相關基因亦存在差異性,因此兩者雖為同種,但在演化上已產生分歧,可認定兩者正在分化的進程中。本研究鑑定兩者的保守基因乙醯膽鹼酯酶基因 (Ace),得知在A. besseyi上共有三種Ace基因,包含Ace-1、 Ace-2 及 Ace-3。Fm與Rl的Ace-1基因可轉譯出628胺基酸 (a.a.)的蛋白序列;Ace-2基因則可分依序別轉譯出635 a.a.和500 a.a.的蛋白序列;Ace-3基因可轉譯出628 a.a.的蛋白序列,經過序列比對Fm與Rl兩品系的同一種乙醯膽鹼酯酶,發現三者皆存在差異性,其中以AChE2差異最大。乙醯膽鹼酯酶 (AChE)為有機磷劑與胺基甲酸鹽類殺線蟲劑的作用標的,屬於乙醯膽鹼酯酶抑制劑,為常見的殺蟲劑。昆蟲方面的研究顯示對乙醯膽鹼酯酶抑制劑類藥劑產生抗藥性的機制以作用點對藥劑敏感度下降為主,而導致作用點對藥劑敏感度下降的主要原因為單核苷酸多態性Single nucleotide polymorphisms (SNPs)。因Fm與Rl兩品系之乙醯膽鹼酯酶已知有差異,為探討差異是否與對乙醯膽鹼酯酶抑制劑這類殺線蟲劑的感受性之間有相關性,再以芬滅松、加保扶及毆殺滅處理線蟲後計算其致死率。三種藥劑分別對Fm之半數致死濃度依序分別為103.6 ppm、226.7 ppm及116.2 ppm;而Rl則依序分別為167.2 ppm、2087.2 ppm及1166.7 ppm,顯示兩品系對乙醯膽鹼酯酶抑制劑類藥劑之感受性具顯著差異。以原位雜合法得知在Fm上Ace-1與Ace-2表現於體壁肌肉、食道部及生殖腺體,Ace-3則表現於食道部,前人研究已知Bursaphelenchus xylophilus與Caenorhbditis elegans的各種Ace基因功能性與表現位置,經比對與本研究之兩線蟲品系之表現位置相似,Ace-1與Ace-2與線蟲移行、取食行為相關,進而推測發生於兩基因的差異與乙醯膽鹼酯酶抑制劑的感受性較具相關性;Ace-3功能則尚未有相關研究及報導,且B. xylophilus的Ace-3研究顯示其對乙醯膽鹼酯酶抑制劑感受性低,因此推測它與A. besseyi產生抗藥性之機制相關性較低。
Rice white tip nematodes, Aphelenchoides besseyi, had been found on a wide range of plants, including rice, bird’s-nest fern, strawberry, and chrysanthemum. Two different host originated A. besseyi Fm and Rl isolates, collected from bird’s-nest fern and rice, were reported to have many differences, such as host ranges, modes of reproduction, native environments, as well as some parasitism associated genes. The house-keeping genes, acetylcholinesterase (Ace) genes were identified from A. besseyi Fm and Rl isolates in this study. Three Ace genes, Ace-1, Ace-2, and Ace-3 that encoded AChE1, AChE2 and AChE3, were found in both isolates of A. besseyi. After sequences alignment, there were some differences between the same Ace gene from two isolates, and Ace-2 gene showed the greatest differences. Several insecticide studies showed that target site insensitivity is the main resistance mechanism to organophosphates (OPs) and carbamates pesticides, a kind of acetylcholinesterase inhibitors. Single nucleotide polymorphisms (SNPs) is found to be responsible for the OP and cabamate target sites insensitivity. Three nematicides including fenamiphos, carbofuran and oxamyl were used to treat these two nematode isolates. The LD50 of Fm isolate to fenamiphos, carbofuran and oxamyl are 103.6 ppm, 226.7 ppm, and 116.2 ppm, respectively. The LD50 of Rl isolate to fenamiphos, carbofuran and oxamyl are 167.2 ppm, 2087.2 ppm, and 1166.7 ppm, respectively. The results showed that two isolates had different sensitivity to three nematicides tested. In situ hybridization of Ace genes from Fm isolate showed that Ace-1 and Ace-2 expressed in body wall, pharyngeal gland regions and intestine’s muscle cells, and Ace-3 expressed in pharyngeal gland regions. Ace-1 and Ace-2 were reported to associate with to movement and feeding behavior, while the function of Ace-3 is still unknown. In the B. xylophilus study, the affinity of AChE3 to acetylcholinesterase inhibitors was lower than AChE1 and AChE2. Our results also suggested that the differences between AChE1 and AChE2 might associate with the susceptibilities of A. besseyi to acetylcholinesterase inhibitors.
URI: http://hdl.handle.net/11455/95773
文章公開時間: 2020-08-18

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