Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89076
標題: Nicotinic acetylcholine receptor subunit α6 associated with spinosad resistance in Rhyzopertha dominica
榖蠹之尼古丁乙醯膽鹼受器α6與賜諾殺抗性之相關性
作者: 王翰棠
Han-Tang Wang
關鍵字: 榖蠹;尼古丁乙醯膽鹼受器α6;賜諾殺;抗性;Rhyzopertha dominica;nicotinic acetylcholine receptor subunit α6;spinosad;resistance
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摘要: 
在臺灣尚未以賜諾殺做為穀倉中榖蠹 (Rhyzopertha dominica) 防治藥劑,採集自伸港之族群視為相對感性族群並隔離飼育為感性品系,其對賜諾殺之感受性 LC50 為0.14 μg/ml。利用賜諾殺篩選實驗室混合族群,經6次篩選後其 LC50 為12.0 μg/ml,相較於感性品系有約86倍抗性產生。以即時定量 PCR 測試感、抗品系之解毒酵素 Rdcyp6b 表現量,結果顯示兩品系間 Rdcyp6b 表現量無顯著差異,另外賜諾殺加入協力劑 PBO 抑制解毒酵素,抗性榖蠹的死亡率亦無顯著下降,顯示解毒酵素非榖蠹對賜諾殺產生抗性之主要因子。進一步選殖並分析穀蠹之尼古丁乙醯膽鹼受器α6亞單位 (RdnAChRα6) 在感、抗性品系間之差異。利用 Reverse transcription-PCR、Rapid amplification of cDNA ends 從感性穀蠹選殖得到全長 2133 bp cDNA,序列中之開放讀架為1497 bp,可轉譯出498個胺基酸。RdnAChRα6具有4個穿膜結構,於N端之胞外區域具有六個配體結合位,RdnAChRα6序列與擬穀盜及多種不同昆蟲之 nAChR α6 有極高之相同度。感性品系及抗性品系 (LC50: 10.3 μg/ml) 尼古丁乙醯膽鹼受器α6亞單位 cDNA 全長序列經比對後有三種不同突變情形,分別為:(1) 第二穿膜區 87 bp 片段缺失;(2) 近N端181 bp 缺失並造成終止密碼子提前出現;及 (3) 於開放讀架第 799 bp 的位置缺失 1 bp 產生移碼突變。另以 real-time quantitative PCR 測試感、抗性品系尼古丁乙醯膽鹼受器α6亞單位表現情形,兩者表現量有顯著差異,抗性品系表現量較感性品系低,尼古丁乙醯膽鹼受器α6亞單位突變及表現量下降皆可能使賜諾殺不易結合而導致榖蠹對其產生抗性。

In Taiwan, spinosad is not applied to control the lesser grain borer (Rhyzopertha dominica) in barns. The population collected from Shenkang were regarded as susceptible population and maintained without selection as susceptible strain in the laboratory. The susceptibility of susceptible strain was with an LC50 value of 0.14 μg/ml. Mixed populations from laboratory were selected with spinosad as a resistant strain. After six times of selection, the resistance strain was with an LC50 value of 12.0 μg/ml while the resistance ratio were about 86 folds compared with the susceptible strain. Real-time quantitative PCR was carried out to detect the expression of the detoxification enzyme, Rdcyp6b, in susceptible and resistance strains. The results showed the expression of Rdcyp6b was not significantly different between two strains. In addition, the mortality of spinosad resistant R. dominica was no significantly decreased after the application of synergist PBO in spinosad to inhibit detoxification enzyme. It suggests that detoxification enzyme was not the main factor for R. dominica developing resistance to spinosad. The nAChR subunit α6 cDNA of R. dominica (RdnAChRα6) was cloned and analyzed using reverse transcription-PCR and rapid amplification of cDNA ends. The full length cDNA of RdnAChRα6 was obtained. The complete 2133 bp cDNA containing and the open reading frame which is 1497 bp and encodes a 498-amino acid protein. There were four transmembrane regions and six ligand binding sites at the N-terminal of the extracellular region in RdnAChRα6. The sequence was highly similar to Tribolium castaneum and many insect nAChR subunit α6. After comparison of RdnAChRα6 sequences between susceptible and resistance strains, there were three different mutation situations, 1) 87 bp truncated in the transmembrane region 2, 2) 181bp truncated in N-terminal results in appearance of the premature stop codons, and 3) 1bp missing at the site 799bp in open reading frame produced frameshift mutation. Except for compared sequence, real-time quantitative PCR was implemented to detect the expression of RdnAChRα6 in susceptible and resistance strains. The results showed the expression of RdnAChRα6 was significantly lower in resistance strain compare with susceptible strain . In conclusion, mutation or reduced expression level of RdnAChRα6 may cause R. dominica resistant to spinosad because of target site insensitivity.
URI: http://hdl.handle.net/11455/89076
其他識別: U0005-2108201516204300
Rights: 同意授權瀏覽/列印電子全文服務,2017-08-25起公開。
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