Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97889
標題: 賜諾殺抗性穀蠹之交互抗性及差異基因表現
Cross-resistance and differential gene expression of spinosad-resistant Rhyzopertha dominica
作者: 王顗鈞
Yi-Chun Wang
關鍵字: 穀蠹
賜諾殺
抗性衰退
交互抗性
cDNA篩減法
Rhyzopertha dominica
spinosad
relaxation
cross-resistance
cDNA subtraction
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摘要: 穀蠹 (Rhyzopertha dominica) 為穀倉內的害蟲,主要以殺蟲劑進行防治,但長期施用相同藥劑容易使族群產生抗性,因此需輪替使用藥劑以減緩抗性產生速率。賜諾殺 (spinosad) 是一種生物性的殺蟲劑,其對環境的汙染較低且對哺乳動物較無毒害,因此適合做為防治穀蠹的藥劑。穀蠹對賜諾殺產生抗性已知是因其尼古丁乙醯膽鹼受器 (nicotinic acetylcholine receptor) α6亞單位產生突變。然而,前人之篩選實驗中顯示穀蠹在兩年內就產生了對賜諾殺的抗性,若只是因基因突變其抗性應不會發展如此迅速。因此,本研究探討穀蠹對賜諾殺產生抗性的其他可能原因,同時也探討抗性衰退及交互抗性在賜諾殺抗性穀蠹上的情形。利用混拌法篩選抗性品系。在抗性衰退實驗的部分,在未經藥劑處理約一年之後,穀蠹對賜諾殺的半致死濃度從29.96 μg/ml下降到3.92 μg/ml,抗性倍率降低了86.92%左右;而在交互抗性測試的部分,顯示賜諾殺抗性穀蠹對於相同作用機制的賜諾特有交互抗性產生,而對於不同作用機制的第滅寧、馬拉松、益達胺、免速達及剋安勃則無交互抗性。另外利用cDNA篩減法 (cDNA subtraction) 篩選出抗性品系或感性品系中表現量較高的基因。從被篩選出的基因中顯示40S ribosomal protein S20在感性品系內之比例為57.21%,較在抗性品系之比例31.07%高。麥芽糖酶 (maltase) 在抗性品系中之比例為17.48%,較在感性品系之比例3.37%高。而croquemort protein在感性品系內之比例為11.54%,在抗性品系則未發現此基因。以即時定量PCR比較麥芽糖酶及croquemort protein在感、抗性穀蠹間的相對表現量,結果顯示此二基因在感、抗性間的表現量皆無顯著差異。而因為麥芽糖酶是分解還原糖的酵素,賜諾殺分子內又具有還原糖,因此使用3,5-二硝基水楊酸 (3,5-dinitrosalicylic acid, DNS) 檢測賜諾殺是否會被穀蠹體內的酵素所分解。將感、抗性穀蠹之粗研磨液及賜諾殺加入DNS反應後,從吸光值判斷研磨液內的還原糖濃度,結果顯示抗性品系的吸光值較感性品系高,且具有顯著性差異,因此推論抗性穀蠹體內具有分解賜諾殺的酵素。透過了解賜諾殺抗性品系之抗性衰退、賜諾殺抗性品系與其他藥劑之交互抗性,以及賜諾殺感、抗性品系之基因表現差異等結果,可提供未來以賜諾殺進行穀蠹防治時之參考資料。
The lesser grain borer, Rhyzopertha dominica, is a barn pest, and mainly controlled by insecticides. It is easy for R. dominica to develop resistance to the same insecticide after the long term usage. Therefore an alternative use of insecticides is necessary. Spinosad is a biological insecticide which is low toxic to environment and mammals. So it is suitable for the control of R. dominica. Resistance of R. dominica to spinosad is due to the mutation of nicotinic acetylcholine receptor α6 subunit. However, R. dominica develops resistance to spinosad after two years of selection, but the resistance should not develop so rapidly as a result of gene mutation only. It leads to this study for further understanding of other resistant mechanisms of R. dominica to spinosad. Furthermore, relaxation and cross-resistance tests were conducted. A resistant strain was selected using a modified grain application method. In the relaxation examine, median lethal concentration of resistant R. dominica decreased from 29.96 μg/ml to 3.92 μg/ml in one year without contact any insecticide. The resistant ratio was decreased 86.92%. In the cross-resistance examine, it showed that there is cross-resistance between spinosad and spinetoram, which has the same mode of action as that of spinosad. On the other hand, there was no cross-resistance shown between spinosad and other insecticides which with different mode of actions including deltamethrin, malathion, imidacloprid, bensultap, and chlorantraniliprole. Moreover, cDNA subtraction was applied to examine the differential gene expression between the susceptible and resistant strains. It appeared that 40S ribosomal protein S20 was 57.21% and 31.07% in the susceptible and resistant strains, respectively. Maltase was 3.37% and 17.48% in the susceptible and resistant strains, respectively. Croquemort protein was 11.54% in the susceptible strain, but not found in the resistant strain. The gene expression of both maltase and croquemort protein was not significantly different between the susceptible and resistant strains by real-time quantitative PCR. Maltase is an enzyme which can hydrolyze reducing sugar. There is a reducing sugar unit in the structure of spinosad. So we can use 3, 5-Dinitrosalicylic acid to detect whether spinosad was hydrolyzed by enzymes from the susceptible and resistant strains. The result showed that optical density was significantly higher from resistant strain sample than that from susceptible one. It suggested that there could be an enzyme from resistant R. dominica hydrolyzing spinosad. The results from this study will provide the information for R. dominica control strategy including the relaxation of resistance, cross-resistance of the spinosad resistant strain, and the differentially expressed genes between susceptible and resistant strains.
URI: http://hdl.handle.net/11455/97889
文章公開時間: 2020-02-12
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