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穀蠹細胞色素P450 cDNA之選殖及其表現與第滅寧藥劑感受性之探討

By Yin-Yin Liao and 廖盈盈

Abstract

To understand the effect of cytochrome P450 on the susceptibility of the lesser grain borer (Rhyzopertha dominica) to deltamethrin, the cytochrome P450 cDNA of R. dominica was cloned and expressed for analysis. Full length cDNA of 1601 bp, which encodes a 505-amino acid protein, was obtained through RT-PCR and RACE. The BLAST result revealed the sequence was highly similar to Tribolium castaneum cyp6b subfamily. Therefore, the cDNA was named Rdcyp6b. The topology of phylogenetic tree indicated Rdcyp6b and T. castaneum cyp6bq9 cluster into a clade. It suggested the possible function of Rdcyp6b is to metabolize deltamethrin as the cyp6bq9 does. Further, the bioassay was carried out to determine the susceptibility of four populations (Hukou, Yangmei, Sansing and Shenkang) to deltamethrin, resulted the LC50 values of 2.50, 4.34, 6.29 and 8.20 μg/ml, respectively. The resistance ratio of Yangmei population was 1.73 to Hukou population. The resistance ratio of Sansing and Shenkang were 2.5 and 3.3 fold, respectively. Using the real-time quantitative PCR, the Rdcyp6b expression of four populations was detected. The results showed Rdcyp6b was highly expressed in Shenkang population as its LC50 value was highest. However, the expression of Rdcyp6b and the LC50 values among the other three populations was not consistent. It could be that non-significant resistance ratio among populations. Moreover, Rdcyp6b was mainly expressed in the thorax.為探討細胞色素P450 (cytochrome P450, CYP) 於穀蠹 (Rhyzopertha dominica) 對第滅寧藥劑感受性的影響,本研究選殖穀蠹細胞色素 P450 cDNA 並分析其表現情形。藉由RT-PCR (reverse transcription-PCR) 及RACE (rapid amplification of cDNA ends) 得到 全長1601 bp 的CYP cDNA,可轉譯505 個胺基酸。比對此cDNA 序 列的結果顯示與擬穀盜 (Tribolium castaneum) cyp6b 亞家族的基因關 係相近,將之命名為Rhyzopertha dominica cyp6b (Rdcyp6b)。建構 Neighbor-joining 親緣關係樹顯示Rdcyp6b 與擬穀盜cyp6bq9 相近,推 測與第滅寧解毒有關。接著,利用混拌法測試湖口、楊梅、三星和伸 港四個穀蠹族群對第滅寧之感受性,其LC50 依序為2.50、4.34、6.29和8.20 μg/ml。相較於感受性最低的湖口族群,楊梅穀蠹相對抗性為1.7 倍,三星與伸港族群的相對抗性分別為2.5 與3.3 倍。進一步以即時定量 (real-time quantitative) PCR偵測四個族群Rdcyp6b之表現量,結果顯示伸港表現量最高,而其餘三個族群之Rdcyp6b 表現情形與預期不符合,未能與第滅寧感受性之結果相對應,推測為其相對抗性之差距不大,未能呈現感性與抗性族群Rdcyp6b 之表現差異。另以real-time quantitative PCR測試穀蠹頭、胸和腹部Rdcyp6 的表現情形,結果顯示於胸部之表現量最高。致謝 ............................................................................................................................................i 摘要 ........................................................................................................................................... ii Abstract .................................................................................................................................... iii 目錄 .......................................................................................................................................... vi 圖次 ......................................................................................................................................... vii 前言 .......................................................................................................................................... 1 前人研究 .................................................................................................................................. 3 一、穀蠹生活習性 ............................................................................................................... 3 二、穀蠹危害情形 ............................................................................................................... 5 三、穀蠹防治情形 ............................................................................................................... 6 四、第滅寧藥劑簡介 ........................................................................................................... 8 五、細胞色素P450 單氧化酶之簡介 ............................................................................... 11 材料與方法 ............................................................................................................................. 14 一、試驗昆蟲之飼育 ......................................................................................................... 14 二、總量RNA 萃取 ........................................................................................................... 14 三、反轉錄作 ..................................................................................................................... 15 四、穀蠹細胞色素P450 單氧化酶之選殖 ....................................................................... 16 五、接合作用 ..................................................................................................................... 18 六、質體轉型 ..................................................................................................................... 18 七、質體純化 ..................................................................................................................... 19 八、選殖株之確認 ............................................................................................................. 20 九、DNA 定序及分析 ....................................................................................................... 20 十、cDNA 末端快速擴增法 .............................................................................................. 21 十一、親緣關係樹之建構及分析 ..................................................................................... 24 十二、穀蠹基因轉錄表現之偵測 ..................................................................................... 24 十三、藥劑測試 ................................................................................................................. 26 十四、半致死濃度分析 ..................................................................................................... 26 結果與討論 ............................................................................................................................. 28 一、穀蠹細胞色素P450 cDNA 選殖與序列分析 ............................................................ 28 二、與其他昆蟲細胞色素P450 之親緣關係 ................................................................... 30 三、穀蠹對第滅寧藥劑之感受性測試 ............................................................................. 31 四、穀蠹細胞色素P450 Rdcyp6b 於不同族群的表現情形 ............................................ 32 五、細胞色素P450 於不同部位的表現情形 ................................................................... 34 六、結論 ............................................................................................................................. 35 參考文獻 ................................................................................................................................ 36 表 ............................................................................................................................................ 45 圖 ............................................................................................................................................ 4

Topics: 穀蠹, 細胞色素P450, 第滅寧, Rhyzopertha dominica, cytochrome P450, deltamethrin
Year: 2016
DOI identifier: 10.1186/1471-2164-9-538
OAI identifier: oai:ir.lib.nchu.edu.tw:123456789/85982
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