第四章 討論
4.2 豌豆蚜蟲的 RNAi 技術應用
本實驗中，藉由選殖體軸決定和眼部特化基因，並進一步做原位雜合實驗去 確認其基因表現的位置，比對不同物種之間同樣基因的表現位置，去推測其基因 在豌豆蚜蟲上可能的功能。依據目前的研究文獻和技術，豌豆蚜蟲還尚未出現適 合用來分析基因功能的技術，但由蚜蟲發展中的 RNAi (RNA interference) 技術可 窺見未來發展的趨勢。
RNAi 主要是使用外來的 dsRNA (double-stranded RNA) 進行基因靜默 (gene silencing) 使目標基因 mRNA 降解，其機制是 Dicer RNaseIII-type enzyme 將細胞 質內的 dsRNA 切成約 21 nt 長的 siRNA (small interfereing RNA)，siRNA 將與 RISC (RNA-inducing silencing complex) 結合，並藉由 antisense siRNA 序列去找到 對應的 mRNA，接著 RISC 將會使其 mRNA 降解讓其基因靜默 (Dykxhoorn et al., 2003)，這種方法主要用於研究目前沒有轉基因技術可用的生物體的基因功能，如 線蟲或昆蟲等 (Fire et al., 1998; Lum et al., 2003; Arakane et al., 2004; Dietzl et al., 2007; Schluns and Crozier, 2007; Chen et al., 2008; Suzuki et al., 2008; Yu et al., 2008;
Tian et al., 2009; Terenius et al., 2011; Na et al., 2013)，一般以昆蟲作為實驗材料時，
主 要 有 三 種 方 法 使 dsRNA 或 siRNA 進 入 個 體 的 細 胞 內 ： (1) 顯 微 注 射 (microinjection)，以注射器將 dsRNA 直接注入體腔或組織上，為目前 RNAi 主要
使用的方法，其優點為可避免生物體中的屏障如腸壁等，讓 dsRNA 作用在目標 的位置上，以及能夠精準計算出注入的體積和量；缺點為影響結果的變因過多，
如針頭形狀、注射位置和注射量可能都會影響個體的死亡率和效率等。(2) 餵食，
將 dsRNA 摻入到昆蟲的食物內或是給予轉基因植物作為食物，優點是能夠儉省 人力、花費且容易執行，因為是非入侵性實驗，所以人為致死率較低且適用於任 何齡期的幼蟲；缺點是效率不如顯微注射，且因為各個昆蟲腸胃環境不同，在有 些昆蟲上無法造成 RNAi，無法正確計算攝取的 dsRNA 量以及這攝取量是否會引 發 RNAi 也不得而之。(3) 浸泡，將個體或是細胞直接放入含有 dsRNA 的液體內，
優點是最為簡單和方便，但昆蟲細胞的效率比個體好，原因是個體本身有多重的 屏障如表皮等等 (Na et al., 2013)。
目前豌豆蚜蟲使用的方法是前兩個：顯微注射和餵食。以顯微注射為的實驗 中發現，不論是注入 dsRNA 或 siRNA 都可以達到類似的效果，表現量大約降低 25~45% 左右 (Mutti et al., 2006; Jaubert-Possamai et al., 2007)，持續時間最長無法 超過七天 (Jaubert-Pssamai et al., 2007)，已測試的基因包含 calreticulin、cathepsin L (Jaubert-Pssamai et al., 2007)、aquaporin (Shakesby et al., 2009) 和 c002 (Mutti et al., 2006, 2008) 等全組織或腸道表現基因，雖然沒有觀察到明顯的表型，致死率、
子代數、攝食行為及體內滲透壓的改變，說明其 RNAi 在蚜蟲上的作用，但無論 如何，至今沒有研究指出其 RNAi 能否有效地由體液進入微卵管內，甚至導致胚 胎發育過程中有任何顯著的缺陷，故目前能否使用 RNAi 顯微注射去研究豌豆蚜 蟲基因在胚胎發育過程中的角色，仍是一個未知數。
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