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RNA干扰(RNAi)是由双链RNA(dsRNA)引发的内源特异性基因转录后沉默机制[1]。dsRNA导入生物体内后,被细胞中称为Dicer的RNase Ⅲ分解成21~23 bp的小干扰RNA (siRNA),siRNA在沉默复合体(RISC)的作用下与目标mRNA结合,序列特异性地降解靶mRNA,阻止相应蛋白产物的合成,造成靶标基因的功能丧失[2]。自从这种快速且直接沉默特定基因的机制被发现以来,RNAi技术迅速在非模式动物的基因功能研究领域得到广泛应用[3]。在多种农林害虫中的成功试验[4-6],也证实了利用RNAi进行害虫防治的可能性和广泛性。
RNAi技术在害虫防治领域的应用需要大剂量dsRNA,目前最常用的试剂盒合成方法价格昂贵、不能持续生产。利用转基因寄主植物表达昆虫源dsRNA对害虫有一定防效[4, 7],但植物转基因难度高、研究周期长,具有很大的局限性。用细菌表达dsRNA的方法,最早由Timmons和Fire开创使用,他们将带有靶基因片段的L4440重组载体转入BL21(DE3)菌株合成dsRNA,饲喂线虫引发RNAi[8]。随后他们从多个菌株中筛选出具有高dsRNA表达效率的HT115菌株,并在线虫中得到应用[9]。RNaseIII是细菌中普遍存在的dsRNA特异性核酸内切酶,由rnc基因编码,HT115菌株为rnc-突变不能降解自身合成的dsRNA[10]。HT115经过修饰,插入一个λDE3噬菌体衍生物,可通过IPTG诱导表达T7 RNA聚合酶[11]。HT115的四环素和Amp抗性,可在培养过程中分别排除rnc+反突变体和空载质粒。L4440载体序列的多克隆位点两侧各有一个相反方向的T7聚合酶启动子,转入HT115菌株后,可经IPTG诱导表达dsRNA[8]。在玉米上喷施表达菌液可抑制花叶病毒的感染[12],饲喂或注射细菌表达的dsRNA也能显著干扰甜菜夜蛾[13]、非洲甘薯象鼻虫[14]等害虫的生长,证明该技术在植物保护方面的应用潜力。
美国白蛾(Hyphantria cunea(Drury))是我国重要检疫性害虫,其幼虫取食范围超过175种植物,包括几乎所有的园林树木、花卉及农作物[15]。美国白蛾繁殖力和传播能力强[16],适生范围广,严重威胁我国生态安全。目前对该害虫的防治手段主要有人工防治、化学防治和利用寄生天敌及性诱剂的生物防治等,这些手段具有一定的防治效果,但总体存在效率低、成本高、对生态环境不友好的问题[17],因此RNA干扰这种靶标性强、环境友好型的技术在害虫防控领域展现较大的应用潜力。几丁质主要存在于昆虫的表皮、中肠围食膜(占4%~20%)中,形成昆虫的外骨骼,起支撑和保护作用[18]。几丁质酶是几丁质水解的关键酶,在昆虫的变态发育中参与新旧表皮的更替和围食膜的降解,选择几丁质酶基因作为RNA干扰靶标,破坏昆虫几丁质表达通路,阻止昆虫的蜕皮、围食膜再生等生理过程,可达到害虫防治的目的[19]。本文以L4440质粒为载体,构建了利用大肠杆菌HT115表达美国白蛾几丁质酶dsRNA的体系,通过对美国白蛾幼虫饲喂细菌表达的dsRNA,检测该方法介导的RNAi在美国白蛾中的可行性。
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