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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。
美国白蛾几丁质酶细菌表达的RNA干扰载体构建及其介导的RNA干扰
Construction of the Expression Vector and RNAi Mediated by Bacteria Expressed dsRNA of Chitinase Gene from Hyphantria cunea
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摘要:
目的 探索细菌表达dsRNA介导的RNAi在美国白蛾中的可行性,为RNAi技术在美国白蛾等林业害虫上的应用提供依据。 方法 选择几丁质酶HcChi基因作为靶标,设计有效的干扰片段,构建到L4440干扰载体上,并转入HT115大肠杆菌菌株。IPTG诱导HT115表达HcChi的干扰片段,用菌液持续饲喂美国白蛾幼虫,观察幼虫生长情况,定量PCR检测HcChi的转录水平。 结果 构建了带有HcChi-L4440表达载体的HT115菌株,经IPTG诱导能够合成HcChi-dsRNA,浓缩菌液持续饲喂幼虫显著抑制HcChi的表达,相对表达量显著下降了76.7%~90.3%,幼虫生长发育迟缓,体重增长量与对照相比显著减小40.7%。 结论 成功构建HcChi RNA干扰载体,通过饲喂法在美国白蛾中获得RNAi效应。该体系首次在美国白蛾中建立,为该物种的基因功能研究和生物防治提供新思路。 Abstract:Objective To explore the feasibility of dsRNA expressed by HT115-mediated RNAi in Hyphantria cunea. Method Hyphantria cunea chitinase gene (HcChi) was chosen as the target gene. An interference fragment was designed and inserted into the expression vector L4440, and then transformed into the Escherichia coli strain HT115. The H. cunea larvae were fed on bacteria solution which was induced by IPTG. The growth of larvae were observed, and the mRNA level of HcChi was detected using qPCR. Result The HT115 strain containing HcChi-L4440 expression vector can express HcChi-dsRNA by IPTG induction. After fed on bacteria solution, the expression level of HcChi in H. cunea decreased significantly by 76.7%-90.3%. The body weight growth decreased about 40.7% compared with the control. Conclusion The RNA interference vector was constructed successfully. The effect of RNAi was observed in H. cunea by feeding method. This is the first time that developing a method of RNAi based on bacteria expressed dsRNA in H. cunea, which provides a new idea on the study of gene function and biological control of H. cunea. -
Key words:
- Hyphantria cunea
- / HT115 strain
- / L4440
- / chitinase
- / RNAi
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