【摘要】 【目的】本研究旨在构建用于小菜蛾Plutella xylostella蛹期特异表达基因Br-Z2/3 dsRNA合成的体外原核表达系统,研究RNAi抑制Br-Z2/3基因对小菜蛾Br-Z2/3和细胞凋亡基因表达及化蛹的影响。【方法】构建小菜蛾L4440-Br-Z2/3重组载体,转化大肠杆菌Escherichia coli HT115感受态细胞,经IPTG诱导大量获得Br-Z2/3 dsRNA,显微注射Br-Z2/3 dsRNA至小菜蛾4龄幼虫进行RNAi; qPCR检测干扰Br-Z2/3基因12和24 h后小菜蛾4龄幼虫Br-Z2/3及其下游细胞凋亡基因reaper,caspase-9和Gadd45g的表达量;观察并统计Br-Z2/3 RNAi后小菜蛾4龄幼虫的化蛹率、平均化蛹时间、蛹畸形率和幼虫死亡率。【结果】成功实现了Br-Z2/3 dsRNA的原核表达。qPCR结果表明,RNAi干扰Br-Z2/3后,小菜蛾4龄幼虫Br-Z2/3基因和相关联的细胞凋亡基因reaper表达量显著下降,但caspase-9和Gadd45g表达量显著上升。注射Br-Z2/3 dsRNA的处理组,化蛹率显著低于对照组,且化蛹高峰期推迟,幼虫死亡率显著高于对照组且畸形蛹率增加。【结论】本研究成功构建了用于小菜蛾Br-Z2/3基因dsRNA合成的体外原核表达系统,利用显微注射法对Br-Z2/3进行RNA干扰,证明Br-Z2/3是调控小菜蛾化蛹的关键基因。更多还原

【Abstract】 【Aim】 This study aims to establish an in vitro prokaryotic expression system for synthesis of the dsRNA of pupa-specific expression gene Br-Z2/3 from the diamondback moth, Plutella xylostella, and to find out the effects of RNAi-mediated suppression of Br-Z2/3 on the expression of Br-Z2/3 and apoptotic genes and the pupation of the moth. 【Methods】 The L4440-Br-Z2/3 recombinant vector was constructed and transformed into competent cells of Escherichia coli HT115. The dsRNA of Br-Z2/3 was obtained after induction with IPTG, and RNAi was carried out by microinjecting Br-Z2/3 dsRNA into the 4 th instar larvae of P. xylostella. At 12 and 24 h after RNAi of Br-Z2/3, the expression levels of Br-Z2/3 and its downstream apoptotic genes, reaper, caspase-9 and Gadd45 g, were detected through qPCR. Simultaneously, the pupation rate, average pupation time, rate of deformed pupae and larval mortality of the 4 th instar larvae of P. xylostella after RNAi of Br-Z2/3 were observed and recorded. 【Results】 The dsRNA of Br-Z2/3 was successfully expressed in the prokaryotic system. The qPCR results showed that the expression levels of Br-Z2/3 and the related apoptotic gene reaper decreased significantly, but those of caspase-9 and Gadd45 g increased significantly after RNAi of Br-Z2/3 in the 4 th instar larvae of P. xylostella. Compared with the control group, the dsBr-Z2/3-injected group showed significantly lower pupation rate with delayed pupation peak, and had significantly higher mortality and higher rate of deformed pupae. 【Conclusion】 An in vitro prokaryotic expression system for synthesis of the dsRNA of Br-Z2/3 from P. xylostella was successfully established. RNAi of Br-Z2/3 by microinjection demonstrates that Br-Z2/3 plays a crucial role in the pupation of P. xylostella.更多还原