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 4th 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 Gadd45g, were detected through qPCR. Simultaneously, the pupation rate, average pupation time, rate of deformed pupae and larval mortality of the 4th 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 Gadd45g increased significantly after RNAi of Br-Z2/3 in the 4th 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.

Key words: Plutella xylostella; Br-Z2/3, apoptotic genes, dsRNA, RNA interference, microinjection, pupation