Recombinant Escherichia Coli Expressing DsRNA Of Arginine Kinase Gene Of Plutella Xylostella And Its RNA-Interference Effect

Arginine kinase (AK) is only found in invertebrate animals where it participatesin energy metabolism. It has been reported that the expression of the PxAK gene inPlutella xylostella is suppressed through the feeding of dsRNA of the PxAK gene andthat the growth and reproduction of P. xylostella are inhibited, even to death. However,due to the high cost of dsRNA synthesis and the ease of degradation of the dsRNAduring the process of conservation and application, it is not practical to use dsRNA inthe control of the diamondback moth. Therefore, a genetically modified Escherichiacoli was developed to efficiently produce and conserve PxAK dsRNA that wassubsequently fed to P. xylostella larvae to examine its effect and provide evidence forthe feasibility of using RNAi techniques in the control of the diamondback moth.The expression of the PxAK gene during the life cycle of the diamondback mothwas measured by quantitative PCR. The results showed that PxAK was expressedthroughout the entire life cycle. The relative expression level of PxAK was below10for the14days after hatching, but reached roughly20on day14when thediamondback moth reached the adult stage, suggesting that adults may need moreenergy to support a more active life style than larvae and pupae.Two PxAK fragments of604bp (AKC) and546bp (AKFC) were amplified usingprimers with Hind III and Not I restriction enzyme sites. The AKC sequence includesthe AK activity sites and the motif CPTNLGT. Both AKC and AKFC fragments werecloned into plasmid L4440following digestion with the Hind III and Not I enzymes.The recombinant plasmid was then transformed into E. coli HT115to express dsRNA.The transgenic dsRNA-expressing E. coli was incubated and fed to DBM larvaeat different concentrations, and the mortality, pupal weight, and laying amount wererecorded. Larval death was observed2days after treatment with bacteria expressingAKC dsRNA, and4days after treatment with bacteria expressing AKFC dsRNA,suggesting that AKC dsRNA is more active than AKFC dsRNA. The RNAi effects onDBM included weight loss, reduced oviposition and larval death.The DBMs were treated with E. coli HT115AKC and HT115AKFC of differentconcentrations, demonstating that the concentration of100×was the most effective.The PxAK gene expression was detected to be significantly suppressed in the DBM feeding bacteria expressing dsRNA using the real-time PCR. The relativeexpression was analyzed in the DBMs between the HT115AKC treatment and theHT115AKFC treatment. It showed that the effect of RNAi was better in theHT115AKC treatment.We first expressed PxAK dsRNA in E. coli and then used the bacteriaexpressing dsRNA to kill DBMs. This study shows that the AK gene in insects couldbe a good target gene for controlling agricultural insect pests and that transgenicbacteria expressing dsRNA could be a new approach to using the RNAi technique ininsect pest management.