The Applied Research Of E. Coli Expressing The DsRNA Of S-adenosyl-L-homocysteine Hydrolase (SAHase) Gene On The Potato Beetle

Leptinotarsa decemlineata(Say), belonging to Chrysomelidae, Coleoptera, is alsocalled Colorado potato beetle which is one kind of cosmopolitan pest quarantine object. Itconstitutes a catastrophic crisis for Potato Production in Xinjiang since1990s.This experiment order to test the effect of the adenosylhomocysteine genes dsRNA(dsSAHase) as a new scientific research tool applied in Integrated Pest Management (IPM).They include the Storage conditions affect toxic effects of E. coli expressing the dsSAHase onthe potato beetle, The toxicity measurement of dsSAHase in different stages of potato beetles,stability of dsRNA in interstitial fluids from Leptinotarsa decemlineata (Say) and thesynergistic effect of polyethyleneimine on dsRNA. The RNAi result will be beneficial toexploiting a new type of pesticide which is efficient, specific and environmentally friendly.The main results were summarized as follows:1、The toxicity measurement of dsSAHase by feeding different doses E. coli expresseddsSAHase to different stages of Colorado Potato Beetles. In this test, we determinated theLC50of dsRNA by liner regression method and observe other RNA interference effects. ThedsSAHase LC50s of the1st-4th stage potato beetle are2.341μg/ml,2.552μg/ml,3.450μg/ml,4.968μg/ml. Feeding dsSAHase caused female adults a negative oviposition responses. Thedevelopment level of ovarian from tested female adults have a positive correlation withfecundity. The LC50of different age larval increased as larval grow. And the LC50of the1stand2nd stage larval significantly lower than the3rd and4th age.2、 Storage conditions affect toxic effects of E. coli expressing the dsRNA ofS-adenosyl-L-homocysteine hydrolase (SAHase) gene on the potato beetle: In the presentpaper, we expressed S-adenosyl-L-homocysteine hydrolase gene dsRNA in E. coli. We storedliving or sterile dsRNA expressing E. coli at different temperature for different times, andcompared the toxic effects on Leptinotarsa decemlineata larvae. The E. coli dissoluted in PBShad higher biological activity after24h of storage under lower temperature than fresh E. coli,and after24h of storage under the higher temperature, the E. coli still have a high biologicalactivity, however, the effect sharply decreased after48h. In the test of storage E. coli in lowertemperature, the difference of biological activity between the dsRNA expressed E. colicultured in the medium and dissolution in PBS is not significant, and the difference of storageE. coli in the higher temperatureis significant. No significant difference was found betweenliving or sterile dsRNA expressing E. coli and different temperature. Twenty-four hoursstorage under-20℃,4℃, room tempreture and37℃has little influence on toxic effect ofdsRNA expressing E. coli.3、Degradation of dsRNA in Colorado Potato Beetles and the protective effect of polyethyleneimine to dsRNA: The degradation was estimated using gel electrophoresis toanalysize E. coli total nucleic acid after incubation with the midgut fluid or the haemolymph.The toxic effects of dsSAHase and PEI-dsRSAHase mixture to Leptinotarsa decemlineatalarvae were tested. Either the midgut fluid or the haemolymph can degrade the dsSAHase.And the stability is not affected by temperature. Moreover, the mortality of Leptinotarsadecemlineata larvae exposed to PEI-dsSAHase mixture is75%, and the mortality ofdsSAHase-treated insects alone is55%.The dsRNA is degraded during delivery. And PEI canprotect dsRNA against enzymatic degradation. This result indicates a potential application ofPEI in dsRNA protection.In conclusion, our experiment confirm that the dsSAHase have a significantly lethalityfor each stage of potato beetle larvae. This effect will reduce as age growth. The ovipositionof Potato beetle female adult can be reduced by feeding dsSAHase. E. coil expressed dsRNAcan be storage in twenty-four hours. Potato beetle body fluid caused a significantlydegradation of dsRNA. Polyethyleneimine have a positive effect on dsRNA.