| As a kind of oligophagous pest that damages cruciferous vegetables,Plutella xylostella has become one of the insects resistant to almost all kinds of pesticides.It causes up to 770 million dollars in agricultural economic losses in China every year.At present,field population of P.xylostella in central China has a level of 250-400-fold to Bt toxin.Previous studies in our laboratory showed that the protein expressed by the toxin binding region fragment of the cadherin and alkaline phosphatase of P.xylostella significantly enhance,the effect on P.xylostella biology and enzyme activity in vivo is not clear.In this study,the cadherin CAD and alkaline phosphatase ALP gene fragments of the diamondback moth Cry toxin binding region were coloned,the expression product PxCAD1 and PxALP was mixed with Cry1 Ac toxin and fed to the 3 instar diamondback moth larvae,observing and record changes in mortality,biological characteristics of surviving larvae and enzyme activity in vivo,the synergistic effect of PxCAD1 and PxALP in the poisoning process of the diamondback moth by Cry1 Ac were initially clarified.The specific results are as follows:1.Two Cry toxin binding region fragments of cadherin genes PxCAD1(751 bp)and PxCAD-m(751 bp)were obtained in this research,both of which can encode 247 amino acids,and there are 6 amino acid differences between them.A kind of alkaline phosphatase gene Cry toxin binding region fragment PxALP(983 bp)was obtained,which can encode322 amino acids.There were 2 N-glycosylation sites in the PxALP peptide,that is Gal NAc.Three responding recombinant protein PxCAD1,PxCAD-m and PxALP were obtained after expressed in E.coli.2.The PxCAD1(or PxCAD-m or PxALP)were mixed with Cry1 Ac toxin and fed to 3instar larvae of P.xylostella.It was found that the PxCAD1 and PxALP had a significant synergistic effect on the Cry1 Ac toxin,the mortality increased by 21.82% and 27.91%,but the PxCAD-m had no significant synergizing effect.3.Compared to the control,after fed on the mixture of PxCAD1(or PxCAD-m or PxALP)and Cry1 Ac toxin:(1)The PxCAD1 treatment group.The pupation rate and the emergence rate of the diamondback moth larvae were reduced,and the adult longevity of female and male and fecundity per female increased,the pupation rate decreased,the adult longevity of female and male increased,and the fecundity per female increased,but there were no significant difference among them;the emergence rate was 48.71%,a significant decrease of 26.76% compared to the control(P<0.05);the hatching rate of progeny diamondback moth eggs was 76.47%,a significant decrease of 21.39% compared to the control(P<0.05).(2)The PxCAD-m treatment group,the pupation rate and emergence rate increased,and the fecundity per female reduced,while there were no big difference among them,hatching rate of progeny diamondback moth eggs was 62.37 %,a significant decrease of 29.49%(P<0.05).(3)The PxALP treatment group.the pupation rate and the emergence rate was reduced,and the adult longevity of female and the fecundity per female was increased,but the difference was not significant.It showed that feeding the mixture of PxCAD1 or PxALP and Cry1 Ac protein significantly influenced the growth and development of P.xylostella larvae,the pupation rate and emergence rate decreased,the emergence rate in PxCAD1 treatment group was significantly reduced.Meanwhile,hatching rate of progeny diamondback moth eggs in the PxCAD1,PxCAD-m treatment group was significantly reduced,which further influenced the population reproduction of the test insects.4.After feeding the mixture of PxCAD1(or PxCAD-m,or PxALP)and Cry1 Ac toxin,the detoxification enzyme activity of P.xylostella larvae at the three time points of 24 h,48h and 72 h were as follows:(1)Carboxylesterase(Car E).In the PxCAD1 and PxALP treatment groups,Car E activities were significantly higher than that in the control(P<0.05),but at 72 h,there was no significant difference of the Car E activity between the PxCAD1 treatment group and control;Car E activity was significantly lower in the PxCAD-m treatment group at 24 h and 72 h than control(P<0.05),and significantly higher than the treatment group at 48h(P<0.05);(2)Glutathione S-transferases(GSTs).In the PxCAD1,PxCAD-m and PxALP treatment groups,GSTs activity was significantly lower than control(P<0.05),except the activity of GSTs in the PxALP treatment group which showed significantly higher than control at 72h(P<0.05).It shows that the mixture of PxCAD1 and Cry1 Ac toxin can induce the increase of Car E activity in the test insects,but has a significant inhibitory effect on the activity of GSTs;while the mixture of PxCAD-m and Cry1 Ac toxin has a certain inhibitory effect on the activity of Car E and GSTs in the test insects.The mixture of PxALP and Cry1 Ac toxin can induce the increase of Car E and GSTs activity in the test insects.5.After feeding the mixture of PxCAD1(or PxCAD-m,or PxALP)and Cry1 Ac toxin,the protective enzymes of P.xylostella larvae showed that(1)Superoxide dismutase(SOD).In the PxCAD1,PxCAD-m and PxALP treatment groups,the SOD activity was significantly higher than control at 24h(P<0.05),and there was no significant difference at48 h and 72 h in the control at the corresponding time points(P<0.05);(2)Catalase(CAT).At 24 h,the SOD activity of the PxCAD1 treatment group was significantly higher than that of the control(P<0.05),there was no significant difference between PxCAD-m treatment group and control(P<0.05),and the PxALP treatment group was significantly lower than control(P<0.05).But the three treatment groups were significantly lower than control at48 h,and no significant difference from the control at 72 h.Contrast to control,the protective enzyme activity in the treatment group was significantly influenced after fed on the mixture of receptor peptide and Cry1 Ac toxin.The results showed that after fed on the mixture of PxCAD1(PxCAD-m and PxALP)and Cry1 Ac,the SOD and CAT activities of Plutella xylostella were inhibited as the treatment time extended.6.After fed on the mixture of PxCAD1 and PxCAD-m with Cry1 Ac toxin,the digestive enzymes in diamondback moth larvae showed that(1)At 24 h,the total protease activity of the larvae in the PxCAD1 treatment group was significantly lower than that in the control(P<0.05),but there was no significant difference between the PxCAD-m treatment group and control;At 48 h and 72 h,there was no significant difference in total protease activity between the treatment group and control at the corresponding time point.(2)At 24 h,48h and 72 h,the activity of trypsin in the treatment group was not significantly different from that of the control at the corresponding time point.This indicates that the mixed protein of PxCAD1 and Cry1 Ac has an inhibitory effect on the total protease activity in the test insects in the early treatment.The results showed that PxCAD1 and PxALP played a significant synergistic effect on Cry1 Ac toxin,and the mortality of P.xylostella larvae was significantly increased.Feeding on PxCAD1 will lead to a decrease in the pupation rate,and the emergence rate of P.xylostella was significantly reduced,the mixture of PxALP and Cry1 Ac protein will lead to a decrease in the pupation rate and the emergence rate of diamondback moth larvae,while feeding on PxCAD1 and the mixture of PxCAD-m and Cry1 Ac toxin will cause hatching rate of progeny diamondback moth eggs of P.xylostella was significantly reduced.The reduction further affected the population reproduction of diamondback moth;After feeding on the mixture,the detoxification,protective and digestive enzymes in the diamondback moth larvae are also affected in different degree,showing a significant increase or decrease compared to the control.This study provides a theoretical basis for the development of newly efficient and safe Bt synergist,which is of great significance to delaying the development of the resistance of diamondback moth to Bt,and also provide a new theoretical basis for the comprehensive management of P.xylostella. |
PDF Full Text Request