| Because the resistance of cotton bollworm Helicoverpa armigera (Hübner) to Bt (Bacillusthuringiensis) is the key factor for the persistently planting transgenic cotton, in order to delay theresistance, multi-gene strategy and other effective resistance management strategies have been used inmany countries in the world, for example, the transgenic cotton which expressed both CrylAc andCry2Ab has been cultivated in some countries. But Cry2Ab which is the important protein in BollgardⅡ (transgenic cotton expressed Cry1Ac+Cry2Ab), itsmode of action has been undefined. In thisdissertation, the action mode of interaction between Cry1Ac and Cry2Ab about their effecting on H.armigera was studied using Bt protein and transgenic cotton. The insecticidal spectrums of Cry1Ac andCry2Ab to Lepidopera insects were tested. The pathological changes in larval midgut of H. armigerafed on artificial diet containing Cry2Ab protein were observed by transmission electron microscope.The effects of Cry2Ab, Cry1Ac, Cry1Ac+Cry2Ab on major detoxifying enzymes activities in midgut ofH. armigera were compared, the synergistic effects of specific enzyme inhibitor on the toxicities werealso tested. The resistances to target insect, expressed protein contents in different transgenic cotton(expressed Cry1AC, Cry2Ab, Cry1Ac+Cry2Ab respectively) were compared, and the feeding andovipositional preferences of H. armigera on transgenic cotton were also compared. The results were asfollows:1. The susceptibility of H. armigera, Spodoptera exigua, Spodoptera litura, Agrotis ypsilon andOstrinia furnacalis to Cry2Ab and Cry1Ac were tested using bioassay in laboratory. The resultsrevealed that H. armigera, S. exigua and P. nubilalisto larvae were very sensitive to Cry1Ac, with LC50values of0.02μg/cm2,0.25μg/cm2and0.06μg/cm2, while Cry1Ac had lower effect on S. litura and A.ypsilon larvae with LC50values of6.04μg/cm2and12.07μg/cm2. Howeve, H. armigera, S. exigua, S.litura and A. ypsilon were susceptible to Cry2Ab, with LC50values of0.83μg/cm2,0.32μg/cm2,0.15μg/cm2and0.90μg/cm2, respectively, but P. nubilalisto was not susceptible to Cry2Ab, with LC50values of2.74μg/cm2. Compare with Bt cotton which expressed Cry1Ac, the transgenic cottonexpressed Cry1Ac+Cry2Ab expanded its insecticidal spectrum.2. The pathological changes in the midgut of the H. armigera, fed on artificial diet containingCry2Ab protein were observed by transmission electron microscope, and the changes induced by fed onartificial diet containing Cry1Ac or Cry2Ab protein were compared. The results showed the midgut celland organelle both exhibited obvious pathological changes, and the pathological changes became moreobviously with extension of feeding time. Compared with H. armigera fed on artificial diet containingCry1Ac protein, the pathological changes caused by Cry2Ab protein was slower.3. The activities of total esterase, carboxylesterase (CarE), acetylcholinesterase (ACHE) andmixed-functional oxidase (MFO) in the midgut of H. armigera which respective treated by Cry1Ac,Cry2Ab and Cry1Ac+Cry2Ab were compared by the kinetic analysis. The results showed that activitiesof esterase were obviously increased after fed on artificial diet containing Cry1Ac or Cry2Ab, but therewere no obvious correlation with treated concentration. The activities of esterase, CarE and ACHE in H. armigera fed on diet containing13.34μg/g Cry2Ab+6.67μg/g Cry1Ac after12h, were significantlyhigher than those in other treatments and control. The activities of MFO in H. armigera fed on dietcontaining2.22μg/g Cry2Ab+13.34μg/g Cry1Ac after24h, were significantly higher than those inother treatments and control. The specific enzyme inhibitor, PBO had obviously synergized action onCry2Ab, while DEM had obviously synergized action on Cry1Ac. It indicated that Cry2Ab and Cry1Achad different effect on H. armigera midgut enzymes, Cry2Ab and Cry1Ac maybe had different actionmode, there were more influence on midgut enzymes when using Cry1Ac and Cry2Ab together.4. The resistances to target insect, expressed protein contents in different transgenic cotton(expressed Cry1Ac, Cry2Ab, Cry1Ac+Cry2Ab respectively) were compared using bioassay and enzymelinked immunoabsorbent assay (Elisa). The results showed the transgenic cotton had obviously toxiceffect on H. armigera, the order of corrected larval mortality from small to large was: BollgardⅡ(expressed Cry1Ac+Cry2Ab)>DP33B (expressed Cry1Ac)>cotton (expressed Cry2Ab). Theexpressed protein content and corrected larval mortality in different development period weresignificant different. The expressed protein content and corrected mortality rate were the highest inseedling stage, secondly was bud stage, the protein content and larval mortality were the lowest in bollstage. So, the transgenic cotton expressed two Bt different kind of protein is a trend of development inthe future, according to the consideration of toxic efficiency and resistant management.5. The feeding and ovipositional preference of H. armigera between conventional cotton andtransgenic cotton were studied. The feeding preference had significant difference between transgeniccotton and conventional cotton, larvae number and leaf injury situation on conventional cotton werehigher than that on transgenic cotton, while there were no significant difference between transgeniccotton expressed Cry2Ab or Cry1Ac. The ovipositional amount had no significant differences betweenconventional cotton and transgenic cotton, and there were also had no obviously difference amongtransgenic cotton. It demonstrated that the H. armigera larvae had feeding preference betweenconventional cotton and transgenic cotton, while the moth had no ovipositional preference. |
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