Effects Of Elevated CO₂ On Expression Of Exogenous Insecticidal Protein Of Transgenic Bt Rice And The Responsive Mechanisms Of Chilo Suppressalis

The planting of transgenic Bt rice can effectively control the occurrence of lepidoptera pests such as rice stemborer,Chilo suppressalis,and thus provides a new method for the control of lepidoptera pests.Numerous studies have shown that elevated CO2 can increase plant photosynthesis and productivity,promote plant growth and development,and dilute the plant tissues N contents,which affects the exogenous Bt protein content of transgenic Bt crops,thus in turn affects its resistance to target insect pests.In addition,soil nitrogen(N)fertilizer level can also affect the exogenous Bt protein content of transgenic Bt crops,and also affect it’s resistance to target insect pests.To reveal the resistance mechanism of target insect pests to transgenic Bt rice under the background of climate change(elevated CO2),in this dissertation,we carried out the following experiments:(1)explore the effects of N-fertilizer rates(i.e.,1/4 N,1 N and 2 N;1N=1.25 m mol/L NH4NO3)on foliar exogenous Bt-toxin protein content,expression level and methylation level of exogenous Bt gene in transgenic Bt rice(i.e.,transgenic Bt rice expressing CrylAb/Cry1Ac;c.v.,Huahui-1)under elevated CO2(i.e.,800 ppm vs.400 ppm);(2)investigate the survival of target insect pest,C.suppressalis larvae fed on transgene Bt rice during different growth stages;and(3)measure the changes of the midgut protease activity and expression levels of exogenous Bt protein receptor genes of C.suppressalis(including APNs and CADs)under elevated CO2.Main results for this study were as follows:1.Effects of elevated CO2 on foliar exogenous-Bt-toxin protein content,expression level and methylation level of Bt transgene in transgenic Bt rice under different N-fertilizer levelsIn this study,the effects of elevated CO2(double-ambient CO2 vs.ambient CO2)on foliar exogenous-Bt-toxin protein content,and transgcne expression and methylation levels in promoterregion and codingregion of transgenic Bt rice during tillering and heading stages were evaluated under different N-fertilizer rates(1/4,1 and 2N levels).The results indicated that the aboveground and belowground biomass,and foliar exogenous Bt protein content of transgenic Bt rice during the tillering and heading stages all significantly increased with the augmentation of N-fertilizer rates.Elevated CO2 significantly increased belowground biomass and foliar total soluble protein content of transgenic Bt rice grown at 1N level,and aboveground and belowground biomass at 2N level.In addition,elevated CO2 significantly decreased the foliar exogenous Bt protein content of transgenic Bt rice during tillering stage at 1N level.During the tillering stage of transgenic Bt rice,elevated CO2 significantly increased transgene methylation levels in promoterregion(P1)and in total of promoterregion and codingregion(P1+P2+P3)at 1N level.During the heading stage of transgenic Bt rice,elevated CO2 significantly increased transgene methylation levels in promoterregion(P1)at 1N level.Under ambient CO2,the transgene methylation level in total of promoterregion and codingregion(P1+P2+P3)were positively correlated with the Bt-transgene expression level.Under elevated CO2,the transgene methylation level in total of promoterregion and codingregion(P1+P2+P3)was negatively correlated with the Bt-transgene expression level.These results suggest that elevated CO2 can reduced the foliar exogenous Bt protein content under normal N-fertilizer level(i.e.,1N)and increased N-fertilizer augmentation(i.e.,2N)will increase the foliar exogenous Bt protein content of transgenic Bt rice during the tillering and heading stages.2.Effects of elevated CO2 on the fitness of C.suppressails larvae fed on transgenic Bt rice during different growth stagesIn this study,the transgenic Bt rice HH1 and its conrresponding non-transformed near isoline MH63 and Chilo suppressalis were used as the research objects,and the effects of elevated CO2 on the exogenous Bt protein content of transgenic Bt rice during the seedling,tillering and heading stages,and the mortality and relative growth rate of the 4th instar larvae of C.suppressails larvae fed on transgenic Bt rice were investigated.The results showed that the foliar exogenous Bt protein content of transgenic Bt rice was highest at the seedling stage,followed by at the tillering stage,and lowest at the heading stage.Compared with ambient CO2,elevated CO2 increased the foliar exogenous Bt protein content of transgenic Bt rice at the seedling stage,and decreased that at the tillering and heading stage respectively,and significantly reduced the foliar exogenous Bt protein content of transgenic Bt rice at the tillering stage.The bioassay test showed that the mortality of the 4th instar larvae of C.suppressails was 100%after fed on transgenic Bt rice for 8,19 and 33 days during the seeding,tillering and heading stages,respectively.The half-lethal time of C.suppressails larvae fed on transgenic Bt rice during the tillering stage was the shortest(ambient CO2:3.76 d,elevated CO2:3.48 d),and that fed on transgenic Bt rice during the heading stage was the longest(ambient CO2:10.72 d,elevated CO2:11.15 d).In conclusion,the foliar exogenous Bt protein content of transgenic Bt rice decreased during the the tillering and heading stages,and the half-lethal time of C.suppressails larvae was prolonged.And elevated CO2 decreased the foliar exogenous Bt protein content of transgenic Bt rice especially at the tillering stage,and simultaneously prolonged the half-lethal time of C.suppressails larvae fed on transgenic Bt rice.3.Effects of elevated CO2 on the midgut protease activities and the expression level of APNs and CADs of C.suppressails larvae fed on transgenic Bt rice at different growth stagesIn this study,the total protease,tryptase-like enzyme,chymotrypsin-like enzyme,aminopeptidase activity,and the expression level of APNs and CADs of C.suppressails larvae fed on transgenic Bt rice at different time(0,1,2,3,4 days)were studied.Compared with ambient CO2,elevated CO2 significantly increased the activity of total protease,tryptase-like enzyme and aminopeptidase of C.suppressails larvae fed on transgenic Bt rice during the seeding stage,and significantly reduced the activity of tryptase-like enzyme and aminopeptidase of C.suppressails larvae fed on transgenic Bt rice during the tillering and heading stages.Compared with ambient CO2,elevated CO2 significantly increased the expression level of APN1 and APN5 of C.suppressails larvae fed on transgenic Bt rice during the seeding stage,and significantly reduced the APN5 expression level of C.suppressails larvae fed on transgenic Bt rice during the tillering and heading stages.In addition,elevated CO2 significantly increased the expression level of CAD1 of C.suppressails larvae fed on transgenic Bt rice during the seeding stage,and significantly reduced the CAD1 expression level of C.suppressails larvae fed on transgenic Bt rice during the tillering and heading stages.In general,the midgut protease activity and expression level of exogenous-Bt-toxin protein receptor of C.suppressails larvae changed after fed on transgenic Bt rice.C.suppressails larvae can respond to the change of foliar exogenous-Bt-toxin protein content of transgenic Bt rice under elevated CO2 through changing the midgut protease activity and expression level of exogenous-Bt-toxin protein receptors.In summary,elevated CO2 significantly decreased the foliar exogenous Bt protein content of transgenic Bt rice,and augmentation of N-fertilizer increased the biomass,foliar soluble protein and exogenous Bt protein content of transgenic Bt rice.During the seeding stage,elevated CO2 increased the resistance of transgenic Bt rice to the target pest,C.suppressails.During the tillering and heading stages,elevated CO2 decreased the decreased resistance of transgenic Bt rice to the target pest.In addition,C.suppressails larvae can respond to the changes of the foliar exogenous Bt protein content of transgenic Bt rice under elevated CO2 situation by changing the activity of tryptase-like enzyme and aminopeptidase,and the expression level of APNs and CADs.Under elevated CO2,the resistance of transgenic Bt rice to target insect pests showed a downward trend during the tillering and heading stages.It’s necessary to strength monitor and control of target insect pests.This study provided a theoretical basis for the sustainable use of transgenic Bt rice under the climate change environment in the future.