Insecticidal Protein Regulation And Physiological Mechanism Of Reproductive Organ In Bt Cotton

In order to uncover the effect of exogenous regulation on the expression of insecticidal protein in the reproductive organs of Bt cotton,the relationship between the reproductive organs formation and insecticidal protein contents was investigated under different varieties,mepiquat chloride(MC)rates,N fertilizer rates and planting densities.Sikang 1 and Sikang 3,two Bt cotton varieties,were used as experimental materials to study the impact of exogenous regulator which could affect protein synthesis and decomposition on insecticidal protein expression in reproductive organs(squares,flowers and bolls)and related physiological mechanism.The main results are as follows:1.Among different Bt cotton varieties,the varieties with higher individual square biomass or whole plant square biomass exhibited higher insecticidal protein contents.And the individual and whole plant square biomass,as well as insecticidal protein contents were significantly increased by the application of MC.On the contrary,N deficiency decreased the individual and whole plant square biomass and the insecticidal protein contents.Correlation analysis showed that there was a significant positive correlation between square biomass and insecticidal protein contents.Therefore,promoting the square development would be beneficial to elevate the insect resistance of cotton squares.The analysis of the relationship between boll formation and boll shell insecticidal protein contents showed that higher boll setting rate together with lower insecticidal protein contents in boll shell was observed under low planting density,whereas lower boll setting rate and higher insecticidal protein contents were found under high planting density.Also,higher insecticidal protein concentrations were associated with higher soluble protein contents,glutamic oxaloacetate transaminase(GOT)and glutamic-pyruvic transaminase(GPT)activities,but lower amino acid contents,and protease and peptidase activities.This study demonstrated that the enhanced protein synthesis and reduced protein degradation were the main physiological reasons for increasd boll shell insecticidal protein concentrations.2.The application of protein synthesis promoter and protein decomposition inhibitor both increased the insecticidal protein contents in reproductive organs of Bt cotton plants.Under different levels of urea,the contents of insecticidal protein in squares,flowers and bolls were the highest under 6%-8%urea,and the increase was 10.5%-37.4%compared to the untreated control.The increased contents of soluble protein and free amino acid,and enhanced activities of key enzymes glutamine synthetase(GS),glutamate synthase(GOGAT),GOT and GPT all contributed to the elevated insecticidal protein contents.Correlation analysis and principal component analysis further showed that the insecticidal protein content was mainly related to the content of main substances and the activities of key enzymes involved in protein synthesis,but not significantly related to the activities of protease and peptidase.Therefore,the enhancement of protein synthesis ability was the main reason for increased insecticidal protein concentrations under urea regulation.The application of protein decomposition inhibitors ethylenediaminetetraacetic acid(EDTA),leuppeptin and phenylmethylsulfonyl fluoride(PMSF)can also increase the insecticidal protein content in reproductive organs,and the effect was stronger when the two inhibitors were used together.Under the combined application of EDTA and leuppeptin,the contents of insecticidal protein in squares,flowers and bolls were basically the highest,and the increase was 19.2%-27.8%compared to the untreated control.The decreased activities of key enzymes involved in protein decomposition(protease and peptidase)contributed to the elevated insecticidal protein contents.Although the contents of free amino acid and the activities of GS,GOGAT,GOT and GPT decreased,the contents of soluble protein increased.Therefore,under the regulation of protein decomposition inhibitors,the increase of protein content caused by the decrease of protein decomposition ability exceeded the decrease of protein content caused by the decrease of protein synthesis ability,resulting in the increase of soluble protein concentrations,including the increased insecticidal protein contents.Namely,the decrease of protein decomposing ability was the main reason for increased insecticidal protein concentrations in reproductive organs under protein decomposition inhibitors regulation.This was further confirmed by correlation analysi's and principal component analysis.3.The application of protein synthesis inhibitor and protein decomposition promoter both reduced the insecticidal protein contents in the reproductive organs of Bt cotton.Under the application of protein synthesis inhibitors chloramphenicol(CAP),cycloheximide(CHM)and L-methionine sulfoximine(MSO),the concentrations of insecticidal protein in squares,flowers and bolls were lowest under the combination sparying with CHM and MSO,and the decrease was 20.4%-39.4%compared to the untreated control.The decreased contents of soluble protein and free amino acid,and declined activities of key enzymes GS,GOGAT,GOT,GPT,protease and peptidase all contributed to the reduced insecticidal protein contents.Therefore,under the regulation of protein synthesis inhibitors,the decrease of protein contents caused by the decrease of protein synthesis abilities exceeded the increase of protein content caused by the decrease of protein decomposition ability,resulting in the decrease of soluble protein concentration,including the decreased insecticidal protein content.Namely,the decrease of protein synthesis abilities was the main reason for decreased insecticidal protein concentrations in reproductive organs under protein synthesis inhibitors regulation.This was further confirmed by correlation analysis and principal component analysis.The application of protein decomposition promoter(Ca2+ and Mg2+)can also reduce the insecticidal protein content in reproductive organs,and under the combined application of Ca2+and Mg2+,the contents of insecticidal protein in squares,flowers and bolls were the lowest,and the decrease was 20.4%-32.8%compared to the untreated control.The increased activities of key enzymes involved in protein decomposition(protease and peptidase)contributed to the decreased insecticidal protein contents.Although the contents of free amino acid and the activities of GS,GOGAT,GOT and GPT increased,the contents of soluble protein decreased.Therefore,under the regulation of protein decomposition promoters,the decrease of protein contents caused by the improvement of protein decomposition abilities exceeded the increase of protein contents caused by the improvement of protein synthesis abilities,resulting in the decrease of soluble protein concentrations,including the decreased insecticidal protein contents.Namely,the increase of protein decomposing abilities was the main reason for the decreased insecticidal protein concentrations in reproductive organs under protein decomposition promoter regulation.This was further confirmed by correlation analysis and principal component analysis.In conclusion,promoting the formation and development of square would enhance insect resistance in reproductive organs,and moderately promoting the formation of bolls would balance the yield and insect resistance.Additionally,the exogenous regulator that would promote protein synthesis or inhibit protein decomposition,especially protein synthesis promotes,were beneficial improving the insect resistance of reproductive organs in Bt cotton plants.