Charactieristics And Mechanism Of Carbon And Nitogen Distribution In Transgenic Cry2A~* Rice

As one of the most important food crops in the world,rice feed about 40%of the world’s population.Introduction of exogenous Bt(Bacillus Thuringiensis)gene through transgenic technology can effectively improve the insect resistance of rice.Nevertheless,as a soluble protein,Bt protein will consume additional nitrogen sources and energy in the process of synthesis and metabolism in plants,which may affect the allocation and metabolism of carbon and nitrogen in plants.And changes in carbon and nitrogen metabolic characteristics of rice will affect agronomic traits,yield and potential ecological risks.At present,many studies have found that the changes in agronomic traits and physiological metabolic processes of Bt transgenic crops compared with their control lines.Therefore,it is of great significance to study the differences in carbon and nitrogen metabolism between transgenic rice and non-transgenic rice lines,and the mechanism of their changes.In this study,different N levels were set for transgenic Cry2A~*rice lines and their non-transgenic parent control lines,including MH63(Cry2A~*)and MH63,MH86(Cry2A~*)and MH86,KY131(Cry2A~*)-1,KY131(Cry2A~*)-2 and their control line KY131).In our study,expressions of Bt proteins in different transgenic Cry2A~*rice materials under different N element conditions;main agronomic characters and yield differences of transgenic Cry2A~*rice lines and their respective control lines;differences in the allocation and accumulation of carbon and nitrogen and the related indicators of carbon and nitrogen metabolism between the rice line and the control line of Cry2A~*gene were studied.Besides,we try to explore the mechanism of changes in carbon and nitrogen distribution and metabolic characteristics between transgenic Cry2A~*rice and their control lines.And main research results were as follows:(1)Cry2A~*protein content in different transgenic Cry2A~*lines were different,and the expression range was 0.81~11.96μg·g~-11 fresh weight.At different nitrogen levels,Cry2A~*protein expression in different Cry2A~*lines showed differences,among which the Cry2A~*protein content in MH86(Cry2A~*)was the lowest,followed by KY131(Cry2A~*)-1,while Cry2A~*protein content in KY131(Cry2A~*)-2 and MH63(Cry2A~*)were affected by nitrogen application.The change trend of Cry2A~*protein in transgenic rice line was different with that of total protein at late growth stage.(2)There were some differences in plant height,dry matter distribution,photosynthetic characteristics and carbon(C)accumulation between the transgenic Cry2A~*rice line and its control line under different N application levels,but there were no regular differences.Under different nitrogen application levels,there was no significant difference between the total N accumulation of Cry2A~*transgenic rice and the control line.In terms of agronomic traits and C and N metabolism,the differences between different materials are not consistent.Under different nitrogen application conditions,ATP content in mitochondria of MH63(Cry2A~*)leaves was significantly lower than that of MH63.The ATP content in the mitochondria of MH86(Cry2A~*)leaves was significantly higher than that of the control line MH86.ATP levels in mitochondria of KY131(Cry2A~*)-1 were higher than those of KY131(Cry2A~*)-1 and its control line.In addition,the response of the characteristic indexes of carbon and nitrogen metabolism to nitrogen fertilizer in transgenic rice lines was different,but the results showed no regularity.This indicates that agronomic characteristics are also affected by the genetic background of transgenic materials.(3)Based on the findings that ATP content in mitochondria of MH63(Cry2A~*)leaves was significantly lower than that of the receptor parent MH63 under N0 treatment,proteomics analysis was performed on MH63(Cry2A~*)and the control line MH63 at different nitrogen application conditions.The results showed that there were 55~61different proteins related to carbon and nitrogen metabolism in the two lines under different N fertilizer conditions,involving energy metabolism,carbohydrate and lipid metabolism,nucleotide metabolism,amino acid metabolism,carbohydrate synthesis and metabolism,terpenoid and polyketone metabolism and other secondary metabolites.The analysis results of KEGG metabolic pathways showed that the differences of energy metabolism were mainly related to the synthesis of ATP.The differentially expressed proteins on the electron transport chain were ND4,an enzyme located in the mitochondrial endometrium catalyzed electrons from NADH to coenzyme Q;Cyt1,a cytochrome reductase,specifically the ubiquitin-cytochrome c reductase cytochrome b/c1subunit,which catalyzes electrons from coenzyme Q to cytochrome c;COX6B,a subunit of cytochrome c oxidase cbb3,is the last enzyme of electron transport in the oxidative respiratory chain;COX15,a pyrophosphorylase,is involved in energy metabolism and oxidative phosphorylation.By comparing the metabolic pathways of oxidative phosphorylation under different N application conditions,it was found that the difference in mitochondrial ATP content under different nitrogen application conditions might be at the end of oxidative phosphorylation process,thus resulting in the difference in ATP content.Combined with the proteomics analysis results of MH63(Cry2A~*)and the control line,we speculated that the change of C and N metabolism characteristics of transgenic materials under the condition of N0 might be related to the energy metabolism pathways.However,different Cry2A~*transgenic lines showed different responses.For example,the transgenic line MH86(Cry2A~*)with no marker,and whose insertion site is intergenic region,had higher N metabolic enzyme activities and ATP content than the control line under different nitrogen application conditions.C and N distribution and energy metabolism in MH63(Cry2A~*)were not significantly different from those of MH63 under RN condition.Therefore,the evaluation of transgenic materials should be carried out case by case.The expression of Cry2A~*protein in transgenic Cry2A~*rice lines and the change trend of Cry2A~*protein and total protein during the later growth stage were also different.The transgenic Cry2A~*rice line and its control line had some differences in agronomic characteristics and carbon and nitrogen metabolism under different N application levels,but showed no regular difference,indicating that the expression of Cry2A~*protein in the material used in this study did not reach the difference threshold.However,the energy metabolism index can be used as the evaluation index in the cultivation of transgenic materials.But the specific correlation needs more systematic research and demonstration using more transgenic lines and more indicators.