| Wheat is a C3 crop,with lower photosynthetic efficiency and optimum growth temperature,whereas C4 crops such as maize and sorghum have higher photosynthetic efficiency,greater biomass and better tolerance to abiotic stress than C3 plants.Since C4 plants have these advantages,botanists have always attempted to transform the genes of key enzymes in the C4 pathway into C3 plants to increase the photosynthetic efficiency and yield of C3 crops.In our previous studies,two transgenic wheat lines(PC27 and PC51)containing maize PEPC gene were obtained,and their net photosynthetic rate were significantly improved compared to those of their genetic control.Based on the former results,we took PC27 and PC51 as the matirals,which were used to study physiological characteristics,expression of endogenous photosynthesis-related genes,and the metabolomic changes under high temperature stress,besides that,the molecular characteristics,yield characters and grain filling characters of the transgenic wheat were also studied.The main resuslts of this study are organized as follows:1.The results of PCR analysis suggested that the exogenous PEPC gene was integrated into the genome of the two new transgenic wheat lines;Southern blot indicated that PEPC gene was integrated into wheat genome in single copy pattern;the results of western blot results showed that PEPC gene had translated into protein correctly.2.Compared with the WT,transgenic wheat exhibited significant changes in the expression of the exogenous PEPC gene and the endogenous CA,PEPCK and NADP-MDH gene under non-stress treatment and high temperature stress at 3 growth stages(jointing stage,heading stage and grain filling stage).After high temperature treatment,the expression levels of PEPC,CA,PEPCK and NADP-MDH gene was significantly reduced,however,the expression in the transgenic wheat remained raletively higher level,and were still increased by 32.09,22.46,14.76,19.8,12.07 and 10.58 fold,respectively.The results indicated that a C4-pathway established in C3-plant by engineering approaches,and upregulated endogenous photosynthetic related genes.3.After high temperature treatment,although the Pn(net photosynthetic rate)of transgenic wheat and the control both significantly decreased,the magnitude of the decrease in transgenic wheat was smaller,and Pn of the two transgenic wheat lines were 15.98?mol CO2 m-2 s-1 and 15.78?molCO2 m-2 s-1,respectively 16.26%and 13.45%higher than that of the control;Fv/Fm of the two transgenic wheat lines were 0.723 and 0.720,respectively 5.01%and 4.55%higher than that of the control;The valure of Fm decline in two transgenic wheat lines were1296,1088 and 1079,respectively 16.04%and 16.74%higher than that of the control;chlorophyll(a+b)content of the two transgenic wheat lines were 2.428 and 2.352,respectively 19.57%and 15.83%higher than that of the control;Chlorophyll(a+b)content of the two transgenic wheat lines were 21.52?mol g-1 FW and 20.20?mol g-1 FW 2.352,respectively 11.44%and 15.37%higher than that of the control;the activity of SOD 502.17unit g-1 FW and 506.12unit g-1 FW,respectively 9.63%and 10.49%higher than that of the control;Compared with the WT,two transgenic lines had relatively better Gs,Tr,Ci,?PS?,qp,NPQ,OJIP cuvre,Wk,Vj,chlorophyll a/b value,rate of superoxide anion production,the content of hydrogen peroxide and malonaldehyde(MDA),the activities of superoxide dismutase(SOD),catalase(CAT),and peroxidase(POD),than that of control.The results indicated that he transgenic wheat had a higher Pn and better tolerance than wild-type control,and was more significant when under high temperature stress.Heat tolerance in transgenic wheat was enhanced by improving photochemical efficiency,increasing resistance oxidase activity,maintaining chlorophyll content.4.Metabolome of the PC27 and WT were analysised by GC-MS,and 161 metabolites were identified.The content of 31 metabolites were different between PC27 and WT under NT and HT,these metabolites can be divided into three categories:responsive metabolite to PEPC gene,responsive metabolite to high temperature,responsive metabolite to PEPC gene and high temperature.There were 13 metabolites respond to PEPC gene,5 metabolites upregulated and 8 downregulate than that of control among these metabolites.There were 28 metabolites respond to high temperature,21 metabolites upregulated and 7 downregulate than that of control among these metabolites.There were 18 metabolites respond to PEPC gene and high temperature,9 metabolites upregulated and 9 downregulate than that of control among these metabolites.These results indicated that PEPC gene can influence nitrogen metabolism,glycolysis and tricarboxylic acid cycle,increased free amino acid and sugar content,the upregulation of these metabolic pathways relieved the occurrence of membrane lipid peroxidation and supplied more energy for carbon assimilation,which played an important role in matain realative higher photosynthetic rate under high temperature stresses.5.The diurnal variation of canopy photosynthetic rate in transgenic lines and WT showed bimodal curves,the max canopy photosynthetic rate of the transgenic lines were 28.50?mol·m-2·s-1 and 28.13?mol·m-2·s-1,respectively 7.10%and 7.34%higher than that of the control;the most significant increment was observed at 10:00 to 13:00;The biomass and grain yield of the transgenic lines were significantly higher than WT,biomass of the two transgenic wheat lines were 1514 kg/667 m2 and 1549 kg/667 m2,respectively 3.92%and 6.31%higher than that of the control;grain yield of the two transgenic wheat lines were 627.18kg/667 m2 and 612.06 kg/667 m2,respectively 7.24%and 4.79%higher than that of the control;The thousand kernel weights of the two transgenic lines were significantly higher than wild-type control,which was contributed to their higher grain yield.Compared with WT,both the two transgnic lines had higher theoretical maximum hundred kernel weight,average grain filling rate,increment of grain weight and grain filling rate at useful grain filling period,which were contributed to their higher thousand kernel · weight.Transgenic lines had better photosysthesis characters and better hot torlerence than WT,which were the physiological basis for high thousand kernel weight and yield. |
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