Expression Analysis Of Maize Phosphoenolpyruvate Carboxylase Gene In Arabidopsis And Tobacco

The phosphoenolpyruvate carboxylase pepc gene is a key enzyme gene in the C₄ pathway.To investigate the effect of pepc on photosynthetic characteristics of C₃ Arabidopsis thaliana plants,this experiment used transgenic C₄ pepc Arabidopsis thaliana and wild-type Arabidopsis thaliana as material for17 T₃ homozygous pepc transgenic Arabidopsis thaliana lines.Molecular biology identification was performed.Real-time fluorescent quantitative PCR was used to determine the expression and expression levels of pepc gene in transgenic Arabidopsis thaliana.The physiological and biochemical characteristics of pepc transgenic Arabidopsis thaliana and wild type plants such as photosynthetic enzyme activity,chlorophyll content,chlorophyll fluorescence and net photosynthetic rate were determined at budding stage.And under drought stress conditions,the pepc transgenic Arabidopsis thaliana antioxidative enzyme activity,soluble protein,soluble sugar and other physiological characteristics were determined.The effect of pepc,a key enzyme gene of maize C₄ photosynthetic pathway,on photosynthetic characteristics of Arabidopsis thaliana was studied.The main results are as follows:?1?T₂ transgenic pepc Arabidopsis transformed plants were screened to obtain 17 highly expressed T3 homozygous pepc transgenic Arabidopsis lines.?2?PCR analysis of 17 highly expressed T₃ homozygous pepc transgenic Arabidopsis lines showed that the pepc gene was successfully transformed into Arabidopsis thaliana and pepc transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana plants were extracted respectively.The RNA was reversely transcribed to generate cDNA for RT-PCR,and the pepc transgenic Arabidopsis plant and positive plasmid DNA were able to amplify the 200 bp target band.The expression level of pepc gene in transgenic Arabidopsis thaliana was determined by real-time quantitative PCR.The pepc gene expression levels of PC2,PC3,PC5,PC7,PC15,PC16,and PC18 lines were 13.60,12.23,and 11.57 higher than that of PC17,respectively 10.34,2.20,3.70,6.34 times,reaching significant levels.?3?Physiological and biochemical characteristics of 17 high-expressing T₃ homozygous pepc transgenic Arabidopsis lines and wild-type plants were tested during the budding stage,and the PEPC enzyme activities of the pepc transgenic lines PC2,PC3,PC5,and PC9 lines were analyzed.They were552.16,438.48,389.76,and 194.88 U/g respectively,34,27,24,and 12 times more than wild-type Arabidopsis plants;and chlorophyll content of PC2,PC3,PC5,PC11,PC12,PC13,and PC18 strains Compared with wild-type Arabidopsis plants,they increased by 53.17%,57.56%,44.88%,42.44%,49.27%,45.61%,and 47.07%,respectively;Rubisco of pepc transgenic lines PC2,PC3,PC5,PC9,PC11,and PC12lines.The enzyme activities were increased by 24.07%,23.77%,24.27%,22.09%,22.33%,and 24%respectively,compared with wild-type Arabidopsis plants.PC2,PC3,PC5,PC7,PC9,PC11,PC12,PC13,PC18 lines Net photosynthetic rate increased by 51.85%,43.82%,41.41%,41.58%,36.77%,37.96%,38.97%,36.28%,and 34.08%,respectively,compared with wild-type Arabidopsis plants,and the increase rate reached a significant level.?4?The changes of the physiological characteristics of transgenic pepc genes were investigated under drought stress conditions.The results showed that the activities of SOD,POD,and CAT enzymes in all lines increased under drought stress than in normal conditions;The activities of SOD enzymes of PC1,PC2,PC4,PC8,PC12 and PC13 strains were 5.57,5.39,5.26,4.47,4.96 and 4.11 U/mg?pro?under drought stress,and the activity of SOD enzyme increased significantly;PC1,PC2,PC4,The POD enzyme activities of PC8,PC12,and PC13 were 12.7,12.84,11.89,10.54,10.32,and 9.88 KU/mg?pro?/h,the activity of POD enzyme was increased in pepc transgenic lines under drought stress treatment,and PC1 was significantly higher than that of other transgenic Arabidopsis thaliana plants.Under normal conditions and drought stress,SOD enzyme activity of pepc transgenic lines was higher than that of wild type Arabidopsis plants;CAT enzyme activity of PC1,PC2,PC4,PC8,PC12,PC13 was 1.41,1.38,1.34,1.27,1.25,1.19KU/mg?pro?/h,pepc transgenic lines had increased CAT enzyme activity under drought stress treatment,and PC1 was significantly higher than other transgenic Arabidopsis plants.The CAT activity of pepc transgenic lines was higher than that of wild type Arabidopsis plants under normal conditions and drought treatment.There was no significant difference in soluble sugar and soluble protein between the T3generation homozygous pepc transgenic Arabidopsis line and the wild-type,and its malondialdehyde content decreased.The pEXPR-PEPC expression vector carrying the key enzyme gene pepc of maize C4 photosynthetic pathway was introduced into the young tobacco leaves of NC89 through Agrobacterium tumefaciens-mediated method,and the regenerated plants were obtained through co-culture,selective culture and rooting culture.CTAB method was used to extract the genomic DNA of tobacco leaves,PCR and RT-PCR amplifications were carried out,the tobacco plants were positive,the pepc gene was introduced into tobacco plants and expressed in tobacco.