Expression Analysis Of Maize PEPC Gene In Tobacco And Alfalfa

Food is a necessity for human survival.With the continuous growth of global population and the deterioration of ecological environment,food security is a problem that human beings must deal with.Improving crop photosynthetic efficiency and crop yield has always been one of the hot researches at home and abroad,but the improvement effect of conventional breeding methods on crop photosynthetic efficiency is weak.C₄plants generally have higher photosynthetic efficiency than C₃plants.Using modern biotechnology to transfer the key photosynthetic enzyme genes of C₄plants into C₃plants to improve the photosynthetic efficiency of C₃plants and then increase the yield of C₃plants has become a research hotspot at home and abroad.In this study,maize PEPC gene was transferred into tobacco using Agrobacterium tumefaciens mediated method,and T3 transgenic maize PEPC gene tobacco plants were obtained through continuous planting screening.PCR,RT-PCR and other molecular biological technologies were used to identify T3transgenic maize PEPC gene tobacco plants.Tested positive for the molecular identification of T3generation to turn the maize PEPC gene of tobacco plants in tobacco group tree stage and budding stage,respectively the PEPC enzyme activity,Rubisco enzyme activity,chlorophyll content,chlorophyll fluorescence parameters and coefficient of net photosynthetic rate,transpiration,stomatal conductance,intercellular CO2 concentration and agronomic traits and physiological and biochemical indexes of maize PEPC gene was studied on the influence of the photosynthetic efficiency of tobacco.The chlorophyll content,net photosynthetic rate,SOD activity,POD activity,CAT activity,MDA content and soluble sugar content of transgenic and non-transgenic tobacco were measured under drought stress,and the effects of maize PEPC gene on drought resistance of tobacco were studied.The main research results are as follows:1.The PEPC gene of corn was transferred into tobacco by Agrobacterium-mediated method,and transgenic tobacco plants were obtained.After continuous planting and screening,tobacco plants with PEPC gene of T3 transgenic corn were obtained.2.PCR identification of T3 transgenic maize PEPC gene in tobacco showed that the size of transgenic tobacco DNA as template PCR product fragment was consistent with that of the positive control（E.coli plasmid DNA containing corn PEPC gene as template）.Transgenic tobacco RNA was extracted and reverse transcribed to c DNA as template for RT-PCR,and the fragment with the same size as the positive control（E.coli plasmid DNA containing corn PEPC gene as template）was obtained.3.Physiological and biochemical indexes of T3 transgenic maize PEPC gene tobacco and non-transgenic tobacco were detected at the cluster stage and bud stage,respectively.The average PEPC enzyme activity of transgenic tobacco was 3.5 times higher than that of non-transgenic tobacco at cluster stage and 3.6 times higher than that of non-transgenic tobacco at bud stage.The average Rubisco enzyme activity was 53.33%higher than that of non-transgenic tobacco in the clumping stage,and 50.77%higher than that of non-transgenic tobacco in the budding stage.The average chlorophyll content was 32.86%higher than that of non-transgenic tobacco at the clumping stage and 28.95%higher than that of non-transgenic tobacco at the budding stage.The average chlorophyll fluorescence parameter was 4.94%higher than that of non-transgenic tobacco at the cluster stage and 4.88%higher than that of non-transgenic tobacco at the budding stage.The average net photosynthetic rate was 38.46%higher than that of non-transgenic tobacco at the clumping stage and 46.67%higher than that of non-transgenic tobacco at the budding stage.The average transpiration coefficient was 27.28%higher than that of non-transgenic tobacco in the cluster stage,and 31.58%higher than that of non-transgenic tobacco in the budding stage.The stomatal conductance was 55.56%higher than that of non-transgenic tobacco in the clumping stage,and63.16%higher than that of non-transgenic tobacco in the budding stage.The average plant height,stem circumference,number of leaves,maximum leaf length and maximum leaf width of transgenic tobacco were 15.19%,3.13%,16.17%,8.01%and 23.87%higher than those of non-transgenic tobacco in the cluster stage,and 3.95%,2.63%,13.04%,3.05%and 4.93%higher than those of non-transgenic tobacco in the bud stage,respectively.4.Under drought stress,the net photosynthetic rate of transgenic tobacco was 67.50%higher than that of non-transgenic tobacco,the chlorophyll content was 37.50%higher than that of non-transgenic tobacco,the activities of SOD,POD and CAT were 58.62%,30.44%and 64.71%higher than that of non-transgenic tobacco,respectively,the MDA content was 35.71%lower than that of non-transgenic tobacco,and the soluble sugar content of transgenic tobacco and non-transgenic tobacco had no significant change.5.The corn PEPC gene was successfully transferred into alfalfa by Aturobacterium-mediated method,and alfalfa transgenic plants were obtained.PCR,RT-PCR and other molecular biological techniques were used to amplify the fragments with the same size as the positive control（E.coli plasmid DNA containing corn PEPC gene was used as the template）.