Abstract:

Objective: Phosphoenol pyruvate carboxylase(PEPC) and phosphopyruvate dikinase(PPDK) are the key enzymes to ensure efficient photosynthesis in C₄ plants. Poplar,as a C₃ plant with low photosynthetic efficiency,has low light energy utilization. In this study,maize PEPC gene and PPDK gene were used as target genes for genetic transformation in poplar,respectively,to obtain highly expressed transgenic plants,and molecular detection and functional analysis were carried out to provide a basis for breeding new poplar varieties with high light efficiency. Method: The PEPC and PPDK genes of maize were screened through bioinformatics. With Gateway system,PEPC and PPDK genes were constructed into plant expression vector pGWB406,respectively,and transformed into 'Nanlin 895' poplars(Populus deltoides×P. euramericana 'Nanlin 895') by Agrobacterium-mediated transformation. The photosynthetic physiological characteristics of transgenic plants and control('Nanlin 895' poplars) were compared and analyzed. Result: The cloned PEPC and PPDK genes all encode typical key enzymes of C₄ photosynthesis. There were 5 lines transformed with PEPC gene and 3 lines transformed with PPDK gene. There were differences in gene expression among different lines of PEPC gene,up to 1.79 times,but there was no significant difference in the expression of proteins in leaves of different lines,and no significant difference in gene and protein expression among lines of PPDK gene; the expression of the two genes in leaves were higher than that in stems,and increased with time during plant growth and development; the expression of PEPC gene and PPDK gene were affected by light conditions,and increased 2-3 times under high illumination; the net photosynthetic rate of the transgenic lines showed a double peak at 11:00—14:00,which were significantly higher than that of the control group,with a maximum of 16.2%(PEPC-5 line); the stomatal conductance were higher at 12:00—16:00 than that of the control group; except PPDK-2 line,higher intercellular CO₂ concentrations were detected in all transgenic lines than in the control group,and except for PEPC-1 and PEPC-4 lines,the CO₂ saturation points of the transgenic lines were lower than that of the control group; the CO₂ compensation points of all transgenic lines were lower than that of the control group,and the photosynthetic rate at light saturation point and light saturation point were higher than that of the control group. The growth parameters of PEPC-3 and PEPC-4 lines were significantly larger than those of the control group(P < 0.05). The average tree height was 5.6% and 2.9% higher than that of the control group,the leaf area was 13.8% and 8.9% higher than that of the control group,the leaf weight was 23.6% and 19.8% higher than that of the control group,and the ground diameter was 13.5% and 5.4% higher than that of the control group,respectively. The leaf biomass measurements showed that the dominant lines were PEPC-3,PEPC-5 and PEPC-4,which were 34.8%,15.4% and 6.3% higher than those of the control group respectively. Conclusion: The introduction of PEPC and PPDK genes made the transgenic plants have obvious advantages in light utilization,significantly improved the photosynthetic capability of the plants,and the growth performance was better than that of the control group. The genetic transformation of poplar using the key enzymes coding genes of photosynthesis in C₄ plants is of great significance for improving the photosynthetic efficiency of poplar and even other woody plants.

Key words: PEPC, PPDK, transgenic poplar, gene expression, photosynthesis