Molecular Cloning Of Maize C₄Key Enzyme Genes （PPDK And NADP-ME） And Expression Analysis Of PPDK And PEPC In Arabidopsis

ABSTRACT:The efficiency of photosynthesis is related with the crop biomass, since the harvest index and leaf area index for many crops, such as wheat and rice, is approaching a ceiling value, it has been a focus that an increase in yield potential should come from improved photosynthesis. According to differences of mechanism in carbon assimilation pathways in photosynthesis, three major photosynthetic types may be distinguished among green plants:C3, C4and Crassulacean acid metabolism plants. C4plants were evolved from C3plants, they adapt to high light, high tempretaure, lower CO2concentration and achieve higher photosynthetic capacity compared with C3plants（Black,1973）. Consequently, the transfer of C4enzyme to C3plants such as rice and wheat is one strategy being adopted for improving their yield. In this experiment, PPDK with an ORF of971amino acids was isolated from maize self lines Z1194and was introduced to Arabidopsis with PEPC, systematical studies on their transcription, translation, eneyzme activities and photosynthetic performences were conducted, which could offer candidate gene and theory for engineering the C4photosynthetic pathway into C3crops such as wheat.The results were as follows:1. The full-length cDNA for PPDK and NADP-ME were isolated from Zea mays by homology-based cloning technology. The full-length PPDK and NADP-ME cDNA replaced the (3-glucuronidse（GUS） coding region of the pCAMBIA3301（p3301） or pCAMBIA1391（p1391）. The positive clone was named p3301-PPDK, p3301-ME and p1391-ME, respectively. In this study, we have obtained a3004bp cDNA fragment of PPDK from maize Z1194（Zea mays L.）, PPDK has a2916bp open reading frame （ORF） and encodes971amino acids with a calculated molecular mass of105kDa polypeptide（GenBank accession number:GU363532）. Comparison of GU363532to the C4PPDK gene （GenBank accession number:BT054438） by DNAMAN indicated99.4%similarity between the amino acid sequences. We have also obtained a2127bp cDNA fragment of NADP-ME from maize Zl194, containing one ORF with1911bp which coded a peotide of636amino acids. The identity of the cloned fragment with NADP-ME gene from NM₀01111843.1was99.4%by homology alignment with software DNAMAN.2. The integration of maize PPDK and PEPC gene in transgenic Arabidopsis plants was confirmed by Southern blotting analysis, the transgentic rate and co-transgentic rate was0.69%and9.5%, the ratio of linked-inheritance was66.7%. Arabidopsis plants were transformed by the floral dipping method with tumefaciens GV3101, including p3301-PPDK, p3301-PEPC, p3301-PPDK+p3301-PEPC（PPDK:PEPC was3:2）, and p3301. Based on the segregation pattern of the transgenic seedlings by Basta-tolerant and PCR assay. A total of327transgentic plants were obtained, plants containing the PPDK gene, the PEPC gene, the PPDK+PEPC gene, or GUS gene were separated into their respective groups, indicating the following distribution of lines:twenty-four homogenous PPDK lines from175PPDK transgentic lines, twenty-six homogenous PEPC lines from125PPDK transgentic lines, eight homogenous lines linked inheritance with both PPDK and PEPC from12double transgentic lines, and three homogenous p3301lines from15transgentic lines of p3301.3. Analyses by RT-PCR, quantitative real-time PCR and Western blotting all confirmed the effective expression of maize C4-PPDK and C4-PEPC in Arabidopsis. The expression levels of PPDK or PEPC among different transgenic lines varied, which ranged from1-to4.24-fold and1-to13.33-fold by quantitative real-time PCR, respectively. Volume analysis by software Quantity One indicated that the expression levels of PPDK were ranged from1.9-to2.5-fold relevant to the NegCtrl, the expression levels of PEPC were ranged from1.6-to3.7-fold relevant to the NegCtrl, respectively.4. The photosynthetic performance could be improved by PPDK or PEPC alone, and what’s more, Pn （PKC）> Pn （PC）> Pn（PK）. In this study, the enzyme activities and the Pn of T3transgentic plants were determined.The enzyme activity of C4-PPDK and C4-PEPC were elevated by0.61-3.63and0.77-4.81times relevant to the controls, the average value was1.89and1.48folds, respectively. The Pn of Ca-PPDK, C4-PEPC and PPDK+PEPC transgenic lines were higher than those of the controls by3.3-25.3%,6.5-35.1%and12.6-45.7%.5. Tthe introduction of C4-PPDK or C4-PEPC into Arabidopsis could contribute to the strengthen of C4-mini cycle. In this study, the enzyme activities of T4transgentic Arabidopsis were determined. The PEPC enzyme activities of PK17-1-2and PK26-3-2were18.65%and46.37%higher than those of the controls, respectively; the PPDK enzyme activities of PC65-4-6and PC73-1-3were12.96%and26.54%higher than those of the controls, respectively, the PPDK and PEPC enzyme activities could significantly increased by transgentic PPDK and PEPC Arabidopsis. The Pn of PK17-1-2, PK26-3-2, PC65-4-6and PC73-1-3were13.14%,23.29%,18.14%and36.44%higher than those of the controls, respectively.6. The coexpression of PPDK and PEPC had synergistic effect on photosynthesis rate. To our interest, the Pn of the line PKC6-5-1were the highest among the T4transgentic Arabidopsis, however, its PPDK enzyme activities or its PEPC enzyme activities were not the highest, although the PPDK or PEPC eneyzme activities of the line PKC17-1-3was the lowest among the T4transgentic Arabidopsis, the Pn of the line PKC17-1-3were higher than those of the line PK17-1-2and PC65-4-6.The results show that both are candidate functional genes in the improvement of Pn of C3Arabidopsis and coexpression of C4-PPDK and C4-PEPC genes had synergistic effects on Pn, which could be of great importance in the agricultural field, especially when considering the limited methods that we currently have in improving crop yields.