Cloning And Complementary Vector Constructing Of Maize PPDK&the Effect Of Nitrogen Treatments On The Expression Of PPDK Protein

As the world population grows, the demand for food is further increased. How to improve crop photosynthetic efficiency and to improve their yields cause more and more attention. The current strategy to improve crop yields is supplementing more nitrogen fertilizer. However, heavy use of nitrogen fertilizer will seriously pollute the environment and destroy the ecological balance. Therefore, the study of how to improve the photosynthesis of crops under conditions of less nitrogen supplementation is very important. C4plants compared to C3plants have higher photosynthetic efficiency and nitrogen use efficiency. Thus, chracterizing the function of key C4enzyme PPDK and its relationship with nitrogen metabolism has very important significance. Previous studies of PPDK participated in nitrogen metabolism focus on C3plant such as Arabidopsis thaliana. However, the study in C4plant has not yet been reported. The main studies are as follows:(1) There exist big genome variations among different maize inbreds. The number of introns and exons of PPDK, especially the PPDK of B73, the inbred that has been genome sequenced, were still unclear. In this study, the cDNA of C4PPDKgsne was cloned from the leaf of maize B73. The detailed analysis confirmed that the PPDK gene of maize B73has19exons and18introns.(2) Maize PPDK gene has a dual promoter system. C4PPDKZml and CyPPDKZml share the same gene locus. The differences of them only between different splicing modes and transcription start site on5’noncoding regions. We had screened many C4PPDKZml (only have C4PPDKZml promoter) and CyPPDKZml (only have CyPPDKZml promoter) mutant using Mutator transpson system by PCR.(3) To further study the function of maize PPDK gene, the C4PPDKZmlgene and the CyPPDKZml gene must be studied separately. In this study, we successfully constructed fusion expression vector of C4PPDKZml-GUSplus (only have C4PPDKZml promoter). That lay a foundation for constructing the vector of OPPDKZml (only have C4PPDKZml promoter).(4) In this study, we successfully constructed the vector of C4PPDKZml (only have C4PPDKZml promoter). The vector has been transformed into maize by collebrators in China Agricultural University.(5) Preliminary study showed maize PPDK protein had differential expression under high and low-N conditions. There was no significant phenotypic difference between the wild type and heterzoyous mutants, no matter under high nitrogen or low nitrogen conditions. The PPDK protein accumulation in wild type was two times higher than that of heterozygote under both high and low-N condition. The expression of wild type PPDK proteins were increased significantly faster than that of heterozygotes. The results showed that PPDK was indeed involved in N response.