Analysis Of The Function Of PEPC And PPDK Promoter And Codon Bias Of PPDK Gene In Amaranthus Hypochondriacus L.

Amaranthus hypochondriacus is a dicotyledonous annual herb which belongs to Amaranthaceae Amaranthus. In accordance with the photosynthetic pathway, Amaranthus belongs to C4 plant. Our laboratory has been cloned pyruvate orthophosphate dikinase(PPDK) gene and phosphoenol pyravate carboxylase (PEPC) gene from the A.hypochondriacus,which are the two key enzyme genes in C4 pathway, and obtained their upstream promoter sequences.The study was researched on the function of the two promoters, in order to lay the foundation for theapplication of these 2 promoters in transgenic plants. The study aslo was reseasched on the codon bias of PPDK gene in A.hypochondriacus, which would provide basis for the genetic improvement of plants.1.The study was constructed three PEPC promoters, respectively are 5’-flanking deletion、lack of 600bp and lack of 1200bp. With these promoters replaced the CaMV35S promoter which is upstream in plant expression vector pCAMBIA3301 GUS gene, import the Agrobacterium LBA4404 by tumefaciens. With the Agrobacterium and tobacco leaf discs were cultured, and then detected the instantaneous expression of GUS gene. The results indicated that the three promoters can drive GUS gene transient expression in tobacco leaves.2.Replaced the PPDK promoter with the CaMV35S promoter which is upstream in pCAMBIA3301 GUS gene, and import the agrobacterium LBA4404 by tumefaciens. Using agrobacterium and tobacco leaf disc coculture, and detecting their GUS gene transient expression. Preliminary results showed that the PPDK promoter can drive GUS gene transient expression in tobacco leaf.3.The PEPC promoter 1200bp and PPDK promoter of the transgenic tobacco plants have been got. The leaves of the transgenic tobacco seedlings were analysised by the GUS histochemical staining.The results showed that all of these promoters can drive GUS gene expression, and they have no significant difference in different promoters. Under the lighting,their expression quantity seemed to be increased, but the difference was not significant.4.PPDK is one of the most important C4 key enzymes. In this paper, the complete coding sequence of PPDK gene of grain amaranthus (A.hypochondriacus) was analyzed by CHIPS, CUSP and Codon W programs online for confirming codon bias, and then compared with PPDK genes from different plant species. The results showed that PPDK gene of A.hypochondriacus was bias toward the synonymous codons with A and T at the third codon position. The phylogenic analysis suggested that PPDK gene of A.hypochondriacus was evolutionarily closer to dicotyledons, such as S.tuberosum, R.communis, Medicago truncatula and so on, than to monocotyledons Z.mays, S.bicolor, E.crusgalli. But O.sativa is an exception, which codon bias of PPDK gene is closer to that of A.hypochondriacus than to that of Z.mays or S.bicolor. The codon bias of PPDK gene of A.hypochondriacus was different from that of E.coli or yeast. There are 22 codons showing significantly usage differences between PPDK gene of A.hypochondriacus and genome of E.coli, and 7 between PPDK gene of A.hypochondriacus and genome of yeast. Therefore the yeast expression system may be more suitable for the expression of PPDK gene of A.hypochondriacus.