Cloning Of PPDK Gene From Amaranthus Hypochondriacus L. And Transformation Of Medicago Sativa L.

The production of forage grass, an effective way to provide the livestock with feed in high quality and quantity, is also playing an important role in saving the foodstuffs, improving the environment, reformation of agriculture production, coordination of the proportion of the agriculture forest and feed, and making agriculture develop continually. As with many advantage traits, forage is the resource of a lot of valuable genes. To clone valuable genes can provide useful objectives for plant engineering.Amaranthus hypochondriacus L. has higher phothosynthesis rate than some common C4 plants such as Zea mays L.and Saccharum sinense Roxb. Pyruvate orthophosphate dikinase (PPDK), a ratelimiting enzyme in C4 photosynthesis and crassulacean acid metabolism (CAM), catalyzes the synthesis reaction of phosphoenolpyruvate using ATP and pyruvate. Some research indicated that over expressing the gene encoding PPDK could improve the photosynthesis rate and the productiong of crops.In this study, the C4type-PPDK gene in Amaranthus hypochondriacus L., a plant material with high photosynthesis rate, was cloned and transformed into Medicago sativa L., which is a kind of important forage and C3 plants.The experiment results are as follows:The gene encoding C4 type-PPDK in Amaranthus hypochondriacus L. has been cloned, with the help of"Rapid Amplification of cDNA Ends"(RACE). It was indicated that the PPDK cDNA from Amaranthus hypochondriacus L. was 3.2 kb, including an ORF of 2895 bp, which encoding a peptide of 947 AA of 106kD. Comparing with PPDK genes in other plants, we found that there is a highest similarity with Mesembryanthemum crystallinum of 82%.Plant expression vector containing the full length of C4-type PPDK gene was constructed and transgenic Medicago sativa plants were obtained. The effect of the foreign C4type-PPDK gene in C3 plant Medicago.sativa will be analyzed.A sequence about 500 bp in the upstream of the C4 type-PPDK cDNA was cloned with reverse PCR and LA-PCR. Analyzed with PLANT CARE, it was found that there are several Cis-elements in this sequence such as TATA-box, CAAT-box, GT1-motif and sp1.A plant expression vector harboring Gus gene promoted by a part of the 500 bp fragment was constructed and transformed into tobacco. Transient GUS expression had been detected in callus of tobacco.