Clone Of Safe Selectable Marker Gene In Transgenic Plants And Clone Of NAMP/Bt Fusion Gene And Its Construction Of Prokaryotic And Plant Expression Vector

To speed up the genetic improvement of crops process, the foreign gene was transformed into plants and a very small number of transformed cells were obtained. A set of efficient and safe selection method was very important.In the past, there was a selectable marker gene in the transgenic plants, and the selectable marker gene may be transferred to weeds or microbiology. So that weeds or microbiology would get the ablity of antibiotic or herbicide resistance, which would affect and damage the environment and human health.D-serine has a certain toxicity and the D-serine dehydrate gene from Escherichia coli can encode the D-serine ammonia lyase which can relieve the toxicity of D-serine on plant. There are several D-amino acid itself is toxic to plants even in the relative low concentration of D-amino acids because many plants lack the normal channels of D-amino acid oxidation deamidation, Therefore, we can use D-amino acid lyase having the toxicity to plant as a safe selectable marker. D-serine can be dehydratased into pyruvate (or fat), water and ammonia, and transgenic seeds with dsdA can be selected easily from the wild-type.The dsdA is a good safety markings because amino acids can be easily digested into water, ammonia and pyruvate (or fat) which have no environmental pollution.D-serine dehydratase gene (dsdA) from the bacteria Escherichia coli was cloned by polymerase chain reaction with specific primers in this research, which was ligated to the T-vector to analysis sequences. The result is that the consensus between the cloned sequence and sequence from reference is 99.93%. The analysised dsdA gene was ligated to the prokaryotic expression vector pET-28a, induced by IPTG and detected by polyacrylamide electrophoresis (SDS-PAGE) .In the prokaryotes dsdA made a very good expression, the molecular weight of protein is 48.0KDa. Then, the dsdA was bigated into the plant expression vector pBI-121 with selectable marker kan, and the plant expression vector was transformed into the, then, dsdA and kan were used respectively to obtain transgenic tobacco and test the effect of dsdA. The result showed that when dsdA was used as a selectable marker, a lot of D-serine was used and efficient was low, it was not as sensitiveas kanamycin in the preliminary step.