The Establishment Of Soybean Safety Marker System Using P5CS Gene

In order to avoid genetic transformation experiments using resistance marker genes may lead to food safety and ecological Safety problem, the experiment created Agrobacterium-mediated soybean cotyledon nodes transformation method safety marker system Prd29A-P5CS/mannitol, soybean Δ’-pyrroline-5-carboxylate synthase gene (P5CS) gene as a marker gene, with mannitol or NaCl as a screening agent; In the appropriate selection pressure, inducible rd29A promoter driving P5CS expression, so P5CS gene can improves the transgenic plant resistance, transgenic plants can be screened out from non-transgenic plants. This safety mark system put the security of transgenic plants and the resistance of plants very good together, prove to be a feasible screening system for soybean transgenic.P5CS is a rate-limiting enzyme in proline synthesis pathway, when plant is in drought, salinity, temperature, and other conditions, plant can synthesis of a large number of proline, thereby improving plant drought tolerance, salt tolerance and resistance to low temperature. Using P5CS gene as a screening marker gene and construction of plant expression vector, not only to avoid the resistance marker genes of potential safety problems, while improving the transgenic plant resistance.rd29A promoter sequences that contain conserved sequences DRE, ABA stress regulation, can induces the expression of downstream genes when the plant at a low temperature, drought, high salt environment. rd29A promoter instead of constitutive promoter35S promoter, then avoid P5CS gene in transgenic plants over-expressing bring negative effects to plants by starting the downstream genes when induceding by stress.Clone rd29A promoter and P5CS gene isolated from Arabidopsis thaliana and soybean, respectively, to replace the pCAMBIA1301plasmid35S promoter and hygromycin gene, constructing pCAMBIA1301-Prd29A-P5CS security screening marker expression vector.Experimental SI culture medium NaCl, mannitol selection pressure of identificat of five soybean varieties (HanJianYiHao, Jiyu72, Ji Nong24, Williams, black bean) of cotyledon node regeneration. The experimental results show that the mannitol selection pressure of five kinds of soybean cotyledon node festival90%death is300mM,350mM,350mM,200mM,300mM; NaCl selection pressure is75mM,65mM,65mM,75mM,65mM; and regeneration bud screening from NaCl screening pressure has obvious yellow phenomenon comparing with regeneration bud growth screening from mannitol screening pressure; the cotyledon node regeneration growth of Ji Nong24, Jiyu72soybean are significantly better than that of other three varieties; so select Ji Nong24, Jiyu72soybean for transgenic experiment, mannitol as a screening agent.Agrobacterium-mediated (with pCAMBIA1301-Prd29A-P5CS plasmid) transformation Ji Nong24, Jiyu72soybean varieties, mannitol as selective agent; which Agrobacterium-mediated (with bar gene) transformation Ji Nong24, basta as selective agen; compare transgenic plant growth status and conversion rate of these two screening system. Experiment got PCR positive seedlings, and Prd29A-P5CS/mannitol screening system was superior to the transformation system which selection marker gene is bar.The experimental set up the plant expressione security marker Prd29A-P5CS/mannitol vector, also study the transgenic soybean varieties and selection pressure as well as the conversion rate, did many preparation and exploration for following verification of this soybean transgenic experiments and soybean screening system..