Creating Of Transgenic Soybean Co-expressing AtDCGS And ?-Zein And Its Effect On The Accumulation Of Methionine

Soybean(Glycine max(L.)Meer.)is one of the most vital crops for its abundance in protein and oil in the world.As it has high content and good quality of proteins,soybean provides an essential source of vegetable protein for humans and livestock with considerably economic value.However,similar to other proteins derived from plants,soybean protein has a low content of methionine which is deficient to meet the nutritional requirements of humans and livestock.Therefore,the increasing of the methionine content in soybean protein has important practical value for improving the soybean nutritional quality,human dietary nutrition and promoting the development of animal husbandry.In this study,the transgenic soybean overexpressing the methionine synthesis regulating enzyme gene(At DCGS)and the transgenic soybean overexpressing the methionine-rich storage protein gene(?-Zein)were crossed to integrate these two genes into one plant.Moreover,two binary expression vector containing both the At DCGS and ?-Zein genes were constructed and transformed to obtain the transgenic co-expressing two exogenous genes soybean.Resistance screening,PCR detection,q RT-PCR and southern blot were preformed to select the positive transgenic soybean co-expressing this two foreign gene.Currently,10 F3 generation transgenic soybean lines and 45 T3 generations transgenic soybean lines were obtained.Two transgenic soybean lines CZ-6 and CZ-9 which have high relative expression level and low copy number of foreign genes were selected for further study.The content of the methionine in some of the T2 and F2 generation transgenic soybean as well as their parents were measured.The results exhibited that the free methionine content in the mature seeds driving from transgenic generations over-expressing At DCGS was increased by maximal 3.3-fold,and the gene expressed under seed-specific manner was more higher than those under constitutive expression manner.In the transgenic soybean co-expressing the two foreign genes,the free methionine content was significantly lower than that of the parent that only expressing the At DCGS,and the total methionine content was further increased compared to the parent that only expressing the ?-Zein,implying that the partial loss of free methionine might be used for the accumulation of exogenous storage proteins and total proteins.Besides,the total methionine content of transgenic soybean seeds co-expressing At DCGS and ?-Zein increased by maximal 22% comparing with those of the wild type.The above results showed that the binary crossing progeny could combine both synthesis and storage pathways of methionine to effectively increase the accumulation of methionine content in soybean.The result of the determination of total protein and lipid content of wild-type and transgenic soybean seeds revealed that the accumulation of the methionine content significantly leaded to the increasing of total protein and the decreasing of the lipid content,suggesting that the free methionine content is a key limiting factor for the accumulation of soybean protein.Therefore,the enhancement of the synthetic ability of soybean methionine has a positive effect on the improvement of the content and quality of soybean protein.In this study,the methionine content and the protein quality were improved by regulating both the synthesis and storage pathways of methionine with the genetic engineering methods in soybean seeds,which provided new sights for the quality improvement of other crops.