Studies On Transformation Of HrpZ_Psg12 Gene Into Soybean Mediated By Agrobacterium Tumefaciens

The soybean [Glycine max (L.) Merr] is an important oil and food crop, is the main source of vegetable protein, has an irreplaceable role in the world. However, in the soybean production, the annual yield losses were of10%-30%due to Cercospora leaf blight, Phytophthora sojae, soybean mosaic virus disease and other diseases, which directly restricting the yield and quality improved. New soybean varieties with disease resistance, high yield, good quality are extremely urgent。 Conventional breeding due to long breeding cycle, lack of quality resources, and gene linkage and exchange among geges, made a slow progress in the breeding of improved varieties. The application of genetic engineering technology has overcomed barriers in distant hybridization and made many excellent resistance gene into soybean with acquired resistance, and opened up a new way for soybean breeding for disease resistance.hrpLpsg12gene, coming from Pseudomoas syringae pv. glycinea, can encode harpin protein, which could cause hyperensensitive response in the non-host plants. Many plants could accept systemic acquired resistance with the help of HR.D-xylose cannot be used in many plants. Xylose isomerase, encoded by xylose isomerase gene(xylA) could transform D-xylose into xylulose. The latter can be applied to glycolytic and pentose phosphate pathway in most plants. The products that xylA encoded are harmless to host plants, therefore, xylA has a wide application prospects as a select marker used for soybean genetic transformation.With gene manipulation, gene hrpZpsg12fragment was transferred into soybean with the help of select marker xylA, the transgenic plants possessed ability of disease resistance.Main manipulations are as follows:According to sequence of gene xylA on GenBank, a pair of specific primers were designed for amplification of xylAfrom genome DNA of E.coli top10. The amplificated fragments were connected with pMD18-T vector; According to sequence of gene hrpZpsg12, a pair of specific primers were designed for amplification of gene hrpZpsg12and modified the end with Xba I or Sac I seperately. pGM-hrpZpsg12plasmid was employed as the templates. The amplificated fragments were connected with pMD18-T vector;pCAMBIA3301and pMD18-hrpZpsg12plasmid were digested by Xba Ⅰ and Sac Ⅰ, recycling target fragments. The recombinant expressed vector was successfully established after target fragments connected together by T4DNA Ligase.pMD18-xylA and pCAMBIA3301-hrpZpsg12plasmid were digested by Xho Ⅰ, recycling targets fragments. The recombinant expressed vector pCAMBIA3301-xylA-hrpZpsg12was successfully constructed after target fragments connected together by T4DNA Ligase.Several factors effecting embryonic tip culture were optimized. The adventitious buds were successfully induced by adding3.5mg/L6-BA and0.1mg/L IBA into the shoot regeneration medium.0.5mg/L IBA+1mg/L NAA was suitable for roots inducing and the root frequency could be reached to94.41%.In order to demonstrate D-xylA was whether applied to soybean, certain amount of D-xylose were added into shoot regeneration medium to observe the growth and development of adventitious buds. hrpZpsg12fragments were transferred into soybean, by using Agrobacterium-mediated transformation method and D-xylose as the selective agent. two regeneration plants were testified positive by PCR.