Prokaryotic Expression Of Fusion Gene SAI, CHS8 And CHIâ…¡ Of Soybean And Construction Of Their Plant Expression Vector And Transformation

Isoflavones is a crucial secondary metabolite in metabolism of soybean,which has importantly impressed on human health.It plays a significant role in the prevention and treatment of osteoporosis sickness, cardiovascular disease and cancer. Research found that there are tow enzymes, including chalcone synthase and chalcone isomerase, to catalysis the biosynthesis of isoflavones sequentially.But there are no ideal results of increasing content of isoflavones by transformation of single key enzyme through genetic engineering.However, transformation for several times is a very difficult subject in genetic transformation system of soybean,therefore fusion gene is a very efficient method to solve this problem.Because the fusion protein may have influenced on the expression or folding, selection of linker is very important to construct fusion gene vector. 2A linker is 20 amino acids peptide from the food-and-mouth disease virus,which will be cut by itself in the eukaryotic cell, as a result,the fusion protein is divided into two independent proteins.Therefore,2A linker not only can help the transformation of multi-genes,but also facilitate the separation of the fusion protein to exercise their respective functions in plant. So far,2A linker has been successfully used in corn of crops,if it will be successfully used in dicotyledonous plants such as soybean, it can contribute a lot to increase the expression of quality genes of soybean.We cloned the genes of chalcone synthase and chalcone isomerase in this study,and got their fusion genes SAI and SI using 2A linker and GS-rich linker respectively.Then we constructed the prokaryotic expression vector of each gene and analysised their expression in E.coli,the results showed that 2A linker had no influence on expression of target proteins in prokaryotic and it was not cut by itself in E.coli.At the same time,we constructed the eukaryocyte expression vector of each gene and transferred them into Agrobacterium. Additionally,KOZAK was added to the upstream region of the single gene CHS and CHI respectively to facilitate the expression of target protein. there exists a fragment which is called KOZAK in mRNA of eukaryotes.Genotype screening, optimization of infection condition which including period of growth of Agrobacterium,method of transformation and time of infection,were exercised when we transferred them into soybean cotyledonary nodes using Agrobacterium-mediated transformation.Additionally, the best selective concentration of hyg was also determined in the later of transformation. Now we have gained the resistant transgenic plants using the transformation system of soybean cotyledonary nodes which has been optimized. As stated previously,our study provided the foundation for increasing the biosynthesis of the isoflavones and for the further study.