Obtaining And Identification Of Transgenic Soybean With The Gene Of BoTMT

Tocopherols, commonly known as vitamin E, are a group of lipid-soluble antioxidants synthesized only by plants and other photosynthetic organisms. In addition to their role as antioxidants, tocopherols are essential micronutrients needed for human health. Of the four naturally occurring tocopherols, α -tocopherol has the highest vitamin E activity and is preferentially absorbed and distributed throughout the body. Plant oil is the main dietary source of tocopherols. However, a -tocopherol is present only as a minor component while its biosynthetic precursor, y -tocopherol, is at high level. These observations suggest that the enzyme of Y -tocopherol methyltransferase (γ -TMT) in tocopherol biosynthetic pathway, which catalyzed the methylation of γ-tocopherol to form a -tocopherol, is likely limited in the seeds of most agriculturally important oil crops. To increase α - tocopherol of the oil plants, the full-length cDNA (BoTMT) of Y -TMT was cloned from Brassica oleracea by the Institute of Microbiology, the Chinese Academy of Sciences. Enzymetoc activity assay indicated that the BoTMT expression at translational level highly converted γ-tocopherol to α -tocopherol in vitro. Attempting to increase α -tocopherol level in oil crops, they also constructed plant constitutive-expression vector pBin-TMTL and the plant seed-specific expression vector p7S-TMTL for BoTMT.To obtain α-tocopherol enhanced soybeans, the transgenic work was conducted in the present study. By using cotyledonary node as the explant, we transferred BoTMT constructed in seed specific expression vector p7S-TMTL and plant constitutive-expression vector pBin-TMTL to soybean through Agrobacterium-medialed co-transformation system. The total of 330 fertile regenerated plants were obtained including 127 with pBin-TMTL. Fifty-eight Paromomycin-resistant plants were found to be positive in PCR screening, and 4 transgenic plants were further confirmed by Western blot, indicating that γ-TMT was possibly integrated into the genome of soybean and was successfully expressed. PCR tests showed that 10 T₁ plants were positive, indicating that the transferred BoTMT inherited successfully.Meanwhile, we also optimized the Agmbacterium-mediated cotyledonary node transformation system of soybean. The results showed that chlorine was more efficient than HgCl₂ for seed sterilization. It was showed that Jilinxiaoli No.l had the highest regeneration frequency among the genotypes we used, followed by H6001 and NF58. The application of cystine, DTT and Na₂S₂O₃ during co-cultivation along with light exposure could decrease the browning of the cotyledonary nodes.