Development Of Transgenic Soybean And Screening Pure Line For High α-tocopherol

Tocopherols, commonly known as vitamin E, are a class 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. However,α-tocopherol is present only as a minor component while its biosynthetic precursor, γ-tocopherol, is present at high level. These observations suggest that the important enzymeγ-tocopherol methyltransferase (γ-TMT) of theα-tocopherol biosynthetic pathway, which catalyzed the methylation of γ-tocopherol to form α-tocopherol, is likely limited in the seeds of most agriculturally important oil crops. With the deeper understanding of tocopherol biosynthesis, it becomes possible that α-tocopherol level can be increased through manipulating essential gene in its biosynthesis. Attempting to increase α-tocopherol level in oil crops, the full-length cDNA (BoTMT) of γ-TMT had been obtained from Brassica oleracea. The plant constitutive-expression vector pBin-TMTL and the plant seed-specific expression vector p7S-TMTL for BoTMT had been constructed also. And the former vector has been transformed into soybean successfully.The study included two sections as following. One is transformation of soybean cotyledonary node with seed specific expression vector p7S-TMTL through Agrobacterium-mediated co-transformation. Twenty-six Kanamycin-resistant plants were examined by PCR with specific primers, in which four plants showed positive signals, indicating preliminary thatγ-TMT was transformed into the genome of these four plants. Meanwhile, cotyledonary node transformation was supplemented and optimized, which provided the novel idea and method for cotyledonary node transformation. The other section was the progenies of transgenic soybeans transformed with the vector pBin-TMTL and examined by PCR and Western blot. Results showed that BoTMT inherited and expressed successfully. There was no difference on physiology character between transgenic soybeans and wild types except the character of number of seeds and yields of single plant, on which transgenic soybeans are better than wild types. In additional, the seed-soaked with kanamycin has been used to screen transgenic soybean in the latter generation. Results indicated that soybean cultivar "JILINXIAOLI" No.1 can be screened by this method and kanamycin concentration was optimized at 400-600mg/L.