Tocopherols are amphipathic, lipid-soluble antioxidants that are produced only by photosynthetic organisms. They play an important roles in human and animal nutrition. Gamma-tocopherol methyltransferase (G-TMT), one of the key enzymes in tocopherol biosynthetic pathway in plants, catalyzes the methylation of delta- and gamma-tocopherol, converts them into beta-, alpha-tocopherols, which is a committed step to elevate vitamin E activity and nutrition value. Introduction of G-TMT gene into plant could be a promising approach to improve a-tocopherol content in the seed. Here, the authors present the results on the cloning of G-TMT gene from Arabidopsis thaliana and constructing the expression vector pBinGlyBarl carrying cloned G-TMT gene. Total RNA was extracted from Arabidopsis thaliana leaves using TRIZOL reagent and treated with DNase I according to the manufacturer's instructions. The first-strand cDNA was synthesized by using RevertAid H Minus Reverse Transcriptase according to the manufacturer's instructions. Then, PCR product using G-TMT primers was inserted into pJET1.2/Blunt vector and the cloned G-TMT fragments were sequenced. The comparission of the cloned gene with the published G-TMT gene (accession # NM_l 05171.3) showed 99 percent similarity. The cloned G-TMT gene was inserted into expression pBinGlyBarl vector and the plant transformation vector pBinGlyBarl-G-TMT was constructed. The structure of the vector was verified by colony PCR and restriction digestion using EcoR1 and Xhol restriction enzymes.
Lọc theo danh mục
Lượt truy cập :
23,880,145
Xem toàn văn