Obtention And Identification Of ZmTMT, GmTMT&GmHPT Transgenic Maize

Tocopherols, known as vitamin E, which is rich in plant tissues, are lipid-soluble and essential inthe human and animals diet. Vitamin E deficiency in human and animals will results in variousphysical diseases, because human and animals cannot synthesize vitamin E themselves, which is onlysynthesized by photosynthetic microorganisms and plants. Alpha-tocopherol displays the greatestefficacy for human and animals uptake, however-tocopherol often present only as a minor componentin crop seeds. Considering the dramatic consumption of the nature vitamin E, it’s very important forvitamin E to increase the contents in crop seed through breeding and transgenic plants.HPT and TMT are the key enzymes in the tocopherol biosynthetic pathway. HPT functions in theupstream of the vitamin E pathway and regulates the total tocopherols, whereas TMT functions in thedownstream and regulates-tocopherol contents. ZmTMT, GmTMT and GmHPT were isolated frommaize and soybean in our lab and the patents were obtained. Here, we introduced these genes into maizeby particle bombardment. The main conclusions are as follows:1, Plant expression vectors containing ZmTMT, GmTMT and GmHPT for maize transformationwere constructed, and subsequently the expression cassette and BAR gene cassette were introduced intomaize immature embryos and callus by co-bombardment. We obtained2transgenic positive lines forZmTMT gene,6for GmTMT,9for GmHPT. Transgenic ZmTMT lines were selfcrossed for5generation.Transgenic GmTMT and GmHPT lines were backcrossed for2generation by the inbred Zheng58.2, Tocopherol contents of transgenic lines were determined by HPLC. In the transgenic lines,ZmTMT promoted by Glb1promotor, which specially expressed in embryo and transformedγ-tocopherol into-tocopherol, resulting in-tocopherol contents2-4times higher than control. TheGmTMT and GmHPT promoted by Ubi promotor, resulting in-tocopherol contents were2-3timeshigher in transgenic GmTMT lines than control, and the total contents of tocopherols were1.2-1.5foldhigher in transgenic GmHPT than the control.3, Copy numbers of the transgenic lines were determined by Southern Blotting, and the flanksequences were obtained by Hi-TAIL PCR. The ZT1line from ZmTMT transgenic maize had more than10copies, and one of the insertion sites located at Chromosome1or4, three at Chromosome8. TheGT-1, GT-2and GH-3lines were from GmTMT transgenic maize. GT-1had7copies, and two of theinsertion sites located at Chromosome3and5. GT-3had3or4copies and two of the insertion siteslocated at Chromosome2or4. GT-5had5copies, one of the insertion sites located at Chromosome5.GH-1, GH-2and GH-5were from GmHPT transgenic lines. GH-1had1copy at Chromosome1, GH-2had2at Chromosome2or5. GH-5had3copies, one of the insertion site located at Chromosome5.The above results demonstrated that overexpression of HPT and γ-TMT, which are the key genesinvolved in tocopherol biosynthetic pathway, dramatically increased the contents of vitamin E and-tocopherol in transgenic maize seeds.