Improving Tocopherols Content And Composition Through Overexpression Of Tocoehromanol Biosvnthesis Genes In Barlev

Photosynthetic organisms synthesize a group of amphipathic antioxidants called vitamin E, which is an essential component of the human diet. Tocopherol and tocotrienol comprise the vitamin E class in plants. Recently, plant derived oils are the major sources of vitamin E in the human diet. Therefore, it would be very important to increase vitamin E content and activity in plants. Barley (Hordeum vulgare) ranks the fourth among cereals in terms of planting area worldwide. Agrobacterium-mediated genetic transformation is an effective way for gene function analysis and quality improvement in barley. In this study, an optimized transformation protocol was used to genetic modify vitamin E content and composition in barley, in order to provide a new direction for barley breeding. The main results were as follows:1. Molecular identification and vitamin E determination of HvHPPD and ScPDH coexpression transgenic barleyp-hydroxyphenolpyruvate dioxygenase (HPPD) and prephenate dehydrogenase (PDH) are two upstream enzymes in vitamin E biosynthesis pathway. Previous studies indicated that overexpression of HPPD and PDH can enhance vitamin E content via increasing substrate level of HGA. Yeast (Saccharomyces cerevisiae) PDH and barley HPPD were introduced into barley through Agrobacterium-mediated transformation. 10 regenerated plants were obtained, and 8 lines were confirmed to be positive transgenic plants by PCR identification. And qRT-PCR indicated that expression level of HPPD was significantly higher than that of wild type. There was a slight increase in β-tocotrienol content in HPPD-PDH#1 and #2 transgenic barley. However, HPPD/PDH coexpression plants showed no significant difference on Tocopherols content in leaves from two-month-old seedlings, compared with wild type. It suggested that composition of VE might vary among different tissues during development. Further studies would determine HGA and tocochromanol content in seeds and leaves from T1 transgenic plants to investigate the effect of increased HGA on VE biosynthesis in different tissues during development.2. Molecular identification and quality analysis of 35S:AtHPT and proHor3: HvHGGT transgenic barleyHomogentisate phytyltransferase (HPT) and homogentisic acid geranylgeranyl transferase (HGGT) are rate-limiting enzymes in tocopherol and tocotrienol biosynthetic pathway, respectively. Our previous work constructed transgenic barley by overexpression Arabidopsis HPT and barley HGGT, which were termed as 35S: AtHPT and proHor3:HvHGGT, respectively. Molecular identification and quality analysis of transgenic T1 and T2 lines have been performed. PCR analysis combined with Southern blot showed that HGGT#1 had single copy of target gene, while HPT#1 had four copies of target gene. Semi-qRT-PCR showed that the levels of target genes were significantly higher in proHor3:HGGT and 35S:HPT transgenic barley plants than those in wild type. RT-PCR also showed that overexpression of HGGT upregulated HvHPPD (an upstream gene) and repressed the expression of HvTMT (a downstream gene). It suggested that overexpression of HvHGGT could influence the expression levels of other related enzymes in vitamin E synthesis pathway.Tocochromanol content was measured using seeds and leaves of seedlings. Tocopherols content increased by 82%-112% (seeds) and 41%-58% (leaves) in 35S: AtHPT transgenic barley plants, while increased by 82%-94% (seeds) and 11% (leaves) in proHor3:HvHGGT plants, mainly due to the production of y-tocopherol and a-tocopherol. Meanwhile, tocotrienol content of proHor3:HGGT transgenic lines decreased in seeds and leaves, which was contrary to expectation. Tocotrienol content in 35S:HPT transgenic barley also decreased slightly. These findings indicated that, besides enzymes, substrate concentration might be also important for limiting vitamin E production.Moreover, HPT and HGGT transgenic plant exhibited altered phenotypes relative to the control plant, with increased thousand grain weight, larger leaves and accelerated growth during early development stage. It suggested that overexpression of HPT and HGGT could influence the development of barley.Overall, overexpression of HPT and HGGT enhanced tocopherols content significantly in both seeds and leaves, which would have potential applications for quality improvement in barley.