Identification,Transformation And Function Analysis Of Vitamin E Biosynthesis Genes γ-TMT,HPT And HPPD From Medicago Sativa L.

Alfalfa (Medicago sativa L.) is an out-crossing autotetraploid and possesses a large genome, which limited the breeding efforts in the discovery and utilization of novel gene resources in alfalfa. With an aim to increase the nutritional quality, optimize the vitamin E content and composition in alfalfa, we have identified and characterized the function of y-tocopherol methyltransferase (y-TMT), homogentisate phytyltransferase (HPT) and P-hydroxyphenylpyruvate dioxygenase (HPPD). The progresses are as follows:1. y-TMT was cloned from Medicago Sativa L.using rapid-amplification of cDNA ends. Phylogenetic tree analysis indicates that MsTMT is closely related to MtTMT from Medicago truncatula indicating that we have successfully cloned y-TMT from alfalfa. Quantative real-time PCR results showed that MsTMT was expressed through out the whole plant, the expression levels were most abundant in leaves, and least abundant in flowers. NaCl,15% PEG, dark and ABA could all induce the expression of MsTMT, however, cold inhibited the expression of MsTMT. α-tocopherol levels increased significantly in transgenic Arabidopsis seeds, no difference was observed in transgenic Arabidopsis leaves. The endogenous vitamin E biosynthesis genes were not altered in transgenic Arabidopsis. Under mannitol treatment, seed germination rate, biomass, CAT and POD activities and osmotic stress marker genes expression of transgenic Arabidopsis were significantly higher than the corresponding control, which indicates that stress tolerance of transgenic Arabidopsis is higher than the corresponding control, a-tocopherol, a-tocotrienol and crude protein levels were increased significantly in transgenic alfalfa. The leaf senescence was inhibited in transgenic alfalfa under dark treatment suggesting that MsTMT regulates transgenic alfalfa leaf senescence and can be used as a candidate gene in alfalfa nutrional quality improvement.2. Homogentisate phytyltransferase (HPT) was cloned from Medicago Sativa L. Using rapid-amplification of cDNA ends. It encoded a protein of 411 amino acid and belonged to PT_UbiA Superfamily. Multiple sequence alignment result showed that the similarity between MsHPT protein and other HPT were 80%. Promoter sequence of the gene was cloned using genome walking technology. Analysis result of MsHPT promoter showed that defense and stress responsiveness cis-element, hormones (MeJA, ABA, GA and ethylene) responsiveness cis-elements and light responsiveness elements were presented in the promoter. Quantitative Real-time PCR results revealed that MsHPT was expressed throughout the plant, and mainly expressed in leaf. Expression of MsHPT was increased significantly in response to NaCl, PEG, GA3 and ABA treatments. The flexible expression of MsHPT in response to different treatments suggesting MsHPT might involve in alfalfa stress resistence.3. P-hydroxyphenylpyruvate dioxygenase (HPPD) was cloned from Medicago Sativa L.using rapid-amplification of cDNA ends. It contained a 1305 bp Opening Reading Frame (ORF), which encoded a protein of 434 amino acids. It belonged to Glo_EDI_BRP_like superfamily containing an HPPD N like and an HPPD C like domains. The active sites were abundant in C-terminal of this protein. Multiple sequence alignment result revealed that the similarity between alfalfa HPPD and other HPPDs was 82.46%. Three Fe binding sites, which were essential for the activity of this enzyme were conserved among tested HPPD sequences. Quantitative real-time PCR results revealed that MsHPPD transcripts were detected in all the organs that tested. It was most abundant in leaves and then roots and seeds, the transcripts were lowest in early flowers. The expression levels in leaves were about 100 times higher than in early flowers. We also cloned the promoter of MsHPPD and fused with GUS, GUS expression levels were found in transgenic Arabidopsis cotyledons, primary roots, sepals, petals, stigma, filament tubes, pollens and the ends of seeds. No GUS activities were observed in root tip, hypocotyl and true leaves. Expression levels of MsHPPD could be induced by NaCl, ABA, PEG and darkness. β-tocotrienol and total vitamin E content were increased significantly in transgenic Arabidopsis seeds. Under normal condition, the germination time of transgenic Arabidopsis was significantly earier than control. Under mannitol and NaCl treatments, the seed germination rate was significantly higher than the corresponding control, however the transgenic Arabidopsis were insensitive to ABA treatment. In dry seeds, ABA content in transgenic Arabidopsis and wild type were same, however, when seeds were imbibed in water for 36h, ABA content in transgenic lines were significantly lower than wild type. In dry seeds, expression levels of ABA biosynthesis genes NCED3,5,9 and ABA signaling pathway genes ABI3 and ABI5 were not significantly different in transgenic Arabidopsis and wild type, however, when seeds were imbibed in water, the expression levels of these genes were significantly reduced in transgenic Arabidopsis. pBIl21-MsHPPD-GUS was transformed into alfalfa, no difference was observed in terms of vitamin E content in transgenic alfalfa leaves, however, crude protein content was significantly increased in transgenic alfalfa.