| Vitamin E, an antioxidant, plays a very important role in plant, animal and human being. Plant is the main resource of the vitamin E for human being. So, it is important for us to clone the relative enzyme genes and improve vitamin E content in plant. In this paper, we used the EST BLAST method and molecular cloning method to clone Glycine max homogentisate phytyltransferase(HPT) and γ — tocopherol methyltransferase(γ-TMT) genes. Then, the HPT gene was transformed to potato leave discs and stem segments by Agrobacterium tumefaciens of LBA4404. The aim of this study is to obtain the highly quality of potatoes that will overexpresses vitamin E though metabolic engineering.· Cloning and analysis of Glycine max HPT and γ-TMT genesUsing the amino acids sequence of Arabidopsis thaliana homogentisate phytyltransferase(Gen--eBank Accession, NM-179653) and γ — tocopherol methyltransferase(GeneBank Accession, AY090280) as a query probe to blast Glycine max EST database, the novel soybean HPT gene cDNA of 1777bp and γ-TMT gene cDNA of 1296bp were obtained. The two sequences were confirmed by RT-PCR, molecular cloning and sequencing, the result confirmed that the in silico cloning cDNA sequences were accurate. HPT gene contained a complete ORF, from 173bp to 1408bp, encoded 411 amino acids; a complete ORF of γ-TMT gene, which from 49bp to 1101bp, encoded 350 amino acids. The HPT gene and γ-TMT were accessed by GenBank, accession numbers are AY956421 and AY960126, respectively.The deduced amino acid sequence of Glycine max HPT was aligned against related HPT sequences of other species such as Triticum aestivum(AAP43912), Hordeum vulgare(AAP43911), Medicago sativa(AAU14795), Arabidopsis thaliana (NP849984), Oryza sativa(XP478828) , Chlamydomonas reinhardtii(AAV32452), the results showed that Gm had a highly homologous with Ms(the identity was 72.32%), had a lowly homologous with Cr(the identity was 8.93% only), the results also showed the identity of 39.73%, 39.96%, 57.59% and 23.88% with Ta, Hv, At and Os respectively. Phylogenetic tree of plant HPTs is mainly consistent with the classification system of these plants.The deduced amino acid sequence of Glycine max γ-TMT compared with Triticum aestivum (AAZ67143), Medicago truncatula(AAX63740), Arabidopsis thaliana(NP849984), Oryza sativa(XP-467331), Chlamydomonas reinhardtii(CAI59122), Brassica oleracea(AAO13806), we found the amino acid was almost conserved and the multiple sequences alignment showed theidentity of 56.46%, 52.12%, 56.20%, 56.71%, 45.57%, 56.71%, respectively. Phylogenetic tree of plant γ-TMTs is mainly consistent with the classification system of these plants.· Semiquantitative RT-PCR of Glycine max HPT and γ-TMT and expression analysis in different tissuesUsing semiquantitative RT-PCR, we could found the illumination and nutrient limitation impact the expression of HPT, with the abiotic stress time increasing, the expression of HPT will be increased obviously, while, in this abiotic stress, the expression of the γ-TMT has not increases clearly.In this papar, we also analysised the expression of HPT and γ-TMT in different tissues. The results showed that HPT espressed in leaves, stems and roots, and the expression in leaves were higher than stems and roors, meanwhile, γ-TMT expression in soybean leaves are higher than stems, rarely γ-TMT expression in soybean roors was found.· potato genetic transformation of Glycine max HPT geneIn this part, we constructed a potato transformation vector PBI121HPT. through mediated by Agrobacterium tumefaciens LBA4404, Glycine max HPT gene was transformed into potato leave discs and stem sebments. 45 explants were regenerated on selective media with kanamysin. One of them was confirmed by PCR as transgenic plant, primarily. |
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