Folate Fortification In Plants By Overexpression Of PTR1 And GTPCHI Gene

Plant foods are the single most important source of folate. However, many plant food sources such as rice, wheat and maize are poor in this vitamin. So far, folate metabolic engineering in food crops is confined to E.coli GTP cyclohydrolase I (GTPCHI), Mammalian GTP cyclohydrolaseâ… (GTPCHI), A. thaliana GTP cyclohydrolase I (GTPCHI) and Aminodeoxychorismate synthase (ADCS). The aim of this study is to elucidate whether Leishmania major PTR1(NADPH-dependent pterin reductase) which reduce oxidized pterins to their bioactive forms and Glycine max folate synthesis pathway enzyme GTP cyclohydrolase I (GTPCHI) will increase folate content in plants (Nicotiana tabacum and A. thaliana). The main results obtained as follows:1 We constructed plant expression vectors promotered by double CaMV35S: pCAMBIA3301-PTR1 and pCAMBIA3301-MT-PTR1(PTR1 was fused with the mitochondria targeting sequence from A. thaliana MDH gene), which was transformed into Nicotiana tabacum, to obtain transgenic plants.2 Systematically study of the PTR1 gene from Leishmania was carried out on transgenic plant:(1)The analysis by Semi-Quantitative RT-PCR showed that the PTR1 which was located in cytosol and MT-PTR1 which was located in Mitochondria was expressed in Tobacco. (2)Folate measurement was conducted on young green leaves both PTR1 and MT-PTR1 transgenic lines. In young green leaves, the folate content of PTR1 transgenic lines both in cytosol and Mitochondria was 1085.86,802.18 ng/gfw, respectively, and that of the wild type plants was 522.65 ng/gfw.3 Two homologous GTPCHI were isolated by RACE:two complete GTPCHI cDNA sequences were isolated and cloned by RACE. The two homologous GTPCHI were designated as Gm03g21540 and Gm08g46160, lengthes at 1380bp and 1374bp. Analysis of the amino acid sequences Align of the two GTPCHI with Solanum lycopersicum showed that positive was 66% and 74.9%, identity was 59.6% and 68.2% respectively; Analysis of the amino acid sequences align of the two GTPCHI with A. thaliana showed that positive was 66% and 74.9%, identity was 59.6% and 68.2% respectively.4 The analysis by Semi-Quantitative RT-PCR showed that Gm03g21540 and Gm08g46160 were expressed in Glycine max root, shoot and leaf. Gm03g21540 had highest expression in root, then foll wed by shoot and leaf. Gm08g46160 had highest expression in shoot, then foll wed by root and leaf.5 Plant expression vectors promotered by double CaMV35S:Gm03g21540 and Gm08g46160 were constructed and then transfomed into the plants.