关键词: 叶酸, 二氢喋呤合成酶, FolP, 生物合成, 代谢工程

Abstract:

Folate cannot be synthesized de novo in human body. Plants are the main source of dietary folates for Human, many fruits，tubers and seeds are poor in this vitamin. Inadequate intake is accociated with the increased risk of anemia, neural tube defect of new births, cardiovascular disease and even some kinds of cancers. Folate deficiency is a worldwide problem，and metabolic engineering provides an opportunity to enhance folate content in food crops.
Folate is synthesized from pteridine,p-aminobenzoate(PABA) and glutamate moieties in plants. The branches of peterin and PABA synthesis in plants is supposed to be located in cyctosol and plastid, respectively,while the final five steps of folate synthesis is proved in mitochondria. Dihydropetorate synthase(DHPS) catalyses the formation of dihydropetorate from 6-hydroxymethldihydropterin and p-aminobenzoate(PABA). To elucidate how DHPS controls the flux of the folate biosynthesis in plants, FolP gene encoding dihydropetorate synthase from Escherichia coli was overexpressed in Arabidopsis thaliana. FolP gene sequence was cloned with E.coli genomic DNA by PCR. Since DHPS is located in mitochondria in plants, it was targeted by a mitochondrial tageting sequence from yeast CoXIV gene, and was driven by constitutively expression promoter of double Cauliflower mosaic virus(CaMV) 35S. Competent cells of Agrobacterium tumefaciens AGL0 were transformed with the binary vector containing FolP. Kanamycin selection was conducted in each generation. PCR was performed on the primary transformant plants (T₁) and T₃ plants with primers based on sequence of CaMV 35S and FolP, while Northern blotting with probe based on FolP sequence and folate analysis by microbial assay with Lactobacillus casei were carried out on T₃ seedlings. It was indicated from PCR detection that the expression cassette containing CaMV 35S and FolP gene was integrated into A. thaliana genome. A specific signal was detected by Northern in transgenic lines but not in wild type plants, which demonstrated that E.coli FolP gene was expressed in Arabidopsis . As shown in Fig.6, the mean values of total folate of nontransgenic and transgenic lines are 0.58 µg/g FW and 0.87 µg/g FW, respectively. The total folate content of transgenic plants was increased by 48% in the three lines tested, when compared with that of non-transgenic plants(wild type). Values for the transgenic lines were significantly higher than that of the non-transgenic lines at P<0.05 by t test. The result indicated that DHPS has contributed to increasing the flux of folate biosynthesis of Arabidopsis.

Key words: Folate, Dihydropetorate synthase, FolP, Biosynthesis, Metabolic engineering