Elevation Of The Folate Content Of Arabidopsis Plants By Overexpression Of The Bacterial FolC And FolP Gene

Unlike plants and microorganisms, humans can not synthesize folate de novo and depend entirely on their dietary supply. 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.Though the folate biosynthesis pathway of plants is now largely elucidated, little is known about the control of flux and accumulation of folate. The first two pioneering trails of the regulation of folate biosynthesis foucued on GTP cyclohydrolase I (GCHI). The aim of this study is to elucicate whether the other two enzymes of the folate biosynthesis pathway , i.e. dihydrofolate synthase(DHFS)/folypolyglutamate synthase(FPGS) and dihydropteroate synthase (DHPS) will increased the folate content in A. thaliana through genetic modification. DHFS/FPGS and DHPS in E.coli was encoded by FolC and FolP gene, respectively. Each of FolC and FolP gene was fused to the mitochondria targeting sequence from yeast CoXIV gene, and was promoted by double CaMV 35S, respectively. The main results obtained as follows:1. Systematically study of the FolC gene from E.coli was carried out on transgenic Arabidopsis: (1) The analysis by Northern blot and Taqman RT-PCR showed that the FolC gene from E.coli was expressed in Arabidopsis. (2) Folate measurement was conducted both on seedlings and young green leaves. In seedlings, the folate content of homozygous FolC transgenic lines C4-3-3 , C11-6-8 and C12-13-1 was 2.23, 2.46 and 2.51 nmol /gfw, respectively, and that of the wild type plants was 1.77 nmol /gfw. While in young green leaves , the folate content was 5.07, 5.54 and 4.31 nmol/gfw, respectively, and that of the wild type plants was 3.09 nmol /gfw. Therefore the folate content of the transgenic line was significantly higher than that of wild type plants both in seedlings and young green leaves. (3) The highest expresser of FolC (C11-6-8) also showed the highest folate content, while line C4-3-3 and C12-13-1, which the expression of FolC was lower than that of C11-6-8, showed a relatively lower folate content. It was thun indicated the mRNA level of FolC gene was correlated with the folate level of FolC transgenic lines. (4) The overexpression of FolC gene did not promote the expression of the endogenous genes encoding DHFS/FPGS in FolC transgenic lines.Therefore the overexpression of foreign FolC gene resulted in the enhancement of folate content in Arabidopsis, and it was proved that the increase folate content was from the contribution of foreign FolC gene but not the endogenous genes encoding DHFS/FPGS .2. The profiling of the expression of the endogenous genes encoding DHFS/FPGS was conducted on...