The Biological Functional Study On Folylpolyglutamate Synthase AtDFB In Arabidopsis

Folate are essential small-molecule metabolites for life activities. They participatie inbiosynthesis of nucleotide, amino acid and pantothenic acid by working as the donor of singlecarbon units. Folate is composed of three parts: pteridine, p-aminobenzoic acid and poly-glutamicacid residues. During the process of folate synthesis, folylpolyglutamate synthetase (FPGS) catalyzethe addition of glutamic acid residues to the γ-carboxyl group of single glutamate tetrahydrofolate(THF), whereby form a multi-tailed tetrahydrofolate. FPGS have three isozymes: AtDFB, AtDFCand AtDFD, which are in plastids, mitochondria and the cytoplasm respectively to perform theirfunction. Our lab have obtaineda DBF gene mutant from Arabidopsis. Its T-DNA inserted in the6thintron of DBF gene. When cultured with9.4N medium,root length of atdfb-3mutant is only about30%of wild-type strain; with0.3N medium, atdfb-3mutant barely grow and and the color of cotyledonturn purple. RT-PCR analysis of atdfb-3mutant and wild-type shows that the transcriptional level ofAtDFC gene is significantly increased in atdfb-3mutant, which suggests that AtDFC and AtDFB exitfunctional complementation under low nitrogen circumstances. Simultaneously,5-F-THF content ofatdfb-3mutant are lower obviously than WT and5-M-THF content of atdfb-3mutant are lower sligthlythan WT.There are two different transcripts of AtDFB gene in Arabidopsis thaliana(AtDFB CDS.1/AtDFBCDS.2). These two transcripts differ from each other by174base pair and58amino acids. To verifywhether these two transcripts have difference in biological function, we constructed two vectors(PH2GW7-DFB.1/PH2GW7-DFB.2)with CaMV35S promoter,which overexpress these two transcriptsrespectively. We then transform the overexpression vectors into wild-type Arabidopsis thaliana andatdfb mutant, and obtained the GM plant of Arabidopsis thaliana by resistance and PCR identificationscreening. Under bothcondition of9.4N and0.3N, the fresh weight and root length of over expressionplant and complementary plant are all close to wild-type Arabidopsis thaliana;the content of5-M-THFand5-F-THF of complementary plant is recovered in some extent.The results shown above suggest that AtDFB gene plays a very important role in folatebiosynthesis pathway. Under low nitrogen conditions, the lack of AtDFB gene directly results in rapidshrink of roots and decrease of folate content; while after overexpression of AtDFB gene, under oflow nitrogen condition, the phenotype of atdfb-3mutant is recovered to wild-type level, and the contentof folate has also increased. Both of these two transcripts of AtDFB gene functions on the recovery offresh weight, root length and folate content of atdfb-3mutant.