The Effect Of Co-Expression Of Glycine Max GmGCHI And GmADCS Genes On The Folate Content Of Arabidopsis Thaniana

Folate plays key role in one-carbon transfer reactions and maintaining the organism’s life. It is a tripartite molecule composed of pterin, p-aminobenzoate (pABA) and glutamate moieties. Three different subcellular organelles involved in folate biosynthesis, cytoplasm responsible for pterin biosynthesis, chloroplast responsible for pABA biosynthesis, mitochondria responsible for folate biosynthesis. GTP cyclohydrolase I gene (commonly short for GCHI) encodes the committing enzyme of pterin synthesis in plants. Aminodeoxychorismate synthase gene encodes the committing enzyme of pABA synthesis in plants. The two enzymes exert major control over flux through the folate pathway. In this research, the effects of co-expression of glycine max GmGCHI and GmADCS genes on Arabidopsis thaliana folate biofortificationon were investigated.1. The transgenic GmGCHI Arabidopsis pure lines are obtained by hygromycin resistance and PCR determination. The GmGCHI expression of different lines identified By RT-PCR analysis. The measurement of folate content by HPLC-MS/MS showed that total folate of transgenic lines and5-methyltetrahydrofolate (5-CH3-THF) and5-formyltetrahydrofolate (5-CHO-THF) are both higher than the wild type, especially GmGCHI122and127displayed significant differences compared with the wild type. Its total folate average content is higher than wild type by40%and the highest increasing level is57%. The5-CH3-THF average content is higher than the wild type by43%and the highest increasing level is56%, and the5-CHO-THF average content is higher than the wild type by69%and the highest increasing level is94%.2. The transgenic GmADCS Arabidopsis pure lines are obtained by hygromycin resistance and PCR determination. The GmADCS expression of different lines identified By RT-PCR analysis.3. Co-expression Arabidopsis lines were obtained and confirmed by hybrided and PCR determination of two genes co-existence. HPLC-MS/MS showed that the total folate content and5-CH3-THF and5-CHO-THF of co-expression lines are both notable higher than the wild type and GmGCHI introduced lines. The total folate average content of co-expression transgenic lines is higher than WT and GmGCHI introduced lines by85%and21%respectively, and the highest enhanced level is111%and37%respectively. The5-CH3-THF average content of co-expression transgenic lines is higher than WT and GmGCHI introduced lines by83%and19%respectively, and the highest enhanced level is113%and36%. The5-CHO-THF average content of co-expression transgenic lines is higher than WT and GmGCHI introduced lines by128%and27%respectively, and the highest enhanced level is156%and55%. It suggests that co-expressing GmGCHI GmADCS genes in Arabidopsis thaliana can enhance the content of folate obviously. 4. Recombinant transformation vectors of pV11393-GmGCHI and pV11393-GmADCS in silkworm expression system have been constructed for further expressing ptoteins of GmGCHI and GmADCS and the analysis of the activities of both enzymes.