The Study On Enhancing Folate Content In Plants By Overexpression Of GCHI And ADCS Genes

Unlike plants and microorganisms, humans and mammals are unable to synthesize folate de novo. Plants are the major dietary source of folates for human beings. But many cereals, such as wheat, maize lack of this vitamin. In recent years, biofortification is an economic and effective way to improve folate contents. The full length genes of the key enzymes of folate biosynthesis GCHI (GTP Cyclohydrolase I) and ADCS(4-Amino-4-Deoxychorismate Synthase) from Glysine max have been cloned. Moreover,15independent GCHI transgenic plants were got by overexpression of GCHI gene; the expression vector Gluc pro-GmGTPCHI-ADCS were constructed and introduced into Triticum aestivum through particle bombardment,500To generation plants were regenerated from immature embryos callus. This study mainly focused on the molecular identification of transformants, folate analysis in positive transformants and preparation of the antibodies of GmGCHI and GmADCS to detect protein expression level. The main results are as follows:1.The content of folate in the hygromycin resistent, PCR positive GmGCHI Arabidopsis thaliana was analysed. The folate contents were increased in7lines out of15transgenic lines, and were significantly increased by28.75%,23.35%and47.94%, respectively in3lines (8g-32,8g-38and8g-39). Meanwhile, we have obtained8independent GmADCS transgenic plants.2.By hybridizing GmGCHI Arabidopsis thaliana with GmADCS Arabidopsis thaliana that were all hygromycin resistent and PCR positive, we obtained16hybrids of GmGCHI+/GmADCS+Arabidopsis thaliana. The folates contents in GCHI+/ADCS+plants were measured. The results indicated that the folate contents in8g-39-7×ADCS19-4,8g-39-7×ADCS20-5-2,8g-39-7×ADCS23-3-2were increased by100%,100%and93%, respectively, compared with wide type and increased by40%,40%,32%, respectively, compared to GmGCHI transformants.3.The DNA of To GCHI/ADCS Triticum aestivum was extracted by the method of CTAB. We obtained47Bar+/GmGCHI+/GmADCS+Triticum aestivums from500transformants by PCR identification. The folates contents of GmGCHI+/GmADCS+Triticum aestivum T1generation seeds were assayed by LC-MS-MS. The data showed that the folate levels in4lines (CY1-134, CY1-143, CY1-112, CY1-113) were increased by32%,46.78%,48.23%and62.45%, respectively, compared to that in control.4.The specific amino acid sequences of Triticum aestivum GmGCHI, GmADCS were obtained based on sequence alignments. The specific sequence of Triticum aestivum GmGCHI, GmADCS were amplified and subcloned into pet30a to produce pet30a-GmGCHI711, pet30a-GmADCS849constructs. The expression of both fusion proteins were nearly100%in inclusion bodies, and the protein amount was low. Recombinant proteins were purified by nickle chelate chromatograph, and the mouse-sourced antibodies were gained and the antibody specificity is under testing.In conclusion, the GCHI and ADCS genes from Glycine max have significantly enhanced the folate contents of transgenic Arabidopsis thaliana leaves and Triticum aestivum seeds. These data indicated that introduction of GmGCHI and GmADCS genes into plants could promote folate biosynthesis in transgenic plants, which has provided a technological foundation for better understanding the basic rule of folate biosynthesis in plants.