Cloning And Functional Analysis Of Folate Binding Protein (SiFBP) Gene In Foxtail Millet

Foxtail millet(Setaria italica(L.)P.Beauv)is one of the important grain crops.It has a long history of cultivation and abundant germplasm resources in China.As one of the favorite foods of northern people,millet has very high edible and nutritional health value.Folic acid is a water-soluble vitamin,which plays an important role in human nutrition and health.It cannot be synthesized by the human body itself and must be absorbed by the daily diet.Numerous studies have shown that natural folate in plants is unstable under natural conditions,and changes in p H and temperature can lead to its degradation.In this study,gene expression pattern analysis,yeast heterologous expression experiment and genetic transformation methods were used to elucidate the function of SiFBP binding and stabilizing folate.The main results were as follows:1.We identified only one SiFBP gene in the foxtail millet genome,and its nucleic acid sequence length was 885 bp,its predicted that the molecular weight was 32 k Da,isoelectric point was 9.12,the coefficient of unstability was 52.16,and the average water was-0.122,we speculate that the protein was unstable,hydrophilic.2.The SiFBP promoter element analysis results showed that,there were multiple abiotic stress and hormones related cis element,abscisic acid response element,gibberellin response element,light response element,anaerobic induce required cis regulatory elements,Fungal induction response elements,cis-acting elements related to meristem expression,and tissue-specific regulatory elements such AS,etc.3.The expression pattern analysis results showed that the relative expression level of SiFBP gene at seedling stage was as follows: roots,leaves,stalk;The relative expression levels of SiFBP gene was higher in tissues from S1 to S5.The tissue-specific expression pattern of SiFBP was taken on analysis by the transgenic plants SiFBPpro::GUS/Col-0,GUS staining result showed that it was expressed in the root tip of lateral roots and developing seeds.The protein was localized in cytoplasm by transient transformation technique in tobacco.4.The yeast heterologous expression vector was constructed to induce the protein expression,and the binding affinity between SiFBP and FA,THF,5-MTHF was verified.It was found that the SiFBP could bind FA,THF and 5-MTHF.5.To analyze the ability of SiFBP stablize folate,we over-express SiFBP in Arabidopsis thaliana.Folate assay was performed on transgenic plants.The results showed that THF content in transgenic lines was increased than wild type.6.The transgenic Arabidopsis thaliana with SiFBP gene was analyzed by metabolic group and transcriptome.It is speculated that the overexpression of SiFBP plays an important role in amino acid biosynthesis,amino acid metabolism,flavonoid biosynthesis and phenylpropanoid biosynthesis.