The Accumulation Of Spinach Folate And Key Gene Analysis

Folate is a B-vitamin,necessary for the human body,yet cannot be synthesized by ourselves.Lackage of folate will hinder certain physiological activities of plants or the human body.Folate deficiency is a problem challenging the world,especially in developing countries.Compared with adding folate fortifiers to food or directly eating folic acid tablets,taking folate through daily diet is a safer and more effective method.Folate biofortification has made positive progress in crops such as potato,tomato and rice,but because of the complexity of folate metabolic pathways,the effect of folate biofortification varies from plant to plant.However,less study has been conducted on spinach folate accumulation.Spinach(Spinacia oleracea L.),a green leafy vegetable containing high proportion of folate,is loved by people domestically and internationally,but currently there are few studies on spinach folate.Based on the evaluation of folate content in spinach germplasm resources,this paper analyzes the spatiotemporal changes of spinach folate accumulation,the response of environmental factors,and screens through bioinformatics analysis,expression analysis,gene cloning and genetic transformation of related candidate genes,to filter possible key gene for spinach folate accumulation.The main results were:1.By homologous sequence alignment,25 genes related to folate accumulation were selected from spinach genome database.They were ADCL and ADCS involved in the synthesis of para-aminobenzoic acid,GCHI,NUDT,DHNA involved in the synthesis of pterin,DHFR,DHFS,HPPK/DHPS involved in the assembly and tailing of folate,and folate polyglutamylation and intracellular transport salvage pathways FPGS,GGH,PTAR,as well as 5FCL,5FCLL,DHC,FTHS,GCSH,GCSL,GCSP,GCST,MS,MTHFR,PURU,SHMT3,SHMT1 a,SHMT1b,which are related to one-carbon metabolism.2.The folate contents of 228 spinach germplasm resources ranged from 10.18μg/g dry weight(DW)to 311.5 μg/g DW,with an average content of 60.78 μg/g DW.The folate contents of spinach leaves were increased significantly under the treatments of 0.5 m M salicylic acid,5 m M serine and 5 m M phenylalanine,with 1.5times,1.1 times and 1.2 times higher than those of the control group,respectively.The folate content of petiole under the 100 m M sodium chloride treatment was also higher(57.21 μg/g DW)than that of the control group.The folate contents of spinach leaves were highest at the 12-leaf stage(151.90 μg/g DW)during the tested growth period,while the leaf folate contents was the lowest at the 8-leaf and 10-leaf stage(average108.45 μg/g DW).Compared with the leaves,the contents of folate in petioles changed smoothly during the growth period(average 73.21 μg/g DW),except the lowest folate content found in the 12-leaf stage petioles(62.39 μg/g DW).3.Phenylalanine elevated the expression of NUDT,FTHS,MS,SHMT1 a,SHMT3 in spinach leaves;serine induced the expression of all tested genes in spinach leaves except ADCS,PTAR,DHFS,HPPK,5FCLL,FTHS,GCSH,PURU;salicylic acid treatment caused ADCL,NUDT,DHNA,GGH,DHC,MS,and MTHR up-regulated,and sodium chloride up-regulated the expression of ADCL,NUDT,DHNA,GGH,PTAR,HPPK,5FCL,DHC,FTHS,GCSP,GCSL,MS,and PURU.Different environmental treatments affected spinach folate-related gene expression,but all of the above treatments significantly induced up-regulation of three genes,NUDT,MS and DHC expression.4.Most of the folate anabolic genes were highly expressed in spinach cotyledons except MTHR;GCHI and MS were mainly expressed in leaves at the six-leaf stage and also in petioles at the six-leaf stage;GCSH was highly expressed in leaves at the eight-leaf stage;HPPK/DHPS,DHFS,PURU,and DHC were mainly expressed in leaves at the twelve-leaf stage;leaf petioles at the ten-and twelve-leaf stages DHFS,5FCL,GCSP,PURU were moderately positively correlated with changes in folate content at the reproductive stage,and FTHS was strongly positively correlated.5.The shoot folic acid content of Arabidopsis strains transduced with So DHNA,So DHFS,So GCHI,So NUDT,and So PTAR increased 1.7-fold,1.6-fold,1.3-fold,1.2-fold,and 1.2-fold,respectively,compared with those of wild-type Arabidopsis.When transiently overexpressed the above five genes in spinach leaves,only So PTAR significantly increased the folate content by 2.5-fold to 24.00 μg/g FW,while So GCHI significantly decreased folate content by 0.8-fold to 13.52 μg/g FW.When So PTAR and So DHNA were transiently co-expressed in spinach leaves,spinach folate content increased by 1.5-fold to 20.22 μg/g FW.