Accumulation Of Oxalate In Spinach And Analysis Of Related Gene Functions

Oxalic acid is a simple dicarboxylic acid,which is widely distributed in plants,accounting for 3%to 10%of the dry weight of plants.It has the functions of regulating plant pH,alleviating heavy metal toxicity,inducing disease resistance and regulating Ca²⁺concentration.However,oxalic acid is a very serious problem for human health,causing kidney stones,high oxalic acid and other related diseases,while spinach is a vegetable with high oxalic acid content.In this study,using spinach as experimental material,the accumulation of oxalic acid in spinach was tested by changing the formation of nitrogen supply,environmental stress and phytohormone spraying.At the same time,13 genes of key enzymes in the Synthesis and degradation pathway of oxalic acid were identified by screening in the spinach genome database.Real-time quantitative polymerase chain reaction?qRT-PCR?was used to analyze the expression pattern of these genes under different conditions.To verify the function of these genes in oxalic acid accumulation,plant expression vectors were constructed and these spinach oxalate metabolism related genes were overexpressed in Arabidopsis thaliana.Additionally,The transgenic Arabidopsis thaliana was treated with exogenous oxalic acid,aluminum stress and salt stress to find out if these genes have any function under abiotic stress.In this paper,the study on the accumulation of oxalic acid in spinach provides some guidance for the low oxalic acid spinach breeding and plants growing management,while the expression analysis of related genes and the study of transgenic arabidopsis laid a theoretical foundation for exploring the molecular mechanism of spinach oxalate accumulation and the biological functions of the key genes.The main findings are as follows:1.The supply of ammonium nitrogen reduced the oxalic acid content of the two spinach materials,which was significantly lower under complete ammonium nitrogen supply than complete nitrate nitrogen supply at 24 h,which was 81.5%and 65.7%of the control,while complete nitrate nitrogen supply only increased the content of oxalic acid in spinach in a short period of time.High temperature stress,oxidative stress,salt stress and drought stress all increased the content of oxalic acid in spinach,and the high temperature stress had the greatest effect,which reached 1.48 times of the control at 12 h.Phytohormones like abscisic acid?ABA?,gibberellic acid?GA?,ethephon?ETH?,indole acetic acid?IAA?,salicylic acid?SA?and6-benzylaminopurine?6-BA?could increase the accumulation of oxalic acid in spinach in a short period of time,while ascorbic acid?AA?and methyl jasmonate?JA?had a sustained effect on the increase of oxalic acid content in spinach.2.Expression analysis by qRT-PCR showed that nitrate nitrogen could induce the expression of SoGLOs and SoOXACs in a short period of time and inhibit SoOXS's expression,while ammonium nitrogen was the opposite.The expression of SoGLO2,SoGLO4 and SoGLO5 was significantly up-regulated under high temperature stress,while the expression of most oxalate-degrading enzyme-related genes was inhibited.Spraying of phytohormone 6-BA,GA and IAA up-regulated the expression of SoGLO1,SoGLO2 and SoGLO3.The expression of SoOXACs are transiently induced by ABA and GA,and AA and IAA can continuously up-regulate the expression of SoOXS.3.12 plant overexpression vectors were successfully constructed.Arabidopsis thaliana was genetically transformed by inflorescence infection method,and T₃generations which can stably express those spinach-oxalate-metabolism-related genes were obtained and identified by PPT selection,PCR and RT-PCR.Overexpression of SoOXAC1 and SoOXAC2 significantly increased the oxalic acid content of Arabidopsis thaliana,while overexpression of SoOXO1 reduced the oxalic acid content of Arabidopsis thaliana to 69.9%of the wild type.Overexpression of SoOXS also reduced the oxalic acid content of Arabidopsis thaliana.The resistance of Arabidopsis leaves overexpressing SoOXDC2?SoOXO1 and SoOXS to exogenous oxalic acid was also significantly enhanced.