Cloning And Functional Analysis Of MtSPX Family Genes From Medicago Truncatula

Phosphorus is an essential element for plant growth and development,but the available phosphorus concentration in soil is usually quite low,which makes it a limiting factor for plant growth,that is,plants are facing phosphorus deficiency.Therefore,how to improve the utilization of phosphorus of plants has become an urgent problem to be solved.Alfalfa is important leguminous forage with high nutrient content and wide cultivation area.As a model plant of leguminous plants,Medicago truncatula has important research significance.Proteins containing SPX domain exist widely in higher eukaryotes,but their functions are still unclear.Some studies have found that they may be involved in phosphorus signal transduction.In this study,real-time fluorescence quantitative technique was used to study the expression level of MtSPX family genes in different tissues of Medicago truncatula under low phosphorus and nitrogen treatment in order to observe the role of MtSPX genes in response to low phosphorus and nitrogen stress.Subcellular localization of MtSPX protein was studied by constructing transient expression vector and Agrobacterium infection.Response to low phosphorus stress.The main results of this study are as follows:1.Real-time fluorescence quantitative detection showed that MtSPX1,MtSPX3 and MtSPX4 genes were expressed in leaves,roots and nodule,and responded to low phosphorus stress and low nitrogen stress.MtSPX3 and MtSPX4 genes are involved in the positive regulation of phosphorus in leaves,MtSPX4 acts positively in regulation of phosphorus in roots,while MtSPX4 gene is also involved in the positive regulation of nitrogen in leaves and roots,and MtSPX3 is involved in the positive regulation of nitrogen in nodules.2.The overexpression vectors and transient expression vectors of MtSPX3 and MtSPX4 genes were constructed.The expression of MtSPX3 gene was detected in the nucleus by Agrobacterium-mediated expression in tobacco leaves.3.Transgenic plants of T2 generation of Arabidopsis thaliana with MtSPX3 and MtSPX4 genes were obtained by Agrobacterium tumefaciens immersion test,which provided materials for the follow-up study of the two genes.MtSPX1 gene knockout vector was successfully constructed.It provides materials for the subsequent construction of mutant plants of the gene.4.In the phenotypic study of low-phosphorus stress,through the comparison of plant phenotype,phosphorus content and aboveground biomass,it was found that MtSPX4 mutant plants absorbed less phosphorus under low-phosphorus stress than wild-type plants,and suffered more toxicity under high-phosphorus stress,that is,they were more sensitive to high-phosphorus stress.The above results showed that MtSPX1,MtSPX3 and MtSPX4 genes played certain roles in the regulation of phosphorus and nitrogen respectively under phosphorus and nitrogen stress.At the cellular level,MtSPX3 gene was expressed in the nucleus.The phosphorus uptake rate of MtSPX4 gene-deleted mutant plants decreased under low phosphorus stress and was seriously affected by phosphorus toxicity under high phosphorus stress.In summary,MtSPX gene may plays an very important role in phosphorus regulation of Medicago truncatula L.