Functional Analysis And Interactional Protein Screening Of GmSPX3 Related To Soybean Tolerance To Low-phosphorus Stress

As one of the essential elements of plants,phosphorus is involved in cell construction,as well as many other physiological and biochemical processes in plants.Although the phosphorus content in soil is not low,little available phosphorus can be used by plants directly.Therefore,it is necessary to tap the autonomous genetic potential of plants,and culture new varieties with high phosphorus efficiency.In recent studies related to plant tolerance to low-phosphorus stress,the SPX domain was discovered.It plays an important role in plant phosphorus balance,yet few reports involving SPX genes in soybeans have been reported.In this paper,in order to study the function of a soybean gene which has high homological relativity with AtSPX1 in Arabidopsis,named GmSPX3,and search for the interaction proteins of it,gene cloning and some related experiments were made.The results of the study are as follows:1.Based on the NCBI database,and using the sequence of AtSPX1 in Arabidopsis as a template,a 60%homologous sequence in soybean,named GmSPX3,was searched.Its length was 870 bp,and it was located on chromosome 4 in the soybean.The Bioinformatics analysis showed that the GmSPX3 in the soybean had the same domain and similar gene structure as Arabidopsis.Treating the Williams 82 soybean under the stress of low P,the RT-PCR analysis of GmSPX3 in the leaf and root both showed that the expression levels increased with time and were diseased very deeply after recovering.We then constructed the fusion expression vector pMDC83-GFP-GmSPX3 and transformed it into onion epidermal cells and Arabidopsis protoplasts.The subcellular localization results showed that the GmSPX3 was located within the nucleus.The plant overexpression vector pCAMBIA3301-GmSPX3 was constructed and transformated into Arabidopsis using the Floral dip method.After determining the Phosphorus concentration,the results showed that the T3 generation of transgenic Arabidopsis with low phosphorus treatment had lower phosphorus concentration than the wild-type in the same medium.2.Treating the Williams 82 soybean with low P,we took the root at the 10th day to construct a yeast hybrid cDNA library using Gateway technology.After screening the cDNA library by the bait of GmSPX3,after several tests 21 positive clones were obtained.Two transcription factors,GmUNE12 and GmPosF21,were selected as the candidate genes.The results of the yeast hybrid system and BiFC showed that the GmUNE12 and GmPosF21 proteins both interacted with the GmSPX3 bait protein.The RT-PCR analysis of GmUNE12 and GmPosF21 under low phosphorus showed that the variation trends were the same as GmSPX3.