| MIP(Major Intrinsic Protein)protein family is a group of transporter proteins widely spread in plants.In soybean plants,researchers identified one nitrogen fixing symbiosis related MIP family protein,and it was named nodulin-26.Through alignment of genomic and protein sequences,we found that in Medicago truncatula,the model legume plants,there is a nodulin-26 ortholog gene,Medtr8g087710(MIP8.1).Furthermore in the nearby chromosome region of MIP8.1,there is a close homolog of MIP8.1,Medtr8g087720(MIP8.2).In the previous studies from our group,by comparing protein accumulation inside the ER of nodule cells between wild type and dnf1,a mutant with defection in the nodule cell signal peptidese complex,we found that MIP8.1 accumulated in the ER of dnf1.This result indicated that MIP8.1and MIP8.2 may be the target proteins of DNF1 signal peptidase complex,and these two proteins may have important roles in the regulation legume nitrogen fixing symbiosis.To gain more detailed and more thorough information of MIP8.1 and MIP8.2,this study analyzed the spatio-temporal expression pattern of MIP8.1 and MIP8.2 by searching the online gene expression databases,the results showed that MIP8.1 and MIP8.2 are expressed exclusively inside the nodules.Meanwhile the investigation of co-expressed genes of MIP8.1 and MIP8.2 showed that MIP8.1 and MIP8.2co-express with many other transporter genes,indicating that MIP8.1 and MIP8.2 may function together with other transporter proteins to regulate the nutrients and energy exchange between symbiosome and the nodule cells.MIP8.1 and MIP8.2 sub-cellular localization can have significant implication for their molecular function.This study generated the recombinant constructs expressing MIP8.1-and MIP8.2-GFP fusion sequences and transformed the plant root cells through argobactium rhizogene Arqua1 mediated transformation,and subsequently obtained the transgenic roots.After inoculation with rhizobia ABS7 strain for 3 weeks,through confocal microscope analysis,this study found that MIP8.1 mainly localizes at the symbiosome membrane inside the nodule cells.This study generated the MIP8.1-and MIP8.2-GUS recombinant constructs and got the transgenic roots through plant transformation.With rhizobia inoculation,this study observed that MIP8.1 and MIP8.2 express almost exclusively inside the nodules.More than that,MIP8.1 and MIP8.2 express most highly in the differentiation zone and fixation zone.MIP proteins form hetero-or homo-complexes to function normally in eukaryotic cells.To confirm the protein interaction of MIP8.1 and MIP8.2 themselves and between each other,this study generated constructs for Bi-fluorescence complementation in Arabidopsis protoplasts.With the successful generation of Arabidopsis protoplast and protoplast transformation,this study will analyze the hetero-or homo-complexes formation of MIP8.1 and MIP8.2.This study made the CRISPR/Cas9 genome editing constructs,and transformed the plant cells to knockout MIP8.1 and MIP8.2 respectively,the results showed that knocking out them separately will not cause any significant defection of nitrogen fixing symbiosis,implicates that MIP8.1 and MIP8.2 may have functional redundancy.This study is going to knockout MIP8.1 and MIP8.2 spontaneously to fully demonstrate the role of these two genes in the regulation of symbiosome differentiation and nitrogen fixiation.Furthermore this study identified the homozygous revserse transposon insertion mutant of MIP8.1,which is very useful in the following analysis of MIP8.1 and MIP8.2 functions.The substrates and signal exchange between the symbiosome and their host nodule cells is vital for the successful nitrogen fixation,this study investigated the molecular function of two homolog transporter genes,MIP8.1 and MIP8.2,which are identified as probable DNF1 target proteins.Further study into the molecular and biochemical function of MIP8.1 and MIP8.2 can shed light into the hidden mechanisms of how plant hosts regulate signal,nutrient and energy exchange between symbiosome and nodule cells to achieve successful nitrogen fixing symbiosis. |
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