Functional Analysis Of Translational Controled Tumor Protein Gene MTR₁g083380 During Symbiotic Nodulation In Medicago Truncatula

Translationally controlled tumor protein?TCTP?,a kind of constitutive protein family founded in the eukaryote,has a high degree of conservatism,and plays an important biological role in many different cellular processes,such as cell growth,cell cycle regulation,cell apoptosis and stress response and so on.Rpf41,a TCTP gene in the leguminous plants Robinia pseudoacacia,was identified during pre-period separation experiment in our lab,and it was first founded that TCTP plays a role in the robinia pseudoacacia-rhizobium symbiotic system.In this paper,to further research the function of TCTP in the robinia pseudoacacia-rhizobium symbiotic system,with Medicago Truncatula,the model plant of leguminous plants as the research object,the function of MTR₁g083380,the homologous gene of Rpf41 gene during Locust nodule formation was identified by subcellular localization,RNA interference?RNAi?and overexpression etc.The research contents and the results are as follows: 1 Construction of MTR₁g083380 gene subcellular localization vector and location study in the onion epidermalHomologous recombination method was used to construct the subcellular localization vector of MTR₁g083380 gene,and it was transformed to onion epidermal cell.The distribution of MTR₁g083380::GFP fusion protein in the onion epidermal cell was observed by laser scanning confocal microscope.The results indicated that MTR₁g083380 protein widely distributed in cell nucleus,cell skeleton,and some cell membrane,which provided a basis for the further research in the function of the MTR₁g083380 gene.2 Construction of MTR₁g083380 gene RNAi plasmid,overexpression vector and phenotypic analysis of transgenic plantRNAi and overexpression binary vectors of MTR₁g083380 gene was constructed from Medicago truncatula,and it was transfered to the damaging roots of Medicago truncatula,via rhizogenes-mediated transformation method.Besides,the influences of MTR₁g083380 gene on the series of processes of rhizobium infection and nodules development,and the growth and development of Medicago Truncatula was observed;it was proved bydetecting expression level of related genes.The results showed that there existed distinct differences between the phenotype of RNAi transplants and control group,RNAi transplants appeared growth retardation,the parts on the ground were small and the leaves were yellow,the number of nodules significantly decreased.Paraffin section and transmission electron microscopy was used to examine the effect of MTR₁g083380 gene RNAi on nodule morphology and ultrastructure,the results indicated that there was a severe premature senility and cortex thickening in the nodules from RNAi transplants after infected rhizobium 30 days,The infected area of cells turned lessening and there was no nitrogen fixation zone.Bacteriods in the infected cells were disorderly,its shapes produced changes,serious deformation and shriveling appeared,the peribacteroid space was increasing and a large amount of vacuole precursor appeared,along with the phenomenon of bacteroid fusion.The expression of leghenmoglobin gene and two nodule-specified marker genes?DMI1,NIN?in the transformed roots was analyzed by using qRT-PCR method.Compared with control group,the expression quantity of three genes reduced in different levels.The TCTP gene of Medicago truncatula was interfered with RNAi,causing the formation of abnormal nodules,and the activity of nitrogen fixation decreased significantly.The overexpression of MTR₁g083380 gene had no obvious effect on the process of rhizobium infection and nodules development.As the time went on,the growth of plants,the number and size of nodules,along with the length of roots were higher than the control group.The aging of nodules?30,45,60dpi?from overexpression group was far less than that of the control group.The nitrogen fixation area of nodules and electronic density in the bacteroid were larger than the control group.The expression quantity of leghenmoglobin gene from overexpressed transformed roots?30dpi?was significantly higher than the control group.In summary,the overexpression of MTR₁g083380 gene can delay the aging process of nodules.3 Construction of MTR₁g083380 gene promoter :: GUS gene fusion vector and detection of expressing genes.MTR₁g083380 gene promoter :: GUS gene fusion vector was constructed,and it was transfered to the roots of Medicago truncatula.Then we test the expression in transformed roots.