| A nodule-inducible gene MtSC20(Medtr0334s0010)had been found in Medicago truncatula in our previous research,which belongs to the phytocyanins family and AGP(Arabinogalactan proteins)protein family.The database query results showed that it was highly expressed in roots and nodules,and may participate in the symbiosis process,but its function was unknown.In this study,the analyses of gene expression,RNA interference and overexpression were carried out in order to indentify the function of MtSC20 in the symbiotic nodulation of Medicago truncatula.Furthermore,the interacting protein of MtSC20 was screened through the membrane system yeast two hybrid and was further identified.The main results are as follows:(1)The results of expression pattern analyses showed that MtSC20 was mainly expressed in roots and nodules in different tissues at 28 dpi(dpi,days after inoculation).Compared with that in the roots treated with nitrogen deficiency,the expression pattern of MtSC20 in the inoculated roots was similar to that in the roots supplied with nitrogen.In the early and middle stages of nodulation(1-14 dpi),MtSC20 expression was induced by nitrogen or rhizobia(except at 5dpi),then was inhibited at 21 dpi and was strongly induced again at 28 dpi,especially in the inoculated roots,indicating that MtSC20 is not only related to rhizobial infection,but also involved in nitrogen response.Tissue expression analysis showed that MtSC20 was expressed in the areas with active cell division in roots and nodules,including lateral root primordia,nodule primordia,and the apical meristem of mature nodules.The subcellular localization results showed that MtSC20 was a membrane system localization protein,which was localized in both the cell membrane and the endoplasmic reticulum.(2)The analyses of RNA interference(RNAi)and overexpression showed that MtSC20 was involved in the extension of Infection threads and the release of rhizobia.After MtSC20 interference,the number of infection threads from the cortex and nodule primordia was decreased significantly,and the number of mature nodules was reduced at 28 dpi.In the RNAi nodules at 28 dpi,the content of leghemoglobin and its gene expression were downregulated and the activity of nitrogenase was decreased.The activities of catalase(POD)and superoxide dismutase(SOD)related to ROS scavenging were reduced,and cysteine protease activity increased,indicating that the root nodules were subjected to oxidative stress and appeared premature aging.After overexpression of MtSC20,the root nodules at 28 dpi appeared pink.The activity of nitrogenase and the content of leghemoglobin increased significantly in the overexpression nodules,showing higher nitrogen fixation capacity.The activities of the enzymes related to ROS scavenging were also increased and the activity of cysteine protease was decreased,suggesting a delay in aging of the nodules.In addition,the submicroscopic structure of root nodules was observed.The morphology of infection threads in the RNAi nodules was significantly different from that in the control,in which the infection threads became wider and filled with rhizobia.The morphology of infection threads of overexpression nodules was similar to the control.(3)Six proteins that may interact with MtSC20 were screened by yeast two hybrid method of membrane system.Medtr3g008840 protein(a member of PMEI protein family)was selected for further verification.Yeast point-to-point,pull-down and BiFC experiments were done and the results showed the interaction between the AGP domain of MtSC20 and Medtr3g008840 protein.In summary,this study proved that MtSC20 was involved in the formation of symbiotic root nodules of Medicago truncatula and may be involved in the extension of infection threads and the release of rhizobia through the interaction with medtr3g008840 protein.These results demonstrated for the first time that the stellacyanins(SC)family protein is involved in the symbiotic nodulation of legumes,and laid a foundation for further elucidating their functions. |
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