Cloning And Plant Expression Vector Construction Of MtERN1-3 And Other Nodulin Genes From Medicago Truncatula

Nodulin Gene is a kind of symbiotic nitrogen fixation genes, which only exist in the root nodule cells infected by rhizobia. Nodulin Gene are very important for the formation legume root nodule, maintaining the function of nitrogen fixtion and the metabolism of products. So far several key nodulin genes have been found that they were induced at the early stage of legume root nodule formation. It played an important role in the information communication between plants and rhizobia, the process of infection and the formation of root nodule. However, the work principle and Whether these genes would play corresponding function when transducted into non-legumes need further research.Through the study of leguminous plant mutants, signaling pathways of symbiotic nitrogen-fixing system is revealed and the functions of many proteins or gene related symbiotic nitrogen-fixing is cleared. DMI1 encodes a ligand-gated ion channel that localizes to the nuclear membrane. DMI2 encodes a leucine-rich-repeat receptor kinase that is localized to the membrane and is required for tight root hair curling around the bacteria. DMI3 encodes a Ca2+-calmodulin-dependent protein kinase that is required for the induction of cell division in the root cortex and for the thanscriptional changes required for the establishment of the symbiosis. Two GRAS family transcriptional regulators, nodulation signaling pathway 1 (Nspl) and Nsp2, are also required for Nod-factor-induced transcriptional changes. The response to NF also involves Ca2+ spiking. ERN1, ERN2 and ERN3 belong to the same family genes. ERN1, ERN2 and ERN3 are applied to the EN0D11 promoter, as Transcriptional regulatory factors in signal transduction pathway. EFD is a key control transcription factors, which connected early and late stage of nodule formation. It can inhibit cytokinins primary receptor gene MtRR4 and nodule formation, meanwhile, it also promote cell differentiation of the nodule meristematic tissue. The proteins, which is produced by MtLYK3 and LjNFR5 etc gene, as nodular factor receptor, combine with nodular factor, and control the formation of the invading line and root hair curling.Biological nitrogen fixation is studied widespreadly. Many scholars transduced nitrogen fixation genes into non-leguminous or leguminous plants to to explore the function of a single gene and the effects of biological nitrogen-fixing function. But if the symbiotic nitrogen-fixing mechanism in leguminous plant is transplanted into non-leguminous plants, indispensable genes related symbiotic nitrogen-fixing mechanism in leguminous plants must be transgened into non-leguminous plants. This experiment is based on early signal transduction mechanism of invading line formation in leguminous plants.7 genes that control signal transduction are cloned from Medicago truncatula,and same of them construct multiple genes plant expression vector. The main results are as follow:1,Nodulin gene MtNSP1 (3617 bp),MtNSP2 (3381 bp),MtDMI3 (5903 bp),MtENOD40 (4119 bp),MtERNl (3951 bp),MtERN2 (3123 bp) and MtERN3 (2561 bp) are amplified via PCR with PrimStar HS DNA polymerase or LA Taq DNA polymerase from Medicago truncatula. Analysis shows that they are consistent with the recorded sequence by GenBank.2,Nodulin gene MtDMI3,MtERN1,MtERN2,MtERN3 and nuclear matrix attachment region NtMAR-pS4 are inserted into vector pCAMBIA1302, to construct multiple nodulin genes expression vector pVCT1313.