Indentification And Symbiotic Function Characterization Of AsDJL1Interacting With Lipid Transfer Protein AsE246in Astragalus Sinicus

In former work, some nodule specific expresse genes were isolated from nodules cDNA library of Astragalus sinicus L constructed by our lab. Among these genes, AsE246only express in infected roots of Astragalus sinicus L, it encodes a lipid transfer protein. Experiment datas showed that:the AsE246has a regulatory function in nodule development and nitrogen fixation procedure.In the preliminary studies, basing on yeast two-hybrid system, we have screened and obtained a candidate target protein AsDJLl interacting with bait protein AsE246. AsDJL1is homologous genes of DnaJ-like,DnaJ protein which is the supplementary protein of the molecular chaperone protein HSP70. AsDJL1has many biological functions such as activating ATPase activity, protein folding and further processing, enhancing the biological resistance, protecting cell membranes from damage of heavy metals.This project focus on the molecular interacting between AsDJLl and AsE246, and the role of AsDJLl in nitrogen fixation procedure. At first, the full length cDNA of AsDJLlgene was cloned by RACE (rapid amplification of cDNA ends). In order to corroborate the interaction between AsE246and AsDJLl, six truncated mutants of AsDJLl sequence were constructed and cloned into plasmid vector for yeast two-hybrid. By target protein expression and purification and pull-down experiment, we verified the interaction of AsE246and AsDJL1in vitro. Using bimolecular fluorescence complementation (BiFC) method, we did the subcellular localization and identified the interacting between AsE246and AsDJL1in tobacco plant living cells.To study the role of AsDJL1in nitrogen fixation procedure, two recombinanted vectors for the AsDJLl gene overexpression and RNAi were constructed. They are transfered into Astragalus sinicus tissue by Agrobacterium. The expression of AsDJLl was improved and silence respectively in transfer plants. They showed different phenotype in symbiotic procedure. This work can give a basis for the further investigation on the function of the AsDJL1protein during the nodulation and symbiotic nitrogen fixation.