Identification Of Proteins Interacting With CASTOR In Lotus Japonicus Symbiotic Signal Pathway

The CASTOR gene encodes predictable ion channel protein that is involved in Nod factor signal transduction in Lotus japonicus. CASTOR mutants undergo root hairs deformation, but do not response to Nod factors with intracellular calcium spiking.In order to investigate Nod factor signal mechanisms, we used Lotus japonicus with inoculation of Mesorhizobium loti for 2, 4, 6, 8 days to extract total RNA, according to BD Matchmarker^TM Library Construction and Screening Kits User Manual of Clontech, we constructed AD-cDNA library of Lotus japonicus, the efficiency of which was 2×10⁶ thansformants/3ug pGADT7-Rec. Colony PCR indicated that the fragments of AD-cDNA library were almost between 0.5 and 2.0kb.We identified 111 positive clones using CASTOR as the bait at pre-screening by yeast two-hybrid approach. After retest the interaction in yeast and X-Gal assay, 99 positive clones were obtained. There were 7 types of proteins including CaM binding translation elongation factor were characterized by sequencing and NCBI blast analysis. More than 30% in total number of positive clones was the CaM binding translation elongation factor with 90% homology in protein, was classified 5 kinds of proteins through DNAMAN assay.By using RT-PCR, further confirmed the expression level of these genes in Lotus japonicus with and without inoculation of Mesorhizobium loti, the results showed that CaM binding translation elongation factor with highest expression after inoculation of Mesorhizobium loti for 6 days and JAB1 whose expression enhanced corresponding to the days of inoculation of Mesorhizobium loti, were the most important proteins with inoculation of Mesorhizobium loti. But there weren't any difference in clpA and ubiquinone oxidoreductase subunit 2 with and without inoculation of Mesorhizobium loti. It was predicted that CASTOR might form compound with these proteins in Nod factor signal pathway.