The Cation Channel Protein POLLUX And Its Interaction With EF1A In Lotus Japonicus

The early signaling transduction of symbiosis system in which the Rhizobium interacting legume plants with each others has been most focused on by global researchers. Multi of signaling key genes has been cloned from the two model plants, Medicago truncatula and Lotus japonicus. To confirm the function and action mechanism, and how to inter-regulate of these genes, are useful for understanding the molecular mechanism of the symbiosis system. It's a useful tool utilizing Yeast two-hybrid assay to find the regulation factor for the signaling genes.Cation channel protein, POLLUX had function as an important role in the upstream symbiotic signaling net. The pollux mutant, could not form nodules and was lack of calcium spiking phenotype, physically. We constructed BD-C-Pollux bait plasmid, and used it to screen the Lotus Yeast two-hybrid cDNA library. There were lots of clones including the translation elongation factor 1A acquired from library interacting with POLLUX. The positive clones were identified by mating in yeast again, then sequenced and searched by program BLAST on NCBI net. We confirmed that the translation elongation factor 1A (EF1A) interacted with C-POLLUX.Then we designed the primers of EF1A according to the sequence on the database of Lotus japonicus, isolated the total RNA, obtained of the translation elongation factor 1A full length cDNA by RT-PCR. To express the protein in E.coli, the fusion tag plasmid were constructed, such as pET28a-EF1A, pET28a-ED219, pET28a-ED267, pGEX-6p1-C-Pollux, and then the fusion proteins were purified with the suitable beads. Pull down and Western blotting assays confirmed the interaction between C-POLLUX and EF1A in vitro. And further more EF1A interacting with other signaling genes were checked by small scale mating in yeast cell, the results were including that NFR1, SYMRK and CASTOR interacted with EF1A in yeast cells while NFR5 or CCaMK did not.