Isolation And Characterization Of Small GTPase Rop6Activating Protein GAP From Lotus Japonicus

The nitrogen-fixing symbiosis between legume plants and Rhizobium bacteria has critical importance in the sustainable development of mondern agriculture. Currently, the emphases of the nitrogen-fixing symbiosis focus on the perception of bacterial signaling factors called Nod factors(NFs) by plant host and the NF signaling pathway, the molecular mechanism underlying the formation of threads and nodules, which are used to reveal the formation of the mystery of Symbiotic nitrogen fixation system. Recent studies found that a small GTPases ROP6plays important role in the signaling pathway of symbiotic. Based on this discovery we identified three GTPase activating proteins related with ROP6by blasting Lotus japonicas database. And using yeast two-hybrid assay and protein pull-down assay in vitro showed that these GAPs were able to interact with ROP6-CA. The main research results are as follows:1. Using the protein sequences in Arabidopsis thaliana to blast the L. japonicas database, we found several RopGAP proteins, and we got three of them by PCR. They are LjRacGAPl, LjRacGAP2and LjRacGAP3. The existing research indicates proteins encoded by three RacGAP genes interact with Racl-CA and Rac2-CA proteins. And the expression pattern for Rac2gene revealed increasing levels during root nodule development.2. ROP6has two mutants, the ROP6-CA (constitutively active) and ROP6-DN (dominant negative) mutants. Using protein pull-down assay in vitro and yeast two-hybrid assay in vivo showed that GAPs were able to interact with ROP6-CA Specifically, while they were’t able to interact with ROP6-DN.3. ROP6is a small GTPase, and GTPase assay in vitro demonstrates ROP6exhibits a weak intrinsic GTPase activity for GTP hydrolysis. The GTPase activity for ROP6enhanced after mixing the purified RacGAP1protein with the reaction system, which indicates RacGAP1accelerates the ROP6GTPase activity.4. By using Real-Time PCR approach, we analyzed the expression patterns for three GAP genes without and with M. loti inoculation. This assay showes three GAPs’ mRNA is all expressed in the roots of L. japonicus with and without M. loti inoculation. The expression for LjRacGAPl gene don’t change with M. loti inoculation; The expression for LjRacGAP2is down-regulated for the first2day with M. loti inoculation and then it tends to be stable; In the2nd day and the6th day with M. loti inoculation the expression for LjRacGAP3is down-regulated substantially, especially in the6th day the expression in the roots of L. japonicas with inoculation is10times as much as that in the roots of L. japonicas without inoculation, which indicates RacGAP3may be involved in nodule development.5. Overexpression of RacGAPl in L japonicas with M.loti inoculation showed the nodule number decreased. Besides, the root nodule size of overexpression RacGAP1plant was smaller than the control plant, which indicates that RacGAP1probably regulates and delays nodule development.