Proteins Interacted With NFR5 Involved In Nodulation Signaling Transduction In Lotus Japonicus

Nod factors are a novel general class of signal molecules produced by rhizobia, which play a key function in the initial steps of nodulation. Both NFR1 and NFR5 participate in the reception and transduction of nod factor Signal, and transmits it to the downstream genes. Then, there were a series of physiological and biochemical changes. This study attempt to identify some proteins which have interaction with NFR5 and to get further functions for the study of nod factor signal pathway.This paper includes two sections as follows:1) Identify the interacting proteins with NFR5(Nod factor receptor 5), using Yeast Two-hybrid System approach.Both NFR5 and NFR1 are involved in signal perception and transduction of Nod factor produced by rhizobia in Lotusjaponicus. In this research, using yeast two-hybrid approach, we constructed AD-cDNA library of L. japonicus and idendified 221 positive clones using the protein kinase domain of NFR5(nfr5-pk) as a bait at preliminary screening. After retest in the yeast and X-Gal assay, 26 positive clones were confirmed. The six kinds of proteins including small G-Protein Rop6 were obtained after sequencing and NCBI blast analysis. These proteins could be participated in the regulating function of NFR5.2) Identification and analysis of the small G-protein ROP6 involved in Nod factor perception and signal transduction in LotusjaponicusThe plasmid pTYB12- Rop6 containing the ROP6 gene fragment was constructed and sequenced. Then it was transformed into E.coli .ER2566, the resulting strain expressed CBD-Rop6 fusion protein, then Rop6 protein for interaction in vitro was purified using Chintin beads.The small G-protein ROP6 directly interacts with NFR5, suggesting that it may participate in the signal transduction network in nodule formation. Roots of L. japonicus were harvested at days 0, 2, 4, 6 and 8 post-induced by rhizobia for total RNA isolation. RT-PCR analysis was performed and the results showed that the expression of ROP6 in native root was very low, however the level of Rop6 expression postively relate with the days after induced, suggesting that expression of the small G-protein ROP6 in L. japonicus depends on induction of rhizobia nod factor.