Study On The Nodulation-Signaling Pathway 2 And The Interacting Protein IPN2 In Lotus Japonicus

The symbiosis between legume and rhizobia is a complicatedly developmental process mediated by exchange signal and energy. The rhizobia can secrete a chitinous polysaccharide so-called Nod factors, which is the key signal of perception each other during the symbiotic interaction. Perception of Nod factors by plant induces genes expression relative to nodulation and leads to depolarization of the plasma membrane, Ca²⁺ spiking in cytosolic, root hair deformation and cortical cell division that ultimately result in development of the nodule. Nodulation Signaling Pathway 2 (NSP2) is one of key components of the Nod factor signal transduction pathway in Lotus japonicus. This research studied the transcriptional activation and capacity of DNA-binding of NSP2 in Lotus japonicus and screened the new protein interacting with NSP2 in the pathway. Some results as follows:1. Using RT-PCR the full-length NSP1 and NSP2 gene were amplified from Lotus japonicus. The detection ofβ-galactosidase activity of the two genes was carried out using the reporter gene LacZ in the yeast strain Y187. The results indicated that NSPs had the ability to activate transcription in vivo, and theβ-galactosidase activity of NSP1 was higher than NSP2.2. Based on the sequence analysis of NSP2, four different mutant constructs were obtained. The same detections indicated that the four mutants have noβ-galactosidase activity and fail to activate transcription, the transcription activating region of NSP2 was located in the N-terminal contained HPS domain.3. Using yeast one-hybrid, the ability to binding DNA of NSPs was identified. The result showed that both NSP1 and NSP2 could bind to the promoter of NIN gene in yeast, rather than that of calcium-binding protein (CBP1).4. We screened the yeast two-hybrid AD-cDNA library using pGBKT7-NSP2-1 /Y187 as the bait and obtained a new protein IPN2 interacted with the NSP2. The bioinformatics analysis indicated that the 1947 bp IPN2 gene composed of six exons and five introns and encoded a protein contained 358 amino acid residues and had a 40.1 kD molecular weight. The IPN2 is a new member of MYB-SANT superfamily with a highly conserved Myb-type DNA binding domain located in the N-terminal (amino acid residues from 37 to 88) in variety plants. We predicted that a Helix-turn-Helixstructure (HTH) could be assembled.5. Using western blotting, the interaction between IPN2 and NSP2 was demonstrated in vitro. The LHR1 domain of the NSP2 was identified as the interaction region between NSP2 and IPN2 in vivo.