The Regulation Of The Transcription Factors NSP1, NSP2 And IPN2 On Nodule Inception In Legume

Nodule formation is initiated by the host plant roots exuding phenolic flavonoid compounds into the rhizosphere.Flavonoid perception attracts the bacteria to the root and activates rhizobia nod(nodulation) gene expression, leading to the production and secretion of nod factors(NF). Nod factors are a key signal molecules of recognitions of rhizobia and legume, which play a key function in the initial steps of symbiotic signals.Genetic dissection of nodulation in Medicago truncatula and Lotus japonicas revealed a signaling pathway necessary for Nod factor signal transduction. Studies have showed that two GRAS family proteins, Nodulation Signaling Pathway1(NSP1) and NSP2, have been shown to participate in the nitrogen fixation symbiosis pathway downstream of CCa MK. In vitro, NSP1 is a DNA binding protein that binds to the promoter of the Nod factor–inducible genes ENOD11, ERN1 and NIN. The in vivo association between NSP1 and the ENOD11 promoter is enhanced by Nod factor elicitation. NSP1 and NSP2 form homopolymers and heteropolymers, and the binding of NSP1 to the ENOD11 pomoter need the action of NSP2. A novel MYB protein named IPN2(Interacting Protein of NSP2) interacting with NSP2 had a strong transcription activation activity, bounding directly to the NIN gene promoter, probably involved in regulation with NSP1 and NSP2. This study had analysis the transcription factors NSP1, NSP2 and IPN2 regulate the NIN Promoter.The main research results are as follows:1. Construct of Dual-Luciferase® expression plasmid of NSP1, IPN2 and NIN. The plasmids were delivered to Nicotiana benthamiana and extracted of proteins. Using dual-luciferase assay detects biochemistry luminescence values of target proteins and then analysis the regulation of the transcription factors NSP1 and IPN2 on NIN promoter.The result show that the activation of NIN promoter by is enhanced by NSP1 and IPN2 and both of two had a strong transcription activation ability.2. We find out the conditions of the leaf derived protopast isolation method using wildtype Arabidopsis Col0 as material and set up a transient expression system based on Arabidopsis mesophyll protoplast. It is show that efficiency of transformation was increased with the PEG concentration and DNA content in a certain range. The experiment got a highest efficiency when the incubating mixture contained 20% PEG, 1-1.5 μg/μL plasmid DNA, enzymolysis 3 hours, 2×105 m L–1 protoplast cells and cultured 12 hours under 25 ℃.3. Based on higher transient expression, the NIN-LUC reporter was co-transformed with Dual-Luciferase® expression plasmid. Using dual-luciferase assay system to measure of target expression. Result shows that that activation of NIN promoter by is enhanced by NSP1, NSP2 and IPN2. Meanwhile, NSP1 had stronger activated ability than IPN2.The ratio of dual-luciferase of NSP1, NSP2 and IPN2 is 11, 10, 2.88, respectively.4. Co-transformation two or more indicated constructs to see the activation of NIN promoter. Result shows that NSP1, NSP2 and IPN2 still had a positive on regulation of NIN(-500 to-1) promoter, but activation decline compared with single transcription factor.5. To explore the transcription factors NSP1, NSP2, IPN2 and ILK(IPN2 LIKE) of the biological function in the process of roots and nodules in Medicago truncatula. We analysed histochemical staining of the four genes promoters to research the biological function.The results as follows:GUS staining showed that the NSP1 and NSP2 genes are dramatically expressed in root vascular bundles, root tips and root hairs after inoculation. GUS activity of NSP1 mainly expressed at the top of young nodule.IPN2 and ILK were expressed very weakly in nodules of the peripheral vascular bundle and moderately in root vascular tissues. But we didn’t observe ILK gene promoter expression inpericycle only a little in Lateral root primordium or nodule primordium.