Screening Of Proteins Interacting With Lipid Transfer Protein AsE246 And Nodulation Receptor Kinase NORK Of Astragalus Sinicus

In previous work of our laboratory, by using suppressive subtractive hybridization (SSH) technique, Chou Minxia has generated a cDNA library of Astragalus sinicus L. genes specifically or enhanced expressed in infected roots. A specific nodules gene AsE246 enhanced expressed only in infected roots of Astragalus sinicus L has been screened from the cDNA library. AsE246 encodes a lipid transfer protein that has high homology with LTPs of many plants. LTP is also named nsLTP, and have two types: nsLTP1 and nsLTP2. AsE246 belongs to nsLTP1 family. The results of Chen Da-song indicate:AsE246 has response to salt stress; expression of AsE246 was inhibited under ammonium stress because of decreased nodule activity.In this study, using yeast two hybrid technique, we screen proteins that may interact with AsE246 from the root nodule cDNA library of Astragalu sinicu L. in order to preliminary study the role of AsE246 in nodulation and nitrogen fixation. One of three transformants that we have screened initially is a positive clone. The in vivo interaction between these two proteins was confirmed through a small-scale yeast hybridization test. The positive gene is named AsDJL1, because it encodes a DnaJ-like protein which contains a DnaJ-C domain. The DnaJ domain can interact with AsE246 by small-scale maiting test. AsDJL1 dose not have self-activating activity, and it does not occur itself interaction.By real-time PCR technique, the temporal and spatial expression characteristics of AsDJL1 during root nodule development and nitrogen fixation process was quantitatively analyzed. We found that:AsDJL1 was specifically-enhancing expressed in nitrogen-fixing root nodules. The expression of AsDJL1 in inoculated roots, stems and leaves was very little; AsDJL1 did not express in uninoculation root and invalid nodules. The test result is the same as AsE246. With the inoculation time longer, expression of AsDJL1 in root nodule increased gradually. From these results we speculate that:AsDJL1 may be involved in the development, nitrogen fixation process and senescenceof root nodules. The different expression characteristics of AsDJL1 during NaCl stress and (NH4)2SO4 stress were quantitatively analyzed by real-time PCR technique. The results showed that: Expression level of AsDJLl was significantly increased under NaCl stress, while decreased under (NH4)2SO4 stress. Experimental results are consistent to AsE246 under stress. From these we speculate that AsDJL1 and AsE246 may form a complex that may be involved in the development of root nodules and nitrogen fixation process, but the specific role of AsDJL1 and AsE246 requires further study and analysis.Symbiosis receptor kinase gene NORK of Astragalus sinicus is homologyly amplified based on SymRK of Lotus japonicus by Chen Da-song, in previous work of our laboratory. NORK encods symbiosis receptor kinase that can received signal in the early stages of nodulation, and is the signal intersection of rhizobia and AM fungal. The structure of NORK is:Leucine rich repeat sequences, the transmembrane part of central and the C-terminal kinase domain. In this experiment, we used gene NORK to constract LRR and PK bait strains. Using yeast two-hybrid systerm, we screened the interacting proteins. The protein NPIP-41 interacting with NORK-PK contains a TIM-phosphate-binding superfamily domain, and may be involved in energy metabolism; NPIP-35 containing a EF1-α-Ⅲdomain is a translation elongation factor,which plays an important role in signal transduction and regulation of translation. NLIP-58,68 and 92 interacting with NORK-LRR are the class containing UBQ domain ubiquitin protein, predicted to participate in NORK ubiquitination. In this study, proteins interacting with NORK and their mechanism with NORK need further research.