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In previous works, we isolated 14 nodule-specific or nodule-enhanced genes from Astragalus sinicus by suppressive subtractive hybridization. In this study, we have further identified the expression patterns of five nodule-specific genes of A. sinicus under salt and ammonium stress. Transcription levels of genes tested were quantified by quantitative fluorescence real-time RT-PCR. Results showed that: (1) About 80 mM NaCl and all stress treatments containing (NH(4))(2)SO(4) significantly inhibited nitrogen-fixing capacity of inoculated plants. About 40 mM NaCl showed relative lighter inhibition. (2) Compare with positive control at normal conditions, the expressions of all genes were significantly reduced by all ammonium stress. (3) Under salt stress without exogenous nitrogen, transcription levels of AsIIA255 and AsE246 were significantly increased after treatment for 3 days. But expressions of AsG2411, AsIIC2512, and AsB2510 were suppressed by 80 mM NaCl and not significantly affected by 40 mM NaCl. (4) Under salt stress with exogenous nitrogen, expressions of AsG2411, AsIIC2512, AsB2510, and AsIIA255 were significantly suppressed. While, the transcription level of AsE246 under 80 mM NaCl containing 1 mM (NH(4))(2)SO(4) was still higher than that of positive control. The correlation of the expression profiles of three cysteine cluster protein (CCP) genes (AsG2411, AsIIC2512, AsIIA255) and one lipid transfer protein (LTP) gene (AsE246) with the nitrogen-fixing capacities of nodules in each treatments may explain the molecular mechanisms of their supposed functions in symbiosis and nitrogen-fixing process. Our results also implied that AsIIA255 and AsE246 might play a role in the response of A. sinicus to salt stress to facilitate the nitrogen-fixation process.

A nodule-enhanced gene, AsD243, was identified from infected roots of Astragalus sinicus using suppressive subtractive hybridization (SSH). It encodes a 20-kD protein related to the bacterial universal stress protein family (Usp). Sequence analysis showed that AsD243 is highly similar to the bacterial MJ0577-type of ATP-binding Usp proteins, which have been proposed to function as a molecular switch. Expression analyses revealed that AsD243 was transcribed in all plant organs, and progressively during all stages of nodulation. Its transcripts increased significantly at 7 days after inoculation, which is 2 days later than the onset of leghemoglobin expression in A. sinicus nodules. AsD243 was expressed more strongly in mature roots than in young roots regardless of inoculation status. We suggest that the AsD243 may have other functions in plant processes besides nodulation.

Thirteen nodule-specific or nodule-enhanced genes have been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of infected and uninfected control roots of Astragalus sinicus. Eleven of them encode small polypeptides showing homology to cysteine cluster proteins (CCPs) that contain a putative signal peptide and conserved cysteine residues. Among these CCP-like genes, AsG257 codes for a homologue of the defensin 2 family and AsD255 contains a scorpion toxin-like domain at the C-terminus. Sequence analysis of a genomic AsD255 fragment which was isolated revealed that one intron separates the first exon encoding the signal peptide from the second exon encoding the cysteine cluster domain of this nodulin. Another two genes, AsE246 and AsIB259, encode two different products similar to lipid transfer proteins (LTPs). Virtual northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the other genes except AsIB259 and AsC2411 were expressed exclusively in inoculated roots and that their expression was 2-4 d later than that of the leghaemoglobin (Lb) gene during nodule development. Transcription of AsIB259 was also detected in uninfected control roots but with a significant decline in expression and a temporal expression similar to Lb. AsC2411 had a basal expression in control roots identified by RT-PCR. Sequence alignment showed that the putative proteins AsE246 and AsIB259 show lower homology with LTPs from legumes than with those from other plants.