To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of Astragalus sinicus genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R is generated using a PCR-based suppressive subtractive hybridization (SSH) technique with two mRNA populations of infected and uninfected control roots. Two nodule-specific genes, AsIIC259 and AsG2511, are identified from infected roots of A. sinicus. The amino acid sequences deduced from the open reading frames (ORFs) reveal that AsIIC259 and AsG2511 encodes a polypeptide with 134 and 58 amino acids, respectively. A signal peptide sequence is predicted with high probability at the N-termini of the AsIIC259 and AsG2511. The motif searches show that the deduced polypeptide of AsIIC259 contains two N-glycosylation sites, a cAMP-and cGMP-dependent protein kinase phosphorylation site and a casein kinase II phosphorylation site. BLASTP searches reveal that AsIIC259 putative protein displays a low degree of similarity to a unique nodulin from Lupinus luteus nodules. No significant identity is displayed over the predicted polypeptides of AsG2511 with any published sequences. Virtual Northern blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicate that the two genes are expressed exclusively in inoculated roots and that their expression is 2-4 d later than that of the leghaemoglobin (Lb) gene during nodule development.nodule-specific genes, Astragalus sinicus L., nodulins, symbiosis, suppressive subtractive hybridizationThe symbiosis between leguminous plants and rhizobia (including the genera Rhizobium, Bradyrhizobium, Azorhizobium, Sinorhizobium and Mesorhizobium) leads to the formation of a novel root organ, the nodule, hosting nitrogen-fixing rhizobia that feed the host plant with ammonium. This symbiotic association is established by the constant communication between both interacted partners: the plant secrets flavonoid molecules into the rhizosphere, which induce the expression of bacterial lipochito-oligosaccharide signaling molecules, the Nod factors (NFs). Responding to NFs, some host plant nodulin genes are expressed and sometimes cortical cell divisions are initiated to form nodules [1] . The products of plant genes with an induced or enhanced expression during nodule development are termed ''nodulins'' [2] . Depending whether their expressions precede or accompany nitrogen fixation, nodulin genes are classified as early and late nodulins [3] .There are two major classes of nodules formed on legume roots: the indeterminate type and determinate type. Indeterminate nodules usually develop on temperate legumes and display a characteristic zonation. The apical meristem is zone I followed by infection zone II, amyloplast-rich interzones II-III, nitrogen-fixing zone