Isolation And Identification Of Nodulin Genes Involved In Symbiotic Nitrogen Fixation Of Astragalus Sinicus L.

To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of A. sinicus genes specifically expressed in infected roots is generated using a PCR-based suppressive subtractive hybridization (SSH) technique with two mRNA populations of infected and uninfected control roots in this study. Initial screening of the cDNAs to remove false positives was performed by dot blotting. The clones corresponding to genes with at least threefold increase in transcript accumulation in infected roots compared with uninfected control roots were sequenced. Fifty-seven different inserts were identified after annotation and redundancy analyses. BLAST searches indicated that 17 cDNA clones showed significant homology with leghemoglobin gene (Lb). Three fragments were identical with A. sinicus asparagine synthetase gene and the other seven fragments were similar to genes of EN0D2, nodulin-26, phosphoenolpyruvate carboxylase of Sesbania rostrata (very low similarities), putative cystein proteinase of Capsicum annuum, diphosphonucleotide phosphatase 1 of Lupinus luteus, RNA helicase of Arabidopsis thaliana and Medicago truncatula BAC clone mth2-10i23, respectively. Moreover, 30 fragments did not show any significant match.To verify the candidates as differentially expressed in the inoculated roots, Virtual Northern blotting were performed to the 16 clones. The results showed that the 14 clones examined were expressed only in the inoculated roots; the remaining two, AsIB259 and AsD243, only displayed stronger signal intensities comparing with control roots. After that, the full-size cDNA sequences of 16 clones or their genomic DNA sequences were obtained by RACE and analyzed by computer. The sequence analysis indicated that the 11 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 homology of the defensin 2 family and AsD255 contains a scorpion toxin-like domain at the C-terminal. Sequence analysis of a genomic AsD255 fragment 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. Among the other five genes, AsE246 and AsIB259, encode two different products similar to lipid transfer proteins (LTPs). Sequence alignment displayed that the putative proteins of AsE246 and AsIB259 show lower homology with LTPs from legumes than that from other plants. AsD243 encodes a 20-kD protein highly similar to the bacterial MJ0577-type of ATP-binding universal stress protein family (Usp), which has been proposed to function as a molecular switch. A very low degree of similarity (35% identities for 74 amino acids of N-terminal) was revealed between AsIIC259 and L1DD4A9, a unique nodulin from Lupinus luteus. No any significant similar sequence or any motif was detected for AsG2511.RT-PCR analysis indicated that the other genes except AsD243, AsIB259 and AsC2411 were expressed exclusively in inoculated roots and that their expressions were...