RESUMEN
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.
Asunto(s)
Planta del Astrágalo/metabolismo , Proteínas de la Membrana/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Planta del Astrágalo/genética , Planta del Astrágalo/microbiología , Clonación Molecular , Biblioteca de Genes , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
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.
