| AsE246 and AsIB259 were two novel nodulin genes, which were isolated for the first time from the symbiotic system between Astragalus sinicus and Mesorhizobium huakuii 7653R by suppression subtractive hybridization (SSH) in previous work of our laboratory. The sequences analyses showed that they encode non specific lipid transfer protein, nsLTP.In this study, in situ hybridization technique was used to determine the tissue expression localization in nodule of AsE246 gene; and by real time quantitative PCR, the temporal-spatial expression patterns of two target genes were investigated under symbiosis and cadmium stress conditions as well.The results showed that, the expression of AsE246 was in the central region of nitrogen fixation zone of the nodule, no expression in the cortex and other regions. The temporal-spatial expression examination showed two target genes were both strictly expressed in root nodules, and reached the peak level at about 22 days after inoculation of M. huakuii 7653R; the two target genes did not express in symbiosis of A. sinicus and AM Fungi. In addition, the expression levels of AsE246 and AsIB259 were significantly increased during the early stage as a response to low-level-cadmium stress. However, either at low- or high-level-cadmium stress conditions, their expression were significantly suppressed during the interim stage of Cd2+ stress treatment than those in normal nodules, and continued to maintain a relative lower level until the terminal stage.In this work, some substantial and convincing data were obtained to confirm that AsE246 and AsIB259 were specifically expressed nsLTP genes in A. sinicus root nodules, they were involved in symbiotic nitrogen fixation, and also very likely participate in response to cadmium stress mechanism. It provides good experimental basis and premise for the further exploration of their function and mechanism involved in symbiotic nitrogen fixation and stress response. |
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