Expression Patterns Of Five Genes Involved In Symbiotic Nitrogen Fixation Of Astragalus Sinicus Under Ammonium And Salt Stress Condition

The interaction between leguminous plants and rhizobia results in the formation of the symbiotic organ, the root nodule. In a mature nodule, rhizobia can fix N₂ from the air into ammonium to supply their host plants. Former studies showed that : various stress factors ,such as salt, drought, alkalinity and nutrient deficiency could suppress the growth and symbiotic characteristics of leguminous plants and rhizobia. In this study, salt and ammonium stress were given to Astragalus sinicus in sand pots which were inoculated with wild type Mesorhizobium huakuii 7653R. The symbiosis phenotype and expression pattern of five nodule-specific genes of Astragalus sinicus under ammonium and salt stress condition were studied.Before this study, we have isolated 14 nodule-specific or nodule-enhanced genes from Astragalus sinicus by suppressive subtractive hybridization technique. In this study, we identify the expression patterns of five nodule-specific genes in nodules of Astragalus sinicus under normal conditions as well as under salt and ammonium stress. Transcription levels of genes tested were quantified by quantitative fluorescence real-time RT-PCR. Results showed that: (1) 80mM NaC1 and all stress treatments containing (NH₄)₂SO₄ significantly inhibited nitrogen-fixing capacity of inoculated plants. 40 mM NaC1 stress showed relative lighter inhibition. (2) Compare with positive control at normal conditions, the expressions of all five genes were significantly reduced by 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 NaC1 and not significantly affected by 40 mM NaC1. (3) Under salt stress with exogenous nitrogen supply, expressions of AsG2411, AsIIC2512, AsB2510 and AsIIA255 were significantly suppressed. While, the transcription level of AsE246 under 80 mM NaC1 containing lmM (NH₄)₂SO₄ was still higher than that of positive control. Correlation of the expressions of three cysteine cluster protein genes (AsG2411, AsIIC2512, AsIIA255) and one lipid transfer protein gene (AsE246) studied here with their nitrogen-fixing activities in nodules may explain the molecular mechanisms of their reported functions in symbiosis and nitrogen-fixing process. Our results also proved that genes of AsIIA255 and AsE246 might play a role in the response of Astragalus sinicus to salt stress to facilitate the nitrogen-fixation process.Besides above works, three whole genes involved in symbiotic nitrogen fixation of Astragalus sinicus were cloned by RT-PCR. They are: (1) Symbiosis receptor-like kinase gene(SYMRK)of Astragalus sinicus. It was involved in perception of the rhizobial lipochitin-oligosaccharide signal.; (2) Phosphoenolpyruvate carboxylase gene of Astragalus sinicus(AsPEPC),It was an nodule-enhanced expression gene involved in nitrogen metabolism in nodules;(3) Diphosphonucleotide phosphatase of Astragalus sinicus(AsPPD). It was an nodule-enhanced expression gene which the function is still needed to study.