Abstract：Legumes are often recognised as the pioneer plants in tailing areas because of their outstanding symbiotic nitrogen fixation. As the copper ions in the environment may lead to unstable expression of housekeeping genes, screening reference genes is important to identify the differentially expressed genes in black medic (Medicago lupulina L.) during the symbiotic nodulation with Sinorhizobium meliloti CCNWSX0020 under copper stress using qRT-PCR. In this study, the eight housekeeping genes(beta actin(ACT), histone H2A(H2A), ribosomal 18S(18S), tubulin beta(TUB), ubiquitin(UBI), elongation factor 1(EF1), glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and cyclophilin(CYP)) from black medic were selected and used to screen the optimal reference genes. The genes were generated from the roots of black medic inoculated rhizobia at 30 d which were planted under the different levels of copper (50, 100, 150 and 200 mg/kg, respectively). The analyses of primers specificity and PCR amplification efficiency showed that all the primers were accordance with the requirements of the stability screening. The results of qRT-PCR indicated that the expression level and stability of candidate genes were various along with the different concentration of copper ion. With geNorm software and the online estimation of gene expression stabilities in all samples, the optimal combination of reference genes was found to be GAPDH and EF1. The expressions of candidate genes under different levels of copper were also online assessed separately and the results displayed that the best reference gene was GAPDH at low concentration of copper (≤50 mg/kg) and UBI at moderate or high copper level (≥100 mg/kg). The conclusions provide the appropriate reference genes for characterizing the genes related to symbiotic nodulation and copper stress of Medicago lupulina L., and basic data for the plant reference genes under other heavy metal stress.
