Abstract：Expression stability of candidate reference genes (h2a、act、18s、tub、ubi、ef1、cyp、gapdh) was tested for qRT-PCR during growth conditions under the different zinc stress. The roots of Medicago lupulina inoculated with rhizobia were harvested in 30days as plant materials. Our results showed that the average expression stability of the eight candidate reference genes from high to low was ef1>ubi>act>h2a>gapdh>cyp>18s>tub overall. Specifically, tub (1.97) and ef1 (2.06) were better reference genes under the low Zn2+ concentration (100mg/kg); The genes ef1 (1.41) and ubi (2.51) were the best genes under Zn2+ concentration (200mg/kg); Under Zn2+ concentration (300mg/kg), ef1 (1.41) and h2a (2.78) were the most comparatively suitable genes; while ef1 (2.45) and act (2.55) were considered as the most reliable reference genes under the high Zn2+ concentration (400mg/kg). In conclusion, All results provide the theoretical foundation for gene expression study in Medicago lupulina under the different Zn2+ concentration.
