天蓝苜蓿锌胁迫下实时定量PCR内参基因筛选

Reference gene selection for quantitative real-time PCR normalization in Medicago Lupulina under zinc stress

选取8个管家基因(h2a、act、18s、tub、ubi、ef1、cyp、gapdh)作为候选内参基因,以不同浓度Zn2+处理下接菌第30d的天蓝苜蓿(Medicago Lupulina L.)接种根为实验材料,筛选用于目的基因实时荧光定量PCR分析的最佳内参基因.研究表明:候选内参基因平均表达稳定性由高到低排序为:ef1>ubi>act>h2a>gapdh>cyp>18s>tub;在不同Zn2+浓度胁迫下,最适内参基因组合各不相同:Zn2+=100mg/kg时,最佳内参基因组合为tub(1.97)和ef1(2.06);Zn2+=200mg/kg时,最佳内参基因组合为ef1(1.41)和ubi(2.51);Zn2+=300mg/kg时,最佳内参基因组合为ef1(1.41)和h2a(2.78);Zn2+=400mg/kg时,最佳内参基因组合为ef1(2.45)和act(2.55).该研究可为重金属污染地天蓝苜蓿抗性基因和共生基因的表达分析提供理论依据.

Expression stability of candidate reference genes（h2a、act、18s、tub、ubi、ef1、cyp、gapdh） was tested for q RT-PCR during growth conditions under the different zinc stress. The roots of Medicago lupulina inoculated with rhizobia were harvested in 30 days as plant materials. Our results showed that the average expression stability of the eight candidate reference genes from high to low was ef1ubiacth2agapdhcyp18stub 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.