Screening and Evaluation of Reference Genes in Eggplant Seedlings by Real-time Fluorescence Quantitative PCR Under Low Temperature and Weak Light Stress

In order to explore the gene expression pattern of Solanum melongena L., the qRT-PCR method was used to analyze the expression differences of mRNA in the 20 candidate reference genes of eggplant, including ACT, EF1α, TUA, TUB, GAPDH, eIF, UBQ, UBI3, PP2A, CYP, RPL28, L25, SAND, TBP, DNAJ, APRT, EXP, CAC, HSP20 and CysPro. The three calculation methods, including GeNorm, NormFinder and BestKeeper, were used to evaluate the expression stability of 20 candidate reference genes in eggplant samples at low temperature, weak light and low temperature and weak light stress. The results showed that the fluorescence quantitative primer amplification efficiency (E) values of 20 candidate reference genes in eggplant ranged from 96.8% to 117.6%, and the correlation coefficient (R²) was between 0.968 8−0.999 9, indicating that the amplification efficiency was good, and the amplified reaction was highly specific. The primers could be specifically amplified, with good specificity, all of which were unimodal. The amplification curves among the samples had strong repeatability, which could be used for qRT-PCR amplification. By analyzing the expression abundance of C_T values of 20 candidate reference genes in eggplant, it was found that the average C_T values of 20 candidate reference genes in eggplant ranged from 19.76 to 31.20. The comprehensive evaluation results of the three calculation methods showed that the TBP gene was the most stable reference gene in all the samples. The study could provide a suitable reference gene for the study of gene-specific expression in eggplant under low temperature stress, weak light stress and low temperature and weak light stress.