Abstract:

The objective of this study was to identify stably expressed internal reference miRNAs and genes under cadmium (Cd) stress in the roots of Ilex asprella, for the purpose of determining the expression levels of Cd stress-responsive miRNAs and their target genes. In this study, total RNA was extracted from Ilex asprella roots subjected to various Cd stress durations, followed by reverse transcription. Subsequently, the Ct values of 9 candidate internal reference miRNAs and 8 candidate internal reference genes were determined through RT-qPCR experiments. The optimal internal reference miRNAs and genes were subsequently identified through a comprehensive and systematic analysis using tools such as geNorm, NormFinder, BestKeeper, and RefFinder. After that, based on the best internal reference miRNAs and genes, this study further determined the expression of 6 miRNAs and their target genes in response to Cd stress in the roots of Ilex asprella. The results showed that although the results of various tools were different due to algorithm differences, in the comprehensive analysis of RefFinder, this study identified UBQ as the best internal reference gene, with the stability ranking of UBQ > Actin > 18S > GAPDH > UBC > EF1-α > 28S > α-TUB, the best internal reference miRNA was U6, and the stability ranking was U6 > 5S > MIR156ab > MIR396f > MIR159d > MIR390j > MIR171t > U4 > MIR166ab. The expression analysis results of 6 miRNAs and their target genes showed that under the Cd stress, the expression of MIR159c decreased gradually, while the expression of MIR171a and MIR393a increased significantly. In addition, the expression levels of MIR167m-5p, MIR399f-5p and MIR529d increased first and then decreased. There was a significant negative correlation between these miRNAs and their target genes, which indicated that under Cd stress environment, Ilex asprella could negatively regulate its target genes through miRNAs to adapt to Cd stress. In conclusion, this study successfully screened the best internal reference gene UBQ and internal reference miRNA U6 in the roots of Ilex asprella under Cd stress, and detected the expression of miRNAs and their target genes in response to Cd stress, which provided a theoretical basis for in-depth study of the regulation mechanism of Cd stress on the expression of miRNAs and their target genes in Ilex asprella.

Key words: Cd stress, Ilex asprella, real time quantitative PCR, internal reference gene, miRNA