Expression stability of internal reference gene in response to Trichoderma polysporum infection in Avena fatua L.

Trichoderma polysporum was a pathogenic fungi which showed strong pathogenicity to Avena fatua L. in recent study. The stress response of A. fatua to T. polysporum is mediated by the regulation of gene expression. Quantification of the expression of genes requires normalizing RT-qPCR data using reference genes with stable expression in the system studied as internal standards. To construct a RT-qPCR system suitable for response of A. fatua to T. polysporum, and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on eight candidate internal reference genes (18S, 28S, TUA, UBC, ACT, GAPDH, TBP and EF-1α) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP, 18S and UBC were the most stable internal reference genes, TBP and TUA, TBP and GAPDH, 18S and TBP, UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua. This is the first study describing a set of reference genes with a stable expression under fungi stress in A. fatua. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in A. fatua.