Study of Some Reference Genes Expression Stability in Rice Using Real-Time PCR Method Under Biotic Stress

Abstract

Objective Gene expression studies by Real-Time PCR constitute a powerful tool to analyze the mechanisms underlying plant biotic-stress tolerance. One of the crucial steps of this technique is the selection and validation of reference genes to normalize target gene expression under different stress conditions. In this study, the expresion of candidate gene in oryza sativa was investigated under biotic stress at different developmental stages. Materials and methods Eight internal control genes consists of eIF-4A, UBQ5, UBC, Actin1, Actin11, GAPDH, Cyclophilin and 18SrRNA which are commonly used as housekeeping genes in plants, were selected and their expression stability were examined in present of Rhizoctonia solani RBL1 strain, potasium silicat as tolerance inducer and different growth stages in three time periods (6 h, 24 h and 72 h) using BestKeeper and NormFinder softwares. Results and Conclusions Based on the results gained through Best Keeper, the UBC has the higher expression than the other genes under biotic stress in rice leaf and also the UBC and Actin11 genes poses the highest correlations with the BestKeeper index (0.97). Additionally, it was shown that the UBC and Actin has the lowest coefficient variation. Also, the evaluation of the reference genes expression using geometric mean of the UBC and Actin11 compared to the Actin1 gene indicated the necessity of appropriate selection of the reference gene. Taken together, it was evidently demonstrated that the UBC and Actin11 genes are the proper reference gene to be employed for the normalization of expression data in the Oryza sativa L. using by Real-Time PCR.

Keywords

• gene expression
• inducer
• reference gene
• rice
• r. solani