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| Selection of Reference Genes for Quantitative Real-time PCR Normalization in Puccinia Striiformis f.sp. tritici |
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Abstract The selection of a suitable reference gene is an important prerequisite for precise gene expression analysis by quantitative Real-time PCR (qRT-PCR). Ten houskeeping genes were choosed for reference genes selecetion based on the research on reference genes. After calculations of PCR efficiencies, eight houskeeping genes were retained and their expression stabilities were evaluated by the computer algorithms geNorm. These genes included ubiquitin-conjugating enzyme(UBC), E2 ubiquitin-conjugating enzyme(UBCE2), ribosomal protein S5(RPS5), α-tubulin (TUBA), β-actin(ACTB), β-tubulin(TUBB), elongation factor1(EF1) and elongation factor3(EF3) . Using qRT-PCR, we investigated the expression stability of 8 housekeeping genes of Puccinia striiformis f.sp. tritici (Pst) in different samples including fresh spores of CYR32 and Pst78, germinated tubes of CYR32 and Pst78, inoculated wheat (Triticum aestivam) leaves sampled at 0.5, 3 and 14 days post inoculation(CYR32/XZ9104 and Pst78/AvS). The analysis with geNorm algorithms revealed that the expression profiles of ACTB, TUBB and TUBA were consistent with the infection process of Pst. The combination of ACTB, TUBB and TUBA can be used as reference genes for gene expression analysis of Pst.
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Received: 29 June 2011
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