Abstract：Selection of a suitable reference gene is an important prerequisite for precise gene expression analysis by Real-time quantitative PCR. Pandora neoaphidis, an obligate aphid pathogenic fungus, can induce a drastic epidemic to cause the collapse of aphid populations on crops. To determine potential reference genes for normalization of Real-time PCR data on P. neoaphidis, the transcript levels of 3 traditional housekeeping genes including 18SrRNA(18S), 28SrRNA(28S) and elongation factor 1 alpha-like protein(EF1), were measured in this study. We investigated the expression stability of 3 candidate reference genes in P. neoaphidis ARSEF 5403 in different developmental stages including conidia stage, germ tubes stage, early hyphae stage and elongated hyphae stage, as well as under different nutrient conditions including OS-SDB medium, GLEN medium and Grace's insect cell culture medium. The expression stability of candidate reference genes was calculated using 3 algorithms including geNorm, NormFinder and BestKeeper. Results from Real-time PCR revealed that designed primers had a good proliferation efficiency and specificity. The analysis with geNorm algorithms revealed that the stability value (M value) of candidate reference genes was 18S(0.457)＞28S(0.534)＞EF1(0.749) under different developmental stages, and 18S(0.389)＞28S(0.557)＞EF1(0.607) under different nutrient conditions. Additionally, the analysis with NormFinder algorithms revealed that M value of candidate reference genes was 18S(0.084)＞28S(0.264)＞EF1(0.509) under different developmental stages, and 18S(0.118)＞28S(0.355)＞EF1(0.403) under different nutrient conditions. 18S was ranked as the most suitable reference gene of the 3 candidate reference genes analyzed by geNorm and NormFinder. However, the analysis with BestKeeper algorithms revealed that 28S was the most suitable reference gene under all conditions examined, followed by 18S, and EF1 was the most unstable reference gene. Comprehensively, the mean rank based on stability banking analyzed by the three algorithms showed that 18S was the most suitable reference gene under all conditions examined. Conclusively, candidate reference gene 18S has the most stable expression levels among all the samples of P. neoaphidis from different developmental stages and different nutrient conditions. Meanwhile, 18S can also be used as a stable reference gene for normalization of Real-time quantitative PCR, and provides supports for the further studies for P. neoaphidis on the expression stability of the developmental or virulence relative genes.
