Screening and validating of endogenous reference genes in Chlorella sp. TLD 6B under abiotic stress

Abstract

Chlorella sp. TLD 6B, a microalgae growing in the Taklamakan Desert, Xinjiang of China, is a good model material for studying the physiological and environmental adaptation mechanisms of plants in their arid habitats, as its adaptation to the harsh desert environment has led to its strong resistance. However, when using real-time quantitative polymerase chain reaction (RT-qPCR) to analyze the gene expression of this algae under abiotic stress, it is essential to find the suitable endogenous reference genes so to obtain reliable results. This study assessed the expression stability of 9 endogenous reference genes of Chlorella sp. TLD 6B under four abiotic stresses (drought, salt, cold and heat). These genes were selected based on the analysis results calculated by the three algorithmic procedures of geNorm, NormFinder, and BestKeeper, which were ranked by refinder. Our research showed that 18S and GTP under drought stress, 18S and IDH under salt stress, CYP and 18S under cold stress, GTP and IDH under heat stress were the most stable endogenous reference genes. Moreover, UBC and 18S were the most suitable endogenous reference gene combinations for all samples. In contrast, GAPDH and α-TUB were the two least stable endogenous reference genes in all experimental samples. Additionally, the selected genes have been verified to be durable and reliable by detecting POD and PXG3 genes using above endogenous reference genes. The identification of reliable endogenous reference genes guarantees more accurate RT-qPCR quantification for Chlorella sp. TLD 6B, facilitating functional genomics studies of deserts Chlorella as well as the mining of resistance genes.

Figure 1

Experimental framework.

Figure 2

Ct value of endogenous reference genes across all treatments under four abiotic stresses. The line on each box represents the median. The little box represents the mean. The lower and upper edges of boxes show the 25th and 75th percentiles, respectively, and the circles represent outliers.

Figure 3

The expression stability values (M) of nine endogenous reference genes were determined by GeNorm software. The most stable and least stable genes are on the right and left. PEG PEG-6000 drought treatment, NaCl 200 mM NaCl salt treatment, 42 °C 42 °C high temperature treatment, 4 °C 4 °C cold treatment, Total Pooled samples from all treatments.

Figure 4

Pairwise variation (V) of endogenous reference genes, as calculated by GeNorm software. V_n/V_n+1 values were used to determine the optimal number of endogenous reference genes. A cut-off of 0.15 (Vn value) is usually applied. PEG PEG-6000 drought treatment, NaCl 200 mM NaCl salt treatment, 42 °C 42 °C high temperature treatment, 4 °C 4 °C cold treatment, Total pooled samples from all treatments.

Figure 5

Relative expression of the POD target gene for validation of selected endogenous reference genes. PEG PEG-6000 drought treatment, NaCl 200 mM NaCl salt treatment, 4 °C 4 °C cold treatment, 42 °C 42 °C high temperature treatment. Different lowercase letters represent statistically significant differences as determined by one-way ANOVA (P < 0.05, Duncan’s multiple range tests).

Figure 6

Relative expression of the PXG3 target gene for validation of selected endogenous reference genes. PEG PEG-6000 drought treatment, NaCl 200 mM NaCl salt treatment, 4 °C 4 °C cold treatment, 42 °C 42 °C high temperature treatment. Different lowercase letters represent statistically significant differences as determined by one-way ANOVA (P < 0.05, Duncan’s multiple range tests).