Reference gene selection for qRT-PCR analysis in the shoots and roots of Camellia sinensis var. sinensis under nutritional stresses

Quantitative real-time polymerase chain reaction (qRT-PCR) is a useful way to evaluate gene expression level, in which the selection of internal references needs to be addressed firstly. However, the stably expressed reference genes in tea plants (Camellia sinensis var. sinensis) under different nutrient deficiency has not been reported. In this study, seven candidate reference genes (i.e., ACTIN7, EF-1 alpha, eIF-4 alpha, GAPDH, PP2A, TBP and TIP41) were employed to evaluate their expression stability using three algorithmic programs (i.e., geNorm, NormFinder and BestKeeper) under six kinds of nutrient deficiency conditions [i.e., ammonium nitrogen (-NH4+-N), nitrate nitrogen (-NO3-N), phosphorus (-P), potassium (-K), calcium (-Ca) and magnesium (-Mg)] in C. sinensis shoots and roots. The results showed that (1) the combinations of PP2A and ACTIN were stably expressed in shoots under -NH4+-N and EF-1 alpha combined with PP2A and TBP were stably expressed in -NH4+-N roots or -P shoots; (2) the combinations of ACTIN and EF-1 alpha or ACTIN and TBP were stable in shoots and roots under -NO3-N; (3) TBP combined with EF-1 alpha and TIP41 was stably expressed in -P roots-Ca roots; (4) eIF-4 alpha and ACTIN exhibited stable expression in -K shoots and the combination of EF-1 alpha and TIP41 was stable in -K roots; (5) GAPDH and EF-1 alpha or EF-1 alpha and eIF-4 alpha were stably expressed in -Ca or -Mg shoots and TIP41 combined with ACTIN and eIF-4 alpha was stably expressed in -Mg roots. Moreover, six nutrient deficiency responsive genes (i.e., CsAMT1;2, CsNRT2;6, CsPT4, CsKT8, CsCTA11 and CsMGT1) were chosen to validate the suitability of the selected reference genes, and the expression levels of these target genes showed significant difference when using the most stable single gene (or combination of genes) and the least stable gene for normalization. These results provide guidelines and facilitate the quantification of gene transcription in C. sinensis shoots and roots under macro- and medium-element deficient conditions.