Selection and Validation of Internal Reference Genes for qRT-PCR Analysis under Fluoride Stress in Camellia sinensis Leaves
In order to screen the internal reference genes for quantitative real-time PCR analysis in tea leaves under fluoride stress,the low-fluoride cultivar'Fuding Dabaicha'and the high-fluoride cultivar'Jinguanyin'were used as experimental materials according to the fluoride evaluation results in these tea cultivars previously.The qRT-PCR technology combined with three Excel-based algorithms(geNorm,NormFinder and BestKeeper)were used to analyze the expression stabilities of eight candidate reference genes(CsACTIN,CsEF-1α,CseIF-4α,CsGAPDH,CsPP2A,CsTBP,CsTIP41 and CsUBC)in tea leaves(shoots and old leaves)under fluoride treatment(0.42 mmol·L-1 NaF)for different time periods(0,1,3,7 d).The results indicate that under fluoride stress,the optimal combination of reference genes in tea shoots was CsEF-1α,CsTIP41,CsTBP and CsACTIN and the optimal combination of reference genes in old leaves was CsPP2A and CsUBC.Moreover,to further confirm the stability of the selected reference genes,the expression levels of CsFEX in tea shoots and old leaves were analyzed using their corresponding optimal internal reference gene combinations.The expression profiles of CsFEX in tea shoots or old leaves between the two cultivars were consistent,indicating that the combinations of four and two internal reference genes were sufficient for normalizing the target gene expression in tea shoots and old leaves under fluoride stress,respectively.
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