Screening and Validation of Internal Reference Genes for miRNA Real-time Quantitative PCR in Response to Nitrogen Levels in Camellia sinensis

Purpose To screen stable reference genes for tea plant nitrogen level responses, and to understand their miRNA expression levels related to nitrogen metabolism.

Methods Using the tea cultivar ‘Shuchazao’ as the material, treatments with 0%, 1%, and 3% urea were applied to examine the effects of different nitrogen levels. The stability of six candidate reference genes (5.8S rRNA, 26S rRNA, U6, 18S rRNA, 5S rRNA, and PC-3p-222) in tea shoots, mature leaves, and roots was systematically evaluated using real-time quantitative PCR (RT-qPCR) combined with BestKeeper, geNorm, and RefFinder software.

Results Except for PC-3p-222, the expression levels and stability of the other five reference genes were significantly higher in leaves than in roots. BestKeeper determined that PC-3p-222 exhibited the highest expression stability in shoots and roots, while U6 in mature leaves; geNorm further clarified that the optimal reference gene for shoots, mature leaves, and roots were 18S rRNA or U6, PC-3p-222 or U6, and 5S rRNA or U6, respectively, and U6 was stably expressed in all tissues; the comprehensive ranking of RefFinder confirmed that the best reference gene in shoots was 18S rRNA, while in mature leaves and roots was PC-3p-222. 5.8S rRNA had the poorest expression stability among all tissues.

Conclusion 18S rRNA, PC-3p-222, and U6 are collectively recommended as reliable reference genes for miRNA expression studies in tea plants under nitrogen response. Validation using gma-miR1507c-3p expression confirms PC-3p-222 as an optimal reference gene choice.