Validation of reference genes for gene expression studies in post-harvest leaves of tea plant (Camellia sinensis)

Zi-Wei Zhou; Hui-Li Deng; Qing-Yang Wu; Bin-Bin Liu; Chuan Yue; Ting-Ting Deng; Zhong-Xiong Lai; Yun Sun

doi:10.7717/peerj.6385

Validation of reference genes for gene expression studies in post-harvest leaves of tea plant (Camellia sinensis)

PeerJ. 2019 Jan 31:7:e6385. doi: 10.7717/peerj.6385. eCollection 2019.

Authors

Zi-Wei Zhou^{1

2}, Hui-Li Deng^{1

2}, Qing-Yang Wu^{1

2}, Bin-Bin Liu^{1

2}, Chuan Yue¹, Ting-Ting Deng¹, Zhong-Xiong Lai², Yun Sun¹

Affiliations

¹ Key Laboratory of Tea Science in Fujian Province, College of Horticulture Fujian Agriculture and Forestry University, Fuzhou, P. R. China.
² Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, P. R. China.

Abstract

Tea is one of three major non-alcoholic beverages that are popular all around the world. The economic value of tea product largely depends on the post-harvest physiology of tea leaves. The utilization of quantitative reverse transcription polymerase chain reaction is a widely accepted and precise approach to determine the target gene expression of tea plants, and the reliability of results hinges on the selection of suitable reference genes. A few reliable reference genes have been documented using various treatments and different tissues of tea plants, but none has been done on post-harvest leaves during the tea manufacturing process. The present study selected and analyzed 15 candidate reference genes: Cs18SrRNA, CsGADPH, CsACT, CsEF-1α, CsUbi, CsTUA, Cs26SrRNA, CsRuBP, CsCYP, CselF-4α, CsMON1, CsPCS1, CsSAND, CsPPA2, CsTBP. This study made an assessment on the expression stability under two kinds of post-harvest treatment, turn over and withering, using three algorithms-GeNorm, Normfinder, and Bestkeeper. The results indicated that the three commonly used reference genes, CsTUA, Cs18SrRNA, CsRuBP, together with Cs26SrRNA, were the most unstable genes in both the turn over and withering treatments. CsACT, CsEF-1α, CsPPA2, and CsTBP were the top four reference genes in the turn over treatment, while CsTBP, CsPCS1, CsPPA2, CselF-4α, and CsACT were the five best reference genes in the withering group. The expression level of lipoxygenase genes, which were involved in a number of diverse aspects of plant physiology, including wounding, was evaluated to validate the findings. To conclude, we found a basis for the selection of reference genes for accurate transcription normalization in post-harvest leaves of tea plants.

Keywords: Camellia sinensis; CsLOX1; Post-harvest leaves; RT-qPCR; Reference genes.

Grants and funding

This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-19) and the Major Science and Technology Project in Fujian Province (2015NZ0002-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.