Screening and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Tea Plant (Camellia sinensis) under Different Nitrogen Nutrition

LIU Yuan; WANG Liyuan; WEI Kang; CHENG Hao; ZHANG Fen; WU Liyun; HU Juan

doi:10.13305/j.cnki.jts.2016.01.012

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Journal of Tea Science ›› 2016, Vol. 36 ›› Issue (1) : 92-101. DOI: 10.13305/j.cnki.jts.2016.01.012

Screening and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Tea Plant (Camellia sinensis) under Different Nitrogen Nutrition

LIU Yuan, WANG Liyuan^*, WEI Kang, CHENG Hao^*, ZHANG Fen, WU Liyun, HU Juan

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Abstract

The objective of this study was to select the most reliable reference genes for qRT-PCR analysis of target tea plant genes under varying nitrogen source and availability. We chose 6 housekeeping genes which included five commonly used and one new candidates to systematically assess their expression levels at three different tissues (young leaves, mature leaves and roots) under different nitrogen regimes by qRT-PCR. GeNorm and NormFinder software were used to analyze and evaluate the data for reference genes. The results indicated that GAPDH, β-actin and RPL13 are the best reference genes for normalizing target gene expression in tea plant under different nitrogen nutrition, whereas α-tubulin and RuBP are not suitable in many experimental conditions and the best combination (GAPDH+β-actin) was recommended. Meanwhile, the expression levels of CsNRT1.2 and CsAMT1.1 in young leaves of tea plants were analyzed. The results showed that the variation tendency of CsNRT1.2 and CsAMT1.1 are exactly consistent when using GAPDH and GAPDH+β-actin as reference genes. However, the expression levels of these genes are showed significant differences when α-tubulin was used as a reference gene. Thus, validation of suitable reference genes for specific condition can guarantee the accurate quantification of the target genes in qRT-PCR analysis.

Key words

nitrogen treatment / reference gene / qRT-PCR / tea plant (Camellia sinensis)

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LIU Yuan, WANG Liyuan, WEI Kang, CHENG Hao, ZHANG Fen, WU Liyun, HU Juan. Screening and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Tea Plant (Camellia sinensis) under Different Nitrogen Nutrition[J]. Journal of Tea Science. 2016, 36(1): 92-101 https://doi.org/10.13305/j.cnki.jts.2016.01.012

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