Evaluation and selection of suitable qRT-PCR reference genes for light responses in tea plant (Camellia sinensis)

Abstract Background: Tea plant (Camellia sinensis) is an important woody economic crop used for processing leaf-type beverages. Tea has been proved to be beneficial to human health because it is rich in tea polyphenols and other active ingredients. Numerous studies have shown that light is a necessary environmental condition to control the growth and metabolism of C. sinensis. Gene expression experiments are always performed to explore the transcriptional regulation mechanism of plants widely based on the technique of quantitative real time polymerase chain reaction (qRT-PCR). The screening and application of reference genes are necessary for the normalization of gene expression under specific conditions. However, the reference genes for systematic analysis of light-induced transcription mechanisms are still not available in C. sinensis.Results: In this research, we identified actin family genes that are always used as reference genes with high frequency and without distinction for various expression experiments in C. sinensis. Six pairs of distinctive primers (corresponding to CsACT1, CsACT2, CsACT(3-4), CsACT(5-6), CsACT(7-8), and CsACT(9-10) genes) were designed to evaluate their expression stability in response to light quality (LQ), light intensity (LI), and photoperiod (PD). Simultaneously, six other family members (CsUBC1, CsCLATHRIN1, CsGAPDH, CsTBP, CsTIP41, and CseIF-4α) of C. sinensis commonly used as reference genes were also investigated. The stability rankings of gene expression were calculated by the statistical algorithms of geNorm, BestKeeper, NormFinder, and RefFinder softwares. Conclusions: CsACT(5-6), CsTIP41, and CsACT(3-4) were the most stable genes for light quality (LQ), light intensity (LI), and photoperiod (PD) treatments, respectively. This study provides a basis for the selection of reference genes for future research on the transcription mechanism of light response in C. sinensis. Moreover, the analysis of actin family members of C. sinensis will help to understand the individual transcription mechanism of housekeeping family.

Download Full-text

Selection of reference genes suitable for qRT-PCR expression profiling of biotic stress, nutrient deficiency and plant hormone responsive genes in bread wheat

Indian Journal of Plant Physiology ◽

10.1007/s40502-017-0282-3 ◽

2017 ◽

Vol 22 (1) ◽

pp. 101-106 ◽

Cited By ~ 6

Author(s):

S. Lekshmy ◽

S. K. Jha

Keyword(s):

Bread Wheat ◽

Biotic Stress ◽

Expression Profiling ◽

Reference Genes ◽

Plant Hormone ◽

Nutrient Deficiency ◽

Qrt Pcr ◽

Selection Of

Download Full-text

Low Caffeine Content in Novel Grafted Tea with Camellia sinensis as Scions and Camellia oleifera as Stocks

Natural Product Communications ◽

10.1177/1934578x1501000522 ◽

2015 ◽

Vol 10 (5) ◽

pp. 1934578X1501000 ◽

Cited By ~ 2

Author(s):

Wei-Wei Deng ◽

Min Li ◽

Chen-Chen Gu ◽

Da-Xiang Li ◽

Lin-Long Ma ◽

...

Keyword(s):

Camellia Sinensis ◽

Expression Patterns ◽

Tea Plant ◽

Protein Accumulation ◽

Tea Leaves ◽

Qrt Pcr ◽

Caffeine Content ◽

Caffeine Synthase ◽

Specificity And Sensitivity ◽

Tea Plants

Caffeine, a purine alkaloid, is a major secondary metabolite in tea leaves. The demand for low caffeine tea is increasing in recent years, especially for health reasons. We report a novel grafted tea material with low caffeine content. The grafted tea plant had Camellia sinensis as scions and C. oleifera as stocks. The content of purine alkaloids was determined in the leaves of one-year-old grafted tea plants by HPLC. We also characterized caffeine synthase (CS), a key enzyme involved in caffeine biosynthesis in tea plants, at the expression level. The expression patterns of CS were examined in grafted and control leaves by Western blot, using a self-prepared polyclonal antibody with high specificity and sensitivity. The expression of related genes ( TCS1, tea caffeine synthase gene, GenBank accession No. AB031280; sAMS, SAM synthetase gene, AJ277206; TIDH, IMP dehydrogenase gene, EU106658) in the caffeine biosynthetic pathway was investigated by qRT-PCR. HPLC showed that the caffeine content was only 38% as compared with the non-grafted tea leaves. Immunoblotting analysis showed that CS protein decreased by half in the leaves of grafted tea plants. qRT-PCR revealed no significant changes in the expression of two genes in the upstream pathway ( sAMS and TIDH), while the expression of TCS1 was greatly decreased (50%). Taken together, these data revealed that the low caffeine content in the grafted tea leaves is due to low TCS1 expression and CS protein accumulation.

Download Full-text