Abstract
Ginseng is a valuable herb of traditional Chinese medicine and ginsenosides, the main bioactive components of ginseng, have been proven to have multiple functions in human therapies and health. Methyl jasmonate (MeJA) is an elicitor that has been demonstrated to have a vital influence on ginsenoside biosynthesis. Quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in quantification of gene expressions. Here, we report the selection and validation of reference genes desirable for normalization of gene expressions quantified by qRT-PCR in ginseng hairy roots treated with MeJA. Twelve reference genes were selected as candidate genes, and their expressions were quantified by qRT-PCR, and analyzed by geNorm, NormFinder and BestKeeper. CYP and EF-1α were shown to be the most stable reference genes in geNorm, CYP was the most stable reference gene in NormFinder, and 18S was the most stable reference gene in BestKeeper. On this basis, we further quantified the relative expression levels of four genes encoding key enzymes that are involved in ginsenoside biosynthesis using CYP and 18S as the reference genes, respectively. Moreover, correlation analysis was performed between the quantified expressions of four genes and the ginsenoside content in MeJA-treated ginseng hairy roots. The results of relative expressions of the four genes quantified using CYP as the reference gene and their significant correlations with the ginsenoside content were better than those using 18S as the reference gene. The CYP gene, hence, was concluded as the most desirable reference gene for quantification of the expressions of genes in MeJA-treated ginseng hairy roots. This finding, therefore, provides information useful for gene research in ginseng, particularly in MeJA-treated ginseng hairy roots, which includes identification and characterization of genes involved in ginsenoside biosynthesis.
Highlights
This study showed that the content of all the detected mono-ginsenosides and glycosides increased in hairy roots after the Methyl jasmonate (MeJA) treatment
We showed that the accumulation of the PPT-type ginsenosides was faster than that of the PPD-type ginsenosides under the MeJA treatment, which was different from the results reported before
The one may be due to the different plant materials used for the studies and the other could be attributed to different mono-ginsenosides detected
Summary
The tissuespecific content and distributions of ginsenosides could be changed by elicitors through controlling growth conditions, for which the elicitors used for such purposes including fungi[10], N, N-dicyclohexylcarbodiimide (DCCD)[11], salicylic acid (SA)[12] and Methyl jasmonate (MeJA)[13] Among these elicitors, MeJA has been proven to be the most effective and been used most widely. It has been reported that MeJA has a vital influence on the expression of the genes involved in ginsenoside biosynthesis and ginsenoside content in hairy roots [14, 15], adventitious roots[16, 17] and other tissues[18] These elicitors can be potentially used for increasing ginsenoside production, identifying genes involved in ginsenoside biosynthesis, as well as studying the molecular mechanisms of ginsenoside biosynthesis and regulation. The MeJA-treated ginseng hairy roots have been widely used in the study of ginsenoside biosynthesis
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