Reference gene selection in Artemisia annua L., a plant species producing anti-malarial artemisinin

Liu, Wanhong; Zhao, Tengfei; Wang, Huanyan; Zeng, Junlan; Xiang, Lan; Zhu, Shunqin; Chen, Min; Lan, Xiaozhong; Liu, Xiaoqiang; Liao, Zhihua

doi:10.1007/s11240-014-0690-2

Cited by 11 publications

(5 citation statements)

References 55 publications

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“…In the current study, the RefFinder analysis, identified MUB as the most stable gene in the all-samples set. Meanwhile, UBC , α - TUB and β - TUB had relatively poor expression stability values, which is similar to previous results in Artemisia annua L [ 29 ] and Brassica napus [ 46 ].…”

Section: Discussionsupporting

confidence: 89%

“…Consequently, it is essential to systematically evaluate potential reference genes to ensure that they are appropriate for a specific experimental condition [ 23 ]. To date, IGG ( http://icg.big.ac.cn ), a wiki-driven knowledgebase that collects internal reference genes for diverse species, has been integrating a comprehensive collection of more than 150 plants [ 24 ], such as Arabidopsis [ 25 ], cucumber [ 26 ], wheat [ 27 ], rice [ 28 ], Artemisia annua [ 29 ], and Panax ginseng [ 30 ]. However, it has not been used for the systematic selection of a reference gene for qRT-PCR analysis in I. indigotica under hormone treatment or low-nitrogen stress, a factor that impedes functional gene studies.…”

Section: Introductionmentioning

confidence: 99%

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Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune

Miao

Cui

et al. 2019

BMC Molecular Biol

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BackgroundIsatis indigotica, a traditional Chinese medicine, produces a variety of active ingredients. However, little is known about the key genes and corresponding expression profiling involved in the biosynthesis pathways of these ingredients. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful, commonly-used method for gene expression analysis, but the accuracy of the quantitative data produced depends on the appropriate selection of reference genes.ResultsIn this study, the systematic analysis of the reference genes was performed for quantitative real-Time PCR normalization in I. indigotica. We selected nine candidate reference genes, including six traditional housekeeping genes (ACT, α-TUB, β-TUB, UBC, CYP, and EF1-α), and three newly stable internal control genes (MUB, TIP41, and RPL) from a transcriptome dataset of I. indigotica, and evaluated their expression stabilities in different tissues (root, stem, leaf, and petiole) and leaves exposed to three abiotic treatments (low-nitrogen, ABA, and MeJA) using geNorm, NormFinder, BestKeeper, and comprehensive RefFind algorithms. The results demonstrated that MUB and EF1-α were the two most stable reference genes for all samples. TIP41 as the optimal reference gene for low-nitrogen stress and MeJA treatment, while ACT had the highest ranking for ABA treatment and CYP was the most suitable for different tissues.ConclusionsThe results revealed that the selection and validation of appropriate reference genes for normalizing data is mandatory to acquire accurate quantification results. The necessity of specific internal control for specific conditions was also emphasized. Furthermore, this work will provide valuable information to enhance further research in gene function and molecular biology on I. indigotica and other related species.Electronic supplementary materialThe online version of this article (10.1186/s12867-019-0126-y) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune

Miao

Cui

et al. 2019

BMC Molecular Biol

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“…Quantitative PCR (qPCR) was performed on an iQTM5 Multicolor Real‐Time PCR Detection System (Bio‐Rad, USA) in 96‐well plates using GoTaq® qPCR Master Mix (Promega) according to the protocol. The internal control genes corresponded to actin and EF1ɑ, which were previously reported to be stable reference genes under low temperature stress . The specific primers for target genes are shown in Table .…”

Section: Methodsmentioning

confidence: 99%

Cold stress improves the production of artemisinin depending on the increase in endogenous jasmonate

Liu

Wang

Chen

et al. 2016

Biotech and App Biochem

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Previous publications reported that the artemisinin level was increased in Artemisia annua following a night-frost period. However, the molecular mechanism was not clear. In this study, we found that exogenous jasmonate (JA) effectively enhanced the freezing tolerance of A. annua. The JA biosynthetic genes (LOX1, LOX2, allene oxide cyclase [AOC], and jasmonate resistant 1 [JAR1]) were induced by cold stress, leading to an increase in endogenous JA in cold-treated A. annua. Increased endogenous JA enhanced the expression of three JA-responsive transcription factors, ethylene response factor 1, ethylene response factor 2, and octadecanoid-responsive AP2/ERF, all of which were reported to transcriptionally activate the expression of artemisinin biosynthetic genes, such as amorpha-4,11-diene synthase (ADS), CYP71AV1, DBR2, and aldehyde dehydrogenase 1 (ALDH1). Furthermore, the expression levels of the four artemisinin biosynthetic genes were also significantly increased under cold stress. Consequently, the levels of artemisinin and related secondary metabolites, such as dihydroartemisinic acid, artemisinin B, and artemisinic acid, were increased in A. annua under cold stress. Our study points to a molecular mechanism in which the production of artemisinin is regulated by cold stress in A. annua.

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“…The upper part of the figure displays the respiratory chain complexes and the lower portion represents their subunits as rectangles (red ones are the transcripts unique to control sample when compared to stress the bottom part, rectangles indicate the subunits of each respiratory chain complex. Red colored boxes represent the genes that were present exclusively in control sample whereas green boxes represent the genes present in reference organism 65 – 68 . …”

Section: Resultsmentioning

confidence: 99%

Transcriptome changes induced by abiotic stresses in Artemisia annua

Vashisth

Kumar

Rastogi

et al. 2018

Sci Rep

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Artemisia annua is known to be the source of artemisinin worldwide which is an antimalarial compound but is synthesised in very limited amount in the plant. Most research laid emphasis on the methods of enhancing artemisinin but our study has been planned in a way that it may simultaneously address two problems encountered by the plant. Firstly, to know the effect on the artemisinin content in the era of climate change because the secondary metabolites tend to increase under stress. Secondly, to identify some of the stress responsive genes that could help in stress tolerance of the plant under abiotic stress. Hence, the A. annua plants were subjected to four abiotic stresses (salt, cold, drought and water-logging) and it was observed that the artemisinin content increased in all the stress conditions except drought. Next, in order to identify the stress responsive genes, the transcriptome sequencing of the plants under stress was carried out resulting in 89,362 transcripts for control and 81,328, 76,337, 90,470 and 96,493 transcripts for salt, cold, drought, and water logging stresses. This investigation provides new insights for functional studies of genes involved in multiple abiotic stresses and potential candidate genes for multiple stress tolerance in A. annua.

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Reference gene selection in Artemisia annua L., a plant species producing anti-malarial artemisinin

Cited by 11 publications

References 55 publications

Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune

Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune

Cold stress improves the production of artemisinin depending on the increase in endogenous jasmonate

Transcriptome changes induced by abiotic stresses in Artemisia annua

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