Selection and evaluation of reference genes for expression analysis of Cassi

Liu, Zubi; Zhu, Qiankun; Li, Juanjuan; Yu, Jihua; Li, Yangyang; Huang, Xinhe; Wang, Wanjun; Tan, Rui; Zhou, Jiayu; Liao, Hai

doi:10.1080/09168451.2015.1052771

Cited by 6 publications

(12 citation statements)

References 28 publications

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“…In order to validate the accuracy of the sequences obtained from the hybrid platform, two full-length transcripts (F01.PB17587 and F01.PB29801) encoding C. obtusifolia 1-deoxy- d -xylulose 5-phosphate synthase ( CoDXS ) and 1-deoxy- d -xylulose 5-phosphate reductoisomerase ( CoDXR ) were directly chosen for functional analysis, respectively, using bacterial complementation assay [ 107 ]. In higher plant, DXS and DXR played an important role in the MEP pathway and were involved in the biosynthesis of anthraquinone [ 108 ]. The plasmids pAC-BETA and pTrc-AtIPI were purchased from ADDGENE Corporation (USA).…”

Section: Methodsmentioning

confidence: 99%

Full-Length Transcriptome Survey and Expression Analysis of Cassia obtusifolia to Discover Putative Genes Related to Aurantio-Obtusin Biosynthesis, Seed Formation and Development, and Stress Response

Yin

Zheng

Zicheng

et al. 2018

IJMS

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The seed is the pharmaceutical and breeding organ of Cassia obtusifolia, a well-known medical herb containing aurantio-obtusin (a kind of anthraquinone), food, and landscape. In order to understand the molecular mechanism of the biosynthesis of aurantio-obtusin, seed formation and development, and stress response of C. obtusifolia, it is necessary to understand the genomics information. Although previous seed transcriptome of C. obtusifolia has been carried out by short-read next-generation sequencing (NGS) technology, the vast majority of the resulting unigenes did not represent full-length cDNA sequences and supply enough gene expression profile information of the various organs or tissues. In this study, fifteen cDNA libraries, which were constructed from the seed, root, stem, leaf, and flower (three repetitions with each organ) of C. obtusifolia, were sequenced using hybrid approach combining single-molecule real-time (SMRT) and NGS platform. More than 4,315,774 long reads with 9.66 Gb sequencing data and 361,427,021 short reads with 108.13 Gb sequencing data were generated by SMRT and NGS platform, respectively. 67,222 consensus isoforms were clustered from the reads and 81.73% (61,016) of which were longer than 1000 bp. Furthermore, the 67,222 consensus isoforms represented 58,106 nonredundant transcripts, 98.25% (57,092) of which were annotated and 25,573 of which were assigned to specific metabolic pathways by KEGG. CoDXS and CoDXR genes were directly used for functional characterization to validate the accuracy of sequences obtained from transcriptome. A total of 658 seed-specific transcripts indicated their special roles in physiological processes in seed. Analysis of transcripts which were involved in the early stage of anthraquinone biosynthesis suggested that the aurantio-obtusin in C. obtusifolia was mainly generated from isochorismate and Mevalonate/methylerythritol phosphate (MVA/MEP) pathway, and three reactions catalyzed by Menaquinone-specific isochorismate synthase (ICS), 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate (IPPS) might be the limited steps. Several seed-specific CYPs, SAM-dependent methyltransferase, and UDP-glycosyltransferase (UDPG) supplied promising candidate genes in the late stage of anthraquinone biosynthesis. In addition, four seed-specific transcriptional factors including three MYB Transcription Factor (MYB) and one MADS-box Transcription Factor (MADS) transcriptional factors) and alternative splicing might be involved with seed formation and development. Meanwhile, most members of Hsp20 genes showed high expression level in seed and flower; seven of which might have chaperon activities under various abiotic stresses. Finally, the expressional patterns of genes with particular interests showed similar trends in both transcriptome assay and qRT-PCR. In conclusion, this is the first full-length transcriptome sequencing reported in Caesalpiniaceae family, and thus providing a more complete insight into aurantio-obtusin biosynthesis, seed formation ...

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Section: Methodsmentioning

confidence: 99%

Full-Length Transcriptome Survey and Expression Analysis of Cassia obtusifolia to Discover Putative Genes Related to Aurantio-Obtusin Biosynthesis, Seed Formation and Development, and Stress Response

Yin

Zheng

Zicheng

et al. 2018

IJMS

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“…As demonstrated previously, larval midgut played essential role in the resistance to allelochemicals from host plants, it was necessary to understand the molecular mechanism by which COTI alter the physiological balance in migdut of P. rapae . De novo assembly of transcriptome has been widely used to identify the growing and developmental genes, hormone biosynthetic genes, and signaling pathway genes in Lepidoptera insects (Antony et al., ; Liu et al., ; Pan et al., ; Park & Kim, ; Qi et al., ; Xu et al., ). Here, transcriptome sequencing was carried out to obtain systematic information related to response in P. rapae midgut.…”

Section: Discussionmentioning

confidence: 99%

“…Apolipophorin III, occurring in a diversity of insect species, increases the capacity of lipophorins to bind the lipid for delivery, and thereby facilitates energy generation. In Galleria mellonella , Chinese oak silkworm and Tribolium castaneum , apoLp‐III played crucial role in lipid transport and immune response against bacteria, fungi, mosquito, and Bacillus thuringiensis toxic protein (Gupta et al., ; Liu et al., ; Zdybicka‐Barabas, Sowa‐Jasiłek, Stączek, Jakubowicz, & Cytryńska, ). Thus, reduced expression of apolipophorins was likely to indicate lower lipolytic activity and reduced resistance in COTI‐fed insects.…”

Section: Discussionmentioning

confidence: 99%

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Comparative transcriptome analysis provides insights of anti‐insect molecular mechanism of Cassia obtusifolia trypsin inhibitor against Pieris rapae

Xiang

Zhang

Yin

et al. 2017

Arch Insect Biochem Physiol

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Pieris rapae, a serious Lepidoptera pest of cultivated crucifers, utilizes midgut enzymes to digest food and detoxify secondary metabolites from host plants. A recombinant trypsin inhibitor (COTI)1 from nonhost plant, Cassia obtusifolia, significantly decreased activities of trypsin-like proteases in the larval midgut on Pieris rapae and could suppress the growth of larvae. In order to know how COTI took effect, transcriptional profiles of P. rapae midgut in response to COTI was studied. A total of 51,544 unigenes were generated and 45.86% of which had homologs in public databases. Most of the regulated genes associated with digestion, detoxification, homeostasis, and resistance were downregulated after ingestion of COTI. Meanwhile, several unigenes in the integrin signaling pathway might be involved in response to COTI. Furthermore, using comparative transcriptome analysis, we detected differently expressing genes and identified a new reference gene, UPF3, by qRT-polymerase chain reaction (PCR). Therefore, it was suggested that not only proteolysis inhibition, but also suppression of expression of genes involved in metabolism, development, signaling, and defense might account for the anti-insect resistance of COTI. K E Y W O R D SCassia obtusifolia, nonhost plant, physiological response, Pieris rapae, trypsin inhibitor

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“…Among them, ACT was recommended as a stable reference gene in different tissues of C. tetragonoloba 40 , E. songoricum 36 , G. max 37,42 , P. vulgaris 43 . EF was recommended in C. obtusifolia 44 , G. max 37 , M. sativa 45 , and UBQ was recommended in G. inflata (Table 3), E. songoricum 36 , G. max 37 , and L. angustifolius 41 . So, the optimal reference genes for different species in the same family are variable, even for the two proximal species in the same genus (G. uralensis and G. inflata).…”

Section: S R R N a A B C T A C Ti N C A C C O P S 3 C Y P D R E B E Fmentioning

confidence: 99%

Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

Liang

Zhou

et al. 2021

Sci Rep

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Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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Selection and evaluation of reference genes for expression analysis of Cassi

Cited by 6 publications

References 28 publications

Full-Length Transcriptome Survey and Expression Analysis of Cassia obtusifolia to Discover Putative Genes Related to Aurantio-Obtusin Biosynthesis, Seed Formation and Development, and Stress Response

Full-Length Transcriptome Survey and Expression Analysis of Cassia obtusifolia to Discover Putative Genes Related to Aurantio-Obtusin Biosynthesis, Seed Formation and Development, and Stress Response

Comparative transcriptome analysis provides insights of anti‐insect molecular mechanism of Cassia obtusifolia trypsin inhibitor against Pieris rapae

Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza

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