The use of miRNAs as reference genes for miRNA expression normalization during Lilium somatic embryogenesis by real-time reverse transcription PCR analysis

Zhang, Jing; Gai, MeiZhu; Xue, Bingyang; Jia, NaNa; Wang, Chunxia; Wang, JinXia; Sun, Hao

doi:10.1007/s11240-016-1160-9

Cited by 25 publications

(11 citation statements)

References 62 publications

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“…The reactions were incubated in 96-well PCR plates (Corning, NY, USA), and each experiment consisted of three biological and technical replicates. The relative miRNA and target gene expression levels were calculated using the 2 −ΔΔCt method with normalization to lpu-miR159a and the F-box family protein ( FP ), respectively, as endogenous controls (Zhang et al, 2017 ).…”

Section: Methodsmentioning

confidence: 99%

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

et al. 2017

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Plant somatic embryos are widely used in the fields of germplasm conservation, breeding for genetic engineering and artificial seed production. MicroRNAs (miRNAs) play pivotal roles in somatic embryogenesis (SE) regulation. However, their regulatory roles during various stages of SE remain unclear. In this study, six types of embryogenic samples of Lilium pumilum DC. Fisch., including organogenic callus, embryogenic callus induced for 4 weeks, embryogenic callus induced for 6 weeks, globular embryos, torpedo embryos and cotyledon embryos, were prepared for small RNA sequencing. The results revealed a total of 2,378,760 small RNA reads, among which the most common size was 24 nt. Four hundred and fifty-two known miRNAs, belonging to more than 86 families, 57 novel miRNAs and 40 miRNA*s were identified. The 86 known miRNA families were sorted according to an alignment with their homologs across 24 land plants into the following four categories: 23 highly conserved, 4 moderately conserved, 15 less conserved and 44 species-specific miRNAs. Differentially expressed known miRNAs were identified during various stages of SE. Subsequently, the expression levels of 12 differentially expressed miRNAs and 4 targets were validated using qRT-PCR. In addition, six samples were mixed in equal amounts for transcript sequencing, and the sequencing data were used as transcripts for miRNA target prediction. A total of 66,422 unigenes with an average length of 800 bp were assembled from 56,258,974 raw reads. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that 38,004 and 15,497 unigenes were successfully assigned to GO terms and KEGG pathways, respectively. Among the unigenes, 2,182 transcripts were predicted to be targets for 396 known miRNAs. The potential targets of the identified miRNAs were mostly classified into the following GO terms: cell, binding and metabolic process. Enriched KEGG analysis demonstrated that carbohydrate metabolism was the predominant pathway in Lilium SE. Thus, we performed systemic characterization, homology comparisons and profiling of miRNA expression, and we constructed an miRNA-target network during Lilium SE for the first time. Our findings establish a foundation for the further exploration of critical genes and elucidation of SE in Lilium.

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Section: Methodsmentioning

confidence: 99%

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

et al. 2017

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“…As an important reproductive organ of lily, the bulb is crucial to the development of lily cut flower industry. It has been proved that carbohydrate metabolism is closely related to the bulb and shoot growth in the Lilium genus [16,17]. However, the past research mostly focused on temperature, photoperiod and hormones.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome Analysis of Carbohydrate Metabolism Genes and Molecular Regulation of Sucrose Transport Gene LoSUT on the Flowering Process of Developing Oriental Hybrid Lily ‘Sorbonne’ Bulb

Zeng

Wang

et al. 2020

IJMS

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The quality of Lily cut flower was determined by the quality of bulbs. During the process of vernalization and flower bud differentiation, sugar massively accumulated in the bulb, which influenced the bulb development. However, the details of sugar genes’ regulation mechanism for these processes were not fully understood. Here, morphological physiology, transcriptomes and gene engineering technology were used to explore this physiological change. Seventy-two genes of 25 kinds of sugar metabolism-related genes were annotated after re-analyzing transcriptome data of Oriental hybrid lily ‘Sorbonne’ bulbs, which were generated on Hiseq Illumina 2000. The results showed that these genes were closely related to lily bulb vernalization and development. Combining gene expression pattern with gene co-expression network, five genes (Contig5669, Contig13319, Contig7715, Contig1420 and Contig87292) were considered to be the most potential signals, and the sucrose transporter gene (SUT) was the focus of this study. Carbohydrate transport pathway and genes’ regulation mechanism were inferred through a physiological and molecular test. SUT seemed to be the sugar sensor that could sense and regulate sugar concentration, which might have effects on other genes, such as FT, LFY and so on. LoSUT2 and LoSUT4 genes were cloned from Oriental hybrid lily ‘Sorbonne’ by RACE, which was the first time for these genes in Oriental hybrid lily ‘Sorbonne’. The physiological properties of these proteins were analyzed such as hydrophobicity and phosphorylation. In addition, secondary and tertiary structures of proteins were predicted, which indicated the two proteins were membrane proteins. Their cellular locations were verified through positioning the experiment of the fluorescent vector. They were highly expressed in cells around phloem, which illustrated the key role of these genes in sugar transport. Furthermore, transient expression assays showed that overexpressed LoSUT2 and LoSUT4 in Arabidopsis thaliana bloomed significantly earlier than the wild type and the expression of FT, SOC1 and LFY were also affected by LoSUT2 and LoSUT4, which indicated that LoSUT2 and LoSUT4 may regulate plants flowering time.

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“…Gene expression analysis is an important research method to gain insight into the putative functions of specific candidate genes involved in complex regulatory relationships. Among the widely used methods, quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive and reproducible technique with a broad quantification range for measuring gene expression [ 13 – 16 ]. This technique has been recently used to quantify changes in gene expression profiles in response to developmental transitions (anthocyanin biosynthesis, sclereid formation, fruit development) and environmental changes (low temperature, dehydration stress, biotic stress) in pear [ 17 – 22 ].…”

Section: Introductionmentioning

confidence: 99%

Selection and validation of suitable reference genes for qRT-PCR analysis in pear leaf tissues under distinct training systems

et al. 2018

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Training systems generally alter tree architecture, which modulates light microclimate within the canopy, for the purpose of improving photosynthetic efficiency and fruit quality. Gene expression quantification is one of the most important methods for exploring the molecular mechanisms underlying the influence of training systems on pear photosynthesis, and suitable reference genes for gene expression normalization are a prerequisite for this method. In this study, the expression stability of nine common and four novel candidate genes were evaluated in 14 different pear leaf samples in two training systems, including those at four developmental stages (training_period) and from different parts of the trees (training_space), using two distinct algorithms, geNorm and NormFinder. Our results revealed that SKD1 (Suppressor of K+ Transport Growth Defect1)/ YLS8 (Yellow Leaf Specific 8) and ARM (Armadillo) were the most stable single reference genes for the ‘training_period’ and ‘training_space’ subsets, respectively, although these single genes were not as stable as the optimal pairs of reference genes, SKD1+YLS8 and ARM+YLS8, respectively. Furthermore, the expression levels of the PpsAPX (Ascorbate peroxidase) gene showed that the arbitrary use of reference genes without previous testing could lead to misinterpretation of data. This work constitutes the first systematic analysis regarding the selection of superior reference genes in training system studies, facilitating the elucidation of gene function in pear and providing valuable information for similar studies in other higher plants.

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The use of miRNAs as reference genes for miRNA expression normalization during Lilium somatic embryogenesis by real-time reverse transcription PCR analysis

Cited by 25 publications

References 62 publications

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Transcriptome Analysis of Carbohydrate Metabolism Genes and Molecular Regulation of Sucrose Transport Gene LoSUT on the Flowering Process of Developing Oriental Hybrid Lily ‘Sorbonne’ Bulb

Selection and validation of suitable reference genes for qRT-PCR analysis in pear leaf tissues under distinct training systems

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