Dataset Information

Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration.

ABSTRACT: Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis is a powerful analytical technique that requires the use of stable reference genes. Using available S. caninervis transcriptome data, we selected 15 candidate reference genes and analyzed their relative expression stabilities in S. caninervis gametophores exposed to a range of abiotic stresses or a hydration-desiccation-rehydration cycle. The programs geNorm, NormFinder, and RefFinder were used to assess and rank the expression stability of the 15 candidate genes. The stability ranking results of reference genes under each specific experimental condition showed high consistency using different algorithms. For abiotic stress treatments, the combination of two genes (?-TUB2 and CDPK) were sufficient for accurate normalization. For the hydration-desiccation-rehydration process, the combination of two genes (?-TUB1 and CDPK) were sufficient for accurate normalization. 18S was among the least stable genes in all of the experimental sets and was unsuitable as reference gene in S. caninervis. This is the first systematic investigation and comparison of reference gene selection for RT-qPCR work in S. caninervis. This research will facilitate gene expression studies in S. caninervis, related moss species from the Syntrichia complex and other mosses.

SUBMITTER: Li X

PROVIDER: S-EPMC4318276 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration.

Li Xiaoshuang X Zhang Daoyuan D Li Haiyan H Gao Bei B Yang Honglan H Zhang Yuanming Y Wood Andrew J AJ

Frontiers in plant science 20150205

Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) expression analysis is a ...[more]

PMID: 25699066

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Project description:Syntrichia caninervis is a desiccation tolerant moss and is the dominant bryophyte found in biological soil crusts in the Gurbantunggut desert. In this study, we assessed the transcriptome profiles of S. caninervis gametophytes during the dehydration-rehydration (D-R) process (across 9 time points) using Illumina sequencing. In total, 22489 transcripts were identified, including 5337 novel transcripts, that mapped to the reference genome. A total of 12548 transcripts exhibited significant alterations in the D-R samples compared with the control samples. The differentially expressed transcripts (DETs) possessed several enriched Gene Ontology terms, such as "water stress response", "oxidation-reduction process", "membrane metabolism", "photosynthesis", and "transcription factor activity". Moreover, during early dehydration stress, the DETs were significantly enriched in stress-related pathways from the Kyoto Encyclopedia of Genes and Genomes, such as "phenylpropanoid biosynthesis", "alpha-linolenic acid metabolism", and "fructose and mannose metabolism". Photosynthesis-related transcripts (e.g., ScPsa H, ScRubisco, and ScLhcb1) were inhibited during the dehydration treatment and significantly accumulated during the late rehydration period. Most transcripts from the late embryogenesis abundant proteins (LEA) and early light-inducible protein (ELIP) families strongly accumulated at the late dehydration stage. These pathways were positively correlated with the content changes of absolute water content and Fv/Fm values, alongside peroxidase and superoxide dismutase activities. Seven transcription factor families, including AP2-ERF, bHLH, G2-like, MYB, NAC, WRKY, and bZIP, were enriched in DETs during D-R treatment. This study is the first transcriptome analysis using the S. caninervis genome for gene annotation and multigroup D-R treatment points. Our results demonstrated the detailed dynamic changes in the transcriptome of S. caninervis during the D-R process. These results also improve understanding of desiccation tolerant plants' adaptations to desiccation stress at the transcription level and provide promising gene resources for transgenic crop breeding.

Project description:BackgroundSyntrichia caninervis is a desiccation-tolerant moss and the dominant bryophyte of the Biological Soil Crusts (BSCs) found in the Mojave and Gurbantunggut deserts. Next generation high throughput sequencing technologies offer an efficient and economic choice for characterizing non-model organism transcriptomes with little or no prior molecular information available.ResultsIn this study, we employed next generation, high-throughput, Illumina RNA-Seq to analyze the poly-(A)?+?mRNA from hydrated, dehydrating and desiccated S. caninervis gametophores. Approximately 58.0 million paired-end short reads were obtained and 92,240 unigenes were assembled with an average size of 493 bp, N50 value of 662 bp and a total size of 45.48 Mbp. Sequence similarity searches against five public databases (NR, Swiss-Prot, COSMOSS, KEGG and COG) found 54,125 unigenes (58.7%) with significant similarity to an existing sequence (E-value???1e-5) and could be annotated. Gene Ontology (GO) annotation assigned 24,183 unigenes to the three GO terms: Biological Process, Cellular Component or Molecular Function. GO comparison between P. patens and S. caninervis demonstrated similar sequence enrichment across all three GO categories. 29,370 deduced polypeptide sequences were assigned Pfam domain information and categorized into 4,212 Pfam domains/families. Using the PlantTFDB, 778 unigenes were predicted to be involved in the regulation of transcription and were classified into 49 transcription factor families. Annotated unigenes were mapped to the KEGG pathways and further annotated using MapMan. Comparative genomics revealed that 44% of protein families are shared in common by S. caninervis, P. patens and Arabidopsis thaliana and that 80% are shared by both moss species.ConclusionsThis study is one of the first comprehensive transcriptome analyses of the moss S. caninervis. Our data extends our knowledge of bryophyte transcriptomes, provides an insight to plants adapted to the arid regions of central Asia, and continues the development of S. caninervis as a model for understanding the molecular aspects of desiccation-tolerance.

Project description:APETALA2/Ethylene Responsive Factor (AP2/ERF) is a large family of plant transcription factors which play important roles in the control of plant metabolism and development as well as responses to various biotic and abiotic stresses. The desert moss Syntrichia caninervis, due to its robust and comprehensive stress tolerance, is a promising organism for the identification of stress-related genes. Using S. caninervis transcriptome data, 80 AP2/ERF unigenes were identified by HMM modeling and BLASTP searching. Based on the number of AP2 domains, multiple sequence alignment, motif analysis, and gene tree construction, ScAP2/ERF genes were classified into three main subfamilies (including 5 AP2 gene members, 72 ERF gene members, and 1 RAV member) and two Soloist members. We found that the ratio for each subfamily was constant between S. caninervis and the model moss Physcomitrella patens, however, as compared to the angiosperm Arabidopsis, the percentage of ERF subfamily members in both moss species were greatly expanded, while the members of the AP2 and RAV subfamilies were reduced accordingly. The amino acid composition of the AP2 domain of ScAP2/ERFs was conserved as compared with Arabidopsis. Interestingly, most of the identified DREB genes in S. caninervis belonged to the A-5 group which play important roles in stress responses and are rarely reported in the literature. Expression profile analysis of ScDREB genes showed different gene expression patterns under dehydration and rehydration; the majority of ScDREB genes demonstrated a stronger response to dehydration relative to rehydration indicating that ScDREB may play an important role in dehydrated moss tissues. To our knowledge, this is the first study to detail the identification and characterization of the AP2/ERF gene family in a desert moss. Further, this study will lay the foundation for further functional analysis of these genes, provide greater insight to the stress tolerance mechanisms in S. caninervis and provide a reference for AP2/ERF gene family classification in other moss species.

Dataset Information

Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration.

Publications

Characterization of reference genes for RT-qPCR in the desert moss Syntrichia caninervis in response to abiotic stress and desiccation/rehydration.

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