Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments

Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala. Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7 and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable. Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation

Exploration of microRNAs and their targets engaging in the resistance interaction between wheat and stripe rust

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. miRNAs are important regulators, they play very central roles in plant organ development, vegetable phase change and defense responses. In this study, two miRNA libraries from wheat cultivar Xingzi 9104 (XZ) challenged with the avirulent Pst race CYR32 and sterile water were constructed, respectively. A total of 596 miRNA candidates were obtained. 420 wheat-specific candidate miRNAs were screened in adult plants challenged with Pst using microarray-based analyses. We analyzed the abundance of candidate miRNAs, and the levels of a subset of candidate miRNAs were determined by quantitative real time PCR (qRT-PCR). The qRT-PCR results indicated that some miRNAs were involved in the incompatible interaction between wheat and Pst. In addition, we identified some miRNAs differentially expressed in different leaves. Additionally, the target genes of wheat miRNAs were confirmed by using degradome sequencing technology. Most of the annotated target genes are related to signal transduction, energy metabolism, and other functions. We selected some target genes for relative expression analysis using qRT-PCR, and found that RabGAP/TBC domain-containing protein, zinc finger protein and Cysteine-rich receptor-like protein kinase 41 may play important role in the incompatible interaction between XZ and CYR32. Intriguingly, miRNAs and target gene seem to form a complicated regulation network that regulates the wheat-Pst interaction. Our data provide the foundation for evaluating the important regulatory roles of miRNAs in the wheat-Pst interaction

De novo transcriptome assembly of a Chinese locoweed (Oxytropis ochrocephala) species provides insights into genes associated with drought, salinity and cold tolerance

Background: Locoweeds (toxic Oxytropis and Astraglus species), containing the toxic agent swainsonine, pose serious threats to animal husbandry on grasslands in both China and the US. Some locoweeds have evolved adaptations in order to resist various stress conditions such as drought, salt and cold. As a result they replace other plants in their communities and become an ecological problem. Currently very limited genetic information of locoweeds is available and this hinders our understanding in the molecular basis of their environmental plasticity, and the interaction between locoweeds and their symbiotic swainsonine producing endophytes. Next-generation sequencing provides a means of obtaining transcriptomic sequences in a timely manner, which is particularly useful for non-model plants. In this study, we performed transcriptome sequencing of Oxytropis ochrocephala plants followed by a de nove assembly. Our primary aim was to provide an enriched pool of genetic sequences of an Oxytropis sp. for further locoweed research. Results: Transcriptomes of four different O. ochrocephala samples, from control (CK) plants, and those that had experienced either drought (20% PEG), salt (150 mM NaCl) or cold (4 °C) stress were sequenced using an Illumina Hiseq 2000 platform. From 232,209,506 clean reads 23,220,950,600 (~23 G nucleotides), 182,430 transcripts and 88,942 unigenes were retrieved, with an N50 value of 1,237. Differential expression analysis revealed putative genes encoding heat shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins, enzymes in secondary metabolite and plant hormone biosyntheses, and transcription factors which are involved in stress tolerance in O. ochrocephala. In order to validate our sequencing results, we further analyzed the expression profiles of nine genes by quantitative real-time PCR. Finally, we discuss the possible mechanism of O. ochrocephala’s adaptations to stress environment. Conclusion: Our transcriptome sequencing data present useful genetic information of a locoweed species. This genetic information will underpin further research in elucidating the environmental acclimation mechanism in locoweeds and the endophyte-plant association

The microbial opsin homologue sop1 is involved in Sclerotinia sclerotiorum development and environmental stress response

Microbial opsins play a crucial role in responses to various environmental signals. Here, we report that the microbial opsin gene sop1 in the necrotrophic phytopathogenic fungus Sclerotinia sclerotiorum was dramatically up-regulated during infection and sclerotial development compared with the vegetative growth stage. Further study showed sop1 was essential for growth, sclerotial development and full virulence of S. sclerotiorum. Sop1-silenced transformants were more sensitive to high salt stress, fungicides and high osmotic stress. However, they were more tolerant to oxidative stress compared with the wild-type strain, suggesting that sop1 is involved in different stress responses and fungicide resistance, which plays a role in the environmental adaptability of S. sclerotiorum. Furthermore, a Delta blast search showed that microbial opsins are not present in animals and almost all higher plants, indicating that as a predicted transmembrane protein, sop1 is a potential drug target for disease control of S. sclerotiorum