The anti-tumor vindoline and catharanthine alkaloids are naturally existed in Catharanthus roseus (C. roseus), an ornamental plant in many tropical countries. Plant-specific TEOSINTE ...BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play important roles in various plant developmental processes. However, the roles of C. roseus TCPs (CrTCPs) in terpenoid indole alkaloid (TIA) biosynthesis are largely unknown.
Here, a total of 15 CrTCP genes were identified in the newly updated C. roseus genome and were grouped into three major classes (P-type, C-type and CYC/TB1).
Gene structure and protein motif analyses showed that CrTCPs have diverse intron-exon patterns and protein motif distributions. A number of stress responsive cis-elements were identified in promoter regions of CrTCPs. Expression analysis showed that three CrTCP genes (CrTCP2, CrTCP4, and CrTCP7) were expressed specifically in leaves and four CrTCP genes (CrTCP13, CrTCP8, CrTCP6, and CrTCP10) were expressed specifically in flowers. HPLC analysis showed that the contents of three classic TIAs, vindoline, catharanthine and ajmalicine, were significantly increased by ultraviolet-B (UV-B) and methyl jasmonate (MeJA) in leaves. By analyzing the expression patterns under UV-B radiation and MeJA application with qRT-PCR, a number of CrTCP and TIA biosynthesis-related genes were identified to be responsive to UV-B and MeJA treatments. Interestingly, two TCP binding elements (GGNCCCAC and GTGGNCCC) were identified in several TIA biosynthesis-related genes, suggesting that they were potential target genes of CrTCPs.
These results suggest that CrTCPs are involved in the regulation of the biosynthesis of TIAs, and provide a basis for further functional identification of CrTCPs.
Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays ...were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.
The study was aimed to evaluate the prognostic or predictive value of serum exosomal microRNAs (miRNAs) for tumor recurrence and response to adjuvant therapy in stage II and stage III colon cancer.
...145 differentially expressed mature miRNAs were identified (P<0.05) and 10 top hits were carried forward in validation test. MiR-4772-3p was significantly under-expressed in 27 patients with recurrence compared to in 57 patients without recurrence (P=0.002). The reduced expression was significantly related to increased risk of tumor recurrence and risk of death. As a predictor for tumor recurrence, ROC analysis revealed the AUC (95% CI) was 0.72 (0.59-0.85, P=0.001) for lower level of miR-4772-3p compared to 0.63 (0.51-0.75, P=0.062) for tumor site and 0.65 (0.51-0.78,P=0.034) for lymph node status. Among 66/84 patients who received FOLFOX adjuvant therapy, 9/10 (90%) patients with a lower level and 10/56 (18%) patients with a higher level of miR-4772-3p had tumor recurrence (P<0.001).
Blood samples were prospectively collected from84 patients with stage II/III colon cancer after tumor resection and before adjuvant therapy. Serum exosomal miRNA profiles were determined by RNA sequencing. Differentially expressed mature miRNAs were identified between patients with or without tumor recurrence. The top hits were validated in individual RNA samples using quantitative real-time reverse transcription PCR.
Reduced expression of serum exosomal miR-4772-3p is a prognostic biomarker for tumor recurrence in stage II and stage III colon cancer patients. The predictive value of this marker for response to FOLFOX adjuvant therapy needs further investigation.
Aubergine (eggplant) is susceptible to fungal wilts caused by Fusarium oxysporum f. sp. melongenae (Fom) and Verticillium dahliae (Vd). Wild and allied relatives represent a good source of ...resistance, and introgression lines (ILs) have been obtained through introgression of the Rfo‐sa1 locus conferring resistance to Fusarium oxysporum from Solanum aethiopicum into cultivated aubergine. In this work, a deep phenotype characterization was performed according to two parameters: progression of symptoms along the stem and disease severity in leaves. This analysis showed that the Fom‐resistant ILs carrying introgression of the Rfo‐sa1 locus displayed significantly improved tolerance to Verticillium attack after a preliminary inoculation with F. oxysporum. This positive effect was particularly evident when Verticillium inoculation was performed simultaneously or after Fusarium inoculation. Transcript profiling carried out using a combination of suppression subtractive hybridization (SSH), microarray and qRT‐PCR analyses of roots inoculated with selected combinations of fungal pathogens enabled the identification of 164 differentially expressed genes between inoculated and uninoculated plants or between different stages after pathogen infection. Overall, the results highlighted a number of candidate genes putatively involved in early defence responses or signalling pathways activated upon infection of aubergine with either Fom and/or Vd, and thus leading to a broad Rfo‐sa1‐mediated tolerance against both these wilt pathogens.
Fuchs' endothelial corneal dystrophy (FECD) is a progressive vision impairing disease caused by thickening of Descemet's membrane and gradual degeneration and loss of corneal endothelial cells. The ...aim of this study was to identify differentially expressed genes between FECD-affected and unaffected corneal endothelium to gain insight into the pathophysiological mechanisms underlying this disease. Microarray gene expression analysis was performed on total RNA from FECD-affected and unaffected corneal endothelium-Descemet's membrane (CE-DM) specimens using the Illumina HumanHT-12 v4.0 expression array. RNA from pools of FECD-affected (n = 3 per pool) and individual unaffected (n = 3) specimens was used for comparison. Altered expression of a sub-set of differentially expressed genes was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in independent specimens. Bioinformatics analysis was performed using InnateDB to reveal functional relationships among the differentially expressed genes and molecular pathways involved in the disease. A total of 16,513 genes were found expressed in the corneal endothelium of which 142 genes were differentially expressed between FECD-affected and unaffected endothelium (log2 fold-change ≥1.5, corrected p-value ≤0.05). Most of the genes were up-regulated (126) and a small proportion down-regulated (16) in affected corneal endothelium. Of the twelve genes prioritised for validation, differential expression of 10 genes, including those ranked 57th and 81st by significance validated by qRT-PCR (8 up-regulated and 2 downregulated, corrected p ≤ 0.05), one gene showed a trend for up-regulation in affected endothelium, consistent with the microarray analysis and another was up-regulated in an independent study indicating robustness of the differential expression dataset. Bioinformatic analysis revealed significant over-representation of differentially expressed genes in extracellular matrix reorganisation, cellular remodelling, immune response, and inflammation. Network analysis showed functional inter-relatedness of the majority of the dysregulated genes and revealed known direct functional relationships between 20 of the genes; many of these genes have roles in macrophage differentiation, phagocytosis and inflammation. This is the second report of microarray gene expression analysis in FECD. This study revealed a set of highly dysregulated genes in the corneal endothelium in FECD. More than a third of the dysregulated genes in the disease have been discovered for the first time and thus are novel. The dysregulated genes strongly suggest the presence of phagocytic cells, most likely immune cells, and inflammation in corneal endothelium in the disease. This study provides a molecular framework for delineating the mechanisms underlying these cellular processes in FECD.
•Less than 1% of genes expressed in the corneal endothelium are dysregulated in FECD.•Nearly 90% of the dysregulated genes are upregulated in the diseased corneal endothelium.•Levels of dysregulated genes are highly altered in the diseased corneal endothelium.•Dysregulated genes indicate an increase in phagocytic response in FECD.
•Shelter covering for cherry might substantially enhance the expression of Spermine synthase (SPDS) gene.•Shelter covering can alter PAs accumulation, including putrescine (Put), spermidine (Spd), ...and spermine (Spm), which at least partially improved fruitsetting of Chinese cherry in rainy regions.•Subcellular localization detected that CpSPDS gene was located in both nucleus and cytoplasm.
Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), play key roles in plant growth, development, and abiotic/biotic stress tolerance. Their functions appear to be intimately related to their synthesis, which occurs via arginine/ornithine decarboxylase (ADC/ODC), Spd synthase (SPDS) and Spm synthase (SPMS) etc. Previously, the transcriptional regulation of cherry under the shelter covering was carried out, and the PA synthase-related genes, particularly spermidine synthase (SPDS) gene, were differentially expressed in the fruits as exposure to the shelter covering. In the present work, the full-length cDNA sequence of this gene was cloned and named as CpSPDS (GenBank accession: MK045261). Bioinformatics analysis indicated that the cDNA sequence of CpSPDS was 1243 bp in length. Cluster analysis showed that CpSPDS had the highest homologies with those of sweet cherry (XP 021826297, 100 %) and peach (XP 020413638, 99 %). Subcellular localization detected that target gene was located in both nucleus and cytoplasm. The qPCR quantification showed that CpSPDS was differentially expressed in alabastrums, fruitlets, mature fruits and mature leaves of cherry. Interestingly, shelter covering significantly improved the fruitsetting in comparison with those from the shelter-free, gave a positive effect on the expression of CpSPDS gene. The endogenous free PAs were quantified in different tissues by high-performance liquid chromatography (HPLC). The PAs contents of the sheltered trees, i.e. Put, Spm and Spd were diversely higher in comparison to the shelter-free, especially in alabastrums, whose Spd titer of the sheltered was 3.29 folds higher than that of the unsheltered, which somehow collaborated result of mRNA quantification. Therefore, shelter covering for cherry might substantially enhance the expression of CpSPDS gene, leading to altering PAs accumulation, which at least partially contributed to the elevation in fruitsetting in rainy regions.
Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the ...estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.
Epithelial ovarian cancer (EOC) is the most aggressive and frequent malignancy detected among women worldwide. The pathophysiology of OC should, therefore be better understood to identify diagnostic, ...prognostic, and predictive novel biomarkers necessary for early detection, management, and prognostication. In this study, we aimed to investigate transcriptomic landscape and biomarker through RNA-seq data analysis. Further analysis by Protein Protein network identified top 10 Differentially Expressed Genes (DEGs). KEGG pathway enrichment analysis revealed the significant enrichment of DEGs in basal cell carcinoma, cell cycle and FoxO signalling pathway. The RNA-seq results of 10 DEGs were validated by QRT-PCR and TCGA database. Correlation studies were also performed between gene expression and clinical characteristics followed by survival analysis. Finally, 8 DEGs (CDKN1A, BCL6, CDC45, WNT2, TLR5, AQP5) including two novel DEGs (CSN1S1 and NKILA) were identified showing significant correlations with EOC characteristics. These may serve as interesting biomarkers and novel treatment targets and warrant further investigation into the functional outcome of EOC.
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Codonopsis tangshen
Oliv (
C. tangshen
) is an important Chinese traditional medicinal plant with various health benefits. However, the growth of
C. tangshen
are seriously affected by continuous ...cropping, which led to the decrease of the yield and quality. A field experiment was conducted to learn the effects of soil amendments on the growth of
C. tangshen
under continuous cropping condition, and the biological events which occurred at molecular level were investigated. The results indicated that the content of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Car) was significantly higher in SCPM (silicon-calcium-potassium-magnesium fertilizer), SCPMA (SCPM combined with azoxystrobin) and SCPMAOM (SCPM combined with azoxystrobin and organic manure) treatments. Moreover, the yield and the levels of alkaloid, polysaccharide, flavone and total protein in the treatments of SCPM, SCPMA and SCPMAOM were significantly higher than those in the control, and these indexes were all highest in the SCPMAOM treatment. RNA-sequencing (RNA-Seq) is an economical and efficient method to obtain genetic information for species with or without available genome data. In this study, RNA-Seq was performed to understand how continuously cropped
C. tangshen
responded to the soil amendments at the transcriptome level. The number of differentially expressed genes (DEGs) were as follows: CK vs. SCPM (719 up- and 1456 down-), CK vs. SCPMA (1302 up- and 1748 down-), CK vs. SCPMAOM (1274 up- and 1678 down-). The soil amendments affected the growth of
C. tangshen
mainly by regulating the genes involved in pathways of ‘photosynthesis,’ ‘plant hormone signal transduction,’ ‘biosynthesis of unsaturated fatty acids,’ ‘phenylpropanoid biosynthesis,’ and ‘starch and sucrose metabolism,’ etc. qRT-PCR was performed to validate the expressions of 10 target genes such as
CP26
,
PsaF
, and
POX
, etc., which verified the reliability of RNA-Seq results. Overall, this study revealed the roles and underlying mechanisms of the soil amendments in regulating the growth of continuously cropped
C. tangshen
at transcriptome level. These findings are beneficial for improving the continuous cropping tolerance and may be valuable for future genetic improvement of
C. tangshen
.
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