Real-time PCR Expression Analysis Research Articles

Genome-wide identification and salt stress-expression analysis of the dehydrin gene family in Chenopodium quinoa

Dehydrins (DHNs) are essential proteins in the embryonic development and abiotic stress responses of plants. Due to their remarkable ability to confer tolerance to plants in conditions of drought, salinity and extreme temperatures, DHNs have garnered considerable interest. Quinoa (Chenopodium quinoa Willd.), a facultative halophyte plant, can thrive in a wide range of agroecosystems, making it a promising candidate for stress tolerance studies. In this study, we identified eleven DHN genes in the quinoa genome belonging to Y-, F- and H-orthologous groups found in angiosperms. Notably, the H-DHNs lack the K-segment, a feature observed in all Amaranthaceae species, but not in other angiosperms. We identified four DHN structural subgroups: FSKn, YnSKn, SKn-DHNs and the atypical HS-DHN. Phylogenetic analysis indicated that each structural subgroup, except for SK2-DHN, presents two paralogous genes, in accordance with the allotetraploid character of C. quinoa. Quantitative real-time PCR expression analysis revealed that DHN1s (FSK2) and DHN3s (Y2SK2) were expressed in all tissues, while DHN2s (FSK3) were predominant in roots and DHN4s (Y4SK2 and SK2) were predominant in flowers. Salt-response gene expression analysis in seedlings showed that CqDHN4s increase their expression in response to salt stress in all varieties studied, while CqDHN1s reduce their expression in a more salt stress-tolerant variety, suggesting a possible adaptive advantage. In silico analysis of the promoters of CqDHN1s and CqDHN4s supports the involvement of these DHNs in responding to abiotic stress.

Functional Characterization of CsSWEET5a, a Cucumber Hexose Transporter That Mediates the Hexose Supply for Pollen Development and Rescues Male Fertility in Arabidopsis.

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and β-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed.

Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis

Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have used reference genes without proper validation. Here, seven candidate reference genes viz., acidic ribosomal protein (ARP0), glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II (RPo), elongation factor1α (EF1α), α- tubulin (AT), ribosomal protein L 10, and β-actin were evaluated using four algorithms (geNorm, BestKeeper, NormFinder and ∆Ct) followed by a comprehensive gene expression analysis using skin tissue of rohu at varied time points of experimental Argulus siamensis infection. ARP0 and EF1α were found to be the most stable whereas RPo and AT were considered as least stable genes based on basal expression level and variation in expression levels. Validation of candidate reference genes was undertaken by looking into the expression of six immune-related genes using the two most stable and two least stable genes as housekeeping genes in Argulus-infected rohu skin at different time points of infection. An increased expression of immune genes indicated the role of inflammation and the immune modulation process at the site of attachment of parasites in governing infection.

Diagnostics and Therapeutic Potential of miR-205 and miR-34a in Ovarian Cancer Management: A miRNA-Target-Based Analysis.

Epithelial ovarian cancer (EOC) treatment strategies mainly focused on surgery combined with chemotherapy. Recent targeted therapy techniques emerge as milestone and could be used for management of ovarian cancer (OC) progression with more efficacy. The aim is to evaluate the therapeutic and diagnostic potential of microRNA (miRNA) in management of EOC using in silico and quantitative real-time PCR (qRT-PCR) expression analysis. We performed functional enrichment and miRNA-Target genes expression analysis in 48 EOC and 22 normal tissue samples using qRT-PCR and correlated with miRNA expression data in matched samples to evaluate the diagnostic and therapeutic potential of miRNA in OC management. In silico functional enrichment analysis revealed miRNA association with disease. Target genes of miRNAs participate in several biologically important pathways leading to cancer progression. Targets of miRNA-205 and miRNA-34a were significantly downregulated, and upregulated, respectively, in EOC. Moreover, significant negative correlation between relative expression of miRNA-205 and target genes (BCL2, ZEB1, E2F1, and TP53) was observed with r = -0.813; r = -0.755; r = -0.559; and r = -0.767, respectively. Similarly, miRNA-34a also showed higher negative correlation with target genes (MDM4, MAPK3, BRCA1, AREG) with r = -0.840; r = -0.870; r = -0.622; and r = -0.623, respectively. In addition, receiver operating characteristics analysis of combined miRNA panel, miRNA-205-Target gene panel, and miRNA-34a-Target gene panel exhibited higher diagnostics value with area under the curve (AUC) of 92.7 (p < 0.0001), 94.8 (p < 0.0001), and 98.3 (p < 0.0001), respectively. Negative Correlation between miRNA and target genes expression data in matched samples highlights therapeutic potential of miRNA in EOC management. Moreover, combined diagnostic potential of miRNA-target gene panel could predict risk of EOC with higher AUC, sensitivity, and specificity.

Biotic elicitor-induced changes in growth, antioxidative defense, and metabolites in an improved prickleless Solanum viarum.

Solanum viarum serves as a raw material for the steroidal drug industry due to its alkaloid and glycoalkaloid content. Elicitation is well-known for measuring the increase in the yield of bioactive compounds in in vitro cultures. The current study was performed for the accumulation of metabolites viz. solasodine, solanidine, and α-solanine in S. viarum culture using microbial-based elicitors added in 1%, 3%, 5%, and 7% on 25th and 35th day of culture period and harvested on 45th and 50th days of culture cycle. The treatment of 3% Trichoderma reesei and Bacillus tequilensis culture filtrate (CF) significantly increased biomass, alkaloids/glycoalkaloid content, and yield in S. viarum. T. reesei was found to be the best treatment for enhanced growth (GI = 11.65) and glycoalkaloid yield (2.54mg DW plant-1) after the 50th day of the culture cycle when added on the 25th day. The abundance of gene transcripts involved in the biosynthesis of alkaloids/glycoalkaloids, revealed by quantitative real-time PCR expression analysis correlates with the accumulation of their respective metabolites in elicited plants. Biochemical analysis shows that elicited plants inhibited oxidative damage caused by reactive oxygen species by activating enzymes (superoxide dismutase and ascorbate peroxidase) as well as non-enzymatic antioxidant mechanisms (alkaloids, total phenols, total flavonoids, carotenoids, and proline). The findings of this study clearly demonstrate that the application of T. reesei and B. tequilensis CF at a specific dose and time significantly improve biomass as well as upregulates the metabolite biosynthetic pathway in an important medicinal plant- S. viarum. KEY POINTS: • Biotic elicitors stimulated the alkaloids/glycoalkaloid content in S. viarum plant cultures. • T. reesei was found to be most efficient for enhancing the growth and alkaloids content. • Elicited plants activate ROS based-defense mechanism to overcome oxidative damage.

Molecular characterization and expression of the autophagy-related gene Atg14 in WSSV-infected Procambarus clarkii

Atg14 (autophagy-related gene 14), also known as Atg14L or Barkor (Beclin-1 associated autophagy-related key regulator), plays an important role in a variety of biological processes including immunity, development, tumor inhibition, longevity, and protection against some cardiac and neurodegenerative diseases. However, very few studies have characterized Atg14 expression in invertebrates, particularly crustaceans. Here, a novel Atg14 gene from Procambarus clarkii (named PcAtg14) was characterized via RACE technology. Bioinformatics analysis showed that the total length of the PcAtg14 gene sequence was 2,880 bp, and it was predicted to encode 488 amino acids. The results of homology comparison showed that PcAtg14 exhibited the highest homology with crustacean the American lobster (Homarus americanus). Quantitative real-time PCR expression analysis showed that PcAtg14 was expressed in all tissues of P. clarkii, with the hepatopancreas having the highest expression and the eyestalk exhibiting the lowest expression. Upon white spot syndrome virus (WSSV) infection, the relative expression of PcAtg14 in the hepatopancreas, muscle, hemocyte, gill, heart and epidermis were significantly up-regulated at different time periods. After PcAtg14 gene silencing via RNA interference (RNAi), the proliferation of WSSV in P. clarkii was significantly inhibited, which coincided with a significant increase in P. clarkii mortality and an increase in the expression of autophagy-related genes (ATGs). Transmission electron microscopy analysis demonstrated an increase in the number of autophagosomes in the hepatopancreas of the PcAtg14 gene silencing group compared to the control group after WSSV infection. Collectively, these results indicated that PcAtg14 suppressed autophagy by reduce the fusion of autophagosomes and lysosomes, thereby promoting WSSV replication in P. clarkii. The findings here therefore provide novel insights into the immune mechanisms through which P. clarkii responds to WSSV infection.

Transcriptome Analyses of Near Isogenic Lines Reveal Putative Drought Tolerance Controlling Genes in Wheat.

Drought stress, especially at the grain-filling stage, is a major constraint for wheat production. Drought tolerance is a complex trait controlled by a large array of genes and pathways. This study conducted gene expression profiling on two pairs of near-isogenic lines (NILs) for an important qDSI.4B.1 QTL conferring drought tolerance on the short arm of chromosome 4B in wheat. Analysis showed 1,614 genome-wide differentially expressed genes (DEGs) between the tolerant and susceptible isolines in both NIL pairs. Six common DEGs were found between NIL1 and NIL2 at both 7 and 14 days after stress induction, with two of them having single nucleotide polymorphism (SNP) variants. These six genes that were confirmed by quantitative real-time PCR (qRT-PCR) expression analysis are considered candidate genes for drought tolerance mediated by qDSI.4B.1 QTL with their main contributions to gene regulation, cell elongation, protein quality control, secondary metabolism, and hormone signaling. These six candidate genes and the highest number of DEGs and variants (SNPs/indels) were located between 49 and 137 Mbp of 4BS, making this interval the most probable location for the qDSI.4B.1 locus. Additionally, 765 and 84 DEGs were detected as responsive genes to drought stress in tolerant and susceptible isolines, respectively. According to gene ontology (GO), protein phosphorylation, oxidation reduction, and regulation of transcription were top biological processes involved in the drought response and tolerance. These results provide insights into stress responses regulated by the 4BS locus and have identified candidate genes and genetic markers that can be used for fine mapping of the qDSI.4B.1 locus and, ultimately, in wheat breeding programs for drought tolerance.

Genome-Wide Identification of the Bcl-2 Associated Athanogene (BAG) Gene Family in Solanum lycopersicum and the Functional Role of SlBAG9 in Response to Osmotic Stress

The Bcl-2-associated athanogene (BAG) proteins are a family of multi-functional group of co-chaperones regulators, modulating diverse processes from plant growth and development to stress response. Here, 10 members of SlBAG gene family were identified based on the available tomato (Solanum lycopersicum) genomic information and named as SlBAG1-10 according to their chromosomal location. All SlBAG proteins harbor a characteristic BAG domain, categorized into two groups, and SlBAG4, SlBAG7, and SlBAG9 of group I contain a plant-specific isoleucine glutamine (IQ) calmodulin-binding motif located in the N terminus. The quantitative real-time PCR expression analysis revealed that these SlBAG genes had organ-specific expression patterns and most SlBAG genes were differentially expressed in multiple abiotic stresses including drought, salt, high temperature, cold, and cadmium stress as well as abscisic acid and H2O2. In addition, heterologous overexpression of SlBAG9 increased the sensitivity of Arabidopsis to drought, salt, and ABA during seed germination and seedling growth. The decreased tolerance may be due to the downregulation of stress-related genes expression and severe oxidative stress. The expression levels of some stress and ABA-related genes, such as ABI3, RD29A, DREB2A, and P5CS1, were significantly inhibited by SlBAG9 overexpression under osmotic stress. Meanwhile, the overexpression of SlBAG9 inhibited the expression of FSD1 and CAT1 under stress conditions and the decreased levels of superoxide dismutase and catalase enzyme activities were detected accompanying the trends in the expression of both genes, which resulted in H2O2 accumulation and lipid peroxidation. Taken together, these findings lay a foundation for the future study of the biological function of SlBAG genes in tomato.

Genotype-dependent and temperature-induced modulation of secondary metabolites, antioxidative defense and gene expression profile in Solanum viarum Dunal

The medicinal plant, Solanum viarum Dunal includes a number of compounds with important pharmacological effects. The effect of temperature (heat and cold) stress on growth, secondary metabolite levels and defense mechanism in two genotypes (prickled and prickleless) of S. viarum was evaluated. The two thermal regimes (4 °C for cold and 35 °C for heat stress) were found to be the most optimum for the accumulation of metabolites (steroidal alkaloids and glycoalkaloids, phenolic acids as well as flavonoids). Prickleless genotype showed 4.08 and 1.51 fold higher total alkaloids/glycoalkaloid content at 35 °C and 4 °C temperatures respectively, in comparison with prickled plants. Prickleless plants also registered 2.03 and 2.28 higher fresh and dry biomass accumulation respectively, under cold stress over the control plants. The accretion of phenolic and flavonoid compounds under heat and cold stress is mainly contributed by gallic and ferulic acid in both the genotypes. The quantitative real-time PCR expression analysis showed the abundance of gene transcript involve in the biosynthesis of alkaloids/glycoalkaloid and phenolics/flavonoids that corroborates with the accumulation of their respective metabolites in prickleless plants under temperature stress. Results revealed that the prickleless plants inhibiting the reactive oxygen species (ROS) mediated oxidative damage by activating the enzymatic (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase) and non-enzymatic (alkaloids, phenols, flavonoids, carotenoids and proline) antioxidants mechanism that confirms its efficiency to tolerate thermal stress under both the thermal regimes. The overall result showed that the prickleless genotype served as a better accumulator of biomass, secondary metabolites with an improved antioxidative mechanism in comparison to prickled genotype. Our findings suggest that thermal stress responses differ significantly between genotypes, emphasizing the necessity of maintaining genotypic diversity in adaptive evolution as in the context of global warming and climate change. The present study also opens the possibility of exploiting improved prickleless genotype of S. viarum as a potential system that can be cultivated under different temperature stress conditions to attain the phytopharmaceutical benefits.

XSP10 and SlSAMT, Fusarium wilt disease responsive genes of tomato (Solanum lycopersicum L.) express tissue specifically and interact with each other at cytoplasm in vivo.

The online version contains supplementary material available at 10.1007/s12298-021-01025-y.

Gastruloid Development Competence Discriminates Different States of Pluripotency.

SummaryFloating spheroidal aggregates of mouse embryonic stem cells can develop into polarized/elongated organoids, namely gastruloids. We set up a high-performing assay to measure gastruloid formation efficiency (GFE), and found that GFE decreases as pluripotency progresses from naive (GFE ≥ 95%) to primed (GFE = 0) state. Specifically, we show that primed EpiSCs fail to generate proper cell aggregates, while early-primed EpiLCs aggregate but eventually fail to develop into elongated gastruloids. Moreover, we characterized proline-induced cells (PiCs), a LIF-dependent reversible early-primed state of pluripotency, and show that PiCs are able to generate gastruloids (GFE ∼ 50%) and are also competent to differentiate into primordial germ cell-like cells. Thus, we propose the GFE assay as a valuable functional tool to discriminate different states of the pluripotency continuum.

The molecular features of normal and atopic dermatitis skin in infants, children, adolescents, and adults

Although atopic dermatitis (AD) often presents in infancy and persists into adulthood, comparative characterization of AD skin among different pediatric age groups is lacking. We sought to define skin biopsy profiles of lesional and nonlesional AD across different age groups (0-5-year-old infants with disease duration <6 months, 6-11-year-old children, 12-17-year-old adolescents, ≥18-year-old adults) versus age-appropriate controls. We performed gene expression analyses by RNA-sequencing and real-time PCR (RT-PCR) and protein expression analysis using immunohistochemistry. TH2/TH22 skewing, including IL-13, CCL17/thymus and activation-regulated chemokine, IL-22, and S100As, characterized the common AD signature, with a global pathway-level enrichment across all ages. Nevertheless, specific cytokines varied widely. For example, IL-33, IL-1RL1/IL-33R, and IL-9, often associated with early atopic sensitization, showed greatest upregulations in infants. TH17 inflammation presented a 2-peak curve, with highest increases in infants (including IL-17A and IL-17F), followed by adults. TH1 polarization was uniquely detected in adults, even when compared with adolescents, with significant upregulation in adults of IFN-γ and CXCL9/CXCL10/CXCL11. Although all AD age groups had barrier abnormalities, only adults had significant decreases in filaggrin expression. Despite the short duration of the disease, infant AD presented robust downregulations of multiple barrier-related genes in both lesional and nonlesional skin. Clinical severity scores significantly correlated with TH2/TH22-related markers in all pediatric age groups. The shared signature of AD across ages is TH2/TH22-skewed, yet differential expression of specific TH2/TH22-related genes, other TH pathways, and barrier-related genes portray heterogenetic, age-specific molecular fingerprints.

Anesthesia-Sepsis-Associated Alterations in Liver Gene Expression Profiles and Mitochondrial Oxidative Phosphorylation Complexes.

Background: Hepatic dysfunction plays a major role in adverse outcomes in sepsis. Volatile anesthetic agents may protect against organ dysfunction in the setting of critical illness and infection. The goal of this study was to study the impact of Sepsis-inflammation on hepatic subcellular energetics in animals anesthetized with both Propofol (intravenous anesthetic agent and GABA agonist) and Isoflurane (volatile anesthetic i.e., VAA).Methods: Sprague-Dawley rats were anesthetized with Propofol or isoflurane. Rats in each group were randomized to celiotomy and closure (control) or cecal ligation and puncture “CLP” (Sepsis-inflammation) for 8 h.Results: Inflammation led to upregulation in hepatic hypoxia-inducible factor-1 in both groups. Rats anesthetized with isoflurane also exhibited increases in bcl-2, inducible nitric oxide synthase, and heme oxygenase-1(HO-1) during inflammation, whereas rats anesthetized with Propofol did not. In rats anesthetized with isoflurane, decreased mRNA, protein (Complex II, IV, V), and activity levels (Complex II/III,IV,V) were identified for all components of the electron transport chain, leading to a decrease in mitochondrial ATP. In contrast, in rats anesthetized with Propofol, these changes were not identified after exposure to inflammation. RNA-Seq and real-time quantitative PCR (qPCR) expression analysis identified a substantial difference between groups (isoflurane vs. Propofol) in mitogen-activated protein kinase (MAPK) related gene expression following exposure to Sepsis-inflammation.Conclusions: Compared to rats anesthetized with Propofol, those anesthetized with isoflurane exhibit more oxidative stress, decreased oxidative phosphorylation protein expression, and electron transport chain activity and increased expression of organ-protective proteins.

Leucine Supplementation Decreases HDAC4 Expression and Nuclear Localization in Skeletal Muscle Fiber of Rats Submitted to Hindlimb Immobilization.

In this study we surveyed a rat skeletal muscle RNA-Seq for genes that are induced by hindlimb immobilization and, in turn, become attenuated by leucine supplementation. This approach, in search of leucine-atrophy protection mediating genes, identified histone deacetylase 4 (HDAC4) as highly responsive to both hindlimb immobilization and leucine supplementation. We then examined the impact of leucine on HDAC4 expression, tissue localization, and target genes. A total of 76 male Wistar rats (~280 g) were submitted to hindlimb immobilization and/or leucine supplementation for 3, 7 and 12 days. These animals were euthanized, and soleus muscle was removed for further analysis. RNA-Seq analysis of hindlimb immobilized rats indicated a sharp induction (log2 = 3.4) of HDAC4 expression which was attenuated by leucine supplementation (~50%). Real-time PCR and protein expression analysis by Western blot confirmed increased HDAC4 mRNA after 7 days of hindlimb immobilization and mitigation of induction by leucine supplementation. Regarding the HDAC4 localization, the proportion of positive nuclei was higher in the immobilized group and decreased after leucine supplementation. Also, we found a marked decrease of myogenin and MAFbx-atrogin-1 mRNA levels upon leucine supplementation, while CAMKII and DACH2 mRNA levels were increased by leucine supplementation. Our data suggest that HDAC4 inhibition might be involved in the anti-atrophic effects of leucine.

The Expression Levels of MicroRNAs Associated with T and B Cell Differentiation/stimulation in Ankylosing Spondylitis.

Spondyloarthropathies (SpAs), are a group of chronic inflammatory diseases with a number of genetic, physiopathological, clinical and radiological features. Ankylosing spondylitis (AS) is the most common type of spondylo-arthropathies, and >90.0% of patients with ankylosing spondylitis are human leukocyte antigen-B27 (HLA-B2 7)-positive. In recent years, non-HLA genetic factors have been reported to have an effect on ankylosing spondylitis. MicroRNAs (miRNAs), are endogenous non coding RNA molecules containing 18-23 nucleotides that play a role in the post-transcriptional regulation of gene expression. In this study, we aimed to determine the expression levels of miRNAs associated with T- and B-cell differentiation/stimulation in peripheral blood mononuclear cells and their relationship with the etiology of the AS in patients and healthy controls. In a molecular study, peripheral blood mononuclear cell isolation, and total RNA isolation were performed first. In the second step, cDNA synthesis and quantitative real-time PCR (qPCR) expression analysis were completed. Ultimately, in the patient and control group, the expression levels of miR-142-5p and miR-143 were found to be significantly different (p <0.05). According to current knowledge, miR-142-5p andmiR-143 expressions were found to be important for those diseases that share similar etiology with AS. We suggest that miR-142-5p and miR-143 may play a role in the pathogenesis, especially miR- 142-5p may be a potential biomarker and a target molecule for the treatment.

Genome-wide identification and expression analysis of SABATH methyltransferases in tea plant (Camellia sinensis): insights into their roles in plant defense responses

ABSTRACT SABATH methyltransferases convent plant small-molecule metabolites into volatile methyl esters, which play important roles in many biological processes and defense reactions in plants. In this study, a total of 32 SABATH genes were identified in the Camellia sinensis var. sinensis (CSS) genome, which were renamed CsSABATH1 to CsSABATH32. Genome location annotation suggested that tandem duplication was responsible for the expansion of SABATH genes in tea plant. Multiple sequence alignment and phylogenetic analysis showed that the CsSABATHs could be classified into three groups (I, II and III), which were also supported by gene structures and conserved motifs analysis. Group II contained only two CsSABATH proteins, which were closely related to PtIAMT, AtIAMT and OsIAMT. The group III SABATH genes of tea plant exhibited expansion on the CSS genome compared with Camellia sinensis var. assamica (CSA) genome. Based on RNA-seq data, the CsSABATHs exhibited tissue-specific expression patterns, and the members with high expression in buds and young leaves were also obviously upregulated after MeJA treatment. The expression of many transcription factors was significantly correlated with that of different members of the CsSABATH gene family, suggesting a potential regulatory relationship between them. Quantitative real-time PCR (qPCR) expression analysis showed that CsSABATHs could respond to exogenous JA, SA and MeSA treatments in tea plants. RNA-seq data analysis and qPCR validation suggested that CsSABATH8, 11, 16, 25, 29 and 32 might play a special role in plant defense against insect herbivory. These results provide references for evolutionary studies of the plant SABATH family and the exploration of the potential roles of CsSABATHs in tea plant defense responses.

Fine Mapping of a Grain Shape Gene from a Rice Landrace Longliheinuo-Dwarf (Oryza sativa L. ssp. japonica)

Identification of grain shape genes can facilitate breeding of rice cultivars with optimal grain shape and appearance quality. In this study, we selected two rice germplasms, namely Longliheinuo-dwarf (LH) and N643, with different grain shape, to construct a genetic population for quantitative trait locus (QTL) analysis. A major QTL (qGS7), controlling the ratio of grain length to grain width, was mapped on the chromosome 7 in a BC1F4 line. By high-resolution linkage analysis, qGS7 was delimited to a 52.8 kb region including eight predicted genes. Through sequence alignment and real-time PCR expression analysis of these ORFs, ORF3 (LOC_Os07g42410) was selected as the candidate gene for further analysis. Single nucleotide polymorphisms (SNP) diversity analysis of ORF3 revealed that a single nucleotide deletion in the 7th exon resulted in a frameshift in parent LH and the parent in which a premature stop codon was identified. It was a rare mutation that caused grain shape difference. Real-time PCR analyses showed that the expression characteristics of ORF3 was in accordance with the development of spikelets. Of the 18 agronomic traits investigation in qGS7 near isogenic lines (NILs) showed that qGS7 not only changed grain shape but also affected plant height, panicle curvature, panicle length, the length of second leaf from the top, and chalkiness.

Reference gene selection for quantitative real-time PCR (qRT-PCR) expression analysis in Galium aparine L.

To accurately evaluate expression levels of target genes, stable internal reference genes is required for normalization of quantitative real-time PCR (qRT-PCR) data. However, there have been no systematical investigation on the stability of reference genes used in the bedstraw weed, Galium aparine L. (BGA). In this study, the expression profiles of seven traditionally used reference genes, namely 18S, 28S, ACT, GAPDH, EF1α, RPL7 and TBP in BGA were assessed under both biotic (developmental time and tissue), and abiotic (temperature, regions and herbicide) conditions. Four analytical algorithms (geNorm, Normfinder, BestKeeper and the ΔCt method) were used to analyze the suitability of these genes as internal reference genes. RefFinder, a comprehensive analytical software, was used to rank the overall stability of the candidate genes. The optimal normalization internal control genes were ranked as: 28S and RPL7 were best for all the different experimental conditions (developmental stages, tissues, temperature, regions and herbicide treatment); 28S and RPL7 for developmental stages; TBP and GAPDH for different tissues; 28S and GAPDH were relatively stable for different temperature; 28S and TBP were suitable for herbicide treatment. A specific set of reference genes were recommended for each experimental condition in BGA.

A Zinc Finger Transcriptional Repressor Confers Pleiotropic Effects on Rice Growth and Drought Tolerance by Down-Regulating Stress-Responsive Genes.

The C2H2-type zinc finger proteins (ZFPs) are involved in a wide range of plant development and stress responses. Many studies have shown the positive roles of ZFP genes in stress tolerance. However, overexpression of ZFP genes usually leads to the side effect of growth retardation. Here we report a new member of the ZFP family, Oryza sativa drought-responsive zinc finger protein 1 (OsDRZ1), positively regulating both stress tolerance and plant architecture in rice (Oryza sativa L.). OsDRZ1 was expressed throughout all tissues examined and could be induced by multiple abiotic stresses. OsDRZ1 protein was localized mostly in the nucleus. Unlike most reported rice ZFPs functioning as transcriptional activators, OsDRZ1 is a transcriptional repressor. Overexpression of OsDRZ1 in rice increased seedling drought tolerance and the transgenic plants appeared to accumulate more free proline and fewer reactive oxygen species (ROS), and elevate the activities of antioxidant enzymes. In contrast, RNA interference (RNAi) of OsDRZ1 led to lower activities of antioxidative response and more sensitivity to drought. RNA sequencing analysis revealed that the genes down-regulated by OsDRZ1 were mostly down-regulated by drought, implying the critical role of OsDRZ1 in modulating drought-responsive gene expression. A cupin gene OsGLP1 (germin-like protein1) was identified as one of the potential target genes of OsDRZ1, as suggested by real-time PCR and transient expression analysis in rice protoplasts. Moreover, overexpression of OsDRZ1 did not lead to growth inhibition but the promotion of rice growth, implying the potential application prospective of OsDRZ1 in engineering drought-tolerant crops.

Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but for normalization of data requires the use of stable reference genes. However, suitable reference genes are still not known in the case of Phenacoccus solenopsis under variable experimental treatments. The present study focused on the identification of stable housekeeping genes as a reference for analysis under different abiotic and biotic factors in P. solenopsis. We analyzed the relative expression of six commonly used candidate reference genes in different developmental stages, host-feeding assay, temperature treatments and field distribution conditions. Expression stabilities were analyzed by geNorm, NormFinder, and RefFinder. Under developmental and field distribution conditions, β-Tubulin was found to be most stable reference genes followed by rpl32 and α-Tubulin. In the case host feeding treatment conditions, β-Tubulin and α-tubulin identified to be the most stable reference genes, while in temperature stress, a combination of α-Tubulin and rpl32 found to be suitable for normalizing the RT-qPCR data. Further, the above-identified genes were validated using RT-qPCR based gene expression analysis of four objective genes namely, Myoinhibitory peptides (MIPs), Zinc_metalloprotease (Zn_Mp), fatty acid synthase (fas) and alpha-glucosidase. Identified reference genes will facilitate gene expression studies in future under different stress treatments in P. solenopsis.