Differential Display Reverse Transcription-polymerase Chain Research Articles

Identification of Differentially Expressed Genes of Rice Under Cadmium Stress Using DDRT-PCR Approach.

Cadmium (Cd) is one of the hazardous environmental pollutants, and it can be harmful to human health through consumption of food-plants capable of bioaccumulating Cd. Therefore, lowering cadmium accumulation in plants is highly desirable. Here, a rice cultivar 'Qisanzhan' was studied using differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Fifty-six differentially expressed genes were found in the root tips of 4-leaf stage rice seedlings exposed to 4 and 12h of 50µmol/L Cd(NO3)2 in a nutrient solution using DDRT-PCR. Further validation using semi-quantitative RT-PCR showed that the expression patterns of 16 genes were consistent with those found in DDRT-PCR. These genes encode receptor-like protein kinase, pleiotropic drug resistance protein, aquaporin protein, plasma membrane ATPase, etc. The differentially genes identified here can be used to obtain a better understanding of the molecular mechanisms of Cd absorption and accumulation in plants.

Deciphering the transcriptional regulation and spatiotemporal distribution of immunity response in barley to Pyrenophora graminea fungal invasion.

BackgroundBarley leaf stripe disease, caused by the fungus Pyrenophora graminea (Pg), is a worldwide crop disease that results in significant loss of barley yield. The purpose of the present work was to use transcriptomic profiling to highlight barley genes and metabolic pathways affected or altered in response to Pg infection and consequently elucidate their involvement and contribution in resistance to leaf stripe.ResultsOur study examined and compared the transcriptomes of two barley genotypes using an established differential display reverse-transcription polymerase chain reaction (DDRT-PCR) strategy at 14 and 20 days post-inoculation (dpi). A total of 54 significantly modulated expressed sequence tags (ESTs) were identified. The analysis of gene expression changes during the course of infection with Pg suggested the involvement of 15 upregulated genes during the immunity response. By using network-based analyses, we could establish a significant correlation between genes expressed in response to Pg invasion. Microscopic analysis and quantitative PCR (qPCR) profiling of callose synthase and cellulose synthases revealed a direct involvement of cell wall reinforcement and callose deposition in the Pg-resistant phenotype.ConclusionsWe have identified a number of candidate genes possibly involved in the host-pathogen interactions between barley and Pg fungus, 15 of which are specifically expressed in Pg-resistant plants. Collectively, our results suggest that the resistance to leaf stripe in barley proceeds through callose deposition and different oxidation processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2573-x) contains supplementary material, which is available to authorized users.

Molecular analysis of the adaptive response in Salmonella Typhimurium after starvation in salty conditions.

The pathogenic bacterium Salmonella enterica serovar Typhimurium elicits a variety of genetic programs to adapt to stress conditions encountered within hostile environments such as host phagocytes and preserved food. In this work, differential display (DD) methodology was used to investigate the effect of one month starvation in a salty microcosm (0.5 M NaCl) on transcript profiling in a Salmonella Typhimurium LT2 strain. cDNA fragments resulting from differentially expressed mRNA were eluted from the gel, re-amplified, cloned, and then sequenced. A total of 21 differentially expressed bands were detected by DD reverse transcription-polymerase chain reaction (RT-PCR). However, only 12 of them were successfully identified as upregulated genes in stressed cells. Based on the sequencing data and BLAST analysis, these genes were sopA, ssaD, yhhK, gmK, cspC, uspA, ompR, phoP, stcC, fimA, acrA,and yehZ. As a confirmation of the differential expression, RT-PCR was carried out using a set of specific primers. Remarkably, the expression levels of these genes were significantly increased in starved bacteria compared to standard laboratory conditions. Our results indicate that the starvation of Salmonella Typhimurium over one month in a salty microcosm changes the expression of stress proteins, response regulator in a two-component system, outer membrane proteins, effector proteins translocated by Salmonella pathogenicity island SPI1 and SPI2 type III secretion systems (TTSS), several metabolic enzymes, efflux pumps, and transport proteins. This suggests that the expression of the identified genes is important for the response of this pathogen to starvation in salt.

Gene Expression Profiling in Fish Toxicology: A Review.

In this review, we present an overview of transcriptomic responses to chemical exposures in a variety of fish species. We have discussed the use of several molecular approaches such as northern blotting, differential display reverse transcription-polymerase chain reaction (DDRT-PCR), suppression subtractive hybridization (SSH), real time quantitative PCR (RT-qPCR), microarrays, and next-generation sequencing (NGS) for measuring gene expression. These techniques have been mainly used to measure the toxic effects of single compounds or simple mixtures in laboratory conditions. In addition, only few studies have been conducted to examine the biological significance of differentially expressed gene sets following chemical exposure. Therefore, future studies should focus more under field conditions using a multidisciplinary approach (genomics, proteomics and metabolomics) to understand the synergetic effects of multiple environmental stressors and to determine the functional significance of differentially expressed genes. Nevertheless, recent developments in NGS technologies and decreasing costs of sequencing holds the promise to uncover the complexity of anthropogenic impacts and biological effects in wild fish populations.

Physiological adaptations to osmotic stress and characterization of a polyethylene glycol-responsive gene in Braya humilis

<em>Braya humilis</em> (Brassicaceae) is a widely distributed plant in arid and semi-arid regions of northern Asia. This plant is well adapted to extremely arid conditions and is a promising candidate species to discover novel drought tolerance strategies. However, not much information about the mechanism(s) mediating drought resistance in this species is currently available. Therefore, the present study aimed to characterize the physiological traits and expression patterns of a polyethylene glycol (PEG)-responsive gene in <em>B. humilis</em> responding to different levels of osmotic stress induced by PEG-6000. Several important physiological parameters were examined, including the levels of relative water content, soluble protein, malondialdehyde, and antioxidant enzyme activity. A tolerance threshold between 20 and 30% PEG-6000 was identified for <em>B. humilis</em>. The water status and oxidative damage below this threshold were maintained at a relatively constant level during the 12 h of treatment. However, once the threshold was exceeded, the water status and oxidative damage were obviously affected after treatment for 4 h. The soluble protein results suggest that <em>B. humilis</em> maintains a vigorous resistance to osmotic stress and that it may play a greater role in osmotic regulation at late stages of stress. Moreover, superoxide dismutase and catalase may be important at preventing oxidative damage in plants at early stages of stress, while peroxidase may be more involved in some biological processes that resist osmotic stress at the late stage, especially in severely damaged plants. Furthermore, a PEG-responsive gene, <em>BhCIPK12</em>, was identified by differential display reverse transcription-polymerase chain reaction (PCR), cloned, and characterized by quantitative real-time PCR. We hypothesized that this gene may play an important role in mediating osmotic stress or drought resistance in plants. Altogether, these results provide valuable insights into the mechanism(s) mediating drought tolerance in <em>B. humilis</em>.

염과 건조 스트레스 조건에서 톨 페스큐의 종자 발아율과 유전자 발현 변화분석

ABSTRACT Salinity and drought stresses are probably the most significant abiotic factor limiting plant’s growth, also negatively affect seed germination and early seedling development. To study on effect of NaCl and PEG stress on seed germination and gene expression pattern of tall fescue, the levels of NaCl and PEG-induced water stresses were determined in first experiment. Different concentration of NaCl (0 to 350 mM) and PEG (0 to 30%) were used for seed treatment. Seed Germination percentage reduced with increasing osmotic potential of growth medium either due to NaCl or PEG. Seeds were not germinate at 350 mM NaCl or 30% PEG treatment. On the basis of the results, Kentucky31(E-) had more resistant than Fawn in both stress conditions. Furthermore, we have used an annealing control primer-based differential display reverse transcription-polymerase chain reaction method to identify salt- and drought stress-induced differentially expressed genes (DEGs) in tall fescue leaves. Using 120 annealing control primers, a total of 4 genes were identified and sequenced. The possible roles of the identified DEGs are discussed in the context of their putative role during salinity and drought stresses.(

Gene Expressing Difference in Sclerotial Formation of Morchella conica.

The difference of gene expression between sclerotia-producing and non-sclerotia-producing single spore isolates from Morchella conica were preliminary analyzed by mRNA differential display reverse transcription-polymerase chain reaction (RT-PCR) technique and 67 differential gene fragments were obtained. Fifty-eight of their second PCR products were cloned and sequenced. Thirteen special differential gene fragments related to sclerotial formation were validated by semi-quantitative RT-PCR. Some gene fragments had certain homologies with lipoprotein, cyclin-dependent kinase C-3, glycerophosphoryl diester phosphodiesterase, Rho GDP-dissociation inhibitor, gamma-aminobutyrate permease, OmpA family protein, Transcript antisense to ribosomal RNA protein, sodium-calcium exchange protein and keratin-associated proteins 5, 6. In addition, the putative protein of some DNA fragments had higher similarity with hypothetical protein-coding gene in NCBI database, as well as some were only putative gene fragments. All these fragments were speculated to be the functional gene associated with sclerotial formation in morel.

English

To search for genes controlling high prolificacy of Chinese indigenous goats, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to screen differentially expressed cDNA bands in the sexually matured ovaries of 3-year-old prolific Jining Grey goats and monotocous Liaoning Cashmere goats with 24 combinations of three anchored primers and eight arbitrary primers. 22 expressed sequence tags (ESTs) were proved to be the positive bands by Northern hybridization. They comprised 10 known ESTs and 12 ESTs without homologous sequences in the GenBank. These results indicate that several genes such as GATA-4, metallothionein-like protein, CAT genes and unknown ESTs (CV983340 and CV983341) were expressed only in Jining Grey goats. Key words: Differential display reverse transcription-polymerase chain reaction, goat, ovary, prolificacy.

Identification of Differentially Expressed Genes by Gabapentin in Cultured Dorsal Root Ganglion in a Rat Neuropathic Pain Model

Neuropathic pain is a chronic pain disorder caused by nervous system lesions as a direct consequence of a lesion or by disease of the portions of the nervous system that normally signal pain. The spinal nerve ligation (SNL) model in rats that reflect some components of clinical pain have played a crucial role in the understanding of neuropathic pain. To investigate the direct effects of gabapentin on differential gene expression in cultured dorsal root ganglion (DRG) cells of SNL model rats, we performed a differential display reverse transcription-polymerase chain reaction analysis with random priming approach using annealing control primer. Genes encoding metallothionein 1a, transforming growth factor-β1 and palmitoyl-protein thioesterase-2 were up-regulated in gabapentin-treated DRG cells of SNL model rats. The functional roles of these differentially expressed genes were previously suggested as neuroprotective genes. Further study of these genes is expected to reveal potential targets of gabapentin.

The ribosomal protein L19 mRNA is induced by copper exposure in the swordtail fish, Xiphophorus helleri

Teleosts are useful vertebrate model species for understanding copper toxicity due to the dual entry route for copper intake via the gills and intestine. In this present study, we utilized the differential display reverse transcription-polymerase chain reaction to isolate potential novel hepatic genes induced by sublethal copper exposure in the freshwater swordtail fish, Xiphophorus helleri. Full length cloning of a cDNA fragment induced by copper exposure to 1μg/ml during 24h resulted in the positive identification of a hepatic ribosomal protein L19 (RPL19) gene. Further characterization of this gene revealed that its transcriptional expression was dependent on dosage and time of copper exposure. This study describes for the first time the involvement of RPL19 in copper toxicity, probably as a result of increase in ribosome synthesis rate to support activities such as cellular protein translation, transcriptional activation and mRNA stabilization during sublethal copper exposure.

Isolation and Molecular Characterization of a Putative Ascorbate Peroxidase Gene from Citrus

Ascorbate peroxidase is the key enzyme in detoxifying hydrogen peroxide (H2O2) and other reactive oxygen species from chloroplast and cytosol. A cDNA encoding a putative citrus ascorbate peroxidase, APXcit was isolated from mature ‘Dancy’ tangerine (Citrus reticulata Blanco) juice vesicles using differential display reverse transcription-polymerase chain reaction. The full-length APXcit sequence was composed of 1,082 bp nucleotides, including an open reading frame of 753 bp, putatively encoding a protein of 250 amino acids with a predicted molecular mass of 27 kDa. The 5′ untranslated region consisted of 90 nucleotides and the 3′ untranslated region of 239. The genomic clone of 2,457 bp was composed of 8 introns and 9 exons. A homology search for APXcit at the GenBank database showed high similarity to ascorbate peroxidase from several plant species. Southern analysis revealed that there may be 2–4 APXcit gene copies in citrus.

Identification of Differentially Expressed Genes in Human Mesenchymal Stem Cell-Derived Neurons

Mesenchymal stem cells (MSCs) have greater potential for immediate clinical and toxicological applications, due to their ability to self-renew, proliferate, and differentiate into a variety of cell types. To identify novel candidate genes that were specifically expressed during transdifferentiation of human MSCs to neuronal cells, we performed a differential expression analysis with random priming approach using annealing control primer-based differential display reverse transcription-polymerase chain reaction approach. We identified genes for acyl-CoA thioesterase, tissue inhibitor of metalloproteinases-1, brain glycogen phosphorylase, ubiquitin C-terminal hydrolase and aldehyde reductase were up-regualted, whereas genes for transgelin and heparan sulfate proteoglycan were down-regulated in MSC-derived neurons. These differentially expressed genes may have potential role in regulation of neurogenesis. This study could be applied to environmental toxicology in the field of testing the toxicity of a chemical or a physical agent.

Identification of differentially expressed genes in the developmental stages from olive flounder Paralichthys olivaceus using an annealing control primer system

We employed a new and improved differential display reverse transcription–polymerase chain reaction (DDRT-PCR) method, which involves annealing control primers (ACPs), to identify the genes that are specifically or prominently expressed in olive flounder (Paralichthys olivaceus) juveniles (35 days post-hatch; dph) compared to larval-stage (dph 21) flounder. Using 60 ACPs, we identified eight differentially expressed genes (DEGs) and basic local alignment search tool (BLAST) searches revealed eight known genes. Gene expression levels were confirmed by RT-PCR. Phosphoglucose isomerase (PGI) was highly expressed at 21 dph, while nephrosin, myosin light chain (MLC), myosin heavy chain (MHC), carboxypeptidase A, chymotrypsin B, fish-egg protein, and matrix protein were expressed at 35 dph. PGI, MLC, and MHC expression was further analyzed by RT-PCR. The differentially expressed genes identified in this study may provide insights into the molecular basis of development in olive flounder.

Increased ATP generation in the host cell is required for efficient vaccinia virus production

To search for cellular genes up-regulated by vaccinia virus (VV) infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR) assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

Characterization of an acrosome protein VAD1.2/AEP2 which is differentially expressed in spermatogenesis

The release of enzymes from the acrosome of the sperm head (acrosome reaction) starts the fertilization process and enables the spermatozoa to penetrate the zona pellucida of the oocytes. Defective acrosome reaction is one of the important causes of infertility in men. To investigate the molecular regulation of spermatogenesis in vivo, we used differential display reverse transcription-polymerase chain reaction to identify stage-specific genes in a retinol-supplemented vitamin-A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressed protein 2, AEP2) gene, which was expressed strongly in the rat testis from post-natal day 32 to adult stage. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively, with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII, and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 co-localized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with hypospermatogenesis, maturation arrest, undescended testis and Sertoli cell-only syndrome. Co-immunoprecipitation experiments followed by western blotting and mass spectrometry (MS-MS) identified syntaxin 1, beta-actin and myosin heavy chain (MHC) proteins as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin 1) and structural (beta-actin and MHC) proteins suggest that VAD1.2 maybe involved in acrosome formation during spermiogenesis.

Transcriptional up-regulation of PHLDA1 by 17β-estradiol in MCF-7 breast cancer cells

Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ERalpha and ERbeta, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17beta-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17beta-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 microM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17beta-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER.

Identification of replicative senescence-associated genes in human umbilical vein endothelial cells by an annealing control primer system

Cellular senescence is regulated by specific genes in many organisms. The identification and functional analysis of senescence-associated genes could provide valuable insights into the senescence process. Here, we employed a new and improved differential display reverse transcription-polymerase chain reaction (DDRT-PCR) method that involves annealing control primers (ACPs) to identify genes that are differentially expressed in human umbilical endothelial cells during replicative senescence. Using 120 ACPs, we identified 31 differentially expressed genes (DEGs). Basic local alignment search tool (BLAST) search revealed 29 known genes and two unknown genes. Expression levels of the 29 known genes were confirmed by real-time quantitative RT-RCR and by Western blotting for eight of these genes. CD9 antigen, MHC class I chain-related sequence A (MICA) and cell division cycle 37 homolog (CDC37) were up-regulated, and bone morphogenetic protein 4 (BMP4), dickkopf-1 (DKK1), and transcription factor 7-like 1 (TCF7L1) were down-regulated in old cells. Treatment with recombinant human MICA caused a decrease in cell proliferation and an increase in senescence-associated β-galactosidase staining. Further analysis of differentially expressed genes may provide insights into the molecular basis of replicative senescence and vascular diseases associated with cellular senescence.

Characterization of growth-related genes in the south-western Atlantic pink shrimp Farfantepenaeus paulensis (Pérez-Farfante 1967) through a modified DDRT-PCR protocol

An exploratory methodology has been used to identify genes related to growth of the shrimp Farfantepenaeus paulensis (Perez-Farfante, 1967). Six hundred postlarvae, with an average (±SD) initial weight of 0.022 g (±0.008), were reared for 60 days in salinity 30 g L⁻¹. In the end, the 15 heaviest (>1.2 g) and the 15 lightest (<0.6 g) animals were frozen in liquid nitrogen. A modified differential display reverse transcription-polymerase chain reaction technique was used to generate expression profiles for all individuals. The resulting cDNA from reverse transcription were amplified by pairing 31 arbitrary primers with a reverse primer anchored to the cDNA tails. From the comparison between the expression profiles of the organisms in the two size classes, differences could be pinpointed. Bands of interest were collected, purified, cloned and sequenced. Despite the relatively small number of primers used, the methodology allowed the identification of three genes, not described previously for the investigated species and possibly related to growth. These partial sequences are likely to belong to genes that code for myosin heavy chain, cyclophilin and haemocyanin as revealed through an amino acid similarity search conducted using the blastp on-line tool.

Isolation of a drought-responsive alkaline .ALPHA.-galactosidase gene from New Zealand spinach

We isolated a cDNA clone encoding a New Zealand spinach (Tetragonia tetragonioides) alkaline α-galactosidase that we designated TtAGA1. The clone was found by monitoring an up-regulated transcripts accumulation with the differential display reverse transcription–polymerase chain reaction method during a treatment of New Zealand spinach shoots with drought. Deduced amino acid sequence suggests that the gene belongs to a family of plant-specific alkaline α-galactosidases. When detached shoots of New Zealand spinach were treated with abiotic stresses such as drought, administration of mannitol (150 mM), wounding, salinity (200 mM and 400 mM sodium chloride), dark, and cold (4°C), drought most enhanced the accumulation of the TtAGA1 transcripts. Treatments with mannitol, wounding, salinity, and dark increased the transcripts accumulation, but cold did not. It is supposed that the degradation of galactosyl saccharides may correlate with drought responses in New Zealand spinach growing in seashore areas.

Differential Gene Expression in Hepatopancreas of the Shrimp Metapenaeus ensis During Ovarian Maturation

Differentially expressed genes were identified in the hepatopancreas of Metapenaeus ensis during ovarian maturation via differential display reverse transcription-polymerase chain reaction (DDRT-PCR). They are G-protein signaling modulator 2 (GPSM2), glutamate carboxypeptide II (GCPII), ligatin, C-type lectin, O-linked N-acetylglucosamine transferase (O-GlcNAc transferase), 1-acylglycerol-3-phosphate O-acyltransferase 4 (AGPAT4), vitellogenin (Vg), and hemocyanin. The hepatopancreas Vg gene identified in this study shows 92% and 49% amino acid sequence homology, respectively, to MeVg1 and MeVg2 previously isolated from this species, suggesting the identification of a new Vg gene in M. ensis. Vg gene expression was highest when the ovary was actively developing. The two metabolic enzymes, O-GlcNAc transferase and AGPAT4, exhibited a similar trend of expression to Vg gene, suggesting their involvement in Vg synthesis. The signal transduction related genes (GPSM2, GCPII, ligatin, and C-type lectin) were highly expressed in the hepatopancreas in the initial phase of maturation. These genes may be important for the signaling in the hepatopancreas for synthesis and mobilization of vitellogenin and nutrients to the developing ovary. The present work provides candidate genes for further investigation on the role of hepatopancreas in shrimp reproduction.