Ubiquitin-conjugating Enzyme Gene Research Articles

MiR-379-5p affects breast cancer cell behavior by targeting UBE2E3 ubiquitin conjugating enzyme

MicroRNAs (miRNAs) play an increasingly recognized role in modulating cancer development. Due to their function in regulating gene expression, miRNAs can suppress or promote tumorigenesis. miR-379-5p expression is downregulated in multiple human cancers, including breast and bladder cancers. However, the mRNAs targeted by miR-379-5p that promote cancer development have not been fully identified. Our goal was to identify a gene whose expression is regulated by miR-379-5p, and which may contribute to cancer development in cells where miR-379-5p expression is reduced. Bioinformatics analysis showed the UBE2E3 ubiquitin conjugating enzyme gene to be a potential target for miR-379-5p. To verify that UBE2E3 is a target, we transfected normal human epithelial mammary cells and breast adenocarcinoma cell lines with a miR-379-5p mimic. The mimic reduced UBE2E3 mRNA and protein levels, as would be predicted for a miR-379-5p target. To determine if UBE2E3 is a direct target of miR-379-5p, we engineered two luciferase reporter gene constructs to contain either a wild-type putative miR-379-5p binding sequence isolated from the 3’UTR of the UBE2E3 gene, or a scrambled sequence. The luciferase assay showed that the miR-379-5p mimic suppressed luciferase activity for the WT binding sequence reporter, but not for the scrambled reporter, showing that the effect of miR-379-5p on UBE2E3 expression is likely to be direct. Finally, to determine if the effect of miR-379-5p on UBE2E3 is related to cellular behaviors that play a role in cancer development, we measured cell viability by resazurin assay, cell proliferation by BrdU assay, and apoptosis by caspase 3/7 activation assay. The miR-379-5p mimic and silencing UBE2E3 expression both resulted in significantly diminished cell viability, while silencing UBE2E3 demonstrated both higher proliferation and apoptotic rates. Overall, these results suggest that while the overall effect of miR-379-5p is to inhibit breast cell viability and proliferation, the effect of silencing its target UBE2E3 is more complex because it induces both cell proliferation and apoptosis.

Genome-wide characterization of ubiquitin-conjugating enzyme gene family explores its genetic effects on the oil content and yield of Brassica napus.

Ubiquitin-conjugating enzyme (UBC) is a critical part of the ubiquitin-proteasome pathway and plays crucial roles in growth, development and abiotic stress response in plants. Although UBC genes have been detected in several plant species, characterization of this gene family at the whole-genome level has not been conducted in Brassica napus. In the present study, 200 putative BnUBCs were identified in B. napus, which were clustered into 18 subgroups based on phylogenetic analysis. BnUBCs within each subgroup showed relatively conserved gene architectures and motifs. Moreover, the gene expression patterns in various tissues as well as the identification of cis-acting regulatory elements in BnUBC promoters suggested further investigation of their potential functions in plant growth and development. Furthermore, three BnUBCs were predicted as candidate genes for regulating agronomic traits related to oil content and yield through association mapping. In conclusion, this study provided a wealth of information on the UBC family in B. napus and revealed their effects on oil content and yield, which will aid future functional research and genetic breeding of B. napus.

Functional characterization of the pUceS8.3 promoter and its potential use for ectopic gene overexpression.

The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (β-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher β-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression ofthis reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.

Genomewide analysis of sperm whale E2 ubiquitin conjugating enzyme genes

Marine mammals are exposed to the oxidative stress induced by hypoxia/reoxygenation cycles yet resist cellular damage. The availability of high-quality genomes promises to provide insights on how this is achieved. In this study, we considered the ubiquitinconjugating enzymes (E2) gene family, UBE2 genes, which encodes enzymes with critical roles in cellular physiology, including the oxidative stress response. The sperm whale was the first marine mammal with a chromosome-level genome, allowing the study of gene family repertories, phylogenetic relationships, chromosome gene organization, and other evolutionary patterns on a genomewide basis. Here, 39 UBE2 genes (similar to human, including 32 intact genes, one partial gene, six pseudogenes) were identified in sperm whale genome. These genes were found on 17 chromosomes and were assigned into 23 subfamilies, 16 subgroups, and four classes based on structural characteristics and functions, phylogeny and conserved domains, respectively. Although the gene structure and motif distribution of sperm whale UBE2 genes are conserved in each subfamily, motif variation and intron gain/loss may contribute to functional divergence. Segmental duplications were detected in six gene pairs, which could drive UBE2 gene innovation in the sperm whale. Contrasting seven cetaceans and five terrestrial taxa, we found that cetaceans have experienced shifts in selective constraint on UBE2 genes, which may contribute to oxidative stress tolerance during the adaptation to aquatic life. Our results provide the first comprehensive survey of cetacean UBE2 genes.

Molecular characterization of ubiquitin-conjugating enzyme gene ube2h and siRNA-mediated regulation on targeting p53 in turbot, Scophthalmus maximus.

Ubiquitin-conjugating enzymes are key factors in the ubiquitin proteasome pathway (UPP), which play key roles in ubiquitination. These enzymes affect the efficiency of UPP during stress conditions. P53 has important control of cell cycle arrest and apoptosis in response to cellular stress; these modifications are critical for the stability and transcriptional activity of p53 as the protein activates downstream target genes that dictate the cellular response. However, few studies have investigated the effects of thermal stress in turbot (Scophthalmus maximus), specifically the UPP signaling pathway, and the crosstalk between the ube2h and p53. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot UBE2H gene (Sm-ube2h) and perform bioinformatics analysis. Our results showed that the cDNA of the Sm-ube2h was 718 bp in length, encoding a 189amino acid protein, with a theoretical isoelectric point of 4.77. It also contained a catalytic (UBCc) domain. Expression of Sm-ube2h in different tissues was detected and quantified by qPCR, which was highest in the spleen and lowest in the liver. We also investigated the Sm-ube2h expression profiles in the liver and heart after thermal stress, and changes in Sm-ube2h and p53 under thermal stress, upon RNA interference. Our data speculated that Sm-ube2h and p53 exhibited antagonistic effects under normal temperature conditions after ube2h interference, but displayed synergistic effects under thermal stress, suggesting the crosstalk between UPP and p53 signaling pathway. Our results improved our understanding of the underlying molecular mechanism of thermal tolerance in turbot.

Recombinant ubiquitin-conjugating enzyme of Eimeria maxima induces immunogenic maturation in chicken splenic-derived dendritic cells and drives Th1 polarization in-vitro

Dendritic cells (DCs) are key linkages between innate immunity and acquired immunity. The antigens that promote the functions of DCs might be the effective candidates of novel vaccine. In this research, the ability of ubiquitin-conjugating enzyme (UCE), a recognized common antigens among chicken Eimeria species, to stimulate DCs of chickens were evaluated. We cloned UCE gene from Eimeria maxima (EmUCE), and its protein expression was confirmed by SDS-PAGE and western-blot. Immunofluorescence assay confirmed the binding of rEmUCE on the surface of chicken splenic-derived DCs (ChSP-DCs). Flow cytometric analysis showed that rEmUCE-treated ChSP-DCs increased MHCII, CD1.1, CD11c, CD80, and CD86 phenotypes. qRT-PCR indicated that transcript levels of maturation markers CCL5, CCR7, and CD83 in ChSP-DCs were upregulated in response to rEmUCE. Following rEmUCE treatment, chSP-DCs activated TLR signaling and inhibited Wnt signaling. Moreover, rEmUCE promoted DC-mediated T-cell proliferation in DC/T-cell co-incubation. Interestingly, CD3+/CD4+ T-cells were significantly enhanced when rEmUCE-treated chSP-DCs were co-incubated with T-cells. Cytokine secretion pattern of rEmUCE-stimulated ChSP-DCs revealed that the production of IL-12 and IFN-γ was increased whereas IL-10 and TGF-β were unchanged. Likewise, the co-incubation of ChSP-DCs with T-cells indicated increased production of IFN-γ but not IL-4. Collectively, rEmUCE could polarize DCs to immunogenic phenotype and shift the immune cells towards Th1 response. Our observations provide valuable insight for future research aimed at vaccine development against avian coccidiosis.

Pleiotropic effects of Ubi4, a polyubiquitin precursor required for ubiquitin accumulation, conidiation and pathogenicity of a fungal insect pathogen.

Ubi4 is a polyubiquitin precursor well characterized in yeasts but unexplored in insect mycopathogens. Here, we report that orthologous Ubi4 plays a core role in ubiquitin- and asexual lifestyle-required cellular events in Beauveria bassiana. Deletion of ubi4 led to abolished ubiquitin accumulation, blocked autophagic process, severe defects in conidiation and conidial quality, reduced cell tolerance to oxidative, osmotic, cell wall perturbing and heat-shock stresses, decreased transcript levels of development-activating and antioxidant genes, but light effect on radial growth under normal conditions. The deletion mutant lost insect pathogenicity via normal cuticle infection and was severely compromised in virulence via cuticle-bypassing infection due to a block of dimorphic transition critical for acceleration of host mummification. Proteomic and ubiquitylomic analyses revealed 1081 proteins differentially expressed and 639 lysine residues significantly hyper- or hypo-ubiquitylated in the deletion mutant, including dozens of ubiquitin-activating, conjugating and ligating enzymes, core histones, and many more involved in proteasomes, autophagy-lysosome process and protein degradation. Singular deletions of seven ubiquitin-conjugating enzyme genes exerted differential Ubi4-like effects on conidiation level and conidial traits. These findings uncover an essential role of Ubi4 in ubiquitin transfer cascade and its pleiotropic effects on the in vitro and in vivo asexual cycle of B. bassiana.

The Ubiquitin-Conjugating Enzyme Gene Family in Longan (Dimocarpus longan Lour.): Genome-Wide Identification and Gene Expression during Flower Induction and Abiotic Stress Responses.

Ubiquitin-conjugating enzymes (E2s or UBC enzymes) play vital roles in plant development and combat various biotic and abiotic stresses. Longan (Dimocarpus longan Lour.) is an important fruit tree in the subtropical region of Southeast Asia and Australia; however the characteristics of the UBC gene family in longan remain unknown. In this study, 40 D. longan UBC genes (DlUBCs), which were classified into 15 groups, were identified in the longan genome. An RNA-seq based analysis showed that DlUBCs showed distinct expression in nine longan tissues. Genome-wide RNA-seq and qRT-PCR based gene expression analysis revealed that 11 DlUBCs were up- or down-regualted in the cultivar “Sijimi” (SJ), suggesting that these genes may be important for flower induction. Finally, qRT-PCR analysis showed that the mRNA levels of 13 DlUBCs under SA (salicylic acid) treatment, seven under methyl jasmonate (MeJA) treatment, 27 under heat treatment, and 16 under cold treatment were up- or down-regulated, respectively. These results indicated that the DlUBCs may play important roles in responses to abiotic stresses. Taken together, our results provide a comprehensive insight into the organization, phylogeny, and expression patterns of the longan UBC genes, and therefore contribute to the greater understanding of their biological roles in longan.

Identification of Differentially Expressed Genes In Deltamethrin-ResistantCulex pipiens quinquefasciatus

Culex quinquefasciatus is one of China's major house-dwelling mosquito species and an important vector of filariasis and encephalitis. Chemical treatments represent one of the most successful approaches for comprehensive mosquito prevention and control. However, the widespread use of chemical pesticides has led to the occurrence and development of insecticide resistance. Therefore, in-depth studies of resistance to insecticides are of vital importance. In this study, we performed a gene expression analysis to investigate genes from Cx. quinquefasciatus that may confer pyrethroid resistance. We aimed to understand the mechanisms of Cx. quinquefasciatus resistance to pyrethroid insecticides and provide insights into insect resistance management. Using a resistance bioassay, we determined the deltamethrin LC50 values (lethal concentration required to kill 50% of the population) for Cx. quinquefasciatus larvae in the F21, F23, F24, F26, F27, and F30 generations. The 7 tested strains exhibited pesticide resistance that was 25.25 to 87.83 times higher than that of the SanYa strain. Moreover, the expression of the OBPjj7a (odorant-binding protein OBPjj7a), OBP28 (odorant-binding protein OBP28), and E2 (ubiquitin-conjugating enzyme) genes was positively correlated with deltamethrin resistance ( R2 = 0.836, P = 0.011; R2 = 0.788, P = 0.018; and R2 = 0.850, P = 0.009, respectively) in Cx. quinquefasciatus. The expression of 4 additional genes, H/ACA, S19, SAR2, and PGRP, was not correlated with deltamethrin resistance. In summary, this study identified 3 Cx. quinquefasciatus genes with potential involvement in deltamethrin resistance, and these results may provide a theoretical basis for the control of mosquito resistance and insights into resistance detection.

Cyanobacteria Affect Fitness and Genetic Structure of Experimental Daphnia Populations.

Zooplankton communities can be strongly affected by cyanobacterial blooms, especially species of genus Daphnia, which are key-species in lake ecosystems. Here, we explored the effect of microcystin/nonmicrocystin (MC/non-MC) producing cyanobacteria in the diet of experimental Daphnia galeata populations composed of eight genotypes. We used D. galeata clones hatched from ephippia 10 to 60 years old, which were first tested in monocultures, and then exposed for 10 weeks as mixed populations to three food treatments consisting of green algae combined with cyanobacteria able/unable of producing MC. We measured the expression of nine genes potentially involved in Daphnia acclimation to cyanobacteria: six protease genes, one ubiquitin-conjugating enzyme gene, and two rRNA genes, and then we tracked the dynamics of the genotypes in mixed populations. The expression pattern of one protease and the ubiquitin-conjugating enzyme genes was positively correlated with the increased fitness of competing clones in the presence of cyanobacteria, suggesting physiological plasticity. The genotype dynamics in mixed populations was only partially related to the growth rates of clones in monocultures and varied strongly with the food. Our results revealed strong intraspecific differences in the tolerance of D. galeata clones to MC/non-MC-producing cyanobacteria in their diet, suggesting microevolutionary effects.

Molecular cloning and expression analysis of Ubiquitin-conjugating enzyme gene from black tiger shrimp (Penaeus monodon)

Molecular cloning and expression analysis of Ubiquitin-conjugating enzyme gene from black tiger shrimp (Penaeus monodon)

Genome-Wide Identification, Phylogenetic and Expression Analyses of the Ubiquitin-Conjugating Enzyme Gene Family in Maize.

BackgroundUbiquitination is a post-translation modification where ubiquitin is attached to a substrate. Ubiquitin-conjugating enzymes (E2s) play a major role in the ubiquitin transfer pathway, as well as a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays).Methodology/Principal FindingsIn the present study, a total of 75 putative ZmUBC genes have been identified and located in the maize genome. Phylogenetic analysis revealed that ZmUBC proteins could be divided into 15 subfamilies, which include 13 ubiquitin-conjugating enzymes (ZmE2s) and two independent ubiquitin-conjugating enzyme variant (UEV) groups. The predicted ZmUBC genes were distributed across 10 chromosomes at different densities. In addition, analysis of exon-intron junctions and sequence motifs in each candidate gene has revealed high levels of conservation within and between phylogenetic groups. Tissue expression analysis indicated that most ZmUBC genes were expressed in at least one of the tissues, indicating that these are involved in various physiological and developmental processes in maize. Moreover, expression profile analyses of ZmUBC genes under different stress treatments (4°C, 20% PEG6000, and 200 mM NaCl) and various expression patterns indicated that these may play crucial roles in the response of plants to stress.ConclusionsGenome-wide identification, chromosome organization, gene structure, evolutionary and expression analyses of ZmUBC genes have facilitated in the characterization of this gene family, as well as determined its potential involvement in growth, development, and stress responses. This study provides valuable information for better understanding the classification and putative functions of the UBC-encoding genes of maize.

UBE2Q1 in a Human Breast Carcinoma Cell Line: Overexpression and Interaction with p53.

The p53 tumor suppressor protein is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage. p53 is a substrate for the ubiquitin-proteasome system, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination have not been well studied. UBE2Q1 is a novel E2 ubiquitin conjugating enzyme gene. Here, we investigated the effect of UBE2Q1 overexpression on the level of p53 in the MDA-MB-468 breast cancer cell line as well as the interaction between UBE2Q1 and p53. By using a lipofection method, the p53 mutated breast cancer cell line, MDA-MB-468, was transfected with the vector pCMV6-AN-GFP, containing UBE2Q1 ORF. Western blot analysis was employed to verify the overexpression of UBE2Q1 in MDA-MB-468 cells and to evaluate the expression level of p53 before and after cell transfection. Immunoprecipitation and GST pull-down protocols were used to investigate the binding of UBE2Q1 to p53. We established MDA-MB-468 cells that transiently expressed a GFP fusion proteins containing UBE2Q1 (GFP-UBE2Q1). Western blot analysis revealed that levels of p53 were markedly lower in UBE2Q1 transfected MDA-MB-468 cells as compared with control MDA-MB-468 cells. Both in vivo and in vitro data showed that UBE2Q1 co-precipitated with p53 protein. Our data for the first time showed that overexpression of UBE2Q1can lead to the repression of p53 in MDA-MB-468 cells. This repression of p53 may be due to its UBE2Q1 mediated ubiquitination and subsequent proteasome degradation, a process that may involve direct interaction of UBE2Q1with p53.

Characterization of the Ubiquitin-Conjugating Enzyme Gene Family in Rice and Evaluation of Expression Profiles under Abiotic Stresses and Hormone Treatments.

Ubiquitin-conjugating enzyme E2s (UBCs), which catalyze the transfer of ubiquitin to substrate or E3 ligases, are key enzymes in ubiquitination modifications of target proteins. However, little is known about the knowledge of UBC gene family in rice. In this study, a total of 39 UBC encoding genes, which all contained an UBC domain with a cysteine active site, were identified in the rice genome. These were classified into fifteen distinct subfamilies based upon their sequence similarity and phylogenetic relationships. A subset of 19 OsUBC genes exhibited chromosomal duplication; 4 and 15 OsUBC genes were tandemly and segmentally duplicated, respectively. Comprehensive analyses were performed to investigate the expression profiles of OsUBC genes in various stages of vegetative and reproductive development using data from EST, Microarrays, MPSS, and real-time PCR. Many OsUBC genes exhibited abundant and tissue-specific expression patterns. Moreover, 14 OsUBCs were found to be differentially expressed under treatments with drought, or salt stresses. The expression analysis after treatments with IAA, 6-BA, GA and ABA indicated that almost all OsUBC genes were responsive to at least two of the four hormones. Several genes were significantly down-regulated under all of the hormone treatments, and most of the genes reduced by 6-BA were also reduced by GA. This study will facilitate further studies of the OsUBC gene family and provide useful clues for functional validation of OsUBCs in rice.

Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock

To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes – ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) – of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis—wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis.

Cloning and Analysis on Full-Length Cdna Sequence of Ubiquitin-Conjugating Enzyme Gene from <I>Oryza meyeriana</I> Baill.

Cloning and Analysis on Full-Length Cdna Sequence of Ubiquitin-Conjugating Enzyme Gene from <I>Oryza meyeriana</I> Baill.

Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana

A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and contained a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.

Cloning of the Full-Length cDNA of Ubiquitin-Conjugating Enzyme Gene from <I>Camellia Oleifera</I>

Cloning of the Full-Length cDNA of Ubiquitin-Conjugating Enzyme Gene from <I>Camellia Oleifera</I>

Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper ( Capsicum annuum L.)

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technology for gene expression and transcriptome analysis. Normalization is a process that is necessary to accurately analyze qRT-PCR data. Stability of reference gene expression is required for this process. Due to the large variation in expression levels of reference genes obtained from different experimental conditions, gene expression stabilities must be evaluated and identified in all experimental systems. In the present paper, the stability of the expression levels of seven potential reference genes in pepper are assessed using qRT-PCR analysis to determine optimal reference genes. These reference genes are evaluated in different pepper tissues, abiotic stress, and hormonal treatment samples. Three common statistical algorithms, geNorm, NormFinder, and BestKeeper, are used to identify expression stability and provide an accurate selection of reference genes. Two reference genes, beta tubulin and ubiquitin-conjugating protein (UBI-3), showed high stability in sample pools with abiotic stress and hormonal treatments. Among the sample pools tested, UBI-3 and glyceraldehyde-3-phosphate dehydrogenase expression levels were the most stable in different tissues. Therefore, these reference genes are selected for qRT-PCR analysis under the experimental conditions tested in pepper. In contrast, ubiquitin-conjugating enzyme and actin genes are identified as the least stable reference genes in all the groups tested, confirming that they are not suitable for normalization. Validation of these candidate genes could provide useful guidelines for reference gene selection in qRT-PCR studies in pepper.

Differential Gene and Protein Expression in Soybean at Early Stages of Incompatible Interaction with Phytophthora sojae

Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand molecular mechanisms of root and stem rot resistance in soybeans, the gene and protein expression in hypocotyls and stems of variety Suinong 10 carrying resistance genes Rps1a and Rps2 was investigated by using mRNA differential display reverse transcription PCR and two-dimensional electrophoresis at 0, 0.5, 1, 2, and 4 h after inoculation with P. sojae race 1. The results of the comparison of gene and protein expression showed that at least eight differential fragments at the transcriptional level were related to metabolic pathway, phytoalexin, and signal transduction in defense responses. Sequence analyses indicated that these fragments represented cinnamic acid 4-hydroxylase gene, ATP β gene coding ATP synthase β subunit and ubiquitin-conjugating enzyme gene which upregulated at 0.5 h post inoculation, blue copper protein gene and UDP-N-acetyl-α-D-galactosamine gene which upregulated at 2 h post inoculation, TGA-type basic leucine zipper protein TGA1.1 gene, cyclophilin gene, and 14-3-3 protein gene which upregulated at 4 h post inoculation. Three resistance-related proteins, α-subunit and β-subunit of ATP synthase, and cytochrome P450-like protein, were upregulated at 2 h post inoculation. The results suggested that resistance-related multiple proteins and genes were expressed in the recognition between soybean and P. sojae during zoospore germination, penetration and mycelium growth of P. sojae in soybean.