Genes & Genomics

Comparative gene expression profiling reveals key pathways and genes different in skin epidermal stem cells and corneal epithelial cells Abstract Background Corneal epithelial cells (CECs) are required for corneal transparency and visual function, and corneal injuries may cause corneal blindness. Skin epidermal stem cells (SESCs), which share the same origin with CECs and have the potential of multi-directional differentiation are ideal seed cells for tissue engineered corneal construction to treat corneal blindness. Objective This study aims to investigate critical genes and pathways that may modulate the transdifferentiation from SESCs to CECs. Methods Isolated SESCs and CECs were used for gene expression analysis by microarray. GO and KEGG pathway of differently expressed genes (DEGs) were enriched using DAVID. The protein–protein interaction (PPI) network were then constructed using Cytoscape and highly interconnected module was subsequently isolated from the network by Molecular Complex Detection. Expression of the hub genes and other selected genes were then verified by qRT-PCR. Results We found 112 upregulated and 105 downregulated genes in CECs compared with SESCs. These DEGs were significantly enriched in focal adhesion, PI3K–Akt and TNF signaling pathway. Highly interconnected module of PPI network contains ten genes. Further regulatory network of these genes showed that ESR1 and SLC2A4 were hub genes. Conclusion Our study identified gene expression in SESCs and CECs and suggested that several crucial genes and pathways may play critical roles in transdifferentiation from SESCs to CECs. It may help uncover molecular mechanisms and offer a foundation for promoting tissue-engineered cornea into clinical application.

Karyotype and B chromosome variation in Lilium amabile Palibin Abstract Objective Lilium amabile Palibin (2n = 2x = 24) is an endemic lily species in Korea. B chromosomes are supernumerary chromosomes and the presence of B chromosome in L. amabile was known by previous researches. The current research was conducted to characterize the genetical and cytological features of the B chromosome plants in L. amabile. Methods Karyotype and B chromosome cytotype analyses were carried out among 135 L. amabile accessions that were collected from six geographical locations in Korea using conventional aceto-carmine staining as well as FISH technique with ribosomal RNA gene probes. Results The karyotype of L. amabile genome consisted of two large metacentric, four intermediate subtelocentric, and six intermediate to small acrocentric chromosomes in which chromosomes 1, 6 and 7 carried the 45S rRNA gene loci and chromosome 3 carried the 5S rRNA gene. There were 4 types of B chromosomes, two large B chromosomes and two small B chromosomes. The ribosomal RNA gene loci were not present in the B chromosomes. The 135 accessions were classified into 13 cytotypes including diploids and different B chromosome aneuploids. Among the aneuploids, the most frequent cytotype was 24 + 1B, which was followed by 24 + 2B, 24 + 1b, 24 + 1B + 2b, 24 + 1B + 4b, and 24 + 2B + 4b. Conclusion The karyotype of L. amabile was consistent with other species in the genus Lilium without polyploids. The B chromosome cytotypes were highly variable and the occurrences of different cytotypes were random among the six populations, implying that the B and b chromosome occurrence was random in each population.

Whole RNA-sequencing and gene expression analysis of Trichoderma harzianum Tr-92 under chlamydospore-producing condition Abstract Background Trichoderma is one of the most important biocontrol fungi, which could produce mycelia, conidiospores, and chlamydospores three types of propagules under different conditions. Chlamydospores are produced in harsh conditions in various fungi, and may be more resistant to adverse conditions. However, the knowledge associated with the mechanism of chlamydospore formation remained unclear in Trichoderma. Objectives This study is aimed to explore the essential genes and regulatory pathways associated with chlamydospore formation in Trichoderma. Methods The culture condition, survival rate, and biocontrol effects of chlamydospores and conidiospores from Trichoderma.harzianum Tr-92 were determined. Furthermore, the whole transcriptome profiles of T. harzianum Tr-92 under chlamydospore-producing and chlamydospore-nonproducing conditions were performed. Results T. harzianum Tr-92 produced chlamydospores under particular conditions, and chlamydospore-based formulation of T. harzianum Tr-92 exhibited higher biocontrol ability against Botrytis cinerea in cucumber than conidoiospore-based formulation. In the transcriptome analysis, a total of 2,029 differentially expressed genes (DEGs) were identified in T. harzianum Tr-92 under chlamydospore-producing condition, compared to that under chlamydospore-nonproducing condition. GO classification indicated that the DEGs were significantly enriched in 284 terms among biological process, cellular components and molecular function categories. A total of 19 pathways were observed with DEGs by KEGG analysis. Furthermore, fifteen DEGs were verified by quantitative real-time PCR, and the expression profiles were consistent with the transcriptome data. Conclusion The results would provide a basis on the molecular mechanisms underlying Trichoderma sporulation, which would assist the development and application of fungal biocontrol agents.

Identification and expression analysis of ERECTA family genes in grape ( Vitis vinifera L.) Abstract Background ERECTA family (ERf) genes are found in many dicots and monocots, and play important roles in plant developmental processes and stress responses. However, there is little known on ERf genes in grape (Vitis vinifera L.). Objectives The primary objective of this study was to identify the ERf genes in grape, and to analyze their expression profiles in different organs, during development, and in response to hormone treatments and abiotic/biotic stresses. Methods ERf protein sequences of dicots were aligned in the grape genome (V. vinifera cv. Pinot Noir, PN40024, 12X) with Blast server. The locus tags obtained were inputted in NCBI to retrieve corresponding nucleotide and protein accession numbers. The subcellular localization experiment was performed by the transient expression of VvERECTA-GFP and VvERL2-GFP in mesophyll protoplasts of Arabidopsis. The expression levels of ERf genes in grape leaves were detected by qRT-PCR after hormone treatments and abiotic/biotic stresses. Results We first identified the ERf genes in the grape genome, including VvERECTA and VvERL2. Their cDNA full-length sequences were obtained with the accession numbers MG601756 and MG601757. The result of subcellular localization indicated that the fusion proteins of VvERECTA and VvERL2 were localized in the plasma membrane. There were four conserved domains identified in VvERECTA and VvERL2, including a LRRNT-2, a LRR, a transmembrane and a protein kinase domain. The grape ERf genes expressed highly in young aboveground organs. As grape leaves or berries becoming mature, VvERECTA expressed in a declining trend. The transcript abundance of VvERL2 decreased during leaves development, but showed no significant differences during berries development. The hormone treatments of ABA, SA, MeJA and BR could induce the expression of VvERECTA and VvERL2. The treatments of heat, drought, downy and powdery mildew significantly increased the expression levels of the grape ERf genes. Conclusion The grape ERECTA gene family might play crucial roles in response to abiotic and biotic stresses. We provide the first description of the grape ERf genes and the most comprehensive analysis of their expressions in different biological processes.

In vivo demonstration of enhanced binding between β-clamp and DnaE of pol III bearing consensus i-CBM Abstract Background Among several key protein–protein and protein–DNA interactions within the replisome, the interaction between β-clamp and the DNA polymerase (Pol) III is of crucial importance. This interaction is mediated by a five or six-residue conserved sequence of the DnaE subunit of Pol III, referred to as the Clamp Binding Motif (CBM). In E. coli, DnaE contains two CBMs designated as e-CBM and i-CBM. A consensus sequence (QL[S/D]LF) for the CBMs has previously been proposed and studies involving mutagenesis of both the CBMs have evaluated their protein-binding properties. Surface Plasmon Resonance has been used to show that replacing i-CBM in DnaE with the consensus sequence enhances its binding to β-clamp 120-fold. Objective The current study was aimed to evaluate in vivo interaction between DnaE bearing the consensus i-CBM and β-clamp. Method The C-terminal 405 residues of DnaE, bearing either the consensus i-CBM or the WT i-CBM, with β-clamp were co-expressed in E. coli followed by co-purification of the protein complexes. The interaction was assessed by the ability of the co-expressed proteins to form stable complexes during both affinity and gel filtration chromatography. Result The interaction of β-clamp with DnaEΔ755M containing the consensus i-CBM was found to be more stable than with WT DnaEΔ755, consistent with the in vitro data previously reported. Conclusion The presence of the pieces of sheared DNA generated during sonication promote the interaction of DnaEΔ755M with β-clamp by binding the OB-fold of DnaEΔ755M and β-clamp and serves as a bridge between them.

Construction of genetic linkage map and identification of QTLs related to agronomic traits in DH population of maize ( Zea mays L.) using SSR markers Abstract Background In this study, we used phenotypic and genetic analysis to investigate Double haploid (DH) lines derived from normal corn parents (HF1 and 11S6169). DH technology offers an array of advantages in maize genetics and breeding as follows: first, it significantly shortens the breeding cycle by development of completely homozygous lines in two or three generations; and second, it simplifies logistics, including requiring less time, labor, and financial resources for developing new DH lines compared with the conventional RIL population development process. Objectives In our study, we constructed a maize genetic linkage map using SSR markers and a DH population derived from a cross of normal corn (HF1) and normal corn (11S6169). Methods The DH population used in this study was developed by the following methods: we crossed normal corn (HF1) and normal corn (11S6169), which are parent lines of a normal corn cultivar, in 2014; and the next year, the F1hybrids were crossed with a tropicalized haploid inducer line (TAIL), which is homozygous for the dominant marker gene R1-nj (Nanda and Chase in Crop Sci 6:213–215, 1966), and we harvested seeds of the haploid lines. Results A total of 200 SSR markers were assigned to 10 linkage groups that spanned 1145.4 cM with an average genetic distance between markers of 5.7 cM. 68 SSR markers showed Mendelian segregation ratios in the DH population at a 5% significance threshold. A total of 15 quantitative trait loci (QTLs) for plant height (PH), ear height (EH), ear height ratio (ER), leaf length (LL), ear length (EL), set ear length (SEL), set ear ratio (SER), ear width (EW), 100 kernel weight (100 KW), and cob color (CC) were found in the 121 lines in the DH population. Conclusion The results of this study may help to improve the detection and characterization of agronomic traits and provide great opportunities for maize breeders and researchers using a DH population in maize breeding programs.

Phenotyping analysis of p53 knockout mice produced by gene editing and comparison with conventional p53 knockout mice Abstract Background Knockout (KO) mice developed by homologous recombination (HR) have become useful tools to elucidate gene function. However, HR has low KO efficiency and is time-consuming, labor-intensive, and expensive. 'Gene editing' has received much attention for efficient genetic manipulation. Objective As generation of KO mice is simplified, KO mice produced by HR can be feasibly reproduced using gene editing. However, phenotyping analysis and comparison between KO mice produced by these two techniques is necessary. Methods We generated p53 KO mice through gene editing and compared their phenotype with the already reported HR-mediated p53 KO mice. Results Tumors occurred in 36 (73%) of 49 homozygous KO mice and the mean age of occurrence was 23 weeks, with lymphoma (64%) and sarcoma (23%) being the most common. Tumors were also developed in 12 heterozygous mice and the mean age of occurrence was 40 weeks, with sarcoma (54%) and lymphoma (46%) in high proportion. Homozygotes had a mean life span of 157 ± 52 days and developmental abnormalities were found in females compared to in males (P < 0.05, P < 0.001). Conclusion We analyzed the basic phenotype of p53 KO mice and observed no significant difference from the conventional HR-mediated p53 KO mice.

Reversine induces caspase-dependent apoptosis of human osteosarcoma cells through extrinsic and intrinsic apoptotic signaling pathways Abstract Background The 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (reversine) acts as a chemopreventive agent and induces apoptotic cell death in various cancer cells. However, the anticancer effects of reversine on osteosarcoma cells are not clearly established. Objective The purpose of this study was to investigate the effect of reversine on cell proliferation and induction of apoptosis in human osteosarcoma cells. Methods Cell viability assay, histological analysis, DAPI staining, caspase activation analysis, flow cytometric analysis and immunoblotting were carried out in MG-63 osteosarcoma cells. Results Reversine inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Reversine-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was significantly up-regulated by reversine treatment. Moreover, the caspase-8, a part of the extrinsic apoptotic pathway, was activated by reversine treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria dependent intrinsic apoptosis pathway, significantly decreased following reversine treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9 increased by reversine treatments. In addition, reversine activated caspase-3 and Poly (ADP-ribose) polymerase (PARP) to induce cell death. The Z-VAD-fmk significantly inhibited cell death through the suppression of caspase-3 expression in MG-63 cells treated with reversine. Conclusion These results suggest that the reversine may inhibit cell proliferation and induce apoptotic cell death in MG-63 osteosarcoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for the discovery of anti-cancer agents.

Transcriptome analysis for identifying possible causes of post-reproductive death of Sepia esculenta based on brain tissue Abstract Background The subpeduncle lobe/olfactory lobe–optic gland axis is called the endocrine regulation center of cephalopods. However, little is known about the mechanism of the subpeduncle lobe/olfactory lobe-optic gland axis regulate the sexual maturation and post-reproductive death of Sepia esculenta Hoyle. Objectives The primary objective of this study was to provide basic information for revealing the mechanism of the subpeduncle lobe/olfactory lobe–optic axis regulating the rapid post-reproductive death of S. esculenta. Methods In this paper, Illumina sequencing based transcriptome analysis was performed on the brain tissue of female S. esculenta in the three key developmental stages: growth stage (BG), spawning stage (BS), and post-reproductive death stage (BA). Results A total of 66.19 Gb Illumina sequencing data were obtained. A comparative analysis of the three stages showed 2609, 3333, and 170 differentially expressed genes (DEGs) in BG-vs-BA, BG-vs-BA, and BS-vs-BA, respectively. The Gene Ontology (GO) enrichment analysis of DEGs revealed that the regulation of cyclin-dependent protein serine/threonine kinase activity, oxidative phosphorylation, and respiratory chain were significantly enriched. The significant enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway identified pathways associated with the regulation of death, such as the mammalian target of rapamycin (mTOR) signaling pathway, AMPK signaling pathway, oxidative phosphorylation, and cell cycle. Conclusion The post-reproductive death of S. esculenta was found to be a complex energy steady-state regulation network system. The mTOR acted as an energy receptor and had a key role in regulating energy homeostasis.

Whole genome sequencing analysis of horse populations inhabiting the Korean Peninsula and Przewalski's horse Abstract Background The Jeju horse is an indigenous horse breed in Korea. However, there is a severe lack of genomic studies on Korean horse breeds. Objective The objective of this study was to report genomic characteristics of domestic horse populations that inhabit South Korea (Jeju, Jeju crossbred, and Thoroughbred) and a wild horse breed (Przewalski's horse). Results Using the equine reference genome assembly (EquCab 2.0), more than ~ 6.5 billion sequence reads were successfully mapped, which generated an average of 40.87-fold coverage throughout the genome. Using these data, we detected a total of 12.88 million SNPs, of which 73.7% were found to be novel. All the detected SNPs were deeply annotated to retrieve SNPs in gene regions using the RefSeq and Ensemble gene sets. Approximately 27% of the total SNPs were located within genes, whereas the remaining 73% were found in intergenic regions. Using 129,776 coding SNPs, we retrieved a total of 49,171 nonsynonymous SNPs in 12,351 genes. Furthermore, we identified a total of 10,770 deleterious nonsynonymous SNPs which are predicted to affect protein structure or function. Conclusion We showed numerous genomic variants from domestic and wild horse breeds. These results provide a valuable resource for further studies on functions of SNP-containing genes, and can aid in determining the molecular basis underlying variation in economically important traits of horses.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com