Journal, genetic engineering & biotechnology最新文献

Background: Sucrose non-fermenting-1 (SNF1)-related protein kinase 2 (SnRK2), a plant-specific serine/threonine kinase family, is associated with metabolic responses, including abscisic acid signaling under biotic and abiotic stresses. So far, no information on a genome-wide investigation and stress-mediated expression profiling of jute SnRK2 is available. Recent whole-genome sequencing of two Corchorus species prompted to identify and characterize this SnRK2 gene family.

Result: We identified seven SnRK2 genes of each of Corchorus olitorius (Co) and C. capsularis (Cc) genomes, with similar physico-molecular properties and sub-group patterns of other models and related crops. In both species, the SnRK2 gene family showed an evolutionarily distinct trend. Highly variable C-terminal and conserved N-terminal regions were observed. Co- and CcSnRK2.3, Co- and CcSnRk2.5, Co- and CcSnRk2.7, and Co- and CcSnRK2.8 were upregulated in response to drought and salinity stresses. In waterlogging conditions, Co- and CcSnRk2.6 and Co- and CcSnRK2.8 showed higher activity when exposed to hypoxic conditions. Expression analysis in different plant parts showed that SnRK2.5 in both Corchorus species is highly expressed in fiber cells providing evidence of the role of fiber formation.

Conclusion: This is the first comprehensive study of SnRK2 genes in both Corchorus species. All seven genes identified in this study showed an almost similar pattern of gene structures and molecular properties. Gene expression patterns of these genes varied depending on the plant parts and in response to abiotic stresses.

Background: Ziziphus species particularly Ziziphus mauritiana and Ziziphus nummularia constitute an important part of genetic resources in India. They contribute economically as a fruit crop with lots of morphological and pomological variability. In current study, 48 accessions belonging to two wild Ziziphus species, i.e., Z. mauritiana and Z. nummularia, were characterized using SSR markers. In addition, external features were also examined using stereomicroscope.

Results: Present investigation was done to explore the genetic structure of North Indian jujube. In total, 23 SSR markers detected 57 SSR alleles with an average of 2.47 alleles. Highest number of alleles (4) were detected by three primers, namely BFU1178, BFU479, and ZCMS14, while lowest number of alleles (2) were detected by fifteen primers. Highest Polymorphism Information Content (PIC) was 0.500 and shown by two primers, namely BFU528 and BFU1248, while lowest PIC (0.041) was observed in primers BFU286 with mean value of 0.443. Similarly, highest value of marker index (MI) was detected by primer BFU1178 i.e. 1.969, and lowest value of marker index was observed in primer BFU286 i.e. 0.021. Dendrogram generated using SSR markers data and principal component analysis showed two major groups of the analyzed germplasm with intermixing. STRUCTURE analysis also clustered all the accessions into two groups. We did not found correlation between geographic and genetic distances.

Conclusions: The preliminary results suggest that there is high level of gene pool mixing in these species which can be attributed to their cross-pollination habit. However, more such studies with large numbers of samples are required in future to gain concrete insights of the genetic structure in these species.

Background: CRISPR/Cas9 is the most powerful and versatile genome-editing tool that permits multiplexed-targeted gene modifications for the genetic enhancement of oil palm. Multiplex genome-editing has recently been developed for modifying multiple loci in a gene or multiple genes in a genome with high precision. This study focuses on the development of high-oleic oil palm, the primary target trait for healthy low-saturated oil. To achieve this, the fatty acid desaturase 2 (FAD2) and palmitoyl-acyl carrier protein thioesterase (PAT) genes, both of which are associated with fatty acid metabolism biosynthesis pathways in oil palm, need to be knocked out. The knockout of FAD2 and PAT leads to an accumulation of oleic acid content in oil palms.

Results: A total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm.

Conclusion: This technology is the first in oil palm to use CRISPR/Cas9 genome-editing to target high-oleic-associated genes. These findings showed that multiplex genome-editing in oil palm could be achieved using multiple sgRNAs. Targeted mutations detected establish that the CRISPR/Cas9 technology offers a great potential for oil palm.

Background: Angina pectoris (AP) occurs when oxygen and other nutrients are insufficient to meet the metabolic needs of the heart muscle. Stable angina is the most common, while the unstable angina is less frequent. Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine plays a vital function in the immune response regulation. TNF gene cluster contains many polymorphisms; the most commonly investigated polymorphism is the rs1800629 SNP. This SNP, located at - 308 position with regard to the TNF promoter region, replaces guanine (G) with adenine (A), with the allelic types - 308 G/A, and has been linked to a variety of inflammatory condition and autoimmune diseases. The - 308 G/A SNP was investigated in AP and interconnected to the TNF level to figure out the responsibilities of TNF-alpha gene polymorphism in the pathogenesis of AP.

Method: The current work design as a case-control study that involves 300 participant divided to 200 patients evaluated as (stable angina n = 100 and unstable angina n = 100) compared with 100 apparently healthy control subjects. The serum level of TNF-alpha was assessed via enzyme-linked immunosorbent assay (ELISA)/sandwich method. The genotype and allele frequency distribution of TNF-alpha rs1800629 gene polymorphism were investigated by TaqMan probe of allelic discrimination method.

Results: The levels of TNF-alpha were significantly higher in patients with stable and unstable angina pectoris in comparison with controls. The deviation from Hardy-Weinberg equilibrium (HWE) of TNF-alpha genotypes was obvious in control and unstable angina pectoris groups. Moreover, the significant differences between patients with AP and controls under the five genetic models consider the association between TNF-alpha (rs1800629) - 308 G/A and AP with OR > 1. However, data analysis of allelic and genotypic of (rs1800629) - 308 G/A revealed higher significantly differences of GG homozygous and GA heterozygous proportions between stable angina patients and control. The A allele was more represented as etiological allele, and G allele was represented as protective allele. The serum levels of TNF-alpha were significantly higher in subjects with genetically mutated AA genotypes than in subjects with wild GG genotypes in the study groups. ROC curve analysis found the best cutoff value of TNF-alpha level was 77.25 pg/ml.

Conclusion: As the results, our data observed a linked of TNF-alpha (rs1800629) - 308 G/A genetic variant with angina pectoris patients, and the A allele has been linked to the production or expression of TNF-alpha serum level and represented an etiological factor of angina pectoris.

Background: Cynopterus brachyotis (Müller 1838) is a generalist and widespread fruit bat species which inhabits different types of habitats in Southeast Asia. This species plays an essential role as a seed disperser and pollinator. Morphological study and phylogenetic analysis using mtDNA markers (cyt-b and D-loop) revealed that this species had two different forms in peninsular Malaysia and Borneo and six lineages in Southeast Asia that lead to new species formation. In addition, this species is also reported to have high genetic diversity in Malaysia and Thailand based on the D-loop sequence. However, a phylogenetic and genetic variation study of C. brachyotis in Indonesia has not been conducted yet. These two studies are important as additional information for taxonomic and population genetic studies of this species. Thus, we performed the phylogenetic and genetic diversity analysis of the C. brachyotis population collected from seven habitats on Java island, including open-fragmented habitats (urban, coffee and rubber plantations, pine forest, secondary forest, mangrove forest) and closed habitats (natural forest) using the mtDNA D-loop marker.

Results: The phylogenetic tree using the Bayesian inference (BI) and genetic distance using the Kimura-2 parameter (K-2P) demonstrated that 33 individuals of C. brachyotis from seven habitats on Java island overlapped between habitats and could not be distinguished according to their habitats and lineage. Intrapopulation and intraspecies analysis revealed high haplotype diversity of this species on Java island (Hd = 0.933-1.000). The haplotype network was split into two haplogroups, showing haplotype sharing between habitats. These phylogenetic and genetic variations analysis of C. brachyotis bats on Java island indicated that this species is widespread and adapt to different habitats.

Conclusions: This study of C. brachyotis on Java island collected from seven different habitats has overlapped and genetically close and has high genetic variation. Our results provide the first reported study of C. brachyotis on Java island and provide data to understand the phylogenetic and genetic diversity of this species in Indonesia.

Background: In the past, simple sequence repeat (SSR) marker development in coconut is achieved through microsatellite probing in bacterial artificial chromosome (BAC) clones or using previously developed SSR markers from closely related genomes. These coconut SSRs are publicly available in published literatures and online databases; however, the number is quite limited. Here, we used a locally established, coconut genome-wide SSR prediction bioinformatics pipeline to generate a vast amount of coconut SSR markers.

Results: A total of 7139 novel SSR markers were derived from the genome assembly of coconut 'Catigan Green Dwarf' (CATD). A subset of the markers, amounting to 131, were selected for synthesis based on motif filtering, contig distribution, product size exclusion, and success of in silico PCR in the CATD genome assembly. The OligoAnalyzer tool was also employed using the following desired parameters: %GC, 40-60%; minimum ΔG value for hairpin loop, -0.3 kcal/mol; minimum ΔG value for self-dimer, -0.9 kcal/mol; and minimum ΔG value for heterodimer, -0.9 kcal/mol. We have successfully synthesized, optimized, and amplified 131 novel SSR markers in coconut using 'Catigan Green Dwarf' (CATD), 'Laguna Tall' (LAGT), 'West African Tall' (WAT), and SYNVAR (LAGT × WAT) genotypes. Of the 131 SSR markers, 113 were polymorphic among the analyzed coconut genotypes.

Conclusion: The development of novel SSR markers for coconut will serve as a valuable resource for mapping of quantitative trait loci (QTLs), assessment of genetic diversity and population structure, hybridity testing, and other marker-assisted plant breeding applications.

Background: The novel coronavirus (SARS-CoV-2) caused lethal infections worldwide during an unprecedented pandemic. Identification of the candidate viral epitopes is the first step in the design of vaccines against the viral infection. Several immunoinformatic approaches were employed to identify the SARS-CoV-2 epitopes that bind specifically with the major histocompatibility molecules class I (MHC-I). We utilized immunoinformatic tools to analyze the whole viral protein sequences, to identify the SARS-CoV-2 epitopes responsible for binding to the most frequent human leukocyte antigen (HLA) alleles in the Egyptian population. These alleles were also found with high frequency in other populations worldwide.

Results: Molecular docking approach showed that using the co-crystallized MHC-I and T cell receptor (TCR) instead of using MHC-I structure only, significantly enhanced docking scores and stabilized the conformation, as well as the binding affinity of the identified SARS-CoV-2 epitopes. Our approach directly predicts 7 potential vaccine subunits from the available SARS-CoV-2 spike and ORF1ab protein sequence. This prediction has been confirmed by published experimentally validated and in silico predicted spike epitope. On the other hand, we predicted novel epitopes (RDLPQGFSA and FCLEASFNY) showing high docking scores and antigenicity response with both MHC-I and TCR. Moreover, antigenicity, allergenicity, toxicity, and physicochemical properties of the predicted SARS-CoV-2 epitopes were evaluated via state-of-the-art bioinformatic approaches, showing high efficacy of the proposed epitopes as a vaccine candidate.

Conclusion: Our predicted SARS-CoV-2 epitopes can facilitate vaccine development to enhance the immunogenicity against SARS-CoV-2 and provide supportive data for further experimental validation. Our proposed molecular docking approach of exploiting both MHC and TCR structures can be used to identify potential epitopes for most microbial pathogens, provided the crystal structure of MHC co-crystallized with TCR.

Background: α-Galactosidase is widely distributed in plants, microorganisms, and animals, and it is produced by different fungal sources. Many studies have confirmed the valuable applications of α-galactosidase enzymes for various biotechnological purposes, like the processing of soymilk.

Results: Aspergillus niger NRC114 was exploited to produce the extracellular α-galactosidase. One factor per time (OFT) and central composite design (CCD) approaches were applied to determine the optimum parameters and enhance the enzyme production. The CCD model choices of pH 4.73, 1.25% mannose, 0.959% meat extract, and 6-day incubation period have succeeded in obtaining 25.22 U/mL of enzyme compared to the 6.4 U/mL produced using OFT studies. Treatment of soymilk by α-galactosidase caused an increase in total phenols and flavonoids by 27.3% and 19.9%, respectively. Antioxidant measurements revealed a significant increase in the enzyme-treated soymilk. Through HPLC analysis, the appearance of sucrose, fructose, and glucose in the enzyme-treated soymilk was detected due to the degradation of stachyose and raffinose. The main volatile compounds in raw soymilk were acids (45.04%) and aldehydes (34.25%), which showed a remarkable decrease of 7.82% and 20.03% after treatment by α-galactosidase.

Conclusions: To increase α-galactosidase production, the OFT and CCD approaches were used, and CCD was found to be four times more effective than OFT. The produced enzyme proved potent enough to improve the properties of soymilk, avoiding flatulence and undesirable tastes and odors.

Background: No doubt that the corpus luteum (CL) plays a vital role in the regulation of female cyclicity in mammals. The scenarios among microRNAs (miRNAs) and their target genes and steroid hormones {estradiol (E2) and progesterone (P4)} are required for better understanding the molecular regulation of CL during its formation, maturation, and regression. We aimed to (I) study the changes in the relative abundance of miR-205, miR-26a-5p, miR-17-5p, and let-7b-5p and their target genes: LHCGR, CASP3, PCNA, AMH, and PLA2G3, during different stages of corpus luteum in Egyptian buffaloes, and (II) and to address different scenarios between steroid concentrations in the serum and the expression pattern of selected miRNAs and their targets.

Methods: The paired ovaries and blood samples were collected from apparently healthy 50 buffalo cows at a private abattoir. The ovaries bearing CL were macroscopically divided according to their morphological structure and color into hemorrhagic (CLH), developing (CLD), mature (CLM), regressed (CLR), and albicans (CLA). Small pieces from different stages of CL (CLH, CLD, CLM, CLR, and CLA) were cut and immediately kept at - 80 °C for total RNA isolation and qRT-PCR. The serum was separated for steroid level estimation.

Results: The LHCGR was expressed during different stages of CL, and the peak of expression was at the mid-luteal stage. The CASP3 revealed a stage-specific response at different stages of CL. The PCNA has an essential role in cellular proliferation in buffaloes CL. Both expression patterns of PLA2G3 and AMH were found over the various developmental and regression stages. It was noticed that miR-205 is conserved to target LHCGR and CASP3 transcripts. Moreover, CASP3 and AMH were targeted via miR-26a-5p. Additionally, the CASP3 and PLA2G3 were targeted via let-7b-5p. The P4 level reached its peak during CLM. There were positive and negative strong correlations between miRNAs (miR-26a-5p and miR-205), target genes (LHCGR and CASP3) during different stages of CL, and steroid hormones in the serum.

Conclusions: Taken together, the orchestrated pattern among miRNAs, target genes, and steroid hormones is essential for maintaining the proper development and function of CL in buffalo cows.

Aim: The aim of this investigation is to detect potential inhibitor for visceral leishmaniasis through computational analysis.

Background: Leishmaniasis is categorized as a vector born pathogenic infection prevalent in tropical, subtropical, and in Mediterranean zones spread by intra-macrophage protozoa. The clinical syndrome of leishmaniasis is divided into the following type's namely cutaneous leishmaniasis, mucocutaneous leishmaniasis, visceral leishmaniasis, and dermal leishmaniasis. Trypanothione synthetase is a key enzyme involving in glutathione biosynthesis as well as hydrolysis. Trypanothione is one of the promising drug targets for parasites. Parasites are inimitable with concern to their dependence on trypanothione to regulate intracellular thiol-redox balance in fighting against oxidative stress and biochemical anxiety. However, trypanothione synthetase was presumed as the target therapeutic alternate in VL therapy.

Objective: The important objective of this current investigation is to identify or analyze the potential inhibitor for V. leishmaniasis through computational approaches which include virtual screening, molecular docking, ADME prediction, and molecular dynamic simulation.

Methods: An investigation was performed to develop a 3D protein structure, using computational screening among associated similar structured proteins from popular compound database banks such as Specs, Maybridge, and Enamine, to detect novel staging with a series of validation for emerging innovative drugs molecules. Modeled protein ligand complex was further analyzed to know the binding ability of the complex. Molecular dynamics were performed to ascertain its stability at 50 ns.

Results: Trypanothione synthetase overall ability in the outcome of series of analysis. Among three database compounds screened, the compound from the Specs database exhibited the better protein-ligand docking scores and fulfilled the drug-like properties through ADMET analysis, and the docked complexes had better stability throughout the simulation. Besides, the other two database leads fulfilled the pharmacological properties, and the complexes were stable in the simulation.

Conclusion: By analyzing the various compounds from different databases, we concluded that the Specs database compound exhibits potential activity against the target protein and is considered a promising inhibitor for trypanothione synthetase.