Journal of Genetic Engineering and Biotechnology最新文献

Background

Somatic embryogenesis offers a reliable method for cucumber (Cucumis sativus L.) regeneration and genetic enhancement against Fusarium wilt. This study aimed to establish a tailored somatic embryogenesis system for Egyptian cultivars, fostering genetic improvements and Fusarium wilt-resistance lines.

Results

Employing the Optimal Arbitrary Design (OAD) approach, we optimized the induction medium, initiating prolific embryogenic calli (53.3 %) at 1 mg/L 2,4-D. The cotyledonary leaf (CL) was the preferred explant, showing 60 % embryogenic callus development. Bieth Alpha exhibited higher responsiveness, generating ∼ 18 somatic embryos per explant compared to Prince's ∼ 10. Somatic embryogenesis system validation used quantitative RT-PCR, showing Cucumis sativus splicing factor 3B subunit (CUS1) and an embryogenesis marker gene expression exclusively within embryogenic calli and mainly during embryogenesis initiation. Evaluating fungal toxin filtrate concentrations for selecting embryogenic calli, the S2 selection (25 % filtrate, four subculture cycles) was chosen for somatic embryo development. To gauge the ramifications of selection at the genetic stratum, an in-depth analysis was executed. A cluster analysis grounded in ISSR banding patterns revealed a distinct separation between in vivo-cultivated plants of the two cultivars and regenerated plants devoid of pathogen filtrate treatment or those regenerated post-filtrate treatment. This segregation distinctly underscores the discernible genetic impact of the selection process.

Conclusions

The highest embryogenic capacity (53.3%) was achieved in this study by optimizing the induction stage, which demonstrated the optimal concentrations of BA and 2,4-D for induced proembryonic masses. Moreover, consistent gene expression throughout both stages of embryogenesis suggests that our system unequivocally follows the somatic embryogenesis pathway.

Background:

Examining functions and characteristics of DNA sequences is a highly challenging task. When it comes to the human genome, which is made up of exons and introns, this task is more challenging. Human exons and introns contain millions to billions of nucleotides, which contributes to the complexity observed in this sequences. Considering how complicated the subject of genomics is, it is obvious that using signal processing techniques and deep learning tools to build a strong predictive model can be very helpful for the development of the research of the human genome.

Results:

After representing human exons and introns with color images using Frequency Chaos Game Representation, two pre-trained convolutional neural network models (Resnet-50 and GoogleNet) and a proposed CNN model having 13 hidden layers were used to classify our obtained images. We have reached a value of 92% for the accuracy rate for Resnet-50 model in about 7 h for the execution time, a value of 91.5% for the accuracy rate for the GoogleNet model in 2 h and a half for the execution time. For our proposed CNN model, we have reached 91.6% for the accuracy rate in 2 h and 37 min.

Conclusions:

Our proposed CNN model is faster than the Resnet-50 model in terms of execution time. It was able to slightly exceed the GoogleNet model for the accuracy rate value.

Background

Screening and developing novel antifungal agents with minimal environmental impact are needed to maintain and increase crop production, which is constantly threatened by various pathogens. Small peptides with antimicrobial and antifungal activities have been known to play an important role in plant defense both at the pathogen level by suppressing its growth and proliferation as well as at the host level through activation or priming of the plant’s immune system for a faster, more robust response against fungi. Rust fungi (Pucciniales) are plant pathogens that can infect key crops and overcome resistance genes introduced in elite wheat cultivars.

Results

We performed an in vitro screening of 18 peptides predominantly of plant origin with antifungal or antimicrobial activity for their ability to inhibit leaf rust (Puccinia triticina, CCDS-96-14-1 isolate) urediniospore germination. Nine peptides demonstrated significant fungicidal properties compared to the control. Foliar application of the top three candidates, β-purothionin, Purothionin-α2 and Defensin-2, decreased the severity of leaf rust infection in wheat (Triticum aestivum L.) seedlings. Additionally, increased pathogen resistance was paralleled by elevated expression of defense-related genes.

Conclusions

Identified antifungal peptides could potentially be engineered in the wheat genome to provide an alternative source of genetic resistance to leaf rust.

Background

Gymnema sylvestre R.Br. is famous medicinal plant among diabetics for its gymnemic acid content. It also contains flavonoids, which are an essential component in various other products. Though some molecular information on the biosynthesis of gymnemic acid, polyoxypregnane, micro RNAs and photosynthetic efficiency is available, there is no gene level information available on the biosynthesis of flavonoids in this plant. RNA was extracted from winter-collected Gymnema sylvestre leaves and cDNA libraries were prepared and used for next generation sequencing. De novo transcriptome assembly were prepared and Coding DNA Sequences (CDS) of 13 major genes involved in flavonoids biosynthesis were identified from transcriptome data. Phenylalanine ammonia lyase gene containing full-length CDS was employed for in silico protein modelling and subsequent quality assessment. These models were then compared against publicly available databases. To confirm the identification of these genes, a similarity search was conducted using the NCBI BLAST tool.

Results

Therefore, in the present study, an effort has been made to provide molecular insights into flavonoid biosynthesis pathway by examining the expressed transcripts in G.sylvestre. Gene sequences of total thirteen major genes viz., phenylalanine ammonia lyase, 4-coumarate CoA ligase, cinnamic acid 4-hydroxylase, shikimate O-hydroxycinnamoyl transferase, coumaroyl quinate (coumaroyl shikimate) 3′-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, naringenin 3-dioxygenase, flavanol synthase, flavonoid 3′-monooxygenase, Flavanone 7-O-glucoside 2″-O-beta-L-rhyamnosyltransferase and leucoanthocyanidin dioxygenase were identified and a putative pathway of flavonoids biosynthesis has been illustrated based on transcriptome data.

Conclusions

This transcriptome study has contributed gene-level insights into the biosynthesis of flavonoids in plants as a whole and represents the first report within a non-model plant, Gymnema sylvestre perticullarly.

Background

Human papillomavirus (HPV) vaccination is one of the crucial national vaccination programs aimed at reducing the prevalence of the diseases associated with HPV infections, which continue to pose a global health concern. However, a significant disparity exists in the distribution of HPV vaccine, particularly in low-middle income countries where the cost of HPV vaccine becomes a major obstacle. Thus, it is essential to ensure the availability of an economically feasible HPV vaccine, necessitating immediate efforts to enhance the cost-effectiveness of vaccine production. This study aimed to develop an efficient production system for the recombinant HPV type 52 L1 protein as HPV vaccine material using methylotrophic yeast Hansenula polymorpha expression system.

Results

This study presents an in-depth examination of the expression and scale-up production of HPV type 52 L1 protein using DASGIP® parallel bioreactor system. The pHIPX4 plasmid, which is regulated by the MOX promoter, generates stable clones that express the target protein. Cultivation employing the synthetic medium SYN6(10) with controlled parameters (e.g. temperature, pH, feeding strategy, and aeration) produces 0.15 µg/mL of HPV type 52 L1 protein, suggesting a possibility for scaling up to a higher production level.

Conclusion

The scale-up production of HPV type 52 L1 protein using Hansenula polymorpha expression system described in this study provides an opportunity for an economical manufacturing platform for the development of the HPV vaccine.

Background

The Nonstructural Protein (NSP) 4B of Zika virus of 251 amino acids from (ZIKV/Human/POLG_ZIKVF) with accession number (A0A024B7W1), Induces the production of Endoplasmic Reticulum ER-derived membrane vesicles, which are the sites of viral replication. To understand the physical basis of how proteins fold in nature and to solve the challenge of protein structure prediction, Ab-initio and comparative modeling are crucial tools.

Results

The systematic in silico technique, ThreaDom, had only predicted one domain (4 – 190) of NSP4B. I-TASSER, and Alphafold were ranked as the best servers for full-length 3-D protein structure predictions of NSP4B, where the predicted models were evaluated quantitatively using benchmarked metrics including C-score (-3.43), TM-score (0.77949), RMSD (2.73), and Z-score (1.561). The functional and protein binding motifs were realized using motif databases, secondary and surface accessibility predictions combined with Post-Translational Modification Sites (PTMs) prediction. Two highly conserved protein-binding motifs (Flavi NS4B and Bacillus papRprotein), together with three (PTMs) (Casein Kinase II, Myristyl site, and ASN-Glycosylation site) were predicted utilizing the Motif scan and Scanprosite servers. These patterns and PTMs were associated with NSP4B's role in triggering the development of the viral replication complex and its participation in the localization of NS3 and NS5 on the membrane. Only one hit from Structural Classification of Protein (SCOP) matched the protein sequence at positions 10 to 397 and was categorized six-hairpin glycosidases superfamily according to CATH (Class, Architecture, Topology, and Homology). Integrating this NSP4B information with the templates' SCOP and CATH annotations achieves it easier to attribute structure–function/evolution links to both previously known and recently discovered protein structures.

There is no currently approved human vaccine against leishmaniasis. Utilization of immunogenic antigens and their epitopes capable of enhancing immune responses against leishmaniasis is a crucial step for rational in silico vaccine design. The objective of this study was to generate and evaluate a potential vaccine candidate against leishmaniasis, designed by immunodominant proteins from gp46 and gp63 of Leishmania major, which can stimulate helper T-lymphocytes (HTL) and cytotoxic T-lymphocytes (CTL). For this aim, the IFN-γ-inducing MHC-I and MHC-II binders were predicted for each examined protein (gp46 and gp63) and connected with appropriate linkers, along with an adjuvant (Mycobacterium tuberculosis L7/L12) and a histidine tag. The vaccine’s stability, antigenicity, structure, and interaction with the TLR-4 receptor were evaluated in silico. The resulting chimeric vaccine was composed of 344 amino acids and had a molecular weight of 35.64 kDa. Physico-chemical properties indicated that it was thermotolerant, soluble, highly antigenic, and non-allergenic. Predictions of the secondary and tertiary structures were made, and further analyses confirmed that the vaccine construct could interact with the human TLR-4 receptor. Virtual immune simulation demonstrated strong stimulation of T-cell responses, particularly by an increase in IFN-γ, following vaccination. In summary, the in silico data indicated that the vaccine candidate showed high antigenicity in humans. It was also found to trigger significant levels of clearance mechanisms and other components of the cellular immune profile. Nevertheless, further wet experiments are required to properly assess the efficacy of this multi-epitope vaccine candidate against leishmaniasis.

Background

The Arabidopsis “Redox Responsive Transcription Factor1” (RRTF1) promoter is transiently activated by salt stress in roots over 6 h period, followed by an adaptation phase during which its activity returns to baseline levels, even if the salt stress is prolonged. This enables the short-term production of genes that, while initially advantageous to the plant, will have long-term detrimental effects if expressed at high levels indefinitely.

Results

In this paper, we demonstrate that the RRTF1 promoter salt adaption response is a dominant feature of the promoter, that cannot be overwritten by a strong enhancer. While maintaining the transient activation profile of the RRTF1 promoter, linking it to the 35S enhancer results in a significant boost of salt stress induction in roots.

Conclusion

The RRTF1 promoter’s enhanced and still adaptable activity could become a useful tool in plant biotechnology.

Background

The common mudskipper (Periophthalmus kalolo Lesson, 1831) belongs to a group of fish species that exhibit amphibious lifestyles during specific daily periods. However, identifying this species poses a challenge due to its morphological similarities with other mudskipper species. These similarities have occasionally caused misidentifications of mudskippers. In Indonesia, previous studies have examined the genetic variation of common mudskippers, but these investigations have been limited to a few specific areas, particularly along the southern coast of Java. As a result, the available data remain fragmented, and no comprehensive genetic population analysis of common mudskippers on the southern coast of Java has been conducted. Therefore, our study aimed to establish DNA barcodes of COI mtDNA and explore the genetic variation and relationship among these common mudskipper populations from the southern coast of Java. We collected nine specimens from two populations, Cilacap Mangrove Forest and Kondang Bandung Beach, and supplemented our dataset with 38 previously collected COI sequences of common mudskippers from three different populations from the southern coast of Java (Pasir Mendit Beach, Bogowonto Lagoon, and Baros Beach).

Results

The study revealed that 47 common mudskippers from five different populations are separated into three genetically distinct clades (A, B, and C). These clades display genetic divergences ranging from 0.97% to 1.91%. Each clade exhibits high levels of haplotype diversity but relatively low nucleotide diversity, suggesting a previous bottleneck in population followed by a fast expansion. However, the phylogeny, haplotype network, and principal coordinate analysis indicate overlapping populations with no geographic separation within these clades. This suggests the potential occurrence of gene flow among these populations, which might have been facilitated by past geological events.

Conclusions

These results enhance our understanding of common mudskipper biodiversity in Indonesia. Further studies involving common mudskipper populations from various geographical sites in Indonesia are required to further enrich our understanding of the variation and evolution of this species.

Background

Genome association studies have shown that gene-gene interactions or epistasis play a crucial role in identifying the etiology, prognosis, and treatment response of many complex diseases beyond their main effects. Skeletal dysplasias are a heterogeneous group of congenital bone and cartilage disorders with a genetic and gen-gen interaction etiology. The current classification of skeletal dysplasias distinguishes 461 diseases in 42 groups, and the incidence of all skeletal dysplasias is more than 1 in every 5000 newborns. The objective is to present the case of a patient with four variants that generates gen-gen interactions in the skeletal dysplasia.

Case presentation

A 1-year-old male patient was diagnosed with skeletal dysplasia based on prenatal ultrasound showing micromelia and pyelocalyceal dilation. Postnatal physical examination revealed body disproportion and involvement of other organs and systems.

Materials and Methods

A sequencing study and deletions/duplications analysis were performed for 358 candidate genes associated with skeletal dysplasia.

The GeneMANIA interface was used to evaluate the expression network of genes associated with each other for the gen-gen interaction.

Results

Four pathogenic variants were obtained two heterozygous variants with pathogenic significance in SLC26A, one heterozygous pathogenic variant in CLCN7 and another heterozygous pathogenic variant in CEP120.

The GeneMANIA interface reveals 77.64% physical interactions, 8.01% co-expression, 5.37% prediction, 3.63% co-localization, 2.87% genetic interactions, 1.88% route of action, and 0.60% shared protein domains.

Discussion and Conclusions

These results suggest that the interaction between these genes affects the activity of the inorganic anion exchanger, leading to disorganization of collagen fibers, early mineralization, and decreased assembly of fibronectin in the bone extracellular matrix. Identifying gene-gene interactions is a fundamental step in understanding proper cell function and thus understanding the pathophysiology of many complex human diseases, improving diagnosis, and the possibilities of new personalized therapies.