Journal of Genetic Engineering and Biotechnology最新文献

Background

The cabbage webworm, Hellula undalis (Fabricius) (Lepidoptera: Pyralidae), is a significant pest of brassicas and other cruciferous plants in warm regions worldwide. Transcriptome analysis is valuable for investigation of molecular mechanisms underlying the insect development and reproduction. De novo assembly is particularly useful for acquiring complete transcriptome information of insect species when there is no reference genome available. In case of Hellula undalis, only 17 nucleotide records are currently available throughout NCBI nucleotide database. Genes associated with metabolic processes, general development, reproduction, defense and functional genomics were not previously predicted in the Hellula undalis at the genomic level.

Methods & Results

To address this issue, we constructed Hellula undalis transcriptome using Illumina NovaSeq6000 technology. Approximately 48 million 150 bp paired-end reads were obtained from sequencing. A total of 30,451 contigs were generated by de novo assembly of sample and were compared with the sequences in the NCBI non-redundant protein database (Nr). In total, 71 % of contigs were matched to known proteins in public databases including Nr, Gene Ontology (GO), and Cluster Orthologous Gene Database (COG), and then, contigs were mapped to 123 via functional annotation against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, we compared the ortholog gene family of the Hullula undalis, transcriptome to Spodoptera frugiperda, spodotera litura and spodoptera littoralis and found that 391 orthologous gene families are specific to Hullula undalis. A total of 1,913 potential SSRs was discovered in Hullula undalis contigs.

Conclusions

This study is the first transcriptome data for Hullula undalis. Additionally, it serves as a valuable resource for identifying target genes and developing effective and environmentally friendly strategies for pest control.

Background

Brassica oleracea var. acephala, commonly referred to as kale, is a well-documented plant species, a food crop but well recognized for its capacity to endure and manage the accumulation of heavy metals. In this research, the phytoremediation potential of kale was evaluated based on cadmium intake, utilizing three distinct kale varieties originating from Bosnia and Herzegovina. All kales were grown in controlled conditions, with different concentrations of cadmium (Cd), a known strong pollutant found in small concentrations in soil under normal environmental conditions. After the root length analysis and cadmium atomic spectrometry, we utilized quantitative PCR (qPCR) and cycle threshold (Ct) values to calculate the expression levels of five genes associated with Cd heavy metal response: Mitogen-activated protein kinase 2 (MAPK₂), Farnesylated protein 26 and 27 (HIPP₂₆, HIPP₂₇), Natural resistance-associated macrophage protein 6 (RAMP₆), and Heavy metal accumulator 2 (HMA₂).

Results

The atomic reader's analysis of rising cadmium concentrations revealed a proportional decline in the length of kale roots. The gene expression levels corresponded to cadmium stress differently among varieties, but mostly showing notable up-regulations under Cd stress, indicating the strong Cd presence within the plant.

Conclusions

This study demonstrated differences in gene expression behavior among three B. oleracea varieties from Bosnia and Herzegovina, indicating and filtering the Cd-resistant kale, and kale varieties suitable for phytoremediation. For the first time, such a study was conducted on kale varieties from Bosnia and Herzegovina, analyzing the impact of cadmium on the growth and resilience of these species.

Background

Malaria has remained a major health concern for decades among people living in tropical and sub-tropical countries. Plasmodium falciparum is one of the critical species that cause severe malaria and is responsible for major mortality. Moreover, the parasite has generated resistance against all WHO recommended drugs and therapies. Therefore, there is an urgent need for preventive measures in the form of reliable vaccines to achieve the target of a malaria-free world. Surface proteins are the preferable choice for subunit vaccine development because they are rapidly detected and engaged by host immune cells and vaccination-induced antibodies. Additionally, abundant surface or membrane proteins may contribute to the opsonization of pathogens by vaccine-induced antibodies.

Results

In our study, we have listed all those surface proteins from the literature that could be functionally important and essential for infection and immune evasion of the malaria parasite. Eight Plasmodium surface and membrane proteins from the pre-erythrocyte and erythrocyte stages were shortlisted. Thirty-seven epitopes (B-cell, CTL, and HTL epitopes) from these proteins were predicted using immune-informatic tools and joined with suitable peptide linkers to design a vaccine construct. A TLR-4 agonist peptide adjuvant was added at the N-terminus of the multi-epitope series, followed by the PADRE sequence and EAAAK linker. The TLR-4 receptor was docked with the construct’s anticipated model structure. The complex of vaccine and TLR-4, with the lowest energy −1514, was found to be stable under simulated physiological settings.

Conclusion

This study has provided a novel multi-epitope construct that may be exploited further for the development of an efficient vaccine for malaria.

Background

N-ras protein is encoded by the NRAS gene and operates as GDP-GTP-controlled on/off switching. N-ras interacts with cellular signaling networks that regulate various cellular activities including cell proliferation and survival. The nonsynonymous single nucleotide polymorphism (nsSNPs)-mediated alteration can substantially disrupt the structure and activity of the corresponding protein. N-ras has been reported to be associated with numerous diseases including cancers due to the nsSNPs. A comprehensive study on the NRAS gene to unveil the potentially damaging and oncogenic nsSNPs is yet to be accomplished. Hence, this extensive in silico study is intended to identify the disease-associated, specifically oncogenic nsSNPs of the NRAS gene.

Results

Out of 140 missense variants, 7 nsSNPs (I55R, G60E, G60R, Y64D, L79F, D119G, and V152F) were identified to be damaging utilizing 10 computational tools that works based on different algorithms with high accuracy. Among those, G60E, G60R, and D119G variants were further filtered considering their location in the highly conserved region and later identified as oncogenic variants. Interestingly, G60E and G60R variants were revealed to be particularly associated with lung adenocarcinoma, rhabdomyosarcoma, and prostate adenocarcinoma. Therefore, D119G could be subjected to detailed investigation for identifying its association with specific cancer.

Conclusion

This in silico study identified the deleterious and oncogenic missense variants of the human NRAS gene that could be utilized for designing further experimental investigation. The outcomes of this study would be worthwhile in future research for developing personalized medicine.

Arnebiae Radix is an important medicinal and perennial herb found in Western China, particularly in the Xinjiang region. However, the assessment, utilization and conservation of Arnebiae Radix resources are still unexplored. In this study, we evaluated the genetic diversity of three Arnebiae Radix populations across 47 regions (Ae = 16, Ag = 16, Ad = 15) in Xinjiang, China, using inter-simple sequence repeat (ISSR) molecular markers. In total, 48 alleles were amplified by six pairs of primers screened with ISSR markers. The average number of effective alleles (Ne) was 1.5770. The percentage of interspecific genetic polymorphisms in A. guttata (Ag = 89.58 %) was greater than that in A. euchroma. and A. decumbens (Ae = Ad = 87.50 %). Intraspecific genetic polymorphisms, Bo Le (BL) population of A. euchroma exhibited the highest percentage of polymorphic bands (PPB% = 58.33 %, Na = 1.313, Ne = 1.467, I = 0.0.366, H = 0.255), which indicated high genetic diversity. In contrast, the Tuo Li (TL) population of A. guttata had the lowest values for these parameters (PPB% = 0.00 %, Na = 0.313, Ne = 1,000, I = 0.000, H = 0.000). The Arnebiae Radix germplasms were classified into two major groups (I and II) based on UPGMA cluster analysis (Fig. 8a) and principal coordinate analysis (PCOA). In addition, A. decumbens is placed in a separate category due to its high differentiation coefficient. The AMOVA and genetic differentiation coefficient results indicated that the genetic variation in Arnebiae Radix was predominantly due to intrapopulation differences (78 %). Additionally, the gene flow index (Nm) between populations was 2.4128, which further indicated that the genetic diversity of Arnebiae Radix was greater at the intrapopulation level. The destruction of the ecological environment leads to the continuous reduction and degradation of the genetic diversity of Arnebiae Radix germplasm resources. In this study, we used ISSR molecular markers to analyze the genetic diversity and relatedness of Arnebiae Radix, which revealed the genetic relationship of Arnebiae Radix germplasm resources at the molecular level and provided a scientific basis for future research on selecting and breeding good varieties, evaluating the quality of Arnebiae Radix, and conserving and utilizing its resources.

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon–intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

Background

Venomous marine cone snails produce unique neurotoxins called conopeptides or conotoxins, which are valuable for research and drug discovery. Characterizing Conus venom is important, especially for poorly studied species, as these tiny and steady molecules have considerable potential as research tools for detecting new pharmacological applications. In this study, a worm-hunting cone snail, Conus flavidus inhabiting the Red Sea coast were collected, dissected and the venom gland extraction was subjected to proteomic analysis to define the venom composition, and confirm the functional structure of conopeptides.

Results

Analysis of C. flavidus venom identified 117 peptide fragments and assorted them to conotoxin precursors and non-conotoxin proteins. In this procedure, 65 conotoxin precursors were classified and identified to 16 conotoxin precursors and hormone superfamilies. In the venom of C. flavidus, the four conotoxin superfamilies T, A, O2, and M were the most abundant peptides, accounting for 75.8% of the total conotoxin diversity. Additionally, 19 non-conotoxin proteins were specified in the venom, as well as several potentially biologically active peptides with putative applications.

Conclusion

Our research displayed that the structure of the C. flavidus-derived proteome is similar to other Conus species and includes toxins, ionic channel inhibitors, insulin-like peptides, and hyaluronidase. This study provides a foundation for discovering new conopeptides from C. flavidus venom for pharmaceutical use.

Jute (Corchorus sp.), a commercially important and eco-friendly crop, is widely cultivated in Bangladesh, India, and China. Some varieties of this tropical plant such as the Corchorus. olitorius variety accession no. 2015 (acc. 2015) has been found to be low-temperature tolerant. The current study was designed to explore the genome-wide variations present in the tolerant plant acc. 2015 in comparison to the sensitive farmer popular variety Corchorus. olitorius var. O9897 using the whole genome resequencing technique. Among different variations, intergenic Single Nucleotide Polymorphism (SNPs) and Insertion-Deletion (InDels) were found in the highest percentage whereas approximately 3% SNPs and 2% InDels were found in exonic regions in both plants. Gene enrichment analysis indicated the presence of acc. 2015 specific SNPs in the genes encoding peroxidase, ER lumen protein retaining receptor, and hexosyltransferase involved in stress response (GO:0006950) which were not present in sensitive variety O9897. Besides, distinctive copy number variation regions (CNVRs) comprising 120 gene loci were found in acc. 2015 with a gain of function from multiple copy numbers but absent in O9897. Gene ontology analysis revealed these gene loci to possess different receptors like kinases, helicases, phosphatases, transcription factors especially Myb transcription factors, regulatory proteins containing different binding domains, annexin, laccase, acyl carrier protein, potassium transporter, and vesicular transporter proteins that are responsible for low temperature induced adaptation pathways in plants. This work of identifying genomic variations linked to cold stress tolerance traits will help to develop successful markers that will pave the way to develop genetically modified cold-resistant jute lines for year-round cultivation to meet the demand for a sustainable fiber crop economy.

Background

Wheat stripe mosaic virus (WhSMV) is a significant wheat pathogen that causes substantial yield losses in Brazil and other countries. Although several detection methods are available, reliable and efficient tools for on-site WhSMV detection are currently lacking. In this study, a Loop-Mediated Isothermal Amplification (LAMP) method was developed for rapid and reliable field detection of WhSMV. We designed WhSMV-specific primers for the LAMP assay and optimized reaction conditions for increased sensitivity and specificity using infected plant samples.

Results

We have developed a diagnostic method utilizing the Loop-Mediated Isothermal Amplification (LAMP) technique capable of rapidly and reliably detecting WhSMV. The LAMP assay has been optimized to enhance sensitivity, specificity, and cost-effectiveness.

Conclusion

The LAMP assay described here represents a valuable tool for early WhSMV detection, serving to mitigate the adverse economic and social impacts of this viral pathogen. By enabling swift and accurate identification, this assay can significantly improve the sustainability of cereal production systems, safeguarding crop yields against the detrimental effects of WhSMV.