Journal of Genetic Engineering and Biotechnology最新文献

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.

The myostatin (MSTN) gene exhibits significant nucleotide sequence variations in sheep, impacting growth characteristics and muscular traits of the body. However, its influence on specific growth traits in some sheep remains to be further elucidated. This study utilized single nucleotide polymorphism sequence analysis to investigate the role of the MSTN gene in meat production performance across four sheep breeds: Charolais sheep, Australian White sheep, crossbreeds of Australian White and Small-tailed Han, and crossbreeds of Charolais and Small-tailed Han. At a SNP locus of the MSTN gene, the C2361T site was identified, with three genotypes detected: CC, CT, and TT, among which CC predominated. Gene substitution effect analysis revealed that replacing C with T could elevate the phenotypic value. Comparative analysis of data from different genotypes within the same breed highlighted the superiority of CC and TT genotypes in phenotypic values, underscoring the significance of specific genotypes in influencing key traits. Contrasting the performance of different genotypes across breeds, Charolais sheep and Charolais Han hybrids demonstrated superiority across multiple indicators, offering valuable insights for breeding new sheep varieties. Analysis of gender effects on growth characteristics indicated that ewes exhibited significantly wider chest, waist, and hip widths compared to rams, while rams displayed better skeletal growth and muscle development. Additionally, the MSTN gene also exerted certain effects on lamb growth characteristics, with the CC genotype closely associated with weight. These findings not only contribute crucial insights for sheep breeding but also pave the way for future research exploring the interaction of this gene with others.

Background

The citrus vein phloem degeneration (CVPD) disease is one of important diseases that infects citrus plants and threatens global citrus production and quality due to its severe symptoms and rapid spread. In the 2000s, South Sulawesi Province as one of the citrus producers in Indonesia reported CVPD infection. To date, it is still uncertain as to whether the citrus production center has already been rid of the CVPD infection, keeping in mind the low prevalence of certified citrus saplings use and sub-optimal management of plantations by farmers.

Results

Field observation results revealed varied chlorosis symptoms from young to productive cultivation, which certainly makes it appealing to find out the presence of the causative bacterium, as it has yet to be known whether all the leaves with positive chlorosis symptoms carry the bacterium Candidatus Liberibacter asiaticus. Citrus saplings that appear healthy may carry CVPD pathogens as the incubation period of CVPD pathogens in the host plant spans three to five months. Thus, it is necessary to find the right, rapid way to detect the presence of CVPD pathogens in the citrus plant. The most effective method to use is PCR as the bacterium Candidatus L. asiaticus is non-culturable in vitro, but it is detectable using 16S rDNA. Sampling of leaves with CVPD symptoms was conducted purposively from eight varieties in five citrus cultivation locations, i.e., Pangkep, Sidrap, Bantaeng, Luwu Utara, and Kepulauan Selayar Regencies. DNA isolation was carried out following the Genomic DNA Kit (Geneaid) procedure, followed by detection using the specific pathogenic primer pair OI1 (5′ GCG CGT ATG CAA TAC GAG CGG C 3′) and OI2c (5′ GCC TCG CGA CTT CGC AAC CCA T 3′).

Conclusion

The PCR visualization result shows seven positive samples with DNA fragments measuring 1160 bp. The seven samples were samples of the Key lime, tangerine, Mandarin (cv. batu 55), and Mandarin (cv. selayar), each being derived from Sidrap, Luwu Utara, and Bantaeng. The average disease incidence rate was 66.56 %. Based on the field observation results, the insect vector Diaphorina citri was nowhere to be found in the five citrus cultivation locations in South Sulawesi.

Background

Sommeratia caseolaris is considered the most important mangrove species for reforestation and conservation programs. Therefore, the knowledge of genetic diversity and the population structure of the species has important implications both for the conservation of existing genetic resources and development programs. In the present study, the genetic diversity and structure population of eight populations of S. caseolaris from the Northern to the Southern Coast of Vietnam were determined using nine ISSR molecular markers.

Results

Eight populations of the mangrove species Sonneratia caseolaris were sampled across the natural range in Vietnam to evaluate the genetic diversity of the species. Nine ISSR markers were used to analyse 30 individuals from each population. There were moderate to high levels of genetic diversity (I = 0.447; h = 0.300). PCoA analysis gave very similar results to UPGMA dendrogram construction with the eight populations clustered into three genetic groups which mostly aligned with geographical distances among them. AMOVA analysis results indicated that most (81 %) of the genetic variation was within populations.

Conclusion

The current study also indicates the high level of genetic variation existing among and within the natural population of S. caseolaris in Vietnam. These results open new perspectives towards the conservation of the species' genetic resources and their future use in conservation and reforestation programs.