Search Results (8)

In molecular biology studies, suitable vectors are fundamental tools; however, most vectors can only express one target gene, which limits the ability to study multiple genes simultaneously within the same plant tissue. The traditional method for achieving multi-gene co-expression involves co-transferring multiple plasmids into plant tissues, but this approach is often inefficient due to the difficulty of successfully transforming multiple plasmids at once. To overcome this limitation, we have developed a series of vectors, called pMAGs (Multigene Assembly Genetic vectors), capable of simultaneously expressing or silencing two or three different genes in plants. These vectors not only provide an optimal solution for a wide range of biological experiments but also work effectively across numerous plant species. Full article

Bidirectional promoters (BDPs) regulate the transcription of two adjacent, oppositely oriented genes, offering a compact structure with significant potential for multigene expression systems. Although BDPs are evolutionarily conserved, their regulatory roles and sequence characteristics vary across species, with limited studies in fish. Here, we systematically analyzed the distribution, sequence features, and expression patterns of BDPs in the medaka (Oryzias latipes) genome. A total of 1737 divergent gene pairs, representing 13% of medaka genes, were identified as potentially regulated by BDPs. These genes are enriched in essential biological processes, including organelle function, RNA processing, and ribosome biogenesis. Transcriptomic analysis revealed that co-regulation (co-expression and co-silencing) is a prominent feature of these gene pairs, with variability influenced by tissue and sex. Sequence analysis showed that medaka BDPs are compact, with most fragments under 400 bp and an average GC content of 42.06%. Validation experiments confirmed the bidirectional transcriptional activity of three histone-related BDPs in both medaka SG3 cells and embryos, demonstrating effective and robust regulatory efficiency. This study enhances our understanding of the genomic organization and transcriptional regulation in fish and provides a valuable reference for developing species-specific multigene expression systems in fish genetic engineering. Full article

RNA interference (RNAi)-based biotechnology has been previously implemented in decapod crustaceans. Unlike traditional RNAi methodologies that investigate single gene silencing, we employed a multigene silencing approach in decapods based on chimeric double-stranded RNA (dsRNA) molecules coined ‘gene blocks’. Two dsRNA constructs, each targeting three genes of the crustacean hyperglycaemic hormone (CHH) superfamily of neuropeptides, were produced: Type II construct targeting Cq-Molt-inhibiting hormone 1 (MIH1), Cq-MIH-like 1 (MIHL1), and Cq-MIHL2 isoforms and Type I construct targeting Cq-ion transport peptide (Cq-ITP; a putative hybrid of CHH and MIH) and Cq-CHH and Cq-CHH-like (CHHL) isoforms. Both constructs were injected into juvenile redclaw crayfish, Cherax quadricarinatus, to determine the effects of multigene knockdown on molting and developmental processes. A 20-Hydroxyecdysone (20E) enzyme-linked immunosorbent assay (ELISA) and glucose assay were used to determine the effects of RNAi on molting and hemolymph glycemic activities, respectively. Multigene silencing reduced the intermolt interval by 23%. Statistically significant elevated 20E was recorded in treated intermolt individuals, consistent with the reduced intermolt interval as well as unique and abnormal phenotypes related to the molting process, which indicates a shift in 20E-induced cascade. There was no effect of RNAi treatment on hemolymph glucose level or molt increment. Through multigene silencing and subsequent annotation of gene networks, gene blocks may provide a tailored approach to investigate complex polygenic traits with RNAi in a more efficient and scalable manner. Full article

The Cleavage Stimulation Factor (CstF) complex, consisting of three subunits, is essential for the 3′ end processing of precursor messenger RNA (pre-mRNA). In mammals, this complex includes CstF50, CstF64, and CstF77, named according to their molecular weights, and these proteins are conserved across many organisms. However, the functional roles of the three CstF genes (NlCstF50, NlCstF64, and NlCstF77) in Nilaparvata lugens, a major rice pest, have not been fully explored. This study identified and characterized the sequences of these genes, with proteins encoded by NlCstF50, NlCstF64, and NlCstF77 consisting of 439, 419, and 732 amino acids, respectively. These proteins are conserved among various insect species. Spatio-temporal expression analysis revealed that these genes are expressed at all developmental stages and in various tissues, with peak levels in eggs and testes. RNA interference (RNAi) targeting one or all three NlCstF genes resulted in a reduction in gene expression by 68% to 90% at 72 h post-injection, indicating that multi-gene dsRNA can achieve similar silencing outcomes as single-gene dsRNA. Knocking down one or all three NlCstF genes caused significant lethal phenotypes and molting disruptions. Mortality rates increased from 62.5% (dsNlCstF50) to 95.4% (dsNlCstF(50+64+77)). Additionally, silencing these genes reduced the number of eggs laid per female and hatch rates. These results highlight the critical role of NlCstF genes in the development and reproduction of N. lugens, suggesting their potential as targets for RNAi-based pest control strategies. Full article

Tandemly organized rRNA genes are a typical example of a multigene family, where individual members evolve co-ordinately within—but independently between—species due to gene conversion and unequal crossing over. More frequently, in eukaryotic species with an interspecies hybrid origin, expression of unhomogenized rRNA genes from one progenitor is epigenetically silenced because of nucleolus dominance, and distinct rRNA genes may lose functionality and evolve faster. Interestingly, we obtained unusual ribosomal gene sequences from Oryza species that showed great diversity and did not appear in the present rice genomic sequence. The diversity of rDNA sequences indicated that the homogenization in rice is incomplete and explains the introgression of distinct rRNA gene families into ancestral rice genomes before speciation and continent separation. The divergent large subunit (LSU) ribosomal genes are expressed, some of them differentially, depending on the N fertilization of plants. Detection of differential transcripts of the rRNA genes suggested that rRNA gene families are not functionally equivalent. Phylogenetic analysis assigned Oryza species branching order to monocots, and monocot lineages probably have the same ecological origin by molecular clock calculation. Therefore, our results suggested that the geographical distances of continent-separation cause barriers to the gene flow and homogenization among Oryza species which requires further explanation. Full article

Subtelomeres (ST) are chromosome regions that separate telomeres from euchromatin and play relevant roles in various biological processes of the cell. While their functions are conserved, ST structure and genetic compositions are unique to each species. This study aims to identify and characterize the subtelomeric regions of the 13 Toxoplasma gondii chromosomes of the Me49 strain. Here, STs were defined at chromosome ends based on poor gene density. The length of STs ranges from 8.1 to 232.4 kbp, with a gene density of 0.049 genes/kbp, lower than the Me49 genome (0.15 kbp). Chromatin organization showed that H3K9me3, H2A.X, and H3.3 are highly enriched near telomeres and the 5′ end of silenced genes, decaying in intensity towards euchromatin. H3K4me3 and H2A.Z/H2B.Z are shown to be enriched in the 5′ end of the ST genes. Satellite DNA was detected in almost all STs, mainly the sat350 family and a novel satellite named sat240. Beyond the STs, only short dispersed fragments of sat240 and sat350 were found. Within STs, there were 12 functional annotated genes, 59 with unknown functions (Hypothetical proteins), 15 from multigene FamB, and 13 from multigene family FamC. Some genes presented low interstrain synteny associated with the presence of satellite DNA. Orthologues of FamB and FamC were also detected in Neospora caninum and Hammondia hammondi. A re-analysis of previous transcriptomic data indicated that ST gene expression is strongly linked to the adaptation to different situations such as extracellular passage (evolve and resequencing study) and changes in metabolism (lack of acetyl-CoA cofactor). In conclusion, the ST region of the T. gondii chromosomes was defined, the STs genes were determined, and it was possible to associate them with high interstrain plasticity and a role in the adaptability of T. gondii to environmental changes. Full article

Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields. Full article

The NRT2 transporters mediate High Affinity Nitrate/NitriteTransport (HAN/NiT), which are essential for nitrogen acquisition from these inorganic forms. The NRT2 proteins are encoded by a multigene family in plants, and contain 12 transmembrane-spanning domains. Chlamydomonas reinhardtii has six NRT2, two of which (NRT2.5 and NRT2.4) are located in Chromosome III, in tandem head to tail. cDNAs for these genes were isolated and their sequence revealed that they correspond to half-size NRT2 transporters each containing six transmembrane domains. NRT2.5 has long N- and C- termini sequences without known homology. NRT2.4 also contains long termini sequences but smaller than NRT2.5. Expression of both studied genes occurred at a very low level, slightly in darkness, and was not modified by the N or C source. Silencing of NRT2.4 by specific artificial miRNA resulted in the inhibition of nitrite transport in the absence of other HANNiT (NRT2.1/NAR2) in the cell genetic background. Nitrite transport activity in the Hansenula polymorpha Δynt::URA3 Leu2 mutant was restored by expressing CrNRT2.4. These results indicate that half-size NRT2 transporters are present in photosynthetic organisms and that NRT2.4 is a HANiT. Full article