Journal of Applied Genetics最新文献

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are the most promising toolkit of synthetic biology for genetic engineering applications across species. Essentially, the Type II CRISPR system, featuring Cas9 nuclease from Streptococcus pyogenes complexed with sgRNA, introduces targeted DNA cleavage, enabling modifications with exceptional precision. This technology can be utilized for not only editing but also modulating gene expressions, thereby finding widespread utility in various biotechnological applications. Here we discuss strategies to construct a consolidated platform aiming at developing a CRISPR-based gene editing system in microbial hosts such as yeast. Employing the well-known gene editing enzymes, i.e., Cpf1 and dCas9, two independent strategies to develop a one-pot plasmid system have been proposed. Furthermore, approaches to reduce off-target cleavages introduced by non-specific targeting of CRISPR complex have been discussed. Finally, an overarching discussion on advanced strategies to design robust CRISPR components is provided for streamlining future genome editing applications.

Recessive opaque2 (o2) and opaque16 (o16) genes enhance lysine and tryptophan in maize kernels. Though few o2, o16-, and o2o16-based maize genotypes have been developed, the transition of quality attributes and seed morphology through different stages of kernel development has not been studied yet. To understand the role of o2 and o16 genes in the regulation of essential amino acids and kernel opaqueness in maize, we analyzed the accumulation pattern of lysine and tryptophan, and the occurrence of opaqueness in the developing kernels at 15, 30, and 45 days after pollination (DAP) among a set of o2-, o16-, and o2o16-based inbreds. Genotypes with o2o16 possessed significantly higher lysine (0.64%) and tryptophan (0.25%) over o2 (lysine, 0.48%; tryptophan, 0.18%) and o16 (lysine, 0.46%; tryptophan, 0.17%) alone across kernel development stages. A decreasing trend of amino acid accumulation in o2-, o16-, and o2o16-based genotypes was observed through 15-, 30-, and 45-DAP. Kernel opaqueness also showed a similar decreasing trend among o2-, o16-, and o2o16-based inbreds during kernel development. A positive association was observed between lysine and tryptophan (r = 0.95), tryptophan and opaqueness (r = 0.60), and lysine and opaqueness (r = 0.60) across DAPs. Hard endosperm in wild types and o16 genotypes was due to compact starch-granule structures packed with more proteinaceous matrix compared to o2 and o2o16. This is the first report on nutritional quality and opaqueness at different stages of kernel development in o2-, o16-, and o2o16-based genotypes.

Adenosine deaminase acting on RNA 1 (ADAR1) plays an essential role in the development of malignancies by modifying the expression of different oncogenes. ADAR1 presents three distinct activities: adenosine-to-inosine RNA editing, modulating IFN pathways, and response to cellular stress factors. Following stressors such as heat shock, ADAR1p110 isoform relocates from the nucleus to the cytoplasm, where it suppresses RNA degradation which leads to the arrest of apoptosis and cell survival. In this study, we assessed the expression of ADAR1 across different cancer cell lines. We revealed that the presence of ADAR1 varies between cells of different origins and that a high transcript level does not reflect protein abundance. Additionally, we subjected cells to a heat shock in order to evaluate how cellular stress factors affect the expression of ADAR1. Our results indicate that ADAR1 transcript and protein levels are relatively stable and do not change under heat shock in examined cell lines. This research lays a groundwork for future directions on ADAR1-related studies suggesting in which types of cancer ADAR1 may be a promising target for novel therapeutic approaches.

Infertility remains a significant global health challenge. With up to one in seven couples affected worldwide, the inability to conceive has become a major concern for reproductive health. Many causes of infertility are linked to genetic variants that are disruptive for germ cell homeostasis. Using causative variants to model human infertility can improve our understanding of the molecular pathways that regulate germ cell development. One notable example is the germ cell morphogen NANOS, which is highly conserved across species. Variants of NANOS are associated with infertility in various animal models, from Drosophila to humans. Here, we examine how modelling human infertility based on NANOS variants can offer insights into the molecular processes underlying germ cell development. Ultimately, uncovering the molecular basis of human infertility through this approach is vital for developing advanced diagnostic methods and therapeutic options in the future.

This study aimed to improve the restorer line IR 42266-29-3R (A42) for multiple stress tolerance using integrated marker-assisted backcross breeding (MABB) and the doubled haploid (DH) approach. The primary objective was to introduce the abiotic stress tolerant QTL (qDTY1.1 and qHTSF4.1) into the background IR 42266-29-3R, which already harbors three bacterial blight (BB) resistance genes (xa5, xa13, and Xa21). The BC₁F₁ population was derived from crosses between IR 42266-29-3R and N22, leading to the development of 113 true DHs. Efficient callus induction (29.64%) was achieved using N6 medium supplemented with 2.0 mg/l 2,4-D, 0.5 mg/l BAP, and 3% maltose. Green plant regeneration rates were notably high on the MS medium supplemented with 0.5 mg/l NAA, 0.5 mg/l Kn, 2.0 mg/l BAP, and 3% sucrose, reaching 68.6% and 61.9% on the 2nd and 7th days, respectively. From the 113 DHs, 24 were selected based on superior morpho-agronomic traits and maximum gene combinations. These DHs underwent phenotypic evaluation during the reproductive stage for drought and heat stress responses, alongside assessment for BB resistance. Among them, CS65 showed a genetic profile encompassing xa13, Xa21, qDTY1.1, and qHTSF4.1, while six others exhibited xa5, Xa21, qDTY1.1, and qHTSF4.1. Notably, CS1, CS3, CS37, CS64, and CS65 demonstrated low susceptibility to heat and drought stresses, coupled with yields comparable to the recurrent parent and moderate to high resistance against bacterial blight. CS65 emerged as the most promising genotype due to its robust tolerance to multiple stresses and improved yield potential. Moreover, eight out of the 24 DHs tested positive for both Rf3 and Rf4 genes, displaying spikelet fertility rates exceeding 75%. These selected restorer lines will serve as foundational material for developing superior hybrid rice lines, while the non-restorer lines will contribute to the broader pool of rice varieties in future breeding programs.

Drought stress can damage crop growth and lead to a decline in yield, thereby affecting food security, especially in regions vulnerable to climate change. SNAC1 (stress-responsive NAC1), the NAC transcription factor family member, plays a crucial role in stomatal movement regulation. Effective regulation of stomatal movement is essential for protecting plants from water loss during adverse conditions. Our hypothesis revolves around altering HvSNAC1 activity by introducing a point mutation in its encoding gene, thereby influencing stomatal dynamics in barley. Two TILLING mutants, each harboring missense mutations in the NAC domain, exhibited higher stomatal density after drought stress compared to the parent cultivar 'Sebastian'. These mutants also demonstrated distinct patterns of ABA-induced stomatal movement compared to the wild-type (WT). To delve deeper, we conducted a comprehensive analysis of the transcriptomes of these mutants and the parent cultivar 'Sebastian' under both optimal watering conditions and 10 days of drought stress treatment. We identified differentially expressed genes (DEGs) between the mutants and WT plants under control and drought conditions. Furthermore, we pinpointed DEGs specifically expressed in both mutants under drought conditions. Our experiments revealed that the cis-regulatory motif CACG, previously identified in Arabidopsis and rice, is recognized by HvSNAC1 in vitro. Enrichment analysis led to the identification of the cell wall organization category and potential target genes, such as HvEXPA8 (expansin 8), HvXTH (xyloglucan endotransglucosylase/hydrolase), and HvPAE9 (pectin acetylesterase 9), suggesting their regulation by HvSNAC1. These findings suggest that HvSNAC1 may play a role in regulating genes associated with stomatal density, size and reopening.

Mechanical wounding triggers rapid transcriptional and hormonal reprogramming in plants, primarily driven by jasmonate (JA) signalling. While the role of JA, ethylene, and salicylic acid in wound responses is well characterised, the contribution of strigolactones (SLs) remains largely unexplored. Here, for the first time, it was shown that SLs modulate wound-induced transcriptional dynamics in Arabidopsis thaliana. Using transcriptome profiling of wild-type (Columbia-0) and the SL biosynthesis mutant more axillary growth3 (max3), a discrete cohort of genes whose wound induction is SL-dependent was identified. These genes include core JA biosynthetic genes and several JA-responsive transcriptional repressors, indicating that SLs potentiate early JA signalling. Promoter motif and protein-protein interaction analyses revealed that SLs regulate a transcriptional module composed of AP2/ERF, WRKY, and C2H2 zinc-finger factors, which integrate JA signalling, ROS homeostasis, and tissue regeneration. Notably, many of these factors are misregulated in max3 even prior to wounding, suggesting a primed but hypo-responsive state. Presented findings suggest a model in which SLs act upstream of the JA burst, coordinating transcriptional readiness and post-injury activation. This expands the functional scope of SLs in stress response and positions them as potential modulators of hormone crosstalk during wound responses.

Breast cancer (BC) is characterized by the increase of malignant cells in the breast. The malignant cells begin in the lining of the breast milk glands or ducts (ductal epithelium). BC is the most frequent cancer in women, but it may also occur in males. Long non-coding RNAs (lncRNA) have been demonstrated to control the development and incidence of cancer. However, some lncRNAs experience potential changes in BC, but their role has not been well studied. LINC01279 is known as a valuable biomarker in gastric cancer but has not yet been studied in BC. Changes in LINC01279 expression levels in BC samples were investigated by microarray. Q-PCR was also used to evaluate the expression of LINC01279 in the tumor and normal adjacent samples of 30 BC patients. The LINC01279 co-expressed gene module was discovered using weighted gene correlation network analysis (WGCNA) on the relevant dataset. The top ten hub genes were determined using gene ontology (GO) functional enrichments on the co-expressed gene module. The results of the bioinformatics study showed an increase in LINC01279 expression levels (log2FC = 3.228749561, adj.P.Val = 1.69E - 12) in tumor samples compared to normal marginal tissue. Q-PCR results also showed a significant increase in LINC01279 expression (P-value = 0.0005) in tumor samples. WGCNA analysis identified that the black module is the LINC01279 co-expressed module, and functional annotation analysis of black module genes enriched in significant cancer-related pathways and processes, including cell growth and/or maintenance, regulation of immune response, regulation of cell proliferation, and epithelial-to-mesenchymal transition (EMT). Regarding the real-time PCR results, the analysis of expression patterns has illuminated a distinct association between the heightened expression levels of LINC01279, and the stages of cancer progression as well as the metastatic potential of tumors. However, intriguingly, our observations have failed to reveal any statistically significant correlations between the relative expression of LINC01279 and tumor grade classification, or the presence of ER, PR, and HER2 biomarkers. The present study could provide a new perspective on the molecular regulatory. Processes associated with BC pathogenic mechanisms are linked to the LINC01279, although further research is needed on the possible role of this lncRNA in BC.

To elucidate the crucial function of MICAL2 as a potential immunotherapeutic target and a predictive biomarker in PAAD. The expression of MICAL2 in pan-cancer was investigated using public database, and the expression of MICAL2 in PAAD was validated using tissue samples. The diagnostic and prognostic significance of MICAL2 in PAAD was assessed through the application of ROC curves and Kaplan-Meier curves. The correlation between MICAL2 and infiltrating immune cells and immune checkpoints in PAAD was researched using the TIMER and TCGA databases. In vitro studies involved the evaluation of the biological functions of MICAL2 in human PAAD cells through the knockdown of MICAL2 expression using shRNA. Compared to corresponding normal tissues, the expression of MICAL2 exhibits significant differences in various cancers. Specifically, the level of MICAL2 expression is significantly increased in PAAD. Moreover, MICAL2 demonstrates considerable diagnostic potential in PAAD patients, and its elevated expression is indicative of an unfavorable prognosis. The differential expression of MICAL2 is related to infiltrating immune cells, immune cell markers, and immune checkpoints in PAAD. In ASPC-1 and PANC-1 cells, when MICAL2 was knocked down, there was a notable suppression of proliferation, migration, and invasion. MICAL2 functions as a significant predictor and promising immunotherapeutic target for prognosis assessment in PAAD. It has a pivotal function in fostering the growth and migration of PAAD cells.

The Caracu is a taurine breed adapted to Brazil's climatic conditions and has been selected for 40 + years for Body Weight (BW), maintaining performance records across multiple generations in its database. However, selecting young animals for BW can result in excessive growth, potentially impacting carcass and reproductive traits. This study aimed to estimate genetic correlations and, based on these estimates, evaluate how selection for BW (adjusted to 378 and 550 days of age for males and females, respectively) has influenced carcass traits and reproductive efficiency in this breed. The dataset contained records for BW (kg), Ribeye area (REA, cm²), Height-to-width ratio of the Longissimus dorsi muscle (HWR), Backfat thickness (BFT, mm), Rump fat thickness (RFT, mm), Yearly scrotal circumference (SC, cm), and Days to calving (DC, days) from animals born between 1966 and 2022. (Co)variance components were estimated by Bayesian inference using two-trait animal models. Analyses were conducted to estimate genetic parameters for pairs of traits, including BW with carcass traits (BW × REA, BW × HWR, BW × BFT, and BW × RFT) and BW with reproductive traits (BW × DC and BW × SC). Additionally, analyses were performed for pairwise combinations among carcass traits (REA × HWR, REA × BFT, REA × RFT, HWR × BFT, HWR × RFT, and RFT × BFT) and between reproductive traits (DC × SC). The relationship matrix included 4,783 animals, of which 829 were genotyped with the GGP Bovine 100 K SNP panel. Favorable genetic correlations (r_g) were observed between BW and REA (0.51 ± 0.11), HWR (0.45 ± 0.17), SC (0.24 ± 0.13), and RFT (0.15 ± 0.21), with the latter being favorable but low. On the other hand, unfavorable genetic correlations were observed between BW and BFT (-0.07 ± 0.21), which was unfavorable but close to zero relationship, and between BW and DC (0.48 ± 0.15). The significance of carcass and reproductive traits is underscored by the favorable, r_g between BW and the carcass traits REA (BW × REA) and HWR (BW × HWR), while a high, unfavorable r_g was observed between BW and DC cows. These findings emphasize the importance of carefully balancing genetic selection to optimize growth, carcass quality, and adequate reproduction in Caracu cattle.