-
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2022Myostatin gene () encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, homozygous mutants with "double muscle" phenotypic...
Myostatin gene () encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-: 3; M2-: 2; M3-: 2; M7-: 6; M7-: 2). The body weight of homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of wild-type rabbits of the same age ((2 718±120) g . (1 969±53) g, < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm . (1 274.8±327.3) μm, < 0.01), but also significantly higher than that of hemizygous rabbits ((3 512.2±439.2) μm . (2 610.4±604.4) μm, < 0.05). In summary, five homozygous mutants rabbits of gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Muscle, Skeletal; Mutation; Myostatin; Phenotype; Rabbits
PubMed: 35611733
DOI: 10.13345/j.cjb.210541 -
BMC Plant Biology Mar 2022Sophora davidii is an important plant resource in the karst region of Southwest China, but S. davidii plant-height mutants are rarely reported. Therefore, we performed...
Sophora davidii is an important plant resource in the karst region of Southwest China, but S. davidii plant-height mutants are rarely reported. Therefore, we performed phenotypic, anatomic structural, transcriptomic and metabolomic analyses to study the mechanisms responsible for S. davidii plant-height mutants. Phenotypic and anatomical observations showed that compared to the wild type, the dwarf mutant displayed a significant decrease in plant height, while the tall mutant displayed a significant increase in plant height. The dwarf mutant cells were smaller and more densely arranged, while those of the wild type and the tall mutant were larger and loosely arranged. Transcriptomic analysis revealed that differentially expressed genes (DEGs) involved in cell wall biosynthesis, expansion, phytohormone biosynthesis, signal transduction pathways, flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched in the S. davidii plant-height mutants. Metabolomic analysis revealed 57 significantly differential metabolites screened from both the dwarf and tall mutants. A total of 8 significantly different flavonoid compounds were annotated to LIPID MAPS, and three metabolites (chlorogenic acid, kaempferol and scopoletin) were involved in phenylpropanoid biosynthesis and flavonoid biosynthesis. These results shed light on the molecular mechanisms of plant height in S. davidii mutants and provide insight for further molecular breeding programs.
Topics: Gene Expression Profiling; Metabolomics; Plant Growth Regulators; Sophora; Transcriptome
PubMed: 35337273
DOI: 10.1186/s12870-022-03503-1 -
BMC Genomics Feb 2022Gamma-irradiated mutants of Triticum aestivum L., hexaploid wheat, provide novel and agriculturally important traits and are used as breeding materials. However, the...
BACKGROUND
Gamma-irradiated mutants of Triticum aestivum L., hexaploid wheat, provide novel and agriculturally important traits and are used as breeding materials. However, the identification of causative genomic regions of mutant phenotypes is challenging because of the large and complicated genome of hexaploid wheat. Recently, the combined use of high-quality reference genome sequences of common wheat and cost-effective resequencing technologies has made it possible to evaluate genome-wide polymorphisms, even in complex genomes.
RESULTS
To investigate whether the genome sequencing approach can effectively detect structural variations, such as deletions, frequently caused by gamma irradiation, we selected a grain-hardness mutant from the gamma-irradiated population of Japanese elite wheat cultivar "Kitahonami." The Hardness (Ha) locus, including the puroindoline protein-encoding genes Pina-D1 and Pinb-D1 on the short arm of chromosome 5D, primarily regulates the grain hardness variation in common wheat. We performed short-read genome sequencing of wild-type and grain-hardness mutant plants, and subsequently aligned their short reads to the reference genome of the wheat cultivar "Chinese Spring." Genome-wide comparisons of depth-of-coverage between wild-type and mutant strains detected ~ 130 Mbp deletion on the short arm of chromosome 5D in the mutant genome. Molecular markers for this deletion were applied to the progeny populations generated by a cross between the wild-type and the mutant. A large deletion in the region including the Ha locus was associated with the mutant phenotype, indicating that the genome sequencing is a powerful and efficient approach for detecting a deletion marker of a gamma-irradiated mutant phenotype. In addition, we investigated a pre-harvest sprouting tolerance mutant and identified a 67.8 Mbp deletion on chromosome 3B where Viviparous-B1 and GRAS family transcription factors are located. Co-dominant markers designed to detect the deletion-polymorphism confirmed the association with low germination rate, leading to pre-harvest sprouting tolerance.
CONCLUSIONS
Short read-based genome sequencing of gamma-irradiated mutants facilitates the identification of large deletions linked to mutant phenotypes when combined with segregation analyses in progeny populations. This method allows effective application of mutants with agriculturally important traits in breeding using marker-assisted selection.
Topics: Chromosome Mapping; Phenotype; Plant Breeding; Plant Proteins; Triticum
PubMed: 35139819
DOI: 10.1186/s12864-022-08344-8 -
Frontiers in Plant Science 2023Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an...
Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as , and , were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.
PubMed: 37680361
DOI: 10.3389/fpls.2023.1245308 -
Scientific Reports Mar 2022Genetic Leukoencephalopathies (gLEs) are heritable white matter disorders that cause progressive neurological abnormalities. A founder mutation in the human...
Genetic Leukoencephalopathies (gLEs) are heritable white matter disorders that cause progressive neurological abnormalities. A founder mutation in the human endolysosomal trafficking protein VPS11 has been identified in Ashkenazi Jewish patients manifesting classic gLE symptoms of hypomyelination, developmental delay, motor and systemic deficits. In this study, we characterized the visual and sensorimotor function of two zebrafish vps11 mutant lines: the previously reported vps11(plt), and a new vps11(-/-) null mutant line, using behavioral analysis to track larval motor responses to visual and acoustic stimuli. We found that mutant larvae from both vps11(plt) and vps11(-/-) lines were able to visually distinguish light and dark, but showed a progressive loss of a normal sensorimotor response to visual stimuli from 5 days post fertilization (dpf) to 7dpf. Additionally, optokinetic response analysis performed at 5dpf indicated that the mutants were significantly visually impaired. Both mutant lines also displayed a progressively lower sensorimotor response to a singular acoustic stimulus from 5-7dpf. Next, we tested the habituation response of the mutant lines to series of acoustic taps. We found both mutant lines habituated faster than their siblings, and that vps11(plt) mutants habituated faster than the vps11(-/-) mutants. Together, these data suggest that loss of Vps11 function results in progressive visual and sensorimotor abnormalities in the zebrafish vps11(plt) and vps11(-/-) mutant lines. This is the first study to characterize behavioral deficits in a vertebrate model of Vps11-dependent gLE. The mutants and behavioral assays described here could be a valuable model system in which to test potential pharmacological interventions for gLE.
Topics: Animals; Leukoencephalopathies; Nystagmus, Optokinetic; Vesicular Transport Proteins; Vision, Ocular; Zebrafish; Zebrafish Proteins
PubMed: 35241734
DOI: 10.1038/s41598-022-07448-1 -
Genetics Jan 2022Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone...
Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.
Topics: Animals; Caenorhabditis elegans; Zebrafish
PubMed: 34850872
DOI: 10.1093/genetics/iyab209 -
BMC Microbiology Feb 2020Acinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to...
BACKGROUND
Acinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi.
RESULTS
We determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (Rif assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from Rif assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in Rif assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in Rif assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background.
CONCLUSIONS
We demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.
Topics: Acinetobacter; Bacterial Proteins; Drug Resistance, Bacterial; Evolution, Molecular; Gene Expression Regulation; Genetic Fitness; Microbial Viability; MutL Proteins; MutS DNA Mismatch-Binding Protein; Mutation Rate; Rifampin
PubMed: 32111158
DOI: 10.1186/s12866-020-01729-3 -
PloS One 2019Streptococcus mutans primary thrives on the biofilm formation on the tooth surface in sticky biofilms and under certain conditions can lead to carious lesions on the...
Streptococcus mutans primary thrives on the biofilm formation on the tooth surface in sticky biofilms and under certain conditions can lead to carious lesions on the tooth surface. To search for a new preventive material for oral biofilm-associated diseases, including dental caries, we investigated the effects of polypyrrole, which contains an electrochemical polymer and causes protonation and incorporation of anion under low pH condition, on the biofilm formation of S. mutans and other streptococci. In this study, polypyrrole was applied in biofilm formation assays with the S. mutans strains UA159 and its gtfB and gtfC double mutant (gtfBC mutant), S. sanguinis, S. mitis and S. gordonii on human saliva and bovine serum albumin-coated 96-well microtiter plates in tryptic soy broth supplemented with 0.25% sucrose. The effects of polypyrrole on biofilm formation were quantitatively and qualitatively observed. High concentrations of polypyrrole significantly inhibited the biofilm formation of S. mutans UA159 and S. sanguinis. As an inhibition mechanism, polypyrrole attached to the surface of bacterial cells, increased chains and aggregates, and incorporated proteins involving GTF-I and GTF-SI produced by S. mutans. In contrast, the biofilm formation of gtfBC mutant, S. sanguinis, S. mitis and S. gordonii was temporarily induced by the addition of low polypyrrole concentrations on human saliva-coated plate but not on the uncoated and bovine serum albumin-coated plates. Moreover, biofilm formation depended on live cells and, likewise, specific interaction between cells and binding components in saliva. However, these biofilms were easily removed by increased frequency of water washing. In this regard, the physical and electrochemical properties in polypyrrole worked effectively in the removal of streptococci biofilms. Polypyrrole may have the potential to alter the development of biofilms associated with dental diseases.
Topics: Adult; Biofilms; Humans; Polymers; Pyrroles; Saliva; Streptococcus mutans; Young Adult
PubMed: 31774855
DOI: 10.1371/journal.pone.0225584 -
Frontiers in Microbiology 2022and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and...
and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and lanthanide (Ln)-dependent MDH (XoxF), whose expression is regulated by the availability of Ln. A set of a siderophore, TonB-dependent receptor, and an ABC transporter that resembles the machinery for iron uptake is involved in the solubilization and transport of Ln. The transport of Ln into the cytosol enhances XoxF expression. A unique protein named lanmodulin from strain AM1 was identified as a specific Ln-binding protein, and its biological function was implicated to be an Ln shuttle in the periplasm. In contrast, it remains unclear how Ln levels in the cells are maintained, because Ln is potentially deleterious to cellular systems due to its strong affinity to phosphate ions. In this study, we investigated the function of a lanmodulin homolog in strain 22A. The expression of a gene encoding lanmodulin () was induced in response to the presence of La. A recombinant LanM underwent conformational change upon La binding. Phenotypic analyses on deletion mutant and overexpressing strains showed that LanM is not necessary for the wild-type and XoxF-dependent mutant's methylotrophic growth. We found that expression was regulated by MxcQE (a two-component regulator for MxaF) and TonB_Ln (a TonB-dependent receptor for Ln). The expression level of was altered to be negatively dependent on Ln concentration in ∆ whereas it was constant in the wild type. Furthermore, when exposed to La, ∆ showed an aggregating phenotype, cell membrane impairment, La deposition in the periplasm evidenced by electron microscopy, differential expression of proteins involved in membrane integrity and phosphate starvation, and possibly lower La content in the membrane vesicle (MV) fractions. Taken together, we concluded that lanmodulin is involved in the complex regulation mechanism of MDHs and homeostasis of cellular Ln levels by facilitating transport and MV-mediated excretion.
PubMed: 35814700
DOI: 10.3389/fmicb.2022.921636 -
Nature Communications Feb 2024Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and...
Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.
Topics: Animals; Mice; Disease Models, Animal; Mutation; Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Protein Aggregates
PubMed: 38365903
DOI: 10.1038/s41467-024-45748-4