RESUMO
Plant virus-based sgRNA delivery strategy has been widely applied for efficient genome editing across various plant species, leveraging its significant advantages in the rapid expression and expansion of sgRNA through virus replication and movement. However, the efficacy of the virus-induced gene editing (VIGE) tool in tomato has yet to be explored. In this paper, we established a TRV-mediated CRISPR/Cas9 genome editing system in the somatic cells of tomato, reporting the validation of VIGE and evaluating the mutagenesis efficiency in both tomato leaves and fruits using high-throughput sequencing. The results demonstrated an approximate 65% efficiency of VIGE in tomato leaves for the selected target genes, with VIGE efficiency reaching up to 50% in tomato fruits. This research not only introduces an efficient tool for reverse genetics but also reveals substantial potential of VIGE in surpassing traditional tissue culture techniques for creating heritable mutations in tomato.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Vírus de Plantas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Vírus de Plantas/genética , Folhas de Planta/genética , Folhas de Planta/virologia , Genoma de Planta/genética , Frutas/genética , Frutas/virologia , Plantas Geneticamente Modificadas/genéticaRESUMO
The production of anthocyanin is regulated by light and corresponding photoreceptors. In this study, we found that exposure to blue light and overexpression of CRY1a are associated with increased accumulation of anthocyanin in tomato (Solanum lycopersicum L.). These responses are the result of changes in mRNA and the protein levels of SlHY5, which is a transcription factor. In vitro and in vivo experiments using electrophoretic mobility shift assay and ChIP-qPCR assays revealed that SlHY5 could directly recognize and bind to the G-box and ACGT-containing element in the promoters of anthocyanin biosynthesis genes, such as chalcone synthase 1, chalcone synthase 2, and dihydroflavonol 4-reductase. Silencing of SlHY5 in OE-CRY1a lines decreased the accumulation of anthocyanin. The findings presented here not only deepened our understanding of how light controls anthocyanin biosynthesis and associated photoprotection in tomato leaves, but also allowed us to explore potential targets for improving pigment production.
Assuntos
Antocianinas/biossíntese , Criptocromos/metabolismo , Proteínas de Plantas/fisiologia , Solanum lycopersicum/metabolismo , Fatores de Transcrição/fisiologia , Antioxidantes/metabolismo , Western Blotting , Imunoprecipitação da Cromatina , Criptocromos/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Proteínas RecombinantesRESUMO
Blue light photoreceptors, cryptochromes (CRYs), regulate multiple aspects of plant growth and development. However, our knowledge of CRYs is predominantly based on model plant Arabidopsis at early growth stage. In this study, we elucidated functions of CRY1a gene in mature tomato (Solanum lycopersicum) plants by using cry1a mutants and CRY1a-overexpressing lines (OE-CRY1a-1 and OE-CRY1a-2). In comparison with wild-type plants, cry1a mutants are relatively tall, accumulate low biomass, and bear more fruits, whereas OE-CRY1a plants are short stature, and they not only flower lately but also bear less fruits. RNA-seq, qRT-PCR, and LC-MS/MS analysis revealed that biosynthesis of gibberellin, cytokinin, and jasmonic acid was down-regulated by CRY1a. Furthermore, DNA replication was drastically inhibited in leaves of OE-CRY1a lines, but promoted in cry1a mutants with concomitant changes in the expression of cell cycle genes. However, CRY1a positively regulated levels of soluble sugars, phytofluene, phytoene, lycopene, and ß-carotene in the fruits. The results indicate the important role of CRY1a in plant growth and have implications for molecular interventions of CRY1a aimed at improving agronomic traits.
Assuntos
Carotenoides/metabolismo , Criptocromos/genética , Frutas/metabolismo , Genes de Plantas/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Criptocromos/fisiologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Genes de Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/fisiologia , Folhas de Planta/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Brassinosteroids (BRs) play a critical role in plant growth, development and stress response; however, genetic evidence for the BR-mediated integrated regulation of plant growth still remains elusive in crop species. Here, we clarified the function of DWARF (DWF), the key BR biosynthetic gene in tomato, in the regulation of plant growth and architecture, phytohormone homeostasis and fruit development by comparing wild type, d^(im), a weak allele mutant impaired in DWF, and DWF-overexpressing plants in tomato. Results showed that increases in DWF transcripts and endogenous BR level resulted in improved germination, lateral root development, CO2 assimilation and eventually plant growth as characterized by slender and compact plant architecture. However, an increase in DWF transcript down-regulated the accumulation of gibberellin, which was associated with decreases in leaf size and thickness. BRs positively regulated lateral bud outgrowth, which was associated with decreased transcript of Aux/IAA3, and the ethylene-dependent petiole bending and fruit ripening. Notably, overexpression of DWF did not significantly alter fruit yield per plant; however, increases by 57.4% and 95.3% might be estimated in fruit yield per square metre in two transgenic lines due to their compact architecture. Significantly, BR level was positively related with the carotenoid accumulation in the fruits. Taken together, our results demonstrate that BRs are actively involved in the regulation of multiple developmental processes relating to agronomical important traits.
Assuntos
Homeostase/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Solanum lycopersicum/anatomia & histologia , Solanum lycopersicum/metabolismo , Brassinosteroides/biossíntese , Brassinosteroides/metabolismo , Carotenoides/metabolismo , Etilenos/metabolismo , Frutas/efeitos dos fármacos , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Germinação/genética , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (â¼1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.
Assuntos
Dispositivos Lab-On-A-Chip , Linfócitos T/imunologia , Citocinas/metabolismo , Citocinas/fisiologia , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Procedimentos Analíticos em Microchip/métodos , Neoplasias/imunologia , Fenótipo , Linfócitos T Citotóxicos/imunologiaRESUMO
As the tissue that contains the largest representation of the human proteome, blood is the most important fluid for clinical diagnostics. However, although changes of plasma protein profiles reflect physiological or pathological conditions associated with many human diseases, only a handful of plasma proteins are routinely used in clinical tests. Reasons for this include the intrinsic complexity of the plasma proteome, the heterogeneity of human diseases and the rapid degradation of proteins in sampled blood. We report an integrated microfluidic system, the integrated blood barcode chip that can sensitively sample a large panel of protein biomarkers over broad concentration ranges and within 10 min of sample collection. It enables on-chip blood separation and rapid measurement of a panel of plasma proteins from quantities of whole blood as small as those obtained by a finger prick. Our device holds potential for inexpensive, noninvasive and informative clinical diagnoses, particularly in point-of-care settings.