RESUMO
Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.
RESUMO
Genetic male sterility (GMS) facilitates hybrid seed production in crops including cotton (Gossypium hirsutum). However, the genetic and molecular mechanisms specifically involved in this developmental process are poorly understood. In this study, small RNA sequencing, degradome sequencing, and transcriptome sequencing were performed to analyze miRNAs and their target genes during anther development in a GMS mutant ('Dong A') and its fertile wildtype (WT). A total of 80 known and 220 novel miRNAs were identified, 71 of which showed differential expressions during anther development. A further degradome sequencing revealed a total of 117 candidate target genes cleaved by 16 known and 36 novel miRNAs. Based on RNA-seq, 24, 11, and 21 predicted target genes showed expression correlations with the corresponding miRNAs at the meiosis, tetrad and uninucleate stages, respectively. In addition, a large number of differentially expressed genes were identified, most of which were involved in sucrose and starch metabolism, carbohydrate metabolism, and plant hormone signal transduction based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The results of our study provide valuable information for further functional investigations of the important miRNAs and target genes involved in genetic male sterility and advance our understanding of miRNA regulatory functions during cotton anther development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Gossypium/crescimento & desenvolvimento , Gossypium/genética , MicroRNAs/genética , Mutação , Infertilidade das Plantas/genética , Proteínas de Plantas/genética , Flores/genética , Flores/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , TranscriptomaRESUMO
Due to China's rapidly increasing population, the total arable land area has dramatically decreased; as a consequence, the competition for farming land allocated for grain and cotton production has become fierce. Therefore, to overcome the existing contradiction between cotton grain and fiber production and the limited farming land, development of early-maturing cultivars is necessary. In this research, a high-density linkage map of upland cotton was constructed using genotyping by sequencing (GBS) to discover single nucleotide polymorphism (SNP) markers associated with early maturity in 170 F2 individuals derived from a cross between LU28 and ZHONG213. The high-density genetic map, which was composed of 3978 SNP markers across the 26 cotton chromosomes, spanned 2480 cM with an average genetic distance of 0.62 cM. Collinearity analysis showed that the genetic map was of high quality and accurate and agreed well with the Gossypium hirsutum reference genome. Based on this high-density linkage map, QTL analysis was performed on cotton early-maturity traits, including FT, FBP, WGP, NFFB, HNFFB and PH. A total 47 QTLs for the six traits were detected; each of these QTLs explained between 2.61% and 32.57% of the observed phenotypic variation. A major region controlling early-maturity traits in Gossypium hirsutum was identified for FT, FBP, WGP, NFFB and HNFFB on chromosome D03. QTL analyses revealed that phenotypic variation explained (PVE) ranged from 10.42% to 32.57%. Two potential candidate genes, Gh_D03G0885 and Gh_D03G0922, were predicted in a stable QTL region and had higher expression levels in the early-maturity variety ZHONG213 than in the late-maturity variety LU28. However, further evidence is required for functional validation. This study could provide useful information for the dissection of early-maturity traits and guide valuable genetic loci for molecular-assisted selection (MAS) in cotton breeding.
Assuntos
Mapeamento Cromossômico , Gossypium/genética , Locos de Características Quantitativas/genética , Cruzamento , Genoma de Planta/genética , Genótipo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
MicroRNAs (miRNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of miRNAs have been identified in cotton fibers, the functions of miRNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis (DPA) and was subjected to high-throughput sequencing. A total of 95 known miRNAs were detected to be expressed in cotton seeds. The expression pattern of these identified miRNAs was profiled and 48 known miRNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel miRNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known miRNAs were successfully predicted and 900 expressed sequence tag (EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, miRNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5' RACE. This study is the first to show the regulatory network of miRNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these miRNAs in seed development.
Assuntos
Genoma de Planta/genética , Gossypium/genética , MicroRNAs/genética , Sementes/genética , Sequência de Bases , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Gossypium/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Homologia de Sequência do Ácido Nucleico , TranscriptomaRESUMO
Checkpoint kinases CHK1 and CHK2 are activated in response to DNA damage that results in cell cycle arrest, allowing sufficient time for DNA repair. Agents that lead to abrogation of such checkpoints have potential to increase the efficacy of such compounds as chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were identified as inhibitors of CHK1 by high throughput screening. A structure-based approach is described using crystal structures of JNK1 and CHK1 in complex with 1 and 2 and of the CHK1-3b complex. The ribose binding pocket of CHK1 was targeted to generate inhibitors with excellent cellular potency and selectivity over CDK1and IKKß, key features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a. Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a variety of DNA-damaging agents in preclinical models.
Assuntos
Antineoplásicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/metabolismo , Tiofenos/síntese química , Ureia/análogos & derivados , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Dano ao DNA , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Ensaios de Triagem em Larga Escala , Irinotecano , Camundongos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia , Ureia/síntese química , Ureia/química , Ureia/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Checkpoint kinase 1 (Chk1, CHEK1) is a Ser/Thr protein kinase that plays a key role in mediating the cellular response to DNA-damage. Synthesis and evaluation of a previously described class of Chk1 inhibitors, triazoloquinolones/triazolones (TZs) is further described herein. Our investigation of structure-activity relationships led to the identification of potent inhibitors 14c, 14h and 16e. Key challenges included modulation of physicochemical properties and pharmacokinetic (PK) parameters to enable compound testing in a Chk1 specific hollow fiber pharmacodynamic model. In this model, 16e was shown to abrogate topotecan-induced cell cycle arrest in a dose dependent manner. The demonstrated activity of TZs in this model in combination with a chemotherapeutic agent as well as radiotherapy validates this series of Chk1 inhibitors. X-ray crystal structures (PDB code: 2YEX and 2YER) for an initial lead and an optimized analog are also presented.
Assuntos
Antineoplásicos/síntese química , Neoplasias do Colo/terapia , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/metabolismo , Triazóis/síntese química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Neoplasias do Colo/enzimologia , Terapia Combinada , Cristalografia por Raios X , Dano ao DNA , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Relação Estrutura-Atividade , Topotecan/farmacologia , Triazóis/farmacocinética , Triazóis/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Trk receptor tyrosine kinases have been implicated in cancer and pain. A crystal structure of TrkA with AZ-23 (1a) was obtained, and scaffold hopping resulted in two 5/6-bicyclic series comprising either imidazo[4,5-b]pyridines or purines. Further optimization of these two fusion series led to compounds with subnanomolar potencies against TrkA kinase in cellular assays. Antitumor effects in a TrkA-driven mouse allograft model were demonstrated with compounds 2d and 3a.
RESUMO
Checkpoint Kinase-1 (Chk1, CHK1, CHEK1) is a Ser/Thr protein kinase that mediates cellular responses to DNA-damage. A novel class of Chk1 inhibitors, triazoloquinolones/triazolones (TZ's) was identified by high throughput screening. The optimization of these hits to provide a lead series is described.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologia , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Descoberta de Drogas , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Tropomyosin-related kinases (Trks) are a family of receptor tyrosine kinases activated by neurotrophins. Trks play important roles in pain sensation as well as tumour cell growth and survival signaling. Thus, inhibitors of Trk receptor kinases might provide targeted treatments for pain and cancer. OBJECTIVE: This paper reviews those patent applications since 2002 claiming small-molecule inhibitors of Trk receptor kinases. METHODS: Primary literature and patents were searched with SciFinder and Google Scholar. Patents were selected based on their relevance to Trks and were evaluated and representative compounds were listed as examples. RESULTS/CONCLUSION: Several series of Trk inhibitors with excellent in vitro potencies have been reported and a number of compounds have gone into the clinic. It should be noted that few of these inhibitors are Trk selective, demonstrating that targeting Trk kinases for treatment of pain and/or cancer offers a promising but also challenging approach.
Assuntos
Receptor trkA/antagonistas & inibidores , Receptor trkB/antagonistas & inibidores , Receptor trkC/antagonistas & inibidores , Animais , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/fisiopatologia , Dor/tratamento farmacológico , Dor/fisiopatologia , Patentes como Assunto , Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismoRESUMO
The design, synthesis and biological evaluation of a series of 4-aminopyrazolylpyrimidines as potent Trk kinase inhibitors is reported. High-throughput screening identified a promising hit in the 4-aminopyrazolylpyrimidine chemotype. Initial optimization of the series led to more potent Trk inhibitors. Further optimization using two strategies resulted in significant improvement of physical properties and led to the discovery of 10z (AZ-23), a potent, orally bioavailable Trk A/B inhibitor. The compound offers the potential to test the hypothesis that modulation of Trk activity will be of benefit in the treatment of cancer and other indications in vivo.
Assuntos
Aminas/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/farmacologia , Receptor trkA/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Masculino , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Ratos , Ratos Wistar , Receptor trkA/química , Receptor trkA/metabolismo , Relação Estrutura-AtividadeRESUMO
Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described. Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.