RESUMO
Thalassemia is the most common monogenic disorder around the world. Based on the principle of genotype-phenotype correlation, identification of thalassemia mutations is the essential prerequisite for clinical diagnosis and management. Because only common mutations are routinely detected, the identification of rare or undetermined mutations is a challenge for clinical laboratories. Herein, a proband presenting with inconsistent phenotype-genotype correlation after routine molecular screening was investigated by multiplex ligation-dependent probe amplification (MLPA), targeted-next generation sequencing (targeted-NGS), gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. Eventually, a novel 71.8 kb deletion (- -71.8) was identified and characterized, which included HBZ (ζ), HBA2 (α2), and HBA1 (α1) genes and was causing α0-thalassemia (α0-thal). Furthermore, we summarized a practical procedure based on accumulated experience in studies and clinical practice, which can be a guide for molecular screening and clinical diagnosis of thalassemia, especially for identification of undetermined or novel mutations.
Assuntos
Testes Genéticos , Deleção de Sequência , alfa-Globinas/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Alelos , China , Índices de Eritrócitos , Feminino , Estudos de Associação Genética , Testes Genéticos/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Linhagem , Fenótipo , Análise de Sequência de DNA , Talassemia alfa/sangueRESUMO
AtFPF1 (FLOWERING PROMOTING FACTOR 1) is a gene that promotes flowering in Arabidopsis. An expression vector containing AtFPF1 driven by a Ubi-1 promoter was constructed. The gene was introduced into rice callus by Agrobacterium-mediated transformation and fertile plants were obtained. The presence of AtFPF1 in rice plants was confirmed by PCR, Southern and Northern blot analyses, as well as by beta-glucuronidase assay. The results showed that, as in Arabidopsis, AtFPF1 reduced flowering time in rice. Furthermore, introduction of AtFPF1 enhanced adventitious root formation but inhibited root growth in rice during the seedling stage. The results suggest that AtFPF1 promotes flowering time in both dicots and monocots, and plays a role in the initiation of adventitious roots in rice.
Assuntos
Proteínas de Arabidopsis/metabolismo , Flores/crescimento & desenvolvimento , Oryza/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica de Plantas/genética , Vetores Genéticos/genética , Genoma de Planta , Glucuronidase/metabolismo , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Oryza/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Fatores de Tempo , Transformação Genética/genéticaRESUMO
There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.