RESUMO
The purpose of this study is to investigate the function of a novel potassium transporter gene (NrHAK1) isolated from Nicotiana rustica roots using yeast complement and real-time PCR technique. The complementary DNA (cDNA) of NrHAK1, 2 488 bp long, contains an open reading frame (ORF) of 2 334 bp encoding a protein of 777 amino acids (87.6 kDa) with 12 predicted transmembrane domains. The NrHAK1 protein shows a high sequence similarity to those of high-affinity potassium transporters in Mesembryanthemum, Phytolacca acinosa, Arabidopsis thaliana, and so on. We found that the NrHAK1 gene could complement the yeast-mutant defect in K+ uptake. Among several tissues surveyed, the expression level of NrHAK1 was most abundant in the root tip and was up-regulated when exposed to potassium starvation. Moreover, the transcript accumulation was significantly reduced by adding 5 mmol/L NH4+ to the solution. These results suggest that NrHAK1 plays an important role in potassium absorption in N. rustica.
Assuntos
Proteínas de Transporte de Cátions/genética , Nicotiana/genética , Proteínas de Plantas/genética , Potássio/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Compostos de Amônio Quaternário/farmacologia , Sódio/farmacologia , Nicotiana/metabolismoRESUMO
RNAi technique has been proved as a powerful tool for plant breeding. In this paper, the coat protein of tobacco mosaic virus (TMV) was used for constructing the RNAi interference vector. The tobacco varieties K326 and Longjiang 911 were transformed via Agrobacterium tumefaciens-mediated transformation, and transgenic plants were generated. The expression analysis with real-time PCR indicated that TMV RNA had been degraded varied in different transgenic lines. Field assay revealed that 83% and 90 % transgenic plants showed immunity resistance to TMV in K326 and Longjiang 911 respectively.
Assuntos
Proteínas do Capsídeo/genética , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Interferência de RNA , Vírus do Mosaico do Tabaco/genética , Imunidade Inata/genética , Doenças das Plantas/genética , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/virologia , Reação em Cadeia da Polimerase , Nicotiana/virologia , Vírus do Mosaico do Tabaco/crescimento & desenvolvimentoRESUMO
In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.
Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Glucuronidase/genética , beta-Galactosidase/genética , Embaralhamento de DNA/métodos , Escherichia coli/enzimologia , Glucuronidase/química , Glucuronidase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas/métodos , Relação Estrutura-Atividade , Especificidade por Substrato , beta-Galactosidase/química , beta-Galactosidase/metabolismoRESUMO
Using a yeast one-hybrid method, a transcription factor, OsAP25, which interacts specifically with a GCC box was isolated from rice. The OsAP25 protein contained a conserved ethylene-responsive element binding factor (ERF) domain which shared identity with other reported ERF domains. Phylogenetic analysis showed that OsAP25 could be categorized into class III ERF of the previously characterized ERF proteins on an evolutionary relationship. The semi-quantitative RT-PCR analysis revealed that OsAP25 gene was constitutively expressed in leaves, roots, growing points, flower, bolting stage and grain filling stage. In addition, OsAP25 gene was induced by NaCl, cold, drought, abscisic acid and exogenous ethylene.