RESUMO
Nucleolar proteins play important roles in plant cytology, growth, and development. Fibrillarin2 is a nucleolar protein of Nicotiana benthamiana (N. benthamiana). Its cDNA was amplified by RT-PCR and inserted into expression vector pEarley101 labeled with yellow fluorescent protein (YFP). The fusion protein was localized in the nucleolus and Cajal body of leaf epidermal cells of N. benthamiana. The N. benthamiana fibrillarin2 (NbFib2) protein has three functional domains (i.e., glycine and arginine rich domain, RNA-binding domain, and α-helical domain) and a nuclear localization signal (NLS) in C-terminal. The protein 3D structure analysis predicted that NbFib2 is an α/ß protein. In addition, the virus induced gene silencing (VIGS) approach was used to determine the function of NbFib2. Our results showed that symptoms including growth retardation, organ deformation, chlorosis, and necrosis appeared in NbFib2-silenced N. benthamiana.
Assuntos
Metiltransferases/genética , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Metiltransferases/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/química , Nicotiana/genéticaRESUMO
In this study, two polyclonal antibodies were produced against the Omp protein of 'Candidatus Liberibacter asiaticus'. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and ß-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against 'Ca. L. asiaticus'-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect 'Ca. L. asiaticus' in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to 'Ca. L. asiaticus' detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.