RESUMO
A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Brassica rapa/genética , Vetores Genéticos/genética , Plantas Geneticamente Modificadas/genética , Plasmídeos/genética , Mutação Puntual , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/crescimento & desenvolvimento , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Vetores Genéticos/química , Plasmídeos/química , Origem de Replicação , Alinhamento de Sequência , Transformação Genética , TransgenesRESUMO
Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.
Assuntos
Agrobacterium tumefaciens/genética , Produtos Agrícolas/genética , Glycine max/genética , Plantas Geneticamente Modificadas/genética , Origem de Replicação/genética , Fatores de Transcrição/genética , Zea mays/genética , Arabidopsis/genética , Arabidopsis/imunologia , Replicação do DNA , Dosagem de Genes , Vetores Genéticos , Plasmídeos/genéticaRESUMO
BACKGROUND: Some clinicians have questioned the accuracy of rapid diagnosis of group A streptococcal pharyngitis by commercial immunochemical antigen test kits in the setting of recent streptococcal pharyngitis, believing that the false-positive rate was increased because of presumed antigen persistence. METHODS: We studied 443 patients--211 cases--who had clinical pharyngitis diagnosed as group A beta-hemolytic streptococcus infection in the past 28 days and compared them with 232 control patients who had symptoms of pharyngitis but no recent diagnosis of streptococcal pharyngitis. Our aim was narrowly focused to compare the rapid strep test with the culture method we used in our clinical practice. RESULTS: We found that the rapid strep test in this setting showed no difference in specificity (0.96 vs 0.98); hence, the assertion that rapid antigen testing had higher false-positive rates in those with recent infection was not confirmed. We also found that in patients who had recent streptococcal pharyngitis, the rapid strep test appears to be more reliable (0.91 vs 0.70, P < .001) than in those patients who had not had recent streptococcal pharyngitis. CONCLUSIONS: The findings of this study indicate that the rapid strep test is both sensitive and specific in the setting of recent group A beta-hemolytic streptococcal pharyngitis, and its use might allow earlier treatment in this subgroup of patients.
