ABSTRACT
Objective:To study the virulence-related functions and the pathogenic molecular mechanism of the transcription factor OmpR in Vibrio vulnificus. Methods:The Vibrio vulnificus ompR mutant (Δ ompR) strain was constructed using a markerless gene deletion system. Changes in the tolerance to osmotic pressure, swarming mobility and biofilm synthesis were detected by growth curve, swimming assay and crystal violet assay, respectively. Colony counting methods were used to analyze the cytoadherence of the Δ ompR strain. The cytotoxicity of the Δ ompR strain was measured by lactate dehydrogenase (LDH) releasing test. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes including ompN, ompU, ompA and hcp2 at mRNA level in the Δ ompR. Results:The Δ ompR strain was constructed successfully. Compared with the wild-type strain, the mutant strain showed decreased tolerance to high osmotic pressure, suppressed capability of biofilm formation and reduced cytoadherence and cytotoxicity, whereas no significant difference in motility was detected. The expression of ompN gene at mRNA level in the Δ ompR strain was down-regulated ( P<0.05), while the expression of other target genes showed no significant changes. Conclusions:The transcription factor OmpR regulated the tolerance to high osmotic pressure, biofilm formation, cytoadherence and cytotoxicity in Vibrio vulnificus.
ABSTRACT
Targeted replacement genome editing refers to DNA modification and engineering technology that could induce and achieve mutations of targeted gene replacement or knockin at a target gene or DNA region. In this review, the principles, implementation methods, factors that influence efficiency and accuracy, and applications of gene replacement editing were summarized and discussed. It provides the reference for gene functional characterization and genetic improvements through gene replacement strategies in higher plant especially crops.
ABSTRACT
Plant expression cassette for TaDREB from wheat was constructed into plasmid pBIR1.Aloe stems were used as explants for the transformation mediated by Agrobaterium.Infected tissues were selected using G418 to generate transformants.In total,58 resistant plantlets to the antibiotics were obtained from the infected explants.The designed primers according to the selective gene npt II and the target gene TaDREB were used to analyze all of the G418 resistant plantlets.PCR results demonstrated that TaDREB were successful transferred into aloe genomic with the transformation efficiency of 0.5%.The transgenic aloe plants were treated under 4℃ for two weeks and then at-20℃ for 30min.The treatment showed that the leaves of negative plants appeared severe evidence of freeze injury with brown,withered and translucent,while the positive plants appeared good growing condition.The activities of enzymes such as peroxidase(POD)and superoxide dismutase(SOD)of transgenic plants which were stressed for 14 days under low temperature were analyzed.The results indicated that the trend of SOD and POD activities in transgenic plants was down-up-up-up,and that in non-transgenic plants was down-up-down-down.The average value of relative electrical conductivity in the positive plants was 0.456 which was lower than 0.685 in the negative plants.It is supposed that transformation of the kind of gene could improve the resistant ability of aloe to low temperature.