Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae
MetadataShow full item record
To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.
Showing items related by title, author, creator and subject.
Paul, S.; Dixon, Kingsley; Miller, Ben (2014)In addition to population regeneration, the spore phase provides ferns with a capacity for dispersal through space and time (if spores are able to survive for long periods), and buffers their populations against environmental ...
Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogenFarman, M.; Oliver, Richard (1992)Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleor transformant were then retransformed using ...
Solomon, P.; Lee, R.; Wilson, T.; Oliver, Richard (2004)A gene encoding malate synthase, a key enzyme of the glyoxylate cycle, has been cloned and characterized in the necrotrophic wheat pathogen Stagonospora nodorum. Expression studies of Mls1 showed high levels of transcript ...