Curtin University Homepage
  • Library
  • Help
    • Admin

    espace - Curtin’s institutional repository

    JavaScript is disabled for your browser. Some features of this site may not work without it.
    View Item 
    • espace Home
    • espace
    • Curtin Research Publications
    • View Item
    • espace Home
    • espace
    • Curtin Research Publications
    • View Item

    Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae

    Access Status
    Fulltext not available
    Authors
    Zhang, M.
    Jiang, S.
    Zheng, J.
    Zheng, Z.
    Li, Xingjiang
    Pan, L.
    Luo, S.
    Date
    2015
    Type
    Journal Article
    
    Metadata
    Show full item record
    Citation
    Zhang, M. and Jiang, S. and Zheng, J. and Zheng, Z. and Li, X. and Pan, L. and Luo, S. 2015. Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae. Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology. 31 (8): pp. 1203-1218.
    Source Title
    Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology
    DOI
    10.13345/j.cjb.140490
    ISSN
    1000-3061
    School
    Department of Chemical Engineering
    URI
    http://hdl.handle.net/20.500.11937/4904
    Collection
    • Curtin Research Publications
    Abstract

    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.

    Related items

    Showing items related by title, author, creator and subject.

    • The persistence and germination of fern spores in fire-prone, semi-arid environments
      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 pathogen
      Farman, 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 ...
    • Inheritance and alteration of transforming DNA during an induced parasexual cycle in the imperfect fungus Cladosporium fulvum
      Arnau, J.; Oliver, Richard (1993)
      Protoplasts of a pAN7-1-transformed isolated of Cladosporium fulvum race 4, harbouring a tandem duplication of the vector, and an untransformed race 5 isolate were fused, without selection for the presence of vector ...
    Advanced search

    Browse

    Communities & CollectionsIssue DateAuthorTitleSubjectDocument TypeThis CollectionIssue DateAuthorTitleSubjectDocument Type

    My Account

    Admin

    Statistics

    Most Popular ItemsStatistics by CountryMost Popular Authors

    Follow Curtin

    • 
    • 
    • 
    • 
    • 

    CRICOS Provider Code: 00301JABN: 99 143 842 569TEQSA: PRV12158

    Copyright | Disclaimer | Privacy statement | Accessibility

    Curtin would like to pay respect to the Aboriginal and Torres Strait Islander members of our community by acknowledging the traditional owners of the land on which the Perth campus is located, the Whadjuk people of the Nyungar Nation; and on our Kalgoorlie campus, the Wongutha people of the North-Eastern Goldfields.