Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae
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...
| Main Authors: | , , , , , , |
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| Format: | Journal Article |
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Chinese Academy of Sciences
2015
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| Online Access: | http://hdl.handle.net/20.500.11937/4904 |
| _version_ | 1848744646524534784 |
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| author | Zhang, M. Jiang, S. Zheng, J. Zheng, Z. Li, Xingjiang Pan, L. Luo, S. |
| author_facet | Zhang, M. Jiang, S. Zheng, J. Zheng, Z. Li, Xingjiang Pan, L. Luo, S. |
| author_sort | Zhang, M. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T06:04:46Z |
| format | Journal Article |
| id | curtin-20.500.11937-4904 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T06:04:46Z |
| publishDate | 2015 |
| publisher | Chinese Academy of Sciences |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-49042017-09-13T14:46:23Z Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae Zhang, M. Jiang, S. Zheng, J. Zheng, Z. Li, Xingjiang Pan, L. Luo, S. 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. 2015 Journal Article http://hdl.handle.net/20.500.11937/4904 10.13345/j.cjb.140490 Chinese Academy of Sciences restricted |
| spellingShingle | Zhang, M. Jiang, S. Zheng, J. Zheng, Z. Li, Xingjiang Pan, L. Luo, S. Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae |
| title | Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae |
| title_full | Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae |
| title_fullStr | Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae |
| title_full_unstemmed | Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae |
| title_short | Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae |
| title_sort | construction of an integration vector carrying hygromycin b resistance gene and its genetic transformation in rhizopus oryzae |
| url | http://hdl.handle.net/20.500.11937/4904 |