Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene
Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some p...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-08-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.01749/full |
| _version_ | 1819080919662723072 |
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| author | Guanhua Yuan Guanhua Yuan Guanhua Yuan Xiaoying Xu Xiaoying Xu Wei Zhang Wei Zhang Wenlei Zhang Wenlei Zhang Yulin Cui Song Qin Tianzhong Liu Tianzhong Liu |
| author_facet | Guanhua Yuan Guanhua Yuan Guanhua Yuan Xiaoying Xu Xiaoying Xu Wei Zhang Wei Zhang Wenlei Zhang Wenlei Zhang Yulin Cui Song Qin Tianzhong Liu Tianzhong Liu |
| author_sort | Guanhua Yuan |
| collection | DOAJ |
| description | Haematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10–5 per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis. |
| first_indexed | 2024-12-21T19:52:32Z |
| format | Article |
| id | doaj.art-1fcc7472574e4ce9843d339fb4580482 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-21T19:52:32Z |
| publishDate | 2019-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-1fcc7472574e4ce9843d339fb45804822022-12-21T18:52:10ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-08-011010.3389/fmicb.2019.01749468625Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin GeneGuanhua Yuan0Guanhua Yuan1Guanhua Yuan2Xiaoying Xu3Xiaoying Xu4Wei Zhang5Wei Zhang6Wenlei Zhang7Wenlei Zhang8Yulin Cui9Song Qin10Tianzhong Liu11Tianzhong Liu12Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaShandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaUniversity of Chinese Academy of Sciences, Beijing, ChinaKey Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaYantai Marine Economic Research Institute, Yantai, ChinaKey Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaShandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaKey Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaShandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaKey Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, ChinaKey Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, ChinaKey Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaShandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, ChinaHaematococcus pluvialis has high commercial value, yet it displays low development of genetic transformation systems. In this research, the endogenous 5′ and 3′ flanking sequences of the constitutive alpha tubulin (tub) gene were cloned along with its encoding region in H. pluvialis, in which some putative promoter elements and polyadenylation signals were identified, respectively. Three selection markers of tub/aadA, tub/hyr and tub/ble with three different antibiotic-resistance genes fused between the endogenous tub promoter (Ptub) and terminator (Ttub) were constructed and utilized for biolistic transformation of H. pluvialis. Stable resistant colonies with introduced aadA genes were obtained after bombardments of either H. pluvialis NIES144 or SCCAP K0084 with the tub/aadA cassette, the efficiency of which could reach up to 3 × 10–5 per μg DNA through an established manipulation flow. Two key details, including the utilization of culture with motile flagellates dominant and controlled incubation of them on membrane filters during bombardments, were disclosed firstly. In obtained transformants, efficient integration and transcription of the foreign tub/aadA fragments could be identified through genome PCR examination and qPCR analysis, nonetheless with random style instead of homologous crossover in the H. pluvialis genome. The presented selection marker and optimized transforming procedures in this report would strengthen the platform for genetic manipulation and modification of H. pluvialis.https://www.frontiersin.org/article/10.3389/fmicb.2019.01749/fullalpha tubulinbiolistic transformationflanking sequenceHaematococcus pluvialisselection marker |
| spellingShingle | Guanhua Yuan Guanhua Yuan Guanhua Yuan Xiaoying Xu Xiaoying Xu Wei Zhang Wei Zhang Wenlei Zhang Wenlei Zhang Yulin Cui Song Qin Tianzhong Liu Tianzhong Liu Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene Frontiers in Microbiology alpha tubulin biolistic transformation flanking sequence Haematococcus pluvialis selection marker |
| title | Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene |
| title_full | Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene |
| title_fullStr | Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene |
| title_full_unstemmed | Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene |
| title_short | Biolistic Transformation of Haematococcus pluvialis With Constructs Based on the Flanking Sequences of Its Endogenous Alpha Tubulin Gene |
| title_sort | biolistic transformation of haematococcus pluvialis with constructs based on the flanking sequences of its endogenous alpha tubulin gene |
| topic | alpha tubulin biolistic transformation flanking sequence Haematococcus pluvialis selection marker |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2019.01749/full |
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