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| Main Authors: | , , , |
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| Format: | Artículo científico |
| Language: | en |
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BMC plant biology
2025
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| Online Access: | https://pubmed.ncbi.nlm.nih.gov/41053552/ |
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| _version_ | 1868266144182304768 |
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| author | Qin, Zheng Surnido, Whelver Mizuta, Hiroyuki Uji, Toshiki |
| author_facet | Qin, Zheng Surnido, Whelver Mizuta, Hiroyuki Uji, Toshiki Qin, Zheng Surnido, Whelver Mizuta, Hiroyuki Uji, Toshiki |
| collection | PubMed - marine biology |
| contents | Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera. Qin, Zheng Surnido, Whelver Mizuta, Hiroyuki Uji, Toshiki Ulva Gene Knock-In Techniques Transgenes Kanamycin Kinase CRISPR-Cas Systems Hygromycin B Anti-Bacterial Agents Edible Seaweeds Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (aph7"). Integration of aph7" in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7". The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7" cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (UpAPT). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources. |
| format | Artículo científico |
| id | pubmed_41053552 |
| institution | PubMed |
| language | en |
| publishDate | 2025 |
| publisher | BMC plant biology |
| record_format | pubmed |
| spellingShingle | Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera. Qin, Zheng Surnido, Whelver Mizuta, Hiroyuki Uji, Toshiki Ulva Gene Knock-In Techniques Transgenes Kanamycin Kinase CRISPR-Cas Systems Hygromycin B Anti-Bacterial Agents Edible Seaweeds Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera. Qin, Zheng Surnido, Whelver Mizuta, Hiroyuki Uji, Toshiki Ulva Gene Knock-In Techniques Transgenes Kanamycin Kinase CRISPR-Cas Systems Hygromycin B Anti-Bacterial Agents Edible Seaweeds Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (aph7"). Integration of aph7" in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7". The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7" cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (UpAPT). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources. |
| title | Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera. |
| topic | Ulva Gene Knock-In Techniques Transgenes Kanamycin Kinase CRISPR-Cas Systems Hygromycin B Anti-Bacterial Agents Edible Seaweeds |
| url | https://pubmed.ncbi.nlm.nih.gov/41053552/ |