{"title":"通过在 37°C 和 30°C 温度下交替培养转化株,开发出一种无标记的寄生褐藻菌株遗传操作方法。","authors":"Jing Xiao, Yuxin Wang, Dongfang Wu, Yuping Song, Xuwang Cai, Huanchun Chen, Hongbo Zhou, Xiaojuan Xu","doi":"10.1186/s12896-024-00887-w","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.</p><p><strong>Results: </strong>In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the Kan<sup>R</sup> cassette from JS0135ΔnanH::Kan<sup>R</sup>, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.</p><p><strong>Conclusions: </strong>In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"60"},"PeriodicalIF":3.5000,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373133/pdf/","citationCount":"0","resultStr":"{\"title\":\"A marker-free genetic manipulation method for Glaesserella parasuis strains developed by alternately culturing transformants at 37°C and 30°C.\",\"authors\":\"Jing Xiao, Yuxin Wang, Dongfang Wu, Yuping Song, Xuwang Cai, Huanchun Chen, Hongbo Zhou, Xiaojuan Xu\",\"doi\":\"10.1186/s12896-024-00887-w\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.</p><p><strong>Results: </strong>In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the Kan<sup>R</sup> cassette from JS0135ΔnanH::Kan<sup>R</sup>, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.</p><p><strong>Conclusions: </strong>In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.</p>\",\"PeriodicalId\":8905,\"journal\":{\"name\":\"BMC Biotechnology\",\"volume\":\"24 1\",\"pages\":\"60\"},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-09-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373133/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"BMC Biotechnology\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1186/s12896-024-00887-w\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"BMC Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1186/s12896-024-00887-w","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
A marker-free genetic manipulation method for Glaesserella parasuis strains developed by alternately culturing transformants at 37°C and 30°C.
Background: Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.
Results: In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the KanR cassette from JS0135ΔnanH::KanR, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.
Conclusions: In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.
期刊介绍:
BMC Biotechnology is an open access, peer-reviewed journal that considers articles on the manipulation of biological macromolecules or organisms for use in experimental procedures, cellular and tissue engineering or in the pharmaceutical, agricultural biotechnology and allied industries.