野生蓝藻质粒提取的研究

Plasmid Extraction from Wild Cyanobacterial Species

摘要

在蓝藻生态与进化研究中，质粒作为重要的可移动遗传元件，常携带着环境适应、代谢调控及种间互作相关的功能基因，是解析微生物环境响应机制的关键资源. 然而，蓝藻质粒提取过程常因细胞壁结构特殊、多糖干扰及内源酶活性等复杂因素，面临提取效率低、完整性差的技术瓶颈，制约了相关遗传资源的挖掘与功能解析，对于野生海洋蓝藻而言尤为突出. 为此，本研究针对野生海洋蓝藻 Nodosilinea sp. E11(E11)与 cf. Phormidesmis sp. S3-3(S3-3)质粒提取效率低的问题，提出了一种适用于野生蓝藻质粒的提取方法，研究了溶菌酶浓度、水浴时间、超声功率与模式以及生物量等因素对质粒提取浓度与产率的影响. 结果表明：生物量为 0.2 g(湿重)的蓝藻 E11 在溶菌酶 30 mg/mL、37 ℃水浴处理 3 h、400 W 功率间歇超声(工作 3 s/间歇 3 s)180 s 的条件下，质粒质量浓度达 285.50 ng/μL；相同质量的蓝藻 S3-3 在溶菌酶 30 mg/mL、37 ℃水浴处理 3 h、400 W 功率间歇超声(工作 3 s/间歇 3 s)90 s 的条件下，质粒质量浓度达 397.38 ng/μL. 此外，以蓝藻 E11 为例，将生物量增加至 0.6 g(湿重)后，其质粒质量浓度虽升至 361.67 ng/μL，但因细胞碎片与基因组 DNA 干扰，产率降至 41.62%，因此，质粒产率与生物量并不呈现正比例关系. 基于上述针对野生蓝藻质粒提取的方法，本研究成功从蓝藻 E11 中分离获得两个结构完整的质粒. 该方法在有效提升目标菌株质粒提取效率的同时，兼顾了经济成本、时间效率与操作便捷性，为今后野生蓝藻质粒的高通量获取、功能注释及环境适应性研究奠定了可靠的技术基础，对深入理解蓝藻环境适应机制、挖掘新型海洋微生物遗传资源具有重要的科学意义．

Abstract

Plasmids are key mobile genetic elements；they carry genes involved in the functions of environmental adaptation，metabolic regulation，and interactions among cyanobacterial species. Analyzing these genes is crucial for understanding environmental responses in microbes. However，the extraction of plasmids from cyanobacteria，especially wild marine strains，remains technically challenging. Issues such as low extraction efficiency and poor DNA integrity arise due to their structurally unique cell walls，interference by polysaccharides，and activities of endogenous enzymes. These challenges limit the mining and functional analysis of cyanobacterial genetic resources. To address the low extraction efficiency of plasmids from the wild marine cyanobacteria Nodosilinea sp. E11(E11) and cf. Phormidesmis sp. S3-3(S3-3)，this study proposed a suitable novel extraction method. It also investigated the influences of lysozyme concentration，water bath duration，ultrasonic power and mode，and biomass on plasmid extraction concentration and yield. Regarding E11，a biomass of 0.2 g(wet weight)，30 mg/mL lysozyme， water bath treatment at 37 ℃ for 3 h，and intermittent ultrasound at 400 W(3 s on/3 s off) for 180 s，resulted in a plasmid mass concentration of 285.50 ng/µL. Concerning S3-3，a biomass of 0.2 g(wet weight)，30 mg/mL lysozyme，water bath treatment at 37 ℃ for 3 h，and intermittent ultrasound at 400 W(3 s on/3 s off) for 90 s，induced a plasmid mass concentration of 397.38 ng/µL. Additionally，with E11，an increase in biomass up to 0.6 g(wet weight)，enhanced plasmid mass concentration to 361.67 ng/µL，but the yield decreased to 41.62% due to interference from cell debris and genomic DNA. This trend indicates that plasmid yield is not directly proportional to biomass. Employing this plasmid extraction method optimized for wild cyanobacteria，two structurally intact plasmids were isolated from E11. This method not only improves the extraction efficiency of plasmids from target strains significantly but also balances economic cost，efficient use of time，and operational convenience. It provides a reliable technical foundation for the high-throughput acquisition and functional annotation of wild cyanobacterial plasmids，enabling environmental adaptability research. The study contributes important insights into a scientific understanding of cyanobacterial environmental adaptation mechanisms and explores novel marine microbial genetic resources.

关键词

质粒提取 / 蓝藻 / 溶菌酶 / 水浴 / 超声 / 生物量

Key words

plasmid extraction / cyanobacteria / lysozyme / water bath / ultrasound / biomass

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基金资助

国家自然科学基金资助项目(42277374)

国家自然科学基金资助项目(42477409)