为日本隐花(Cryptomeria japonica D. Don)MALE STERILITY 4(MS4)基因开发简单的 DNA 提取和 PCR-RFLP:实现环境友好型方案

Saneyoshi Ueno, Yukiko Ito, Yoichi Hasegawa, Yoshinari Moriguchi
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引用次数: 0

摘要

本研究介绍了一种简单的 DNA 提取方法和一种新型 PCR-RFLP 技术,用于对日本隐花植物的雄性不育性 4(MS4)基因进行基因分型。传统的基于 CTAB 的 DNA 提取方法虽然有效,但涉及有害化学物质,而且需要高速离心,这在许多野外环境中不切实际。我们的方法是利用家用洗洁精缓冲液、氯化钠和聚乙烯吡咯烷酮 K-30 从隐杆线虫针叶组织中提取 DNA。这种方法简便易行,对环境更加友好。提取的 DNA 被成功用于针对 MS4 基因单核苷酸多态性的 PCR-RFLP 分析,证明了其基因分型的有效性。PCR-RFLP 标记可靠地区分了不同的基因型,证实了我们的简单提取方法的实际应用价值,即使对含有抑制物质的针叶树也是如此。这项技术尤其适合在植物园和野外站使用,因为在这些地方使用危险化学品和专业设备的可能性有限。我们的研究为 DNA 提取和遗传分析提供了一种简便、可靠、安全的方法,有助于遗传资源管理。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Developing simple DNA extraction and PCR-RFLP for MALE STERILITY 4 (MS4) gene in Cryptomeria japonica D. Don: Toward an environmentally friendly protocol
This study presents the development of a simple DNA extraction method and a novel PCR-RFLP technique for genotyping the MALE STERILITY 4 (MS4) gene in Cryptomeria japonica. Traditional CTAB-based DNA extraction methods, while effective, involve hazardous chemicals and require high-speed centrifugation, which are impractical in many field settings. Our approach utilizes a household dish detergent-based buffer, sodium chloride, and polyvinylpyrrolidone K-30 to extract DNA from C. japonica needle leaf tissues. The simplicity of this method makes it more accessible and environmentally friendly. The extracted DNA was successfully used in PCR-RFLP analysis, targeting a single nucleotide polymorphism in the MS4 gene, demonstrating its efficacy for genotyping. The PCR-RFLP markers reliably discriminated between individual genotypes, confirming the practical application of our simple extraction method, even for conifers containing inhibitory substances. This technique is particularly advantageous for use in arboretums and field stations, where the use of hazardous chemicals and specialized equipment is limited. Our study contributes to genetic resource management by providing an easy, reliable, and safer method for DNA extraction and genetic analysis.
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