一种水稻叶片基因组DNA简易提取方法  

A Simplified Method for Extracting Genomic DNA from Rice Leaves

作　　者：金素奎 国倩倩 刘巧泉 高继平[2] JIN Su-kui;GUO Qian-qian;LIU Qiao-quan;GAO Ji-ping(Joint International Research Laboratory of Agriculture and Agri-Product Safety,the Ministry of Education,Yangzhou University,Yangzhou 225009;College of Agriculture,Yangzhou University,Yangzhou 225009)

机构地区：[1]扬州大学教育部农业与农产品安全国际联合实验室,扬州225009 [2]扬州大学农学院,扬州225009

出　　处：《生物技术通报》2025年第1期74-84,共11页Biotechnology Bulletin

基　　金：国家重点研发计划(2022YFD1200103);江苏省重点研发计划(BE2022336)。

摘　　要：【目的】为了应对日益增加的大规模遗传材料鉴定需求,急需建立一种快速、简易、高效、低成本的基因组DNA提取方法。【方法】根据水稻遗传鉴定的特点,对传统TPS法进行改进和简化。(1)将特制的钢珠加样条与全自动研磨仪结合使用,提高了研磨水稻叶片组织的速度;(2)使用TPS缓冲液室温研磨裂解,无需液氮冷冻研磨;(3)研磨后的组织匀浆仅需75℃水浴20 min即可完成裂解;(4)室温静置5 min后吸取粗提液上清5μL至96孔PCR板内,使用排枪加入95μL灭菌的去离子水进行稀释,即完成基因组DNA的提取过程。【结果】简易TPS法提取基因组DNA的步骤减少至6步以内,省略了DNA沉淀、离心、洗涤、溶解等过程。提取过程简单、快速,可在30 min内完成96个样本的DNA提取。提取过程中使用的试剂种类较少且廉价易得、安全环保。通过优化DNA提取液的用量,用简易TPS法提取的基因组DNA开展的各类常规分子遗传鉴定的效果与CTAB法及传统TPS法相当。【结论】简易TPS法提高了水稻基因组DNA提取的效率,适合高通量的遗传鉴定。【Objective】In order to cope with the increasing demand for large-scale genetic material identification,it is urgent to establish a fast,simple,efficient and low-cost genomic DNA extraction method.【Method】The traditional TPS method was improved and simplified according to the characteristics of rice genetic identification.1)The tissue grinding speed of rice leaves was improved by combining special steel bar and automatic grinding instrument.2)Directly grinding and lysing using the TPS buffer at room temperature instead of freezing grinding with liquid nitrogen.3)The homogenized tissue after grinding was lysed in a 75℃water bath for 20 min.4)After standing at room temperature for 5 min,took 5μL of the supernatant from the crude extract and transferred it to into a 96-well PCR plate,and added 95μL sterilized deionized water for dilution using a pipette to complete the genomic DNA extraction process.【Result】The simplified TPS method reduced the steps of genomic DNA extraction to less than 6 steps,omitting DNA precipitation,centrifugation,washing,dissolution and so on.The extraction process is simple and fast,and the DNA extraction of 96 samples can be completed within 30 minutes.The reagents used in the extraction process are fewer,cheaper,easy to be obtained,safe and environmental protection.By optimizing the amount of DNA extraction solution,the effect of conventional molecular genetic identification using genomic DNA extracted by simplified TPS method is equivalent to that of CTAB method and traditional TPS method.【Conclusion】The simplified TPS method improves the efficiency of rice genomic DNA extraction and is suitable for rice high-throughput genetic identification.

关 键 词：水稻叶片 基因组DNA CTAB法 传统TPS法 简易TPS法 

分 类 号：Q943.2[生物学—植物学] S511[农业科学—作物学]