生物学杂志

生物学杂志 ›› 2025, Vol. 42 ›› Issue (4): 120-.doi: 10.3969/j.issn.2095-1736.2025.04.120

• 技术方法 • 上一篇    下一篇

一种快速提取真菌基因组进行PCR的方法优化

谭奕阳1， 刘书彤1， 张严化1， 王德培1， 薛鲜丽1,2   

  1. 1. 天津科技大学 生物工程学院， 天津 300457； 2. 工业发酵微生物教育部重点实验室， 天津 300457
  • 出版日期:2025-08-18 发布日期:2025-08-14
  • 通讯作者: 薛鲜丽，博士，副教授，研究方向为丝状真菌遗传改造，E-mail:xuexianli@tust.edu.cn
  • 作者简介:谭奕阳，硕士研究生，研究方向为工业菌株生产有机酸遗传改造，E-mail:18198158936@163.com
  • 基金资助:
    国家重点研发计划项目（2021YFC1808901）

A method for rapid extraction of fungal genome for PCR

TAN Yiyang1, LIU Shutong1, ZHANG Yanhua1, WANG Depei1, XUE Xianli1,2   

  1. 1. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China;
    2. Engineering Research Center of Food Biotechnology of Ministry of Education, Tianjin 300457, China
  • Online:2025-08-18 Published:2025-08-14

PDF (PC)

0

摘要： 研究旨在优化一种快速提取真菌基因组用于聚合酶链式反应（polymerase chain reaction， PCR）的方法。通过对NaOH裂解条件、菌体条件优化及普适性分析，确定制备真菌PCR模板的最佳条件：选取直径为3.5 mm的菌落，用100 μL 0.5 mol/L NaOH于25 ℃静置裂解15 min。此方法提取出的基因组纯度（OD260/OD280）和质量浓度分别在1.8~1.9和150~350 ng/μL，均能满足PCR验证的需求。普适性分析结果显示：黑曲霉大量转化子不同基因PCR验证成功率高达98.6%；此方法也适用于酵母菌、米曲霉及里氏木霉等多种真菌基因的验证，具有较好的普适性。此方法简捷安全，可有效提高真菌基因编辑的效率。

关键词: 基因组提取, 丝状真菌, 碱性裂解, 聚合酶链式反应, 普适性

Abstract: The aim of this study was to optimize a method for rapid extraction of fungal genome for polymerase chain reaction (PCR). The optimal conditions for the preparation of fungal PCR templates were determined by the optimization of NaOH lysis conditions, bacterial conditions and generalizability analysis: the results presented the optimal conditions for the preparation of fungal PCR templates were a diameter of 3.5 mm colonies lysed in 100 μL of 0.5 mol/L NaOH at 25 ℃ for 15 min on a static basis. The purity （OD260/OD280 ） and the mass concentration of genome extracted by this method were 1.8－1.9 and 150－350 ng/μL, which were suitable for PCR verification. The results of universality analysis showed that the success rate of PCR validation of different genes in a large number ofAspergillus nigertransformants was as high as 98.6%, this method was also applicable to the validation of genes in a variety of fungi, such asSaccharomyces cerevisiae,Aspergillus oryzaeandTrichoderma reesei, which has a great universal applicability. This method is simple and safe, and can effectively improve the efficiency of fungal gene editing.

Key words: genome extraction, filamentous fungi, alkaline lysis, polymerase chain reaction, universality

中图分类号: