基于CRISPR/Cas9的蛹虫草无抗性标记转化技术的构建Construction of the CRISPR/Cas9-based marker-free transformation of Cordyceps militaris
李波;邹根;周思池;尹昕;杨占山;鲍大鹏;李晓玲;汪滢;
摘要(Abstract)：
传统的真菌遗传改造方法需要抗性标记，但目前可使用的抗性标记基因非常有限，导致蛹虫草遗传改造面临着抗性基因数量不足的问题，且尚未能实现多个目的基因的连续敲入或敲除，因此在蛹虫草中建立高效的无抗性标记转化技术显得尤为重要。本研究利用CRISPR/Cas9技术对蛹虫草的Cmura5基因进行编辑，通过内源5S-1、5S-2和U6启动子对gRNA进行转录，结果表明使用U6启动子对Cmura5基因的编辑效率达到了100%。在尿嘧啶缺陷型菌株Cmura5–中，回补野生型Cmura5基因可实现正向选择，即野生型菌株可以在基础培养基上生长。利用设计的同源臂对Cmura5基因进行回收，可以实现反向选择，即野生型在含有5-氟乳清酸培养基中生长受到抑制。以尿嘧啶缺陷型Cmura5–为出发菌株，利用无抗性标记转化技术，导入一个重组质粒效率为75%；连续导入2个重组质粒效率为80%；连续导入3个重组质粒效率为100%；连续导入4个重组质粒效率为50%，平均转化效率为75.7%，每一轮的标记回收率均在100%，实现了4个外源基因在蛹虫草中同时表达。
关键词(KeyWords)： CRISPR/Cas9;蛹虫草;无抗性标记;ura5;营养缺陷型标记
基金项目(Foundation): 上海市科技兴农重点攻关项目(2018-02-08-00-12-F01555);; 国家自然科学基金(31902089);; 上海市科学基金委“一带一路”(20310741900)~~
作者(Authors): 李波;邹根;周思池;尹昕;杨占山;鲍大鹏;李晓玲;汪滢;
DOI: 10.13346/j.mycosystema.210457
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