Zhou Lili, Wang Yali, Wang Peilin, Wang Chunling, Wang Jiamin, Wang Xingfen, Cheng Hongmei
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
State Key Laboratory of North China Crop Improvement and Regulation, Hebei Agricultural University, Baoding, China.
Front Plant Sci. 2022 Dec 23;13:1059404. doi: 10.3389/fpls.2022.1059404. eCollection 2022.
CRIPSR/Cas9 gene editing system is an effective tool for genome modification in plants. Multiple target sites are usually designed and the effective target sites are selected for editing. Upland cotton ( L., hereafter cotton) is allotetraploid and is commonly considered as difficult and inefficient to transform, it is important to select the effective target sites that could result in the ideal transgenic plants with the CRISPR-induced mutations. In this study, -mediated hairy root method was optimized to detect the feasibility of the target sites designed in cotton phytoene desaturase () gene. showed the highest hairy root induction (30%) when the bacteria were cultured until OD reached to 0.8. This procedure was successfully applied to induce hairy roots in the other three cultivars (TM-1, Lumian-21, Zhongmian-49) and the mutations were detected in induced by CRISPR/Cas9 system. Different degrees of base deletions at two sgRNAs (sgRNA5 and sgRNA10) designed in were detected in R15 hairy roots. Furthermore, we obtained an albino transgenic cotton seeding containing CRISPR/Cas9-induced gene editing mutations in sgRNA10. The hairy root transformation system established in this study is sufficient for selecting sgRNAs in cotton, providing a technical basis for functional genomics research of cotton.
CRISPR/Cas9基因编辑系统是植物基因组修饰的有效工具。通常会设计多个靶位点，并选择有效的靶位点进行编辑。陆地棉（L.，以下简称棉花）是异源四倍体，通常被认为转化困难且效率低下，选择能产生具有CRISPR诱导突变的理想转基因植株的有效靶位点非常重要。在本研究中，优化了根癌农杆菌介导的毛状根方法，以检测在棉花八氢番茄红素去饱和酶（）基因中设计的靶位点的可行性。当细菌培养至OD达到0.8时，根癌农杆菌诱导毛状根的效率最高（30%）。该方法成功应用于诱导其他三个品种（TM-1、鲁棉21、中棉49）的毛状根，并在由CRISPR/Cas9系统诱导的毛状根中检测到突变。在R15毛状根中检测到在八氢番茄红素去饱和酶中设计的两个sgRNA（sgRNA5和sgRNA10）处有不同程度的碱基缺失。此外，我们获得了一株在sgRNA10中含有CRISPR/Cas9诱导的基因编辑突变的白化转基因棉花幼苗。本研究建立的毛状根转化系统足以用于棉花中sgRNA的筛选，为棉花功能基因组学研究提供了技术基础。
