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The application of CRISPR/Cas9 in hairy roots to explore the functions ofAhNFR1andAhNFR5genes during peanut nodulation

文献类型： 外文期刊

作者： Shu, Hongmei 1 ; Luo, Ziliang 2 ; Peng, Ze 2 ; Wang, Jianping 2 ;

作者机构： 1.Jiangsu Acad Agr Sci, Inst Ind Crops, Nanjing 210014, Peoples R China

2.Univ Florida, Agron Dept, Gainesville, FL 32610 USA

关键词： CRISPR; Cas9; Mutants; Nod factor receptor (NFR) genes; Nodulation; Peanut; Symbiosis

期刊名称：BMC PLANT BIOLOGY （ 影响因子：4.215； 五年影响因子：4.96 ）

ISSN： 1471-2229

年卷期： 2020 年 20 卷 1 期

页码：

收录情况： SCI

摘要： Background Peanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven't been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function ofNFRsin peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function ofNFRgenes. Results We firstly identified fourAhNFR1genes and twoAhNFR5genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the twoAhNFR1and twoAhNFR5genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants ofAhNFR1andAhNFR5genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selectedAhNFR1andAhNFR5genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the twoAhNFR5genes showed Nod- phenotype, whereas mutants with editing in the two selectedAhNFR1genes could still form nodules after rhizobia inoculation. Conclusions This study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanutAhNFRgenes in hairy root transformation system, we validated the function ofAhNFR5genes in nodule formation in peanut.

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