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 of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes. We firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes 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 of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation. 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 peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

中文翻译：

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CRISPR / Cas9在毛状根中的应用探讨花生结节过程中AhNFR1和AhNFR5基因的功能。

花生是世界范围内重要的豆类作物。随着已发布的异源四倍体基因组的进一步研究，花生中基因的功能对作物改良至关重要。CRISPR / Cas9系统正在成为一种功能强大的工具，用于基因功能研究和作物改良，尚未在花生中广泛使用。花生植物形成根瘤，通过与根瘤菌的共生关系固定氮。在模型豆科植物中，植物对根瘤菌的反应是由Nod因子受体（NFR）引发的。但是，关于NFR在花生中的功能的信息仍然有限。在这项研究中，我们将CRISPR / Cas9工具应用于花生毛根转化系统中，以探索NFR基因的功能。我们首先在栽培花生（Tifrunner）中鉴定了四个AhNFR1基因和两个AhNFR5基因。基因表达分析表明，在结瘤形成过程中，两个AhNFR1和两个AhNFR5基因在结瘤（Nod +）品系E5中的表达水平高于非结瘤（Nod-）E4品系，表明它们在花生结瘤中的作用。为了进一步探索它们在花生根瘤中的功能，我们应用了CRISPR技术，使用毛状根转化系统创建了AhNFR1和AhNFR5基因的敲除突变体。这些基因在转基因毛状根中的测序表明，选定的AhNFR1和AhNFR5基因已通过CRISPR系统成功编辑，证明了其对异源四倍体花生靶向突变的功效。在两个AhNFR5基因中具有编辑的突变体表现出Nod表型，而在接种了根瘤菌后，在两个选定的AhNFR1基因中具有编辑的突变体仍可能形成结节。这项研究表明，CRISPR-Cas9可用于花生毛状根转化系统中，用于花生功能基因组学研究，特别是在根部基因功能方面。通过在毛状根转化系统中使用针对花生AhNFR基因的CRISPR-Cas9，我们验证了AhNFR5基因在花生根瘤形成中的功能。