Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China.
Agronomy Department, University of Florida, Gainesville, FL, 32610, USA.
BMC Plant Biol. 2020 Sep 7;20(1):417. doi: 10.1186/s12870-020-02614-x.
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.
花生是一种重要的豆类作物，在全球范围内广泛种植。随着已发表的异源四倍体基因组，进一步研究花生中的基因功能对于作物改良至关重要。CRISPR/Cas9 系统作为一种研究基因功能和作物改良的强大工具，尚未在花生中得到广泛应用。花生植物通过与根瘤菌共生形成根瘤来固定氮。在模式豆科植物中，植物对根瘤菌的反应是由 Nod 因子受体（NFR）启动的。然而，关于 NFR 在花生中的功能信息仍然有限。在这项研究中，我们应用 CRISPR/Cas9 工具在花生毛状根转化系统中探索 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，在形成根瘤的过程中，与非结瘤（Nod-）系 E4 相比，两个 AhNFR1 和两个 AhNFR5 基因在结瘤（Nod+）系 E5 中具有高表达水平，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结瘤中的功能，我们应用 CRISPR 技术，使用毛状根转化系统创建 AhNFR1 和 AhNFR5 基因的敲除突变体。对转基因毛状根中这些基因的测序表明，所选的 AhNFR1 和 AhNFR5 基因已被 CRISPR 系统成功编辑，证明其在异源四倍体花生中靶向突变的有效性。编辑两个 AhNFR5 基因的突变体表现出 Nod-表型，而编辑两个选定的 AhNFR1 基因的突变体在接种根瘤菌后仍能形成根瘤。
本研究表明，CRISPR-Cas9 可用于花生毛状根转化系统进行花生功能基因组研究，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 CRISPR-Cas9 靶向花生 AhNFR 基因，我们验证了 AhNFR5 基因在花生结瘤形成中的功能。
