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| Construction of CRISPR-Cas9 Editing System Based on U6 Promoter in Peanut (Arachis hypogaea) |
| ZHOU Cai1,2, HUANG Jian-Bin1, LI Jing-Jing1, ZHOU Xian-Tao1, LI Xiao-Bei1, ZHANG Kai-Yuan1, WANG Si-Ming1, LI Qiao-Qiao3, WANG Shu-Hua4, TANG Yan-Yan1,2, QIAO Li-Xian1,2,* |
1 College of Agriculture, Qingdao Agricultural University / International Cooperation Laboratory for Peanut Quality Improvement between China and US, Qingdao 266109, China;
2 Academy of Dongying Efficient Agricultural Technology and Industry on Saline and Alkaline Land in Collaboration with Qingdao Agricultural University, Dongying 257091, China;
3 Qingdao Jimo District Agricultural and Rural Development Service Center, Qingdao 266200, China;
4 Human Resources and Social Service Center in Bei'an, Jimo District Qingdao City, Qingdao 266200, China |
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Abstract CRISPR/Cas9 gene editing technology has been successfully applied to various crops, and targeted mutagenesis of target genes as well as targeted improvement of target traits have been achieved. However, no mature and efficient gene editing system has been established in peanut (Arachis hypogaea). In this study, 2 U6 promoters, namely AhA3U6 and AhB9U6, were isolated from the peanut variety 'Huayu 23'. Subsequently, these promoters were employed to substitute the GmU6 promoter in the original vector pBGK041-GmU6. The homologous genes of the peanut AhPEPC1 (PEPC: phosphoenolpyruvate carboxylase) gene, namely Aradu.A52DW and Araip.RUX3H, were selected as the target genes in this study, and the CRISPR-Cas9 recombinant vectors pBGK041-AhA3U6-AhPEPC1 and pBGK041-AhB9U6-AhPEPC1 were successfully constructed. Subsequently, all 3 vectors were introduced into Agrobacterium rhizogenes for the induction of hairy root formation. The transformation positive rates were determined to be 78.4%, 83.5%, and 83.0%, respectively. The results indicated that the gene editing efficiency of pBGK041-GmU6-AhPEPC1 was merely 1.7%, whereas that of pBGK041-AhA3U6-AhPEPC1 and pBGK041-AhB9U6-AhPEPC1 reached 4.2% and 2.6%, respectively. Furthermore, the 3 vectors were introduced into peanut plants via the pollen tube pathway, with conversion efficiencies of 30.0%, 33.7%, and 29.2% being achieved. The sequencing results of transgenic seeds showed that no gene editing was detected in the pBGK041-GmU6-AhPEPC1 vector transgenic seeds, while the editing efficiencies of the pBGK041-AhA3U6-AhPEPC1 and pBGK041-AhB9U6-AhPEPC1 vector transgenic seeds were 2.2% and 1.3%, respectively. The oil content of the transgenic kernels was 56.88% and 57.49%. Replacement of the original GmU6 promoter with endogenous peanut promoters AhA3U6 and AhB9U6 in the pBGK041-GmU6 vector induced detectable mutations in both transgenic peanut hairy roots and kernels. This promoter substitution enhanced editing efficiency and elevates kernel oil content by approximately 4%. This system provides technical support for the implementation of peanut gene editing and new germplasm resources for the breeding of high-oil varieties.
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Received: 07 April 2025
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Corresponding Authors:
* Corresponding author, lxqiao73@163.com
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