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
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.
周材, 黄建斌, 李晶晶, 周贤涛, 李小蓓, 张开元, 王斯铭, 李乔乔, 王淑华, 唐艳艳, 乔利仙. 基于花生U6启动子的CRISPR-Cas9编辑体系构建[J]. 农业生物技术学报, 2025, 33(12): 2745-2756.
ZHOU Cai, HUANG Jian-Bin, LI Jing-Jing, ZHOU Xian-Tao, LI Xiao-Bei, ZHANG Kai-Yuan, WANG Si-Ming, LI Qiao-Qiao, WANG Shu-Hua, TANG Yan-Yan, QIAO Li-Xian. Construction of CRISPR-Cas9 Editing System Based on U6 Promoter in Peanut (Arachis hypogaea). 农业生物技术学报, 2025, 33(12): 2745-2756.
[1] 陈锦清, 郎春秀, 胡张华, 等. 1999. 反义PEP基因调控油菜籽粒蛋白质/油脂含量比率的研究[J]. 农业生物技术学报, 7(4): 316-320. (Chen J Q, Lang C X, Hu Z H, et al.1999. Study on regulating the protein/oil content ratio in rapeseed grains via the antisense PEP gene[J]. Journal of Agricultural Biotechnology, 7(4): 316-320.) [2] 范正琪, 卢孟柱, 李纪元, 等. 2011. 麻疯树pepc基因正义、反义植物表达载体构建与功能初步分析[J]. 林业科学研究, 24(2): 165-170. (Fan Z Q, Lu M Z, Li J Y, et al.2011. Construction of sense and antisense plant expression vectors of Jatropha curcas pepc gene and preliminary functional analysis[J]. Journal of Forestry Research, 24(2): 165-170.) [3] 雷建峰, 李月, 徐新霞, 等. 2016. 棉花不同GbU6启动子截短克隆及功能鉴定[J]. 作物学报, 42(5): 675-683. (Lei J F, Li Y, Xu X X, et al.2016. Cloning and functional identification of different truncated GbU6 promoters in cotton[J]. Acta Agronomica Sinica, 42(5): 675-683.) [4] 刘峰, 李春娟, 许婷婷, 等. 2007. 花生遗传转化的主要方法和研究进展[J]. 生物技术通讯, 4: 719-722. (Liu F, Li C J, Xu T T, et al.2007. Main methods and research progress of peanut genetic transformation[J]. Letters in Biotechnology, 4: 719-722.) [5] 潘雷雷, 纪红昌, 黄建斌, 等. 2020. 花粉管通道和农杆菌介导的花生AhFatB基因编辑[J]. 华北农学报, 35(4): 64-70. (Pan L L, Ji H C, Huang J B, et al.2020. Pollen tube pathway and Agrobacterium-mediated gene editing of peanut AhFatB gene[J]. Acta Agriculturae Boreali-Sinica, 35(4): 64-70.) [6] 潘丽娟, 迟晓元, 陈娜, 等. 2013. 花生PEPC基因反义表达载体构建及对花生的遗传转化[J]. 花生学报, 42(2): 9-13. (Pan L J, Chi X Y, Chen N, et al.2013. Construction of antisense expression vector of peanut PEPC gene and genetic transformation of peanut[J]. Journal of Peanut Science, 42(2): 9-13.) [7] 蒲艳, 刘超, 李继洋, 等. 2018. 番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立[J].中国农业科学, 51(2): 315-326. (Pu Y, Liu C, Li J Y, et al.2018. Cloning of tomato U6 promoter and establishment of CRISPR/Cas9 gene editing system[J]. Scientia Agricultura Sinica, 51(2): 315-326.) [8] 魏绍巍, 黎茵. 2011. 植物磷酸烯醇式丙酮酸羧化酶的功能及其在基因工程中的应用[J]. 生物工程学报, 27(12): 1702-1710. (Wei S W, Li Y.2011. Functions of plant phosphoenolpyruvate carboxylase and their applications in genetic engineering[J]. Chinese Journal of Biotechnology, 27(12): 1702-1710.) [9] 邢蔓, 谭太龙, 李健, 等. 2016. 甘蓝型油菜 PEPC 基因 ihpRNA表达载体的构建与遗传转化研究[J]. 华北农学报, 31(6): 7-11. (Xing M, Tan T L, Li J, et al.2016. Construction and genetic transformation of ihpRNA expression vector for PEPC gene in Brassica napus[J]. Acta Agriculturae Boreali-Sinica, 31(6): 7-11.) [10] 张新友, 汤丰收, 董文召, 等. 2020, 花粉管通道法在花生遗传转化中的效率限制与优化策略[J]. 中国农业科学, 53(12): 2320-2331. (Zhang X Y, Tang F S, Dong W Z, et al.2020. Efficiency limitations and optimization strategies of pollen tube pathway method in peanut genetic transformation[J]. Scientia Agricultura Sinica, 53(12): 2320-2331.) [11] 张银波, 江木兰, 胡小加. 2005. 油菜PEPase基因的克隆及其对应RNAi载体的构建[J]. 中国油料作物学报, 27(1): 1-4. (Zhang Y B, Jiang M L, Hu X J.2005. Cloning of rapeseed PEPase gene and construction of its corresponding RNAi vector[J]. Chinese Journal of Oil Crop Sciences, 27(1): 1-4.) [12] 赵桂兰, 陈锦清, 尹爱萍, 等. 2005. 获得转反义PEP基因超高油大豆新材料[J]. 分子植物育种, 3(6): 792-796. (Zhao G L, Chen J Q, Yin A P, et al.2005. Obtaining new materials of ultra-high oil soybean with transgenic antisense PEP gene[J]. Molecular Plant Breeding, 3(6): 792-796.) [13] Catlin D, Ochoa O, McCormick S, et al.1998. Celery transformation by Agrobacterium tumefaciens: Cytological and genetic analysis of transgenic plants[J]. Plant Cell Reports, 7: 100-103. [14] Cheng Q, Wang Y, Li T.et al.2021, Functional characterization of peanut U6 promoters for CRISPR/Cas9-mediated genome editing[J]. Plant Cell Reports, 40(12): 2305-2316. [15] El-Kereamy A, El-Shershaby A M, Ibrahim S D.et al.2024. Asynchronous cell division in zygotes limits CRISPR editing efficiency in long-cycle crops[J]. New Phytologist, 241(4): 1623-1637. [16] Friedland A E, Tzur Y B, Esvelt K M, et al.2013. Heritable genome editing in C. elegans via a CRISPR-Cas9 system[J]. Nature Methods, 10(8): 741-743. [17] Gomez R A, Silva L M, Pereira A C.et al.2024. Nuclear retention of sgRNA impedes CRISPR efficiency in peanut via reduced HASTY-mediated export[J]. The Plant Cell 36(2): 451-467. [18] Jacobs T B, Lafayette P R, Schmitz R J, et al.2015. Targeted genome modifications in soybean with CRISPR/Cas9[J]. BMC Biotechnology, 15(16): 1-10. [19] Jores T, Tonnies J, Wrightsman T, et al.2021. Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters[J]. Nature Plants, 7(6): 842-855. [20] Li M, Zhou H, Wang K.et al.2022. Barriers to efficient CRISPR editing in legume plants: sgRNA processing defects in peanut[J]. Molecular Plant, 15(7): 1124-1138. [21] Liu F, Chen J, Zhang Y.et al.2023. Engineering hyperactive U6 promoters for CRISPR-Cas9 genome editing in peanut[J]. Nature Communications, 14(1): 5891. [22] Macovei A, Sevilla N R, Cantos C, et al.2018. Novel alleles of rice eIF4G generated by CRISPR/Cas9‐targeted mutagenesis confer resistance to Rice tungro spherical virus[J]. Plant Biotechnology Journal, 16: 1918-1927. [23] O'Leary B, Park J, Plaxton W C.2011. The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): Recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs[J]. Biochemical Journal, 436(1): 15-34. [24] Pan L, Zhang J, Chi X.et al.2016. The antisense expression of AhPEPC1 increases seed oil production in peanuts (Arachis hypogaea L.)[J]. Grasas y Aceites, 67(4): 164-164. [25] Simmen K A, Mattaj I W.1990. Complex requirements for RNA polymerase III transcription of the Xenopus U6 promoter[J]. Nucleic Acids Research, 18(19): 5649-5657. [26] Svitashev S, Schwartz C, Lenderts B, et al.2016. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes[J]. Nature Communications, 7: 13274. [27] Wang Y, Cheng Q, Li T.et al.2021, Functional characterization of peanut (Arachis hypogaea) U6 promoters for CRISPR/Cas9-mediated genome editing[J]. Plant Biotechnology Journal, 19(8): 1531-1543. [28] Wang Y, Cheng X, Shan Q, et al.2014. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew[J]. Nature Biotechnology, 32(9): 947-951. [29] Zhang P, Xu Z, Gao C.et al.2023. tRNA-sgRNA architectures enhance editing efficiency by preventing 3'-end truncation in plants[J]. Nature Plants, 9(2): 114-126.
