白花草木樨毛状根高效基因组编辑体系的建立

doi:10.11983/CBB25037

植物学报

白花草木樨毛状根高效基因组编辑体系的建立

陈龙浩^{1, 2†}, 杨瑞娟^2†, 苑筱一², 邢思年², 臧云², 吴凡³, 张吉宇³, 秦晓春¹, 刘文文^2*, 付春祥^2*

¹济南大学生物科学与技术学院, 济南 250022; ²中国科学院青岛生物能源与过程研究所, 山东能源研究院, 青岛新能源山东省实验室, 青岛 266101; ³兰州大学草地农业科技学院, 草种创新与草地农业生态系统全国重点实验室, 兰州 730000

收稿日期:2025-03-06 修回日期:2025-03-21 出版日期:2025-05-14 发布日期:2025-05-14
通讯作者: 付春祥, 刘文文
基金资助:
国家自然科学基金(No.U23A20216, No.32271752)、青岛新能源山东实验室“抓攻关”项目(No.QNESLKPP202302)、泰山学者项目

Development of High-efficiency Genome Editing System for Hair Roots in Melilotus albus

Longhao Chen^{1, 2}^†, Ruijuan Yang²^†, Xiaoyi Yuan², Sinian Xing², Yun Zang², Fan Wu³, Jiyu Zhang³, Xiaochun Qin¹, Wenwen Liu^2*, Chunxiang Fu^2*

¹Shool of Biological Science and Technology, University of Jinan, Jinan 250022, China; ²Qingdao New Energy Shandong Laboratory, Shandong Energy Institute, Chinese Academy of Sciences,Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences, Qingdao 266101, China; ³State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, China

Received:2025-03-06 Revised:2025-03-21 Online:2025-05-14 Published:2025-05-14
Contact: fucx@qibebt.ac.cn, liuww@qibebt.ac.cn
Supported by:

摘要/Abstract

摘要： 白花草木樨(Melilotus albus)具有粗蛋白含量高、耐逆性强等特点, 是优良饲草、轮作和水土保持作物。然而, 白花草木樨稳定遗传转化体系以及基因编辑体系尚未建立, 限制了其基因功能分析及新种质的创制。本研究基于35S::MtLAP1表达盒可诱导再生体花青苷累积进而裸眼“显红色”的原理, 建立了白花草木樨下胚轴毛状根快速诱导和筛选体系, 毛状根诱导率达62%, 阳性率达30.8%。此外, 利用携带有35S::MtLAP1表达盒且由拟南芥AtUbiquitin-10强启动子启动Cas9和sgRNA模块协同表达的基因编辑载体, 能通过上述毛状根体系实现白花草木樨MaPDS (Phytoene Desaturase)的高效编辑, 编辑效率达42.5%。本研究为白花草木樨基因功能研究及高品质种质新资源培育奠定了基础。

关键词: 白花草木樨, MaPDS基因, 毛状根, 基因组编辑

Abstract:
INTRODUCTION: Melilotus albus (M. albus) is an excellent forage crop, suitable for crop rotation and soil and water conservation. However, the stable genetic transformation system and gene editing system of M. albus have not been reported, which limits its application in gene function analysis and the creation of new germplasm resources.
RATIONALE: The plant can produce hairy roots after infection by Agrobacterium rhizogenes harboring Ri plasmids. MtLAP1 expression can activate the anthocyanin synthesis process and then produce purple/red anthocyanin accumulation in the transgenic hairy roots.
RESULTS: This study verified that the 35S::MtLAP1 expression cassette can induce the accumulation of anthocyanins in regenerants, resulting in red color visible to the naked eye. When using the hypocotyls of Melilotus albus as explants, the induction efficiency of hairy root was as high as 62%, and the positive rate was as high as 30.8%. In addition, the research results showed that the gene editing vector, which carried the 35S::MtLAP1 expression cassette and co-expressed the Cas9 and sgRNA modules driven by the constitutive strong promoter of Arabidopsis Ubiquitin-10, could achieve an editing efficiency of 42.5% targeted to Phytoene Desaturase gene in M. albus.
CONCLUSION: This study successfully established a rapid inducing and screening system for positive hairy roots and developed an efficient genome-editing tool for M. albus, laying a foundation for functional gene studies and the development of high-quality new germplasm resources in this species.

Hairy root induction, the-naked-eye screening system and MaPDS gene editing in Melilotus albus.

Key words: Melilotus albus, MaPDS, hair roots, genome editing

陈龙浩, 杨瑞娟, 苑筱一, 邢思年, 臧云, 吴凡, 张吉宇, 秦晓春, 刘文文, 付春祥. 白花草木樨毛状根高效基因组编辑体系的建立. 植物学报, DOI: 10.11983/CBB25037.

Longhao Chen, Ruijuan Yang, Xiaoyi Yuan, Sinian Xing, Yun Zang, Fan Wu, Jiyu Zhang, Xiaochun Qin, Wenwen Liu, Chunxiang Fu. Development of High-efficiency Genome Editing System for Hair Roots in Melilotus albus. Chinese Bulletin of Botany, DOI: 10.11983/CBB25037.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB25037

参考文献

Ajdanian L, Niazian M, Torkamaneh D (2024). Optimizing ex vitro one-step RUBY-equipped hairy root transformation in drug- and hemp-type Cannabis. Plant Biotechnol J 22(7), 1957-1959.

Butler NM, Jansky SH, Jiang JM (2020). First-generation genome editing in potato using hairy root transformation. Plant Biotechnol J 18(11), 2201-2209.

Bai C, Cao YP, Zhao SY, Wu ZY, Dai SJ, Wang HL, Fu CX (2023). Generation of CRISPR/Cas9-mediated mutants in Monochasma savatieri using a hairy root system. Ind Crops Prod 191, 116008.

Cheng Q, Su P, Hu YT, He YF, Gao W, Huang LQ (2014). RNA interference-mediated repression of SmCPS (copalyldiphosphate synthase) expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS. Biotechnol Lett 36(2), 363-369.

Chen X, Zhong ZH, Tang X, Yang SX, Zhang YH, Wang SD, Liu YQ, Zhang Y, Zheng XL, Zhang Y, Feng XZ (2024). Advancing PAM-less genome editing in soybean using CRISPR-SpRY. Hortic Res 11(8), uhae160.

Doudna JA, Charpentier E (2014). The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213), 1258096.

Gantait S, Mukherjee E (2020). Hairy root culture technology: applications, constraints and prospect. Appl Microbiol Biotechnol 105(1), 1-19.

Hu ZB, Du M (2006). Hairy Root and Its Application in Plant Genetic Engineering. J Integr Plant Biol 48(2), 121-127.

He YB, Zhang T, Sun H, Zhan HD, Zhao YD (2020). A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res 7(1), 802-805.

Jiang SL, Li Q, Meng XX, Huang MX, Yao JY, Wang CY, Fang PP, Tao A, Xu JT, Qi JM, Jin SX, Zhang LW (2024). Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute (Corchorus capsularis). Crop J 12(04), 1266-1270.

Koschmieder J, Fehling-Kaschek M, Schaub P, Ghisla S, Brausemann A, Timmer J, Beyer P (2017). Plant-type phytoene desaturase: Functional evaluation of structural implications. PloS one 12(11), e0187628.

Kumar A, Lin H, Li QJ, Ruan YT, Cousins D, Li FY, Gao S, Jackson K, Wen JQ, Murray JD, Xu P (2022). Anthocyanin pigmentation as a quantitative visual marker for arbuscular mycorrhizal fungal colonization of Medicago truncatula roots. New Phytol 236(5), 1988-1998.

Luo K, Di HY, Zhang JY, Wang YR, Li ZQ (2014). Preliminary evaluation of agronomy and quality traits of nineteen Meliotus accessions. Pratac Sci 31, 2125-2134. (in Chinese)
骆凯, 狄红艳, 张吉宇, 王彦荣, 李治钱 (2014). 19份草木樨种质农艺学与品质性状初步评价. 草业科学 31, 2125-2134.

Luo K, Jahufer MZZ, Zhao H, Zhang R, Wu F, Yan Z, Zhang J, Wang Y (2018). Genetic improvement of key agronomic traits in Melilotus albus. Crop Sci 58(1), 285-294.

Liu L, Gallagher J, Arevalo ED, Chen R, Skopelitis T, Wu QY, Bartlett M, Jackson D (2021). Enhancing grain-yield-related traits by CRISPR–Cas9 promoter editing of maize CLE genes. Nat Plants 7(3), 287-294.

Li SN, Lin DX, Zhang YW, Deng M, Chen YX, Lv B, Li BS, Lei Y, Wang YP, Zhao L, Liang YT, Liu JX, Chen KL, Liu ZY, Xiao J, Qiu JL, Gao CX (2022). Genome-edited powdery mildew resistance in wheat without growth penalties. Nature 602(7897), 455-460.

Li G, Liu R, Xu RF, Varshney RK, Ding HF, Li MW, Yan X, Huang SX, Li J, Wang D, Ji YS, Wang CY, He JG, Luo YF, Gao SH, Wei PC, Zong XX, Yang T (2023). Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea (Pisum sativum L.). Crop J 11(01), 132-139.

Liu L, Qu JH, Wang CY, Liu M, Zhang CM, Zhang XY, Guo C, Wu CG, Yang GD, Huang JG, Yan K, Shu HR, Zheng CC, Zhang SZ (2024). An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion. Plant Biotechnol J 22(8), 2093-2103.

Ma XL, Zhang QY, Zhu QL, Liu W, Chen Y, Qiu R, Wang B, Yang ZF, Li HY, Lin YR, Xie YY, Shen RX, Chen SF, Wang Z, Chen YL, Guo JX, Chen LT, Zhao XC, Dong ZC, Liu YG (2015). A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant 8(8), 1274-1284.

Moroz MA, Zurita J, Moroz A, Nikolov E, Likar Y, Dobrenkov K, Lee J, Shenker L, Blasberg R, Serganova I, Ponomarev V (2021). Introducing a new reporter gene, membrane-anchored Cypridina luciferase, for multiplex bioluminescence imaging. Mol Ther-Oncolytics 21, 15-22.

Peel GJ, Pang YZ, Modolo LV, Dixon RA (2009). The LAP1 MYB transcription factor orchestrates anthocyanidin biosynthesis and glycosylation in Medicago. Plant J 59(1), 136-149.

Rogers ME, Colmer TD, Frost K, Henry D, Cornwall D, Hulm E, Deretic J, Hughes SR, Craig AD (2008). Diversity in the genus Melilotus for tolerance to salinity and waterlogging. Plant Soil 304(1-2), 89-101.

Ron M, Kajala K, Pauluzzi G, Wang DX, Reynoso MA, Zumstein K, Garcha J, Winte S, Masson H, Inagaki S, Federici F, Sinha N, Deal RB, Bailey-Serres J, Brady SM (2014). Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166(2), 455-469.

Sherif AAE (2008). Melilotus indicus (L.) All., a salt-tolerant wild leguminous herb with high potential for use as a forage crop in salt-affected soils. Flora 204(10), 737-746.

Tang X, Zheng XL, Qi YP, Zhang DW,Cheng Y, Tang AT, Daniel FV, Zhang Y (2016). A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in Plants. Mol. Plant 9(7), 1088-1091.

Tang X, Ren QR, Yang LJ, Bao Y, Zhong ZH, He Y, Liu SS, Qi CY, Liu BL, Wang Y, Sretenovic S, Zhang YX, Zheng XL, Zhang T, Qi YP, Zhang Y (2019). Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol J 17(7), 1431-1445.

Wu XW, Bowatte S, Zhang JY ,Hou FJ (2020). Evaluation of traits related to the biological nitrogen fixation in Melilotus albus half-sib families. Pratac Sci 37, 728-735. (in Chinese)
吴兴旺, Bowatte S, 张吉宇, 侯扶江 (2020). 白花草木樨半同胞家系的生物固氮性状评价. 草业科学 37, 728-735.

Wang PL (2021). The study on the second recurrent selection for low coumarin content and detection technology of near-infrared model for quality trait in Melilotus. Master's thesis. Lanzhou: Lanzhou University. pp. 1-59. (in Chinese)
王朋磊 (2021). 草木樨低香豆素第二次轮回选育及品质性状近红外测定技术的研究. 硕士论文. 兰州: 兰州大学. pp. 1-59.

Wu F, Duan Z, Xu P, Yan Q, Meng MH, Cao MS, Jones CS, Zong XF, Zhou P, Wang YM, Luo K, Wang SS, Yan ZZ, Wang PL, Di HY, Ouyang ZF, Wang YR, Zhang JY (2021). Genome and systems biology of Melilotus albus provides insights into coumarins biosynthesis. Plant Biotechnol J 20(3), 592-609.

Wu ZG, Singh SK, Lyu RQ, Pattanaik S, Wang Y, Li YQ, Yuan L, Liu YL (2022). Metabolic engineering to enhance the accumulation of bioactive flavonoids licochalcone A and echinatin in Glycyrrhiza inflata (Licorice) hairy roots. Front Plant Sci 13, 932594-932594.

Xv Y, Cao YP, Wang Y, Fu CX, Dai SJ (2019). Agrobacterium rhizogenes-mediated Transformation System of Spinacia oleracea. Chin Bull Bot 54, 515-521. (in Chinese)
徐悦, 曹英萍, 王玉, 付春祥, 戴绍军 (2019). 发根农杆菌介导的菠菜毛状根遗传转化体系的建立. 植物学报 54, 515-521.

Yi XF, Wang CC, Yuan XQ, Zhang M, Zhang CW, Qin TJ, Wang HY, Xu L, Liu LW, Wang Y (2024). Exploring an economic and highly efficient genetic transformation and genome-editing system for radish through developmental regulators and visible reporter. Plant J 120, 1682-1692.

Zabala JM, Marinoni L, Giavedoni JA, Schrauf GE (2018). Breeding strategies in Melilotus albus Desr., a salt-tolerant forage legume. Euphytica 214 (2), 22.

Zhu ZH , Zhao EK, Qian GT, Sun W, Xue JP ,Shi YH (2019). Hairy Root System and Its Application in Medicinal Plants. Mod Chin Med 21, 1475-14811496. (in Chinese)
朱智慧, 晁二昆, 钱广涛, 孙伟, 薛建平, 师玉华 (2019). 药用植物毛状根研究体系及应用方向. 中国现代中药 21, 1475-14811496.

Zhang HL, Cao YP, Zhang H, Xu Y, Zhou CE, Liu WW, Zhu RF, Shang C, Li Jk, Shen ZB, Guo SY, Hu ZB, Fu CX, Sun DQ (2020). Efficient Generation of CRISPR/Cas9-Mediated Homozygous/Biallelic Medicago truncatula Mutants Using a Hairy Root System. Front Plant Sci 11, 294.

Zong XF, Wang SS, Han YY, Zhao Q, Xu P, Yan Q, Wu F, Zhang JY (2021). Genome-wide profiling of the potential regulatory network of lncRNA and mRNA in Melilotus albus under salt stress. Environ Exp Bot 189, 104548.

Zou JP, Meng XB, Hong ZY, Rao YC, Wang KJ, Li JY, Yu H, Wang C (2024). Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in rice. Trends Biotechnol 43(2), 433-446.

Zhao HX, Zhao SY, Cao YP, Jiang XP, Zhao LJ, Li ZM, Wang MQ, Yang RJ, Zhou CN, Wang ZM, Yuan F, Ma DM, Lin H, Liu WW, Fu CX (2024). Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa. Crop J 12(03), 788-795.

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白花草木樨毛状根高效基因组编辑体系的建立

Development of High-efficiency Genome Editing System for Hair Roots in Melilotus albus

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