利用基因编辑技术提高不结球白菜抗坏血酸的含量

doi:10.16420/j.issn.0513-353x.2025-0036

摘要/Abstract

摘要：

为提高不结球白菜抗坏血酸含量，以不结球白菜品种‘黄玫瑰’为材料，选择不结球白菜GGP基因的uORF序列为靶标，构建CRISPR/Cas9基因组编辑载体，通过发根农杆菌介导的遗传转化方法获得了4种不同基因编辑类型‘黄玫瑰’GGP基因uORF的编辑植株，检测再生植株的T₁代纯合绿色植株的抗坏血酸含量，再与空载转基因再生植株相比，抗坏血酸含量提高了26.2% ~ 67.1%。这为利用CRISPR/Cas9系统在不结球白菜中开展基因编辑和生物育种研究提供了依据。

关键词: 不结球白菜, 上游开放阅读框, 遗传转化, 抗坏血酸, 基因编辑

Abstract:

To increase the ascorbic acid content in non-heading Chinese cabbage，the variety‘Yellow Rose’was used as the experimental material. Targeting the uORF sequence of the GGP gene in non-heading Chinese cabbage，a CRISPR/Cas9 genome editing vector was constructed. Through Agrobacterium rhizogenes-mediated genetic transformation，four different gene-edited types of the GGP gene uORF in‘Yellow Rose’were obtained. The ascorbic acid content was measured in the T₁ generation homozygous green plants of the regenerated edited lines，showing a 26.2% to 67.1% increase compared to empty vector transgenic regenerated plants. This study provides a foundation for applying the CRISPR/Cas9 system in gene editing and molecular breeding research in non-heading Chinese cabbage.

Key words: non-heading Chinese cabbage, upstream open reading frame, genetic transformation, ascorbic acid, gene editing

王文龙, 刘照坤, 鲁文君, 王耀龙, 李晓锋, 朱红芳, 刘同坤, 李英, 侯喜林, 张昌伟. 利用基因编辑技术提高不结球白菜抗坏血酸的含量[J]. 园艺学报, 2025, 52(5): 1317-1325.

WANG Wenlong, LIU Zhaokun, LU Wenjun, WANG Yaolong, LI Xiaofeng, ZHU Hongfang, LIU Tongkun, LI Ying, HOU Xilin, ZHANG Changwei. Enhancing Ascorbic Acid Content in Non-heading Chinese Cabbage Using Gene Editing Technology[J]. Acta Horticulturae Sinica, 2025, 52(5): 1317-1325.

图/表 6

图1 WIP-RUBY-cas9载体

Fig. 1 WIP-RUBY-cas9 gene editing vector

表1 引物序列

Table 1 Primer sequences

引物名称Primer name	引物序列（5′-3′）Primer sequence
靶点sgRNA序列 Target sgRNA sequence	GGAGCTCTGCCGTCTGAAGG
RUBY-F	TGGATCATGCGACCCTCGCC
RUBY-R	GAACACGGCGATCAGCTTGT
Cas9-F	GACAAGAAGTACTCCATCGG
Cas9-R	CTCGATCTTCTTGAAGTAGT
Bra026398-F	AAGCCATTCAATCTCCCCGAATC
Bra026398-R	TCTCAGACAGACAGAAAGACTTC

图2 发根农杆菌介导的转基因‘黄玫瑰’植株生长过程 A：下胚轴准备；B：芽诱导；C；幼苗培养；D：移栽；E：抽薹；F：开花结籽；G：收种；H：播种和发芽；I：T1代植株

Fig. 2 Agrobacterium rhizogenes-mediated growth process of transgenic‘Yellow Rose’plants A：Hypocotyl preparation；B：Bud induction；C：Seedling cultivation；D：Transplant；E：Bolting；F：Flowering and seed setting；G：Harvesting；H：Sowing and germination；I：T1 generation plants

图3 Cas9融合基因PCR检测 M：DL 2000 marker；N：水；WT：野生型；V：空载体；P：阳性质粒；1 ~ 10：抗性植株

Fig. 3 Cas9 fusion gene PCR detection M：DL 2000 marker；N：Water；WT：Wild type；V：Empty vector；P：Positive plasmid；1-10：Resistant plant

图4 CRISPR/Cas9诱导的uORFBrGGP突变类型 A：虚线相连红色方框代表碱基替换，黑色方框加箭头代表碱基缺失（uORFBrGGP1 ~ uORFBrGGP3），相同碱基序列用黑色方框框出，缺失部分用红色下划加箭头线标记（uORFBrGGP4）；B：蓝色代表uORF序列，红色代表碱基替换，连字符代表碱基缺失，下划线标记sgRNA， Hi-TOM 测序结果在右侧表示；D：碱基缺失；SNP：碱基替换。WT：野生型

Fig. 4 Types of uORFBrGGP mutations induced by CRISPR/Cas9 A：The dotted red box represents the base substitution，the black box with the arrow represents the base deletion（uORFBrGGP1 ~ uORFBrGGP3），the same base sequence is framed with the black box，and the missing part is marked with a red underline and arrow line（uORFBrGGP4）；B：Blue represents the uORF sequence，red represents base substitution，hyphen represents base deletion，and underlined sgRNA；Hi-TOM sequencing results are shown on the right，D：Base deletion；SNP：Base substitution. WT：Wild type

图5 T1代纯合绿色植株新生叶片抗坏血酸含量比较 WT为野生型，EV为空载转基因植株，M0为CRISPR/Cas9载体阳性未编辑植株，M1 ~ M4为CRISPR/Cas9载体阳性突变植株uORFBrGGP1 ~ uORFBrGGP4，与空载转基因植株相比，经双尾t检验，*** P < 0.001，ns：无显著性差异

Fig. 5 Comparison of ascorbic acid content in new leaves of T1 generation homozygous green plants. WT was wild-type，EV was an unloaded transgenic plant，M0 was a CRISPR/Cas9 vector positive unedited plant，M1-M4 was a CRISPR/Cas9 vector positive mutant plant uORFBrGGP1-uORFBrGGP4，compared to empty vector transgenic plants，***P < 0.001，ns：no significant difference by two-tailed t-test

参考文献 27

[14]	Ma X L, Zhu Q L, Chen Y L, Liu Y G. 2016. CRISPR/Cas 9 platforms for genome editing in plants:developments and applications. Molecular Plant, 9 (7):961-974.
[15]	Mou R, Niu R X, Yang R Y, Xu G Y. 2024. Engineering crop performance with upstream open reading frames. Trends in Plant Science, 30 (3):1360-1385.
[16]	Niu R X, Zhou Y L, Zhang Y, Mou R, Tang Z J, Wang Z, Zhou G L, Guo S B, Yuan M, Xu G Y. 2020. uORFlight:a vehicle toward uORF-mediated translational regulation mechanisms in eukaryotes. Database,baaa007.
[17]	Ntagkasa N, Woltering E J, Marcelis L F M. 2018. Light regulates ascorbate in plants:an integrated view on physiology and biochemistry. Environ Exp Bot, 147 (1):271-280.
[18]	Pan C T, Li L Y, Liu Q X, He Y J, Wang J, Chen L F, Lu G. 2017. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific Reports, 6:24765.
[19]	Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M. 2011. Global quantification of mammalian gene expression control. Nature, 473:337-342.
[20]	von Arnim A G, Jia Q, Vaughn J N. 2014. Regulation of plant translation by upstream open reading frames. Plant Sci, 214:1-12. doi: 10.1016/j.plantsci.2013.09.006 pmid: 24268158
[21]	Wang Chenyu, Liu Mengjun, Wang Lixin, Liu Zhiguo. 2024. Research progress of CRISPR/Cas 9 technology and its application in horticultural plants. Acta Horticulturae Sinica, 51 (7):1439-1454. (in Chinese)
	王晨雨, 刘孟军, 王立新, 刘志国. 2024. CRISPR/Cas9技术研究进展及其在园艺植物中的应用进展. 园艺学报, 51 (7):1439-1454.
[22]	Wang S J, Wang G, Li H L, Li F, Wang J B. 2023. Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in‘Feizixiao’litchi. Horticultural Plant Journal, 9 (5):947-957.
[23]	Wang Y L, Yang X D, Wang W L, Wang Y, Chen X S, Wu H, Gao Z Y, Xu H H, Liu T K, Li Y, Xiao D, Liu W S, Hou X L, Zhang C W. 2024. Efficient genetic transformation and gene editing of Chinese cabbage using Agrobacterium rhizogenes. Plant Physiology, 197 (2):kiae543.
[24]	Xu G Y, Yuan M, Ai C R, Liu L J, Zhuang E, Karapetyan S, Wang S P, Dong X N. 2017. uORF-mediated translation allows engineered plant disease resistance without fitness costs. Nature, 545 (7655):491-494.
[25]	Zhang H, Zhang J S, Wei P L, Zhang B T, Gou F, Feng Z Y, Mao Y F, Yang L, Zhang H, Xu N F, Zhu J K. 2014. The CRISPR/Cas 9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 12 (6):797-807. doi: 10.1111/pbi.12200 pmid: 24854982
[26]	Zhang H W, Si X M, Ji X, Fan R, Liu J X, Chen K L, Wang D W, Gao C X. 2018. Genome editing of upstream open reading frames enables translational control in plants. Nat Biotechnol, 36:894-898. doi: 10.1038/nbt.4202 pmid: 30080209
[1]	Asensi-Fabado M A, Munné-Bosch S. 2010. Vitamins in plants:occurrence,biosynthesis and antioxidant function. Trends Plant Sci, 15:582-592. doi: 10.1016/j.tplants.2010.07.003 pmid: 20729129
[2]	Cui Y, Cao Q, Li Y P, He M Q, Liu X G. 2023. Advances in cis-element and natural variation-mediated transcriptional regulation and applications in gene editing of major crops. Journal of Experimental Botany, 74 (18):5441-5457.
[3]	Guo Ye, Wan Dongyan, Chai Zhuangzhuang, Wang Yuejin, Wen Yingqiang. 2019. Knock-out analysis of VviPDS1 gene using CRISPR/Cas 9 in grapevine. Acta Horticulturae Sinica, 46 (4):623-634. (in Chinese)
	郭晔, 万东艳, 柴壮壮, 王跃进, 文颖强. 2019. 利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究. 园艺学报, 46 (4):623-634. doi: 10.16420/j.issn.0513-353x.2018-0626
[4]	Hou Xilin, Li Ying, Liu Tongkun. 2022. Advances in genetic breeding and molecular biology of non-heading Chinese cabbage. Journal of Nanjing Agricultural University, 45 (5):864-873. (in Chinese)
	侯喜林, 李英, 刘同坤. 2022. 不结球白菜遗传育种与分子生物学研究进展. 南京农业大学学报, 45 (5):864-873.
[5]	Hu H, Yu F Q. 2024. Studies on the temporal,structural,and interacting features of the clubroot resistance gene Rcr1using CRISPR/Cas9-based systems. Horticultural Plant Journal, 10 (4):1035-1048.
[6]	Hu J H, Miller S M, Geurts M H, Tang W, Chen L, Sun N, Zeina C M, Gao X, Rees H A, Lin Z, Liu D R. 2018. Evolved Cas 9 variants with broad PAM compatibility and high DNA specificity. Nature, 556 (7699):57-63.
[7]	Jiang Rui, Zhou Shengjun, Zhu Yuqiang, Wang Xin, Tan Jihong, Wang Huasen, Zhang Peng. 2024. Research progress of CRISPR/Cas 9 gene editing techniques in melon crops. Acta Horticulturae Sinica, 51 (7):1683-1694. (in Chinese)
	江睿, 周胜军, 朱育强, 王欣, 谭继宏, 王华森, 张鹏. 2024. 瓜类作物CRISPR/Cas9基因编辑技术应用研究进展. 园艺学报, 51 (7):1683-1694.
[8]	Komor A C, Kim Y B, Packer M S, Zuris J A, Liu D R. 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533 (7603):420-424.
[9]	Laing W A, Martínez-Sánchez M, Wright M A, Bulley S M, Brewster D, Dare A P, Rassam M, Wang D, Storey R, Macknight R C, Hellens R P. 2015. An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. The Plant Cell, 27 (3):772-786.
[10]	Liang X H, Shen W, Sun H, Migawa M T, Vickers T A, Crooke S T. 2016. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat Biotechnol, 34:875-880.
[11]	Liu Y, Zhang C L, Wang X F, Li X M, You C X. 2022. CRISPR/Cas 9 technology and its application in horticultural crops. Horticultural Plant Journal, 8 (4):395-407.
[12]	Lu Di, Hu Chunhua, Sheng Ou, Yang Qiaosong, Dou Tongxin, He Weidi, Deng Guiming, Gao Huijun, Liu Siwen, Li Chunyu, Dong Tao, Yi Ganjun, Bi Fangcheng. 2024. Advances in the CRISPR/Cas DNA-free genome editing in plant. Acta Horticulturae Sinica, 51 (8):1927-1948. (in Chinese) doi: 10.16420/j.issn.0513-353x.2023-0894
	陆地, 胡春华, 盛鸥, 杨乔松, 窦同心, 何维弟, 邓贵明, 高慧君, 刘思文, 李春雨, 董涛, 易干军, 毕方铖. 2024. 植物CRISPR/Cas无外源DNA基因组编辑技术研究进展. 园艺学报, 51 (8):1927-1948.
[13]	Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen L Y, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G. 2015. A robust CRISPR/Cas 9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 8 (8):1274-1284.
[27]	Zhu Zongcai, Wang Zhijun, Gao Neng, Wu Dongmei. 2024. Application of CRISPR/Cas 9 gene editing technology in improvement of plant disease resistance:a review. Jiangsu Agricultural Sciences, 52 (3):1-11. (in Chinese)
	朱宗财, 王志军, 高能, 武冬梅. 2024. CRISPR/Cas9基因编辑技术在植物抗病性改良中的应用综述. 江苏农业科学, 52 (3):1-11.