利用CRISPR/Cas9技术敲除青花菜BoSP11创制自交亲和系

doi:10.16420/j.issn.0513-353x.2023-0065

摘要/Abstract

摘要：

通过CRISPR/Cas9技术敲除SRK基因或者SP11基因可以直接将甘蓝类蔬菜自交不亲和材料改造成自交亲和材料。本研究中根据全基因组重测序获得青花菜（Brassica oleracea var. italic）BoSP11基因序列，构建双靶位点的CRISPR/Cas9基因编辑载体，利用农杆菌介导的遗传转化法对BoSP11进行敲除。结果显示，获得的29株转基因植株中有23株发生基因突变，编辑效率为79.3%。TA克隆测序结果表明有4株为纯合突变，19株为杂合突变。统计纯合突变T₀代自交结籽情况发现花期自交亲和指数高达6.82，为目前所报道的能获得的亲和性最高指数。T₁代植株在隔离大棚中通过蜜蜂授粉平均单株采收种子24.8 g，完全实现不需要人工干预完成亲本材料的自交繁育。

关键词: 青花菜, 甘蓝, CRISPR/Cas9, 基因编辑, BoSP11, 自交亲和系

Abstract:

By using CRISPR/Cas9 technology to knock out SRK gene or SP11 gene，the self- incompatible Brassica oleracea vegetables can be directly transformed into self-compatible materials. In this study，a double target CRISPR/Cas9 gene editing vector was constructed based on the sequence of broccoli（Brassica oleracea var. italic）BoSP11 gene obtained by whole genome sequencing，and then used Agrobacterium mediated genetic transformation to knock out the BoSP11 gene. The results showed that 23 of the 29 transgenic plants had gene mutation，and the editing efficiency was 79.3%. The results of TA clone sequencing showed that there were four homozygous mutations and 19 heterozygous mutations. The statistics of the seed setting of T₀ generation of homozygous mutant plants showed that the self-compatibility index at flowering stage was as high as 6.82，which was the highest compatibility index increase reported so far. The T₁generation plants were pollinated by bees in the isolated greenhouses, and the average seed yield per plant was 24.8 g，which completely conducted the self-pollination of parent materials for seed production without any manual involvement.

Key words: broccoli, cabbage, CRISPR/Cas9, gene editing, BoSP11, self-compatible lines

马存发, 武婷, 赵辉, 张天培, 赵立坚, 余永辉, 肖建成, 李军, 巫水钦. 利用CRISPR/Cas9技术敲除青花菜BoSP11创制自交亲和系[J]. 园艺学报, 2024, 51(3): 509-519.

MA Cunfa, WU Ting, ZHAO Hui, ZHANG Tianpei, ZHAO Lijian, YU Yonghui, XIAO Jiancheng, LI Jun, WU Shuiqin. Creation of Self-compatible Broccoli Lines by Knocking out BoSP11 Gene Through CRISPR/Cas9 Technology[J]. Acta Horticulturae Sinica, 2024, 51(3): 509-519.

图/表 8

参考文献 29

[1]	Doughty J, Dixon S, Hiscock S J, Willis A C, Parkin I P, Dickinson H G. 1998. PCP-A1,a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein,is the product of gametophytic gene expression. Plant Cell, 10 (8):1333-1347. pmid: 9707533
[2]	Eggers E J, Burgt A, Heusden S A W, Vries M E, Visser R G F, Bachem C W B, Pim L. 2021. Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato. Nature Communications,12:4141.
[3]	Fan D, Liu T T, Li C F, Jiao B, Li S, Hou Y S, Luo K M. 2015. Efficient CRISPR/Cas9-mediated targeted mutagenesis in populus in the first generation. Scientific Reports,5:12217.
[4]	Fang Zhi-yuan, Sun Pei-tian, Liu Yu-mei, Yang Li-mei. 1987. Preliminary report on breeding of self-incompatible lines of broccoli. China Vegetables,(1):27-29. (in Chinese)
	方智远, 孙培田, 刘玉梅, 杨丽梅. 1987. 青花菜自交不亲和系选育初报. 中国蔬菜,(1):27-29.
[5]	Goring D R, Rothstein S J. 1992. The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. Plant Cell, 4 (10):1273-1281. doi: 10.1105/tpc.4.10.1273 pmid: 1332796
[6]	Hiscock S J, McInnis S M. 2003. Pollen recognition and rejection during the sporophytic self-incompatibility response: Brassica and beyond. Trends in Plant Science, 8 (12):606-613. pmid: 14659710
[7]	Hu B W, Li D W, Liu X, Qi J J, Gao D L, Zhao S Q, Huang S W, Sun J J, Yang L. 2017. Engineering non-transgenic gynoecious cucumber using an improved transformation protocol and optimized CRISPR/Cas9 system. Molecular Plant,10:1575-1578.
[8]	Huang Jingjing, Liu Wei, Liu Yumei, Han Fengqing, Fang Zhiyuan, Yang Limei, Zhuang Mu, Zhang Yangyong, Lü Honghao, Wang Yong, Ji Jialei, Li Zhansheng. 2022. Creation of fertility restorer materials for Ogura CMS broccoli and the study of its genetic background. Acta Horticulturae Sinica, 49 (3):533-547. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0100
	黄静静, 刘伟, 刘玉梅, 韩风庆, 方智远, 杨丽梅, 庄木, 张扬勇, 吕红豪, 王勇, 季家磊, 李占省. 2022. Ogura CMS 青花菜育性恢复材料创制及其遗传背景研究. 园艺学报, 49 (3):533-547. doi: 10.16420/j.issn.0513-353x.2021-0100
[9]	Kachroo A, Schopfer C R, Nasrallah M E, Nasrallah J B. 2001. Allele-specific receptor-ligand interactions in Brassica self-incompatibility. Science,293:1824-1826.
[10]	Li T D, Yang X P, Yu Y, Si X M, Zhai X W, Zhang H W, Dong W X, Gao C X, Xu C. 2018. Domestication of wild tomato is accelerated by genome editing. Nature Biotechnology,36:1160-1163.
[11]	Li X N, Li H Y, Zhao Y Z, Zong P X, Zhan Z X, Piao Z Y. 2021. Establishment of a simple and efficient Agrobacterium-mediated genetic transformation system to Chinese cabbage(Brassica rapa L. ssp. pekinensis). Horticultural Plant Journal, 7 (2):117-128.
[12]	Li Zhan-sheng, Liu Yu-mei, Fang Zhi-yuan, Yang Li-mei, Zhuang Mu, Zhang Yang-yong, Lü Hong-hao, Wang Yong. 2019. Development status,existing problems and countermeasures of broccoli industry in China. China Vegetables,(4):1-5. (in Chinese)
	李占省, 刘玉梅, 方智远, 杨丽梅, 庄木, 张扬勇, 吕红豪, 王勇. 2019. 我国青花菜产业发展现状、存在问题与应对策略. 中国蔬菜,(4):1-5.
[13]	Li Zhan-sheng, Liu Yu-mei, Han Feng-qing, Fang Zhi-yuan, Zhang Yang-yong, Yang Li-mei, Zhuang Mu, Lü Hong-hao, Wang Yong, Ji Jia-lei. 2021. Research progress on broccoli genetic breeding during‘The Thirteenth Five-year Plan’in China. China Vegetables,(1):33-40. (in Chinese)
	李占省, 刘玉梅, 韩风庆, 方智远, 张扬勇, 杨丽梅, 庄木, 吕红豪, 王勇, 季家磊. 2021. “十三五”我国青花菜遗传育种研究进展. 中国蔬菜,(1):33-40.
[14]	Ma C F, Liu M C, Li Q F, Si J, Ren X S, Song H Y. 2019a. Efficient BoPDS gene editing in cabbage by the CRISPR/Cas9 system. Horticultural Plant Journal, 5 (4):164-169.
[15]	Ma C F, Zhu C Z, Zheng M, Liu M C, Zhang D J, Liu B L, Li Q F, Si J, Ren X S, Song H Y. 2019b. CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system. Horticulture Research,6:20.
[16]	Nasrallah J B, Nasrallah M E. 1993. Pollen-stigma signaling in the sporophytic self-incompatibility response. Plant Cell, 5 (10):1325-1335.
[17]	Stone S L, Anderson E M, Mullen R T, Goring D R. 2003. ARC 1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen. Plant Cell, 15 (4):885-898.
[18]	Sun B, Jiang M, Zheng H, Jian Y, Huang W L, Yuan Q, Zheng A H, Chen Q, Zhang Y T, Lin Y X, Wang Y, Wang X R, Wang Q M, Zhang F, Tang H R. 2020. Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO. Horticulture Research,7:161.
[19]	Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K. 2000. The S receptor kinase determines self-incompatibility in Brassica stigma. Nature, 403 (6772):913-916.
[20]	Takayama S, Shiba H, Iwano M, Shimosato H, Che F S, Kai N, Watanabe M, Suzuki G, Hinata K, Isogai A. 2000. The pollen determinant of self-incompatibility in Brassica campestris. Proceedings of the National Academy of Sciences of the United States of America, 97 (4):1920-1925.
[21]	Wang Dan, Wang Mi, Liu Jun, Zhou Xiaohui, Liu Songyu, Yang Yan, Zhuang Yong. 2022. Cloning of U6 promoters and establishment of CRISPR/Cas 9 mediated gene editing system in eggplant. Acta Horticulturae Sinica, 49 (4):791-800. (in Chinese)
	王丹, 王谧, 刘军, 周晓慧, 刘松瑜, 杨艳, 庄勇. 2022. 茄子 U6 启动子克隆及CRISPR/Cas9介导的基因编辑体系建立. 园艺学报, 49 (4):791-800. doi: 10.16420/j.issn.0513-353x.2020-0265
[22]	Wang Fei. 2020. Construction of expression vectors of knocking out BoPDS and SRK15 gene in Brassica oleracea L. by CRISPR/Cas9 system and genetic transformation[M. D. Dissertation]. Lanzhou: Gansu Agricultural University. (in Chinese)
	王飞. 2020. 利用 CRISPR/Cas9系统构建甘蓝BoPDS 和SRK15基因敲除载体及遗传转化[硕士论文]. 兰州: 甘肃农业大学.
[23]	Xu Z S, Feng K, Xiong A S. 2019. CRISPR/Cas9-mediated multiply targeted mutagenesis in orange and purple carrot plants. Molecular Biotechnology,61:191-199.
[24]	Yang X Y, Yan J B, Zhang Z, Lin T, Xin T X, Wang B W, Wang S H, Zhao J C, Zhang Z H, Lucas W J, Li G H, Huang S W. 2020. Regulation of plant architecture by a new histone acetyltransferase targeting gene bodies. Nature Plants, 6 (7):809-822. doi: 10.1038/s41477-020-0715-2 pmid: 32665652
[25]	Yang Yang. 2009. Study on Prokaryotic expression and interaction between determinant factors of self-incompatibility (SI) and exploration on SI related gene in Brassica oleracea L. var. capitata L. [Ph. D. Dissertation]. Chongqing: Southwest University. (in Chinese)
	杨洋. 2009. 甘蓝SI决定因子的原核表达和相互作用研究以及SI相关基因的探讨[博士论文]. 重庆: 西南大学.
[26]	Ye M W, Peng Z, Tang D, Yang Z M, Li D W, Xu Y M, Zhang C Z, Huang S W. 2018. Generation of self-compatible diploid potato by knockout of S-RNase. Nature Plants,4:651-654.
[27]	Zhang Aiping, Liu Jiangna, Yan Jianjun, Zhang Xiying, Bai Yunfeng. 2022. Progress and prospect of genome editing in tomato. Acta Horticulturae Sinica, 49 (1):221-232. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0976
	张爱萍, 刘江娜, 闫建俊, 张西英, 白云凤. 2022. 番茄基因编辑研究进展和前景. 园艺学报, 49 (1):221-232. doi: 10.16420/j.issn.0513-353x.2020-0976
[28]	Zhao X Y, Yuan K W, Liu Y X, Zhang N, Yang L M, Zhang Y Y, Wang Y, Ji J L, Fang Z Y, Han F Q, Lv H H. 2022. In vivo maternal haploid induction based on genome editing of DMP in Brassica oleracea. Plant Biotechnology Journal,20:2242-2244.
[29]	Zheng Aihong, Zhang Fen, Jiang Min, Yuan Qiao, Jiang Leiyu, Chen Qing, Tang Haoru, Sun Bo. 2019. Targeted editing of BoaZDS by CRISPR/Ca 9 technology in Chinese kale. Acta Horticulturae Sinica, 46 (1):57-64. (in Chinese)
	郑爱红, 张芬, 江敏, 袁巧, 江雷雨, 陈清, 汤浩茹, 孙勃. 2019. 利用CRISPR/Ca9技术靶向编辑芥蓝BoaZDS. 园艺学报, 46 (1):57-64. doi: 10.16420/j.issn.0513-353x.2018-0426

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence	产物大小/bp Product size
BoSP11克隆 Cloning the BoSP11	2MSP11-F1	TTTCTGCGAGTAAAAGAGAATC	1 029
BoSP11克隆 Cloning the BoSP11	2MSP11-R1	CTATTTACAATCGCAAGAATAAGTACC
靶序列合成 Construction of target site	sgBoSP11-F1	TGCATGTTTCATATTCATCGTTTC	20
	sgBoSP11-R1	AAACGAAACGATGAATATGAAACA
	sgBoSP11-F2	TGCAGGTAAGCGCAAGAAGCCTTC	20
	sgBoSP11-R2	AAACGAAGGCTTCTTGCGCTTACC
靶位点序列扩增 Amplification of target region	BoSP11-F1	TCTGCGAGTAAAAGAGAATCTTTA	540
	BoSP11-R1	TAGTGATATTGGAAGCGGTACAGG
	BoSP11-F2	AGTAACCAATCGGATGAACTATGT	459
	BoSP11-R2	TCGCAAGAATAAGTACCCATTTGA
转基因检测 Detection of the transformation	Cas9-F1	CAAGTACGTGAACTTCCTCTACC	404
	Cas9-R1	GCTGGGAAAGGTCGATACGAGTC
	Cas9-F2	GACGTCGTCCAATTCTGCTAACCA	464
	Cas9-R2	CGCCGATGAGGTTCTTCTTGATGG

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence	产物大小/bp Product size
BoSP11克隆 Cloning the BoSP11	2MSP11-F1	TTTCTGCGAGTAAAAGAGAATC	1 029
BoSP11克隆 Cloning the BoSP11	2MSP11-R1	CTATTTACAATCGCAAGAATAAGTACC
靶序列合成 Construction of target site	sgBoSP11-F1	TGCATGTTTCATATTCATCGTTTC	20
	sgBoSP11-R1	AAACGAAACGATGAATATGAAACA
	sgBoSP11-F2	TGCAGGTAAGCGCAAGAAGCCTTC	20
	sgBoSP11-R2	AAACGAAGGCTTCTTGCGCTTACC
靶位点序列扩增 Amplification of target region	BoSP11-F1	TCTGCGAGTAAAAGAGAATCTTTA	540
	BoSP11-R1	TAGTGATATTGGAAGCGGTACAGG
	BoSP11-F2	AGTAACCAATCGGATGAACTATGT	459
	BoSP11-R2	TCGCAAGAATAAGTACCCATTTGA
转基因检测 Detection of the transformation	Cas9-F1	CAAGTACGTGAACTTCCTCTACC	404
	Cas9-R1	GCTGGGAAAGGTCGATACGAGTC
	Cas9-F2	GACGTCGTCCAATTCTGCTAACCA	464
	Cas9-R2	CGCCGATGAGGTTCTTCTTGATGG

编号 No.	蕾期自交 Self-pollinated at bud stage			花期自交 Self-pollinated at flowering stage
编号 No.	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index
#7	144	1 172	8.14	187	1 276	6.82
#16	156	1 084	6.95	378	2 071	5.48
#24	96	645	6.72	132	638	4.84
WT	83	387	4.66	122	21	0.17

编号 No.	蕾期自交 Self-pollinated at bud stage			花期自交 Self-pollinated at flowering stage
编号 No.	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index
#7	144	1 172	8.14	187	1 276	6.82
#16	156	1 084	6.95	378	2 071	5.48
#24	96	645	6.72	132	638	4.84
WT	83	387	4.66	122	21	0.17

株系 Line	检测株数 Number of detected plants	无转基因成分株数 Number of transgenic free plants	定植株数 Number of plants planted	采种质量/g Seed harvest weight	平均单株收种质量/g Average seed weight per plant
#7	447	111	86	2 130	24.8
#16	450	107	79	1 580	20.0
#24	444	94	65	1 050	16.2
总计Total	1 341	312	230	4 760	20.7