利用CRISPR/Cas9创建osarf7突变体及其农艺性状调查

doi:10.16819/j.1001-7216.2022.210117

摘要/Abstract

摘要：

【目的】探究OsARF7基因在水稻生长发育中的生物学功能及其对水稻农艺性状的影响。【方法】利用CRISPR/Cas9技术，在粳稻中花11背景下对OsARF7进行定向编辑，获得OsARF7基因突变植株，并考查其农艺性状。【结果】获得22株T₀代转基因株系，经PCR扩增潮霉素基因鉴定出20株阳性植株。提取T₂代转基因植株的基因组DNA，通过对OsARF7的2个编辑位点区域DNA片段进行PCR扩增及测序分析，筛选到15种突变类型的纯合基因型植株。基因表达分析显示，osarf7突变体的OsARF7和生长素转运关键基因OsPIN1b、OsPIN2和OsLAX2的表达量均显著低于野生型。田间调查发现，与野生型相比，osarf7突变体的分蘖成穗率显著降低，其中，分蘖数比野生型增多的osarf7突变体，有效穗数基本保持不变，而分蘖数与野生型相近的osarf7突变体，有效穗数少于野生型，最终表现为无效分蘖明显增多，分蘖成穗率降低。【结论】OsARF7基因与生长素调控水稻分蘖发生相关，并影响分蘖芽的生长发育及成穗;OsARF7基因突变会导致迟发分蘖芽不能正常发育成穗;粳稻OsARF7突变体的创建对揭示OsARF7基因生理功能和探索生长素促控分蘖发生与成穗机理等具有积极意义。

关键词: 水稻, 基因编辑, OsARF7, CRISPR/Cas9, 生长素

Abstract:

【Objective】The purpose is to offer a deep insight into the biological function of OsARF7 and its effect on agronomic traits during the growth and development stage of rice. 【Method】CRISPR/Cas9 technology was employed to edit the gene sequence of OsARF7 and to obtain the mutants of japonica variety Zhonghua 11. The agronomic traits of the mutants were investigated in a field experiment. 【Result】Twenty-two T₀ transgenic lines were obtained through Agrobacterium-mediated infection of japonica rice Zhonghua 11 callus, and twenty positive transgenic lines were distinguished by screening the gene encoding hygromycin. For T₂ transgenic lines, the regions of genomic DNA including two target sites were detected and identified by PCR amplifying and sequencing, and fifteen homozygous mutations were confirmed. Gene expression results displayed that the expression levels of OsARF7, OsPIN1b, OsPIN2, and OsLAX2 in the osarf7 mutants were significantly lower than those in the wild type. In comparison with the wild-type plants, osarf7 mutants showed a significant decrease in the productive panicle percentage. Among them, the osarf7 mutants with more tillers than the wild type showed a similar effective panicle number to the wild type, and the osarf7 mutants with the same tiller number as the wild type presented a decrease in the effective panicle number per plant in paddy field conditions, resulting in increased non-productive tillers and decreased effective panicle percentage. 【Conclusion】OsARF7 was involved in the emergence and development of tiller bud dominated by auxin, and the successful construction of osarf7 mutants plays a positive role in revealing the biological function of OsARF7 and the mechanism of tiller formation and development dominated by auxin.

Key words: rice, gene editing, OsARF7, CRISPR/Cas9, auxin

李兆伟, 孙聪颖, 零东兰, 曾慧玲, 张晓妹, 范凯, 林文雄. 利用CRISPR/Cas9创建osarf7突变体及其农艺性状调查[J]. 中国水稻科学, 2022, 36(3): 237-247.

LI Zhaowei, SUN Congying, LING Donglan, ZENG Huiling, ZHANG Xiaomei, FAN Kai, LIN Wenxiong. Construction of osarf7 Mutants in Rice Based on CRISPR/Cas9 Technology and Investigation on Their Agronomic Traits[J]. Chinese Journal OF Rice Science, 2022, 36(3): 237-247.

图/表 9

参考文献 34

[1]	郭韬, 余泓, 邱杰, 李家洋, 韩斌, 林鸿宣. 中国水稻遗传学研究进展与分子设计育种[J]. 中国科学: 生命科学, 2019, 49: 1-28.
	Guo T, Yu H, Qiu J, Li J Y, Han B, Lin H X. Advances in rice genetics and breeding by molecular design in China[J]. Scientia Sinica Vitae, 2019, 49: 1-28. (in Chinese with English abstract)
[2]	Woodward A W, Bartel B. Auxin: Regulation, action, and interaction[J]. Annals of Botany, 2005, 95(5): 707-735.
[3]	Guilfoyle T J, Hagen G. Auxin response factors[J]. Current Opinion in Plant Biology, 2007, 10: 453-460.
[4]	Simonini S, Deb J, Moubayidin L, Stephenson P, Valluru M, Freire-Rios A, Sorefan K, Weijers D, Friml J, Østergaard L. A noncanonical auxin-sensing mechanism is required for organ morphogenesis in Arabidopsis[J]. Genes & Development, 2016, 30: 2286-2296.
[5]	Guilfoyle T, Hagen G, Ulmasov T, Murfett J. How does auxin turn on genes[J]. Plant Physiology, 1998, 118: 341-347.
[6]	Okushima Y, Overvoorde P J, Arima K, Alonso J M, Chan A, Chang C, Ecker J R, Hughes B, Lui A, Nguyen D, Onodera C, Quach H, Smith A, Yu G, Theologis A. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19[J]. Plant Cell, 2005, 17(2): 444-463.
[7]	Wang D, Pei K, Fu Y, Sun Z, Li S, Liu H, Tang K, Han B, Tao Y. Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa)[J]. Gene, 2007, 394(1): 13-24.
[8]	Xing H, Pudake R N, Guo G, Xing G, Hu Z, Zhang Y, Sun Q, Ni Z. Genome-wide identification and expression profiling of auxin response factor (ARF) gene family in maize[J]. BMC Genomics, 2011, 12(1): 178.
[9]	Ha C V, Le D T, Nishiyama R, Watanabe Y, Sulieman S, Tran U T, Mochida K, Dong N V, Yamaguchi-Shinozaki K, Shinozaki K, Tran LSP. The auxin response factor transcription factor family in soybean: genome-wide identification and expression analyses during development and water stress[J]. DNA Research, 2013, 20(5): 511-524.
[10]	Wu J, Wang F, Cheng L, Kong F, Peng Z, Liu S, Yu X, Lu G. Identification, isolation and expression analysis of auxin response factor (ARF) genes in Solanum lycopersicum[J]. Plant Cell Reports, 2011, 30(11): 2059-2073.
[11]	Liu S Q, Hu L F. Genome-wide analysis of the auxin response factor gene family in cucumber[J]. Genetics and Molecular Research, 2013, 12(4): 4317-4331.
[12]	刘瑞娥, 胡长贵, 孙玉强. 植物生长素反应因子研究进展[J]. 植物生理学报, 2011, 47(7): 669-679.
	Liu R E, Hu C G, Sun Y Q. Advances in plant auxin response factors[J]. Journal of Plant Physiology, 2011, 47(7): 669-679. (in Chinese with English abstract)
[13]	张赛娜. OsARF19调控水稻叶夹角的分子机制[D]. 杭州: 浙江大学, 2014: 32-33.
	Zhang S N. Molecular mechanism of OsARF19 controlling leaf angle in rice (Oryza sativa)[D]. Hangzhou: Zhejiang University, 2014: 32-33. (in Chinese with English abstract)
[14]	Ulmasov T, Hagen G, Guilfoyle T J. Activation and repression of transcription by auxin response factors[J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96: 5844-5849.
[15]	Waller F, Furuya M, Nick P. OsARF1, an auxin response factor from rice, is auxin-regulated and classifies as a primary auxin responsive gene[J]. Plant Molecular Biology, 2002, 50(3): 415-425.
[16]	汪高航. 利用点突变研究水稻OsARF的功能[D]. 杭州: 浙江大学, 2010: 22-24.
	Wang G H. Preliminary study of the functions of OsARFs by mutation[D]. Hangzhou: Zhejiang University, 2010: 22-24. (in Chinese with English abstract)
[17]	淳俊, 王文国, 王胜华, 陈放. 水稻(Oryza sativa L.)愈伤组织发育过程Auxin-miR167-ARF8信号通路的研究[J]. 四川大学学报, 2013, 50(4): 863-868.
	Chun J, Wang W G, Wang S H, Chen F. The study on Auxin-miR167-ARF8 signal pathway during the growth and development process of rice callus[J]. Journal of Sichuan University, 2013, 50(4): 863-868. (in Chinese with English abstract)
[18]	Shen C, Yue R, Yang Y, Zhang L, Sun T, Tie S, Wang H. OsARF16 is involved in cytokinin-mediated inhibition of phosphate transport and phosphate signaling in rice (Oryza sativa L.)[J]. PLoS One, 2014, 9(11): e112906.
[19]	Shen C, Yue R, Sun T, Zhang L, Yang Y, Wang H. OsARF16, a transcription factor regulating auxin redistribution, is required for iron deficiency response in rice (Oryza sativa L.)[J]. Plant Science, 2015, 231: 148-158.
[20]	Sato Y, Nishimura A, Ito M, Ashikari M, Hirano H Y, Matsuoka M. Auxin response factor family in rice[J]. Genes & Genetic Systems, 2001, 76: 373-380.
[21]	Ellis C M, Nagpal P, Young J C, Hagen G, Guilfoyle T J, Reed J W. Auxin response factor1 and Auxin response factor1 regulate senescence and floral organ abscission in Arabidopsis thaliana[J]. Development, 2005, 132(20): 4563-4574.
[22]	Doudna J A, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346(6213): 1258096.
[23]	Belhaj K, Chaparro-Garcia A, Kamoun S, Patron N J, Nekrasov V. Editing plant genomes with CRISPR/ Cas9[J]. Current Opinion in Biotechnology, 2015, 32: 76-84.
[24]	王加峰, 郑才敏, 刘维, 罗文龙, 王慧, 陈志强, 郭涛. 基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建[J]. 作物学报, 2016, 42(8): 1160-1167.
	Wang J F, Zheng C M, Liu W, Luo W L, Wang H, Chen Z Q, Guo T. Construction of tgw6 mutants in rice based on CRISPR/Cas9 technology[J]. Acta Agronomy Sinica, 2016, 42(8): 1160-1167. (in Chinese with English abstract)
[25]	周延彪, 赵新辉, 唐晓丹, 周在为, 庄楚雄, 杨远柱. 基于CRISPR/Cas9技术的水稻反光敏不育基因csa突变体的获得[J]. 杂交水稻, 2018, 33(6): 68-74.
	Zhou Y B, Zhao X H, Tang X D, Zhou Z W, Zhuang C X, Yang Y Z. Acquisition of mutants of the reverse photoperiod-sensitive genic male sterility gene csa in rice based on CRISPR/Cas9 technology[J]. Hybrid Rice, 2018, 33(6): 68-74. (in Chinese with English abstract)
[26]	黄忠明, 周延彪, 唐晓丹, 赵新辉, 周在为, 符星学, 王凯, 史江伟, 李艳锋, 符辰建, 杨远柱. 基于CRISPR/Cas9技术的水稻温敏不育基因tms5突变体的构建[J]. 作物学报, 2018, 44(6): 844-851.
	Huang Z M, Zhou Y B, Tang X D, Zhao X H, Zhou Z W, Fu X X, Wang K, Shi J W, Li Y F, Fu C J, Yang Y Z. Construction of tms5 mutants in rice based on CRISPR/Cas9 technology[J]. Acta Agronomy Sinica, 2018, 44(6): 844-851. (in Chinese with English abstract)
[27]	Ma X L, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y G. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicotplants[J]. Molecular Plant, 2015, 8: 1274-1284.
[28]	Qi Y H, Wang S K, Shen C J, Zhang S N, Chen Y, Xu Y X, Liu Y, Wu Y R, Jiang D A. OsARF12, a transcription activator on auxin response gene, regulates root elongation and affects iron accumulation in rice (Oryza sativa)[J]. New Phytologist, 2012, 193: 109-120.
[29]	Bibikova M, Beumer K, Trautman J K, Carroll D. Enhancing gene targeting with designed zinc finger nucleases[J]. Science, 2003, 300: 764.
[30]	Huang P, Xiao A, Zhou M G, Zhu Z Y, Lin S, Zhang B. Heritable gene targeting in zebrafish using customized TALENs[J]. Nature Biotechnology, 2011, 29: 699-700.
[31]	季新, 李飞, 晏云, 孙红正, 张静, 李俊周, 彭廷, 杜彦修, 赵全志. 基于CRISPR/Cas9系统的水稻光敏色素互作因子OsPIL15基因编辑[J]. 中国农业科学, 2017, 50(15): 2861-2871.
	Ji X, Li F, Yan Y, Sun H Z, Zhang J, Li J Z, Peng T, Du Y X, Zhao Q Z. CRISPR/Cas9 system-based editing of phytochrome-interacting factor OsPIL15[J]. Scientia Agricultura Sinica, 2017, 50(15): 2861-2871. (in Chinese with English abstract)
[32]	唐丽, 李曜魁, 张丹, 毛毕刚, 吕启明, 胡远艺, 韶也, 彭彦, 赵炳然, 夏石头. 基于基因组编辑技术的水稻靶向突变特征及遗传分析[J]. 遗传, 2016, 38(8): 746-755.
	Tang L, Li Y K, Zhang D, Mao B G, Lü Q M, Hu Y Y, Shao Y, Peng Y, Zhao B R, Xia S T. Characteristic and inheritance analysis of targeted mutagenesis mediated by genome editing in rice[J]. Hereditas, 2016, 38(8): 746-755. (in Chinese with English abstract)
[33]	Xu M, Zhu L, Shou H, Wu P. A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice[J]. Plant Cell Physiology, 2005, 46(10): 1674-168.
[34]	Chen Y, Fan X, Song W, Zhang Y, Xu G. Over- expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1[J]. Plant Biotechnology Journal, 2012, 10(2): 139-149.

引物名称Primer name	引物序列Primer sequence (5′-3′)	用途Usage
OsARF7-T1-fwd	gccgACACGAACCTGTGCCACCGC	靶序列1构建
OsARF7-T1-rev	aaacGCGGTGGCACAGGTTCGTGT	Construction of the target sequence 1
OsARF7-T2-fwd	gttGAGCTATGGCATGCTTGCGC	靶序列2构建
OsARF7-T2-rev	aaacGCGCAAGCATGCCATAGCT	Construction of the target sequence 2
Hyp-F	ACGGTGTCGTCCATCACAGTTTGCC	阳性转基因植株鉴定
Hyp-R	TTCCGGAAGTGCTTGACATTGGGGA	Identification of the positive transgenic plant
OsARF7-T1-F	ACCACCACCTCCTCCTCAC	靶点1测序检测
OsARF7-T1-R	TCGCACGAATCAGCACAAC	Sequencing for target 1
OsARF7-T2-F	TTACGGATCTGAGAACCTG	靶点2测序检测
OsARF7-T2-R	TAACTACATTGCCGAAGAA	Sequencing for target 2

引物名称Primer name	引物序列Primer sequence (5′-3′)	用途Usage
OsARF7-T1-fwd	gccgACACGAACCTGTGCCACCGC	靶序列1构建
OsARF7-T1-rev	aaacGCGGTGGCACAGGTTCGTGT	Construction of the target sequence 1
OsARF7-T2-fwd	gttGAGCTATGGCATGCTTGCGC	靶序列2构建
OsARF7-T2-rev	aaacGCGCAAGCATGCCATAGCT	Construction of the target sequence 2
Hyp-F	ACGGTGTCGTCCATCACAGTTTGCC	阳性转基因植株鉴定
Hyp-R	TTCCGGAAGTGCTTGACATTGGGGA	Identification of the positive transgenic plant
OsARF7-T1-F	ACCACCACCTCCTCCTCAC	靶点1测序检测
OsARF7-T1-R	TCGCACGAATCAGCACAAC	Sequencing for target 1
OsARF7-T2-F	TTACGGATCTGAGAACCTG	靶点2测序检测
OsARF7-T2-R	TAACTACATTGCCGAAGAA	Sequencing for target 2

编号 Number	潜在脱靶位置 Putative off-target locus	潜在脱靶序列 Sequence of the putative off-target site (5′-3′)	错配碱基数 No. of mismatched bases	测试株数 No. of plants tested
Off-target 1-1	Chr08: 12898391-12898413	ACCCTAACCTCTCCCACCGC	4	9
Off-target 1-2	Chr10: 15856432-15856454	AAACGGACGTGTGCCGCCGC	4	9
Off-target 1-3	Chr02: 27855035-27855057	AGACGGGCCTGTGCCAACGC	4	9
Off-target 1-4	Chr12: 12787156-12787178	CCAAGAACCTGCGCCGCCGC	4	9
Off-target 1-5	Chr07: 2126716-2126738	ACAGCAGCCGGTGCCACCGC	4	9
Off-target 2-1	Chr12: 26007034-26007056	GAGCTGTGGCATGCATGCGC	2	10
Off-target 2-2	Chr02: 3488312-3488334	GAGTTGTGGCATGCATGCGC	3	9
Off-target 2-3	Chr04: 34285188-34285210	GAGCTATGGCACGCCTGCGC	2	8
Off-target 2-4	Chr06: 4926812-4926834	GAGCTATGGCATGCTTGTGC	1	10
Off-target 2-5	Chr04: 22007551-22007573	GAGCTATGGCATGCTTGTGC	1	10
Off-target 2-6	Chr01: 40696054-40696076	GAGCTGTGGCACGCCTGCGC	3	10

编号 Number	潜在脱靶位置 Putative off-target locus	潜在脱靶序列 Sequence of the putative off-target site (5′-3′)	错配碱基数 No. of mismatched bases	测试株数 No. of plants tested
Off-target 1-1	Chr08: 12898391-12898413	ACCCTAACCTCTCCCACCGC	4	9
Off-target 1-2	Chr10: 15856432-15856454	AAACGGACGTGTGCCGCCGC	4	9
Off-target 1-3	Chr02: 27855035-27855057	AGACGGGCCTGTGCCAACGC	4	9
Off-target 1-4	Chr12: 12787156-12787178	CCAAGAACCTGCGCCGCCGC	4	9
Off-target 1-5	Chr07: 2126716-2126738	ACAGCAGCCGGTGCCACCGC	4	9
Off-target 2-1	Chr12: 26007034-26007056	GAGCTGTGGCATGCATGCGC	2	10
Off-target 2-2	Chr02: 3488312-3488334	GAGTTGTGGCATGCATGCGC	3	9
Off-target 2-3	Chr04: 34285188-34285210	GAGCTATGGCACGCCTGCGC	2	8
Off-target 2-4	Chr06: 4926812-4926834	GAGCTATGGCATGCTTGTGC	1	10
Off-target 2-5	Chr04: 22007551-22007573	GAGCTATGGCATGCTTGTGC	1	10
Off-target 2-6	Chr01: 40696054-40696076	GAGCTGTGGCACGCCTGCGC	3	10

株系 Line	株高 Plant height / cm	总分蘖数 Tiller number per plant	有效穗数 Effective panicle per plant	穗长 Length of main panicle /cm	穗粒数 Grain number per panicle	结实率 Seed setting rate / %	千粒重 1000-grain weight / g	单株产量 Grain yield per plant / g
osarf7-1	73.5±1.6 ab		11.2±1.3 a	24.6±2.1 a	119.3±30.8 ab	63.4±19.0 bc	23.3±2.9 e	8.6±4.6 ab
osarf7-2-1	72.2±3.4 ab	20.6±3.3 a	7.4±1.5 abc	22.8±1.5 ab	135.9±34.9 ab	74.5±14.7 abc	25.5±0.2 bcde	12.9±6.3 ab
osarf7-2-2	67.3±4.9 b	11.0±2.6 e	5.0±1.0 c	23.5±2.5 ab	131.8±35.0 ab	82.5±8.8 ab	23.5±0.3 de	9.3±4.2 ab
osarf7-2-3	72.7±1.5 ab	20.0±1.0 a	7.3±3.5 abc	23.7±1.2 ab	167.5±13.8 a	78.4±5.8 abc	22.6±0.6 e	9.0±3.3 ab
osarf7-5-1	67.3±3.2 b	12.7±1.2 de	5.0±0.9 c	22.6±1.2 ab	119.4±12.1 ab	81.8±5.2 ab	25.5±0.6 bcde	10.4±0.5 ab
osarf7-5-2	70.8±3.2 ab	12.8±1.5 de	6.2±0.8 bc	22.8±2.0 ab	112.8±29.5 b	75.9±9.5 abc	25.2±1.3 cde	9.7±1.2 ab
osarf7-6	68.5±3.6 b	14.5±1.1 bcd	7.6±1.8 bc	23.0±1.5 ab	126.2±23.0 ab	69.9±11.9 abc	24.4±1.2 de	11.3±3.0 ab
osarf7-7	73.0±3.7 ab	20.2±1.8 a	10.2±1.9 ab	23.2±1.4 ab	135.9±21.2 ab	73.7±14.6 abc	25.0±1.5 cde	14.5±4.2 ab
osarf7-8-1	74.8±2.2 a	13.8±0.5 bcde	6.5±2.1 bc	23.7±2.0 ab	144.8±34.6 ab	76.5±10.0 abc	25.4±1.2 bcde	14.1±3.0 ab
osarf7-8-2	75.4±2.4 a	16.0±0.7 bc	7.0±2.6 bc	23.1±1.8 ab	140.5±32.2 ab	83.21±9.3 a	24.7±0.7 cde	12.0±3.3 ab
osarf7-9	72.3±0.8 ab	15.5±1.0 bc	6.8±1.9 bc	22.3±1.6 ab	119.9±23.1 ab	86.5±8.3 a	26.7±1.6 bcd	12.5±2.4 ab
osarf7-12	75.0±2.0 a	17.3±0.6 ab	6.3±1.5 bc	24.0±1.0 ab	139.8±36.0 ab	90.8±3.3 a	23.6±0.8 cde	17.4±9.8 a
osarf7-15-1	75.8±2.4 a	13.7±1.2 de	5.1±1.7 c	21.9±1.3 b	117.2±22.5 b	83.0±5.9 a	28.1±1.1 ab	10.0±3.2 ab
osarf7-15-2	74.7±1.4 a	11.9±1.2 de	4.9±2.3 c	21.8±1.9 b	122.0±24.5 ab	79.1±15.0 ab	27.4±1.3 abc	9.4±3.1 ab
osarf7-16	72.0±2.3ab	15.4±1.6 bc	5.6±0.9 c	21.8±1.3 b	103.0±26.7 b	57.2±26.4 c	30.1±2.2 a	13.0±3.9 b
WT	71.2±1.9 ab	11.3±0.6 de	10.2±1.8 ab	20.8±1.6 b	111.8±15.0 b	88.4±4.4 a	24.3±0.7 de	14.2±4.8 ab