大豆生物钟基因GmLNK1/2、GmRVE4/8和GmTOC1 CRISPR/Cas9组织表达分析及敲除靶点的鉴定

doi:10.3724/SP.J.1006.2020.94169

摘要/Abstract

摘要：

生物钟基因能够参与调控植物的整个生命进程, 对提高作物产量具有重要的作用。LNK1 (NIGHT LIGHT- INDUCIBLE AND CLOCK-REGULATED 1)、LNK2、RVE4 (REVEILLE 4)、RVE8 (REVEILLE 8)和TOC1 (TIMING OF CAB EXPRESSION 1)是植物中重要的生物钟基因。本研究利用BLAST同源比对和进化树分析的方法分别鉴定AtLNK1、AtLNK2、AtRVE4、AtRVE8和AtTOC1在大豆中的同源基因, 通过qRT-PCR实验证明这些生物钟基因在大豆根、茎、叶等组织中均有表达。成功构建这些基因的CRISPR/Cas9敲除载体, 并利用大豆根毛转化体系成功鉴定出13个基因的CRISPR/CAS9有效靶点。为进一步获得稳定的大豆突变体材料及研究其生物钟基因的功能提供了理论基础。

关键词: 大豆, 基因敲除, 生物钟基因, CRISPR/Cas9

Abstract:

Circadian clock genes play an important role in improving crop yield. LNK1, LNK2, RVE4, RVE8, and TOC1 are important circadian clock genes in plants. Homologous genes of AtLNK1, AtLNK2, AtRVE4, AtRVE8, and AtTOC1 in soybean were found by evolutionary tree analysis. These genes were expressed in soybean roots, stems and leaves. The knockout vectors of these genes were successfully constructed by using CRISPR/Cas9 gene editing technology. Transformation system of soybean root hair and RT-PCR were used to identify 13 genes targets effectively. The results of this study provide important target informations for further obtaining soybean mutant materials, and a foundation for further studying the function of circadian clock genes.

Key words: soybean, gene knockout, clock gene, CRISPR/Cas9

甘卓然,石文茜,黎永力,侯智红,李海洋,程群,董利东,刘宝辉,芦思佳. 大豆生物钟基因GmLNK1/2、GmRVE4/8和GmTOC1 CRISPR/Cas9组织表达分析及敲除靶点的鉴定[J]. 作物学报, 2020, 46(8): 1291-1300.

GAN Zhuo-Ran,SHI Wen-Qian,LI Yong-Li,HOU Zhi-Hong,LI Hai-Yang,CHENG Qun,DONG Li-Dong,LIU Bao-Hui,LU Si-Jia. Identification of CRISPR/Cas9 knockout targets and tissue expression analysis of circadian clock genes GmLNK1/2, GmRVE4/8, and GmTOC1 in soybean[J]. Acta Agronomica Sinica, 2020, 46(8): 1291-1300.

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链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2020.94169

https://zwxb.chinacrops.org/CN/Y2020/V46/I8/1291

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参考文献 39

[1]	田志喜, 刘宝辉, 杨艳萍, 李明, 姚远, 任小波, 薛勇彪. 大豆分子设计育种成果与展望. 中国科学院院刊, 2018,33:915-922.
	Tian Z X, Liu B H, Yang Y P, Li M, Yao Y, Ren X B, Xue Y B. Update and prospect of soybean molecular module-based designer breeding in China. Bull Chin Acad Sci, 2018,33:915-922 (in Chinese with English abstract).
[2]	Li M W, Xin D, Gao Y, Li K P, Fan K, Muñoz N B, Yung W S, Lam H M. Using genomic information to improve soybean adaptability to climate change. J Exp Bot, 2017,68:1823-1834. doi: 10.1093/jxb/erw348 pmid: 27660480
[3]	Raju S K K, Shao M R, Sanchez R, Xu Y Z, Sandhu A, Graef G, Mackenzie S. An epigenetic breeding system in soybean for increased yield and stability. Plant Biotechnol J, 2018,16:1836-1847.
[4]	Inoue K, Araki T, Endo M. Circadian clock during plant development. J Plant Res, 2018,131:59-66.
[5]	McClung C R. Plant circadian rhythms. Plant Cell, 2006,18:792-803. doi: 10.1105/tpc.106.040980 pmid: 16595397
[6]	Lu S, Zhao X, Hu Y, Liu S, Nan H, Li X, Fang C, Cao D, Shi X, Kong L, Su T, Zhang F, Li S, Wang Z, Yuan X, Cober E R, Weller J L, Liu B, Hou X, Tian Z, Kong F. Natural variation at the soybean J locus improves adaptation to the tropics and enhances yield. Nat Genet, 2017,49:773-779. doi: 10.1038/ng.3819 pmid: 28319089
[7]	Inoue K, Araki T, Endo M. Circadian clock during plant development. J Plant Res, 2018,131:59-66. pmid: 29134443
[8]	Green R M, Tobin E M. Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression. Proc Natl Acad Sci USA, 1999,96:4176-4179. pmid: 10097183
[9]	Bendix C, Marshall C M, Harmon F G. Circadian clock genes universally control key agricultural traits. Mol Plant, 2015,8:1135-1152. pmid: 25772379
[10]	Gray J A, Shalit-Kaneh A, Chu D N, Hsu P Y, Harmer S L. The REVEILLE clock genes inhibit growth of juvenile and adult plants by control of cell size. Plant Physiol, 2017,173:2308-2322. doi: 10.1104/pp.17.00109 pmid: 28254761
[11]	Xie Q, Wang P, Liu X, Yuan L, Wang L, Zhang C, Li Y, Xing H, Zhi L, Yue Z, Zhao C, McClung C R, Xu X. LNK1 and LNK2 are transcriptional coactivators in the Arabidopsis circadian oscillator. Plant Cell, 2014,26:2843-2857. doi: 10.1105/tpc.114.126573
[12]	Müller N A, Zhang L, Koornneef M Jiménez-Gómez J M. Mutations in EID1 and LNK2 caused light-conditional clock deceleration during tomato domestication. Proc Natl Acad Sci USA, 2015,115:7135-7140. doi: 10.1073/pnas.1801862115 pmid: 29789384
[13]	Müller N A, Wijnen C L, Srinivasan A, Ryngajllo M, Ofner I, Lin T, Ranjan A, West D, Maloof J N, Sinha N R, Huang S, Zamir D, Jiménez-Gómez J M. Domestication selected for deceleration of the circadian clock in cultivated tomato. Nat Genet, 2016,48:89-93. doi: 10.1038/ng.3447 pmid: 26569124
[14]	高耀辉, 马斌, 肖凤洁, 魏光普. CRISPR/Cas9系统在园林植物中的研究展望. 北方园艺, 2019, (15):133-140.
	Gao Y H, Ma B, Xiao F J, Wei G P. Prospect of research on CRISPR/Cas9 system in garden plants. Nor Hortic, 2019, (15):133-140 (in Chinese with English abstract).
[15]	暴会会, 尹竹君, 王少坤, 马瑞红, 谢俊俊, 张杰, 杨正安. CRISPR-Cas9系统在蔬菜育种上应用研究进展. 江西农业学报, 2019,31(7):38-44.
	Bao H H, Yin Z J, Wang S K, Ma R H, Xie J J, Zhang J, Yang Z A. Research advances in application of CRISPR/Cas9 system in vegetable breeding. Acta Agric Jiangxi, 2019,31(7):38-44 (in Chinese with English abstract).
[16]	Zhang J, Zhu Z, Yue W, Li J, Chen Q, Yan Y, Lei A, Hua J. Establishment of CRISPR/Cas9 mediated knock-in system for porcine cells with high efficiency. Appl Biochem Biotechnol, 2019,189:26-36. doi: 10.1007/s12010-019-02984-5 pmid: 30859452
[17]	Lin C Y, Su Y H. Genome editing in sea urchin embryos by using a CRISPR/Cas9 system. Dev Biol, 2016,409:420-428. doi: 10.1016/j.ydbio.2015.11.018 pmid: 26632489
[18]	Cai Y, Wang L, Chen L, Wu T, Liu L, Sun S, Wu C, Yao W, Jiang B, Yuan S, Han T, Hou W. Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean. Plant Biotechnol J, 2020,18:298-309. doi: 10.1111/pbi.13199 pmid: 31240772
[19]	Bao A, Chen H, Chen L, Chen S, Hao Q, Guo W, Qiu D, Shan Z, Yang Z, Yuan S, Zhang C, Zhang X, Liu B, Kong F, Li X, Zhou X, Tran L P, Cao D. CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean. BMC Plant Biol, 2019,19:131. doi: 10.1186/s12870-019-1746-6 pmid: 30961525
[20]	Do P T, Nguyen C X, Bui H T, Tran L T N, Stacey G, Gillman J D, Zhang Z J, Stacey M G. Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean. BMC Plant Biol, 2019,19:311. doi: 10.1186/s12870-019-1906-8 pmid: 31307375
[21]	曾栋昌, 马兴亮, 谢先荣, 祝钦泷, 刘耀光. 植物CRISPR/Cas9多基因编辑载体构建和突变分析的操作方法. 中国科学: 生命科学, 2018,48:783-794.
	Zeng D C, Ma X L, Xie X R, Zhu Q L, Liu Y G. A protocol for CRISPR/Cas9-based multi-gene editing and sequence decoding of mutant sites in plants. Sci Sin Vitae, 2018,48:783-794 (in Chinese with English abstract).
[22]	侯智红, 吴艳, 程群, 董利东, 芦思佳, 南海洋, 甘卓然, 刘宝辉. 利用CRISPR/Cas9技术创制大豆高油酸突变系. 作物学报, 2019,45:839-847.
	Hou Z H, Wu Y, Cheng Q, Dong L D, Lu S J, Nan H Y, Gan Z R, Liu B H. Creation of high oleic acid soybean mutation plants by CRISPR/Cas9. Acta Agron Sin, 2019,45:839-847 (in Chinese with English abstract).
[23]	Cheng Q, Dong L D, Gao T J, Liu T F, Li N H, Wang L, Chang X, Wu J J, Xu P F, Zhang S Z. The bHLH transcription factor GmPIB1 facilitates resistance to Phytophthora sojae in Glycine max. J Exp Bot, 2018,69:2527-2541. doi: 10.1093/jxb/ery103 pmid: 29579245
[24]	解莉楠, 宋凤艳, 张旸. CRISPR/Cas9系统在植物基因组定点编辑中的研究进展. 中国农业科学, 2015,48:1669-1677.
	Xie L N, Song F Y, Zhang Y. Progress in research of CRISPR/Cas9 system in genome targeted editing in plants. Sci Agric Sin, 2015,48:1669-1677 (in Chinese with English abstract).
[25]	Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J A, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337:816-821. doi: 10.1126/science.1225829 pmid: 22745249
[26]	Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet, 2011,45:273-297. doi: 10.1146/annurev-genet-110410-132430
[27]	Wiedenheft B, Sternberg S H, Doudna J A. RNA-guided genetic silencing systems in bacteria and archaea. Nature, 2012,482:331-338. doi: 10.1038/nature10886
[28]	Nekrasov V, Staskawicz B, Weigel D, Jones J D, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol, 2013,31:691-693. doi: 10.1038/nbt.2655 pmid: 23929340
[29]	Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J J, Qiu J L, Gao C. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol, 2013,31:686-688. doi: 10.1038/nbt.2650 pmid: 23929338
[30]	Li J F, Norville J E, Aach J, McCormack M, Zhang D, Bush J, Church G M, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol, 2013,31:688-691. doi: 10.1038/nbt.2654 pmid: 23929339
[31]	Feng Z Y, Zhang B T, Ding W, Liu X D, Yang D L, Wei P L, Cao F Q, Zhu S H, Zhang F, Mao Y F, Zhu J K. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res, 2013,23:1229-1232. doi: 10.1038/cr.2013.114 pmid: 23958582
[32]	Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu L J. Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res, 2013,23:1233-1236. doi: 10.1038/cr.2013.123
[33]	Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks D P. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res, 2013,41:e188. doi: 10.1093/nar/gkt780 pmid: 23999092
[34]	Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One, 2014,9:e93806. pmid: 24710347
[35]	Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J, 2014,79:348-359. doi: 10.1111/tpj.12554 pmid: 24836556
[36]	梁丹, 吴宇轩, 李劲松. CRISPR-Cas9技术在干细胞中的应用. 生命科学, 2015,27(1):93-98.
	Liang D, Wu Y X, Li J S. Progress of CRISPR-Cas9 in stem cell research. Chin Bull Life Sci, 2015,27(1):93-98 (in Chinese with English abstract).
[37]	Chen K, Gao C. Targeted genome modification technologies and their applications in crop improvements. Plant Cell Rep, 2014,33:575-583. doi: 10.1007/s00299-013-1539-6 pmid: 24277082
[38]	Curtin S J, Zhang F, Sander J D, Haun W J, Starker C, Baltes N J, Reyon D, Dahlborg E J, Goodwin M J, Coffman A P, Dobbs D, Joung J K, Voytas D F, Stupar R M. Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol, 2011,156:466-473. doi: 10.1104/pp.111.172981 pmid: 21464476
[39]	Cai Y, Chen L, Liu X, Sun S, Wu C, Jiang B, Han T, Hou W. CRISPR/Cas9-Mediated genome editing in soybean hairy roots. PLoS One, 2015,10:e0136064. pmid: 26284791

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靶点引物 Target primer	引物序列 Primer sequence (5°-3°)	靶点对应基因 Target of gene
LNK1T1F	gtcaGGAGACAAGTGTGTGGTGG	GmLNK1a, GmLNK1b
LNK1T1R	aaacCCACCACACACTTGTCTCC	GmLNK1a, GmLNK1b
LNK1T2F	gtcaAGAGGAGTTCTGCTGGCTC	GmLNK1a, GmLNK1b
LNK1T2R	aaacGAGCCAGCAGAACTCCTCT	GmLNK1a, GmLNK1b
LNK1T3F	attgCTTGGGAGACAAGTGTGTGG	GmLNK1c, GmLNK1d
LNK1T3R	aaacCCACACACTTGTCTCCCAAG	GmLNK1c, GmLNK1d
LNK1T4F	attgAGACTTTGAAGATGTTGAC	GmLNK1c, GmLNK1d
LNK1T4R	aaacGTCAACATCTTCAAAGTCT	GmLNK1c, GmLNK1d
LNK2T1F	gtcaACATAATATGGGGTGAAGG	GmLNK2a, GmLNK2b
LNK2T1R	aaacCCTTCACCCCATATTATGT	GmLNK2a, GmLNK2b
LNK2T2F	gtcaAAACTGATCAGGGTTCCCT	GmLNK2c, GmLNK2d
LNK2T2R	aaacAGGGAACCCTGATCAGTTT	GmLNK2c, GmLNK2d
LNK2T3F	attgTTTGATTGGAACGACGAAG	GmLNK2a, 2b, 2c, 2d
LNK2T3R	aaacCTTCGTCGTTCCAATCAAA	GmLNK2a, 2b, 2c, 2d
LNK2T4F	attgTCATATTGTGCCTTATCCGG	GmLNK2c, GmLNK2d
LNK2T4R	aaacCCGGATAAGGCACAATATGA	GmLNK2c, GmLNK2d
RVE48T1F	gtcaCTTCCCTGCTGATGAATGC	GmRVE4/8b, GmRVE4/8c
RVE48T1R	aaacGCATTCATCAGCAGGGAAG	GmRVE4/8b, GmRVE4/8c
RVE48T2F	gtcaTCATCCCATGTGACATACCC	GmRVE4/8a, GmRVE4/8d
RVE48T2R	aaacGGGTATGTCACATGGGATGA	GmRVE4/8a, GmRVE4/8d
RVE48T3F	attgCAGCTTTGCGCTTTGGACG	GmRVE4/8b, GmRVE4/8c
RVE48T3R	aaacCGTCCAAAGCGCAAAGCTG	GmRVE4/8b, GmRVE4/8c
RVE48T4F	attgAAGCTTTGCGCTTAGGCCG	GmRVE4/8a, GmRVE4/8d
RVE48T4R	aaacCGGCCTAAGCGCAAAGCTT	GmRVE4/8a, GmRVE4/8d
TOC1T1F	gtcaCGATTCCAAGAGTTCTCAAG	GmTOC1a, GmTOC1b
TOC1T1R	aaacCTTGAGAACTCTTGGAATCG	GmTOC1a, GmTOC1b
TOC1T2F	gtcaGTGATGTCCGCACAAGATG	GmTOC1a, GmTOC1b
TOC1T2R	aaacCATCTTGTGCGGACATCAC	GmTOC1a, GmTOC1b
TOC1T3F	attgGTGGGAATAATAGTAAGAG	GmTOC1c, GmTOC1d
TOC1T3R	aaacCTCTTACTATTATTCCCAC	GmTOC1c, GmTOC1d
TOC1T4F	attgTTGTAAAGTGCTTGAGGCT	GmTOC1c, GmTOC1d
TOC1T4R	aaacAGCCTCAAGCACTTTACAA	GmTOC1c, GmTOC1d

培养基类型 Medium type	药品名称 Name of the medicine	药品用量 Medicine dosage
萌发培养基 Germination medium	B5盐 B5 salt mixture	1×
	蔗糖 Sucrose (g L^-1)	20
	琼脂 Agar (g L^-1)	8
发根培养基 Rooting medium	MS合成盐 MS salt mixture	1×
	蔗糖 Sucrose (g L^-1)	30
	2-(4-吗啉)乙磺酸 MES	0.6
	琼脂 Agar (g L^-1)	8
	头孢霉素 Cef (mg L^-1)	250
	羧苄青霉素 Car (mg L^-1)	250

靶点检测引物 Target primer	引物序列 Primer sequence (5°-3°)	靶点检测引物 Target primer	引物序列 Primer sequence (5°-3°)
SP3	GTCGTGCTCCACATGTTGACCGG	LNK2cT4&2R	TCCTGAGGTTAGTAGTTCTCCACT
SP1	GAAGTTATTGCATCTATGTCGGG	LNK2dT3F	CTCTCCGTCGCCGTTATAGCA
LNK1aF	TGACGCCAGGGTATCTTAAA	LNK2dT3R	ACCAAACGAAGCACGAACA
LNK1aR	TCTCCATGTGTGTGTTTTGGTA	LNK2dT4&2F	GGTGGTGGAGGGAGAAGATGAG
LNK1bF	CTCAGGGTAGGGAGGACTTG	LNK2dT4&2R	AGACACTTATTGCCGCTACAACTG
LNK1bR	CGGTAAAGTTGAGCCTTGGT	RVE4/8aF	ACAGCTCTTCAGCTAGGTGTT
LNK1cF	TCATATAGTGCCCCATGCCA	RVE4/8aR	GAGGAGAGGGGGTATGGGTT
LNK1cR	AGTTCTATAGCAGCTCATGACA	RVE4/8bF	AGCGAAGAACTCTGCAATCCA
LNK1dF	GCCCGATCATTGCTTCAAGAG	RVE4/8bR	CTACCACCTTGGGCCGAAAT
LNK1dR	AGTTCTATAGCAGCTCGTGGC	RVE4/8cF	TTCGAAGCCATGCTCAGAAG
LNK2aT3F	GAATTCGGCGATGTGTGAGC	RVE4/8cR	CCAGCAACAAGGTTCGTAGT
LNK2aT3R	ACAGCTACACAAAGACACACA	RVE4/8dF	TTCGTTGGTCATCTTGCTGGT
LNK2aT1F	CCGTCCAAGGAGATTGTCACTGA	RVE4/8dR	CTGGACATGGCCTTCTGTGT
LNK2aT1R	TCCTGAGGTAGGTAGTTCTCCACT	TOC1aF	TCCCTCAACGATGCTG
LNK2bT3F	TGATGGAGTGCGTTTCTCTG	TOC1aR	GCCTCCGTCTCCACAT
LNK2bT3R	CCCTGATTTTCCTGGCGTAA	TOC1bF	TTGAGCAAGTCCAGGGTT
LNK2bT1F	CCGTCCAAGGAGATTGTCACTGA	TOC1bR	ATGGCTGTGATGGTAACTCG
LNK2bT1R	TCCTGAGGTAGGTAGTTCTCCACT	TOC1cF	CTCTAACTAACTATCCAGACCCTA
LNK2cT3F	TTCTCCGTCGATCAGTGAAGTG	TOC1cR	ATGGCTGGTGGGTTGA
LNK2cT3R	GCTAAGAGTCACGCCTCCTTG	TOC1dF	TACGCCCTCCCTCTTT
LNK2cT4&2F	CGTGATGCCAAATTAGTTGGGTAT	TOC1dR	GGGACTTGGGAAATACA