番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立

doi:10.3864/j.issn.0578-1752.2018.02.011

摘要/Abstract

摘要： 【目的】从番茄中克隆高效转录的SlU6启动子，构建CRISPR/Cas9基因编辑载体，并在番茄中建立CRISPR/Cas9系统，为番茄功能基因组学和分子育种研究提供技术基础。【方法】采用PCR方法从‘中蔬四号’番茄品种中克隆4种SlU6启动子，利用Transfer PCR方法分别对4个启动子进行两种不同长度的截短，分别构建8个截短的SlU6启动子驱动GUS的植物融合表达载体。利用农杆菌瞬时转化法分别转染番茄叶片，通过GUS染色筛选出在番茄叶片中转录活性较高的SlU6-2启动子。采用DNA重组技术构建以SlU6-2为启动子驱动sgRNA，以番茄白粉病相关基因MLO1和EDR1为靶序列的CRISPR/Cas9基因组编辑载体。载体构建成功后，采用PEG法转化番茄原生质体，提取基因组DNA，采用酶切/PCR法分析内源基因突变情况；采用测序法分析内源基因突变的类型。利用突变位点频率分布图来验证番茄内源启动子在番茄CRISPR/Cas9系统中的有效性。【结果】经过两轮PCR，共获得4种8个不同长度的番茄U6启动子，其长度分别是452、202、448、206、433、190、448和218 bp，启动子序列比对分析发现番茄U6启动子与拟南芥U6启动子一样，也含有比较保守的两个元件，USE和TATA框。成功构建了8个SlU6启动子分别驱动GUS的植物融合表达载体。番茄叶片染色结果显示转化后的番茄叶片均被染成蓝色，表明克隆的番茄8个SlU6启动子均具有转录活性。选择SlU6-2P4为启动子驱动sgRNA，成功构建番茄白粉病相关基因MLO1和EDR1为靶序列的CRISPR/Cas9基因组编辑载体，验证结果表明番茄内源启动子SlU6-2P4能有效地驱动sgRNA的转录，并成功实现对番茄内源基因的编辑。内源基因突变的类型都为碱基替换，突变热点仅存在于内源基因靶序列区。【结论】成功克隆了4种在番茄叶片中高效转录的SlU6启动子；基于SlU6-2启动子的CRISPR/Cas9基因组编辑载体，在番茄中成功实现对内源基因的编辑。

关键词: CRISPR/Cas9, SlU6启动子, 克隆, 番茄, 基因编辑

Abstract: 【Objective】U6 promoter is a vital element for the transcription of sgRNA in the CRISPR/Cas9 system. It is necessary to clone some endogenous U6 promoters with high transcription activity and construct CRISPR/Cas9 vector, which could provide a strong technical basis for functional genomics and molecular breeding in tomato. 【Method】 Four different tomato U6 promoters were cloned by first round of PCR amplification from tomato cultivar Zhongshu 4. Each U6 promoter with two different length was truncated and used to construct plant expression vector carried SlU6 promoter::GUS, respectively. The Eight GUS fusion expression vectors were transformed into tomato leaves by agroinfiltration. According to the degree of GUS staining, the promoter with high transcription activity was selected to construct the CRISPR/Cas9 gene editing vector with target sequence from powdery mildew-related gene MLO1 and EDR1. These gene editing vectors were transformed into tomato protoplast by PEG method. The mutation of endogenous target genes in each transformed tomato protoplast was analyzed by a restriction enzyme PCR (RE-PCR) assay. Finally, the types of endogenous gene mutation were analyzed by sequencing. The efficiency of the CRISPR/Cas9 system based on tomato endogenous U6 promoter was verified by the frequency distribution map of mutant loci. 【Result】4 kinds and 8 different lengths of tomato U6 promoters were obtained by two rounds of PCR. Their length was 452, 202, 448, 206, 433, 190, 448 and 218 bp, respectively. After sequences analysis, results showed that the four tomato U6 promoters also contained the USE motif and TATA box which were found in Arabidopsis U6 promoters. The construction of GUS fusion expression vectors driven by corresponding truncated tomato U6 promoters were done and transformed into tomato leaves. The GUS histochemical staining showed that the transformed tomato leaves were dyed blue, which indicated that all 8 SlU6 promoters have transcription activity.The SlU6-2P4 promoter was chose to drive sgRNA transcription and construct the CRISPR/Cas9 system with target sequence from MLO1 and EDR1 respectively. The result showed that endogenous SlU6-2P4 promoter could drive sgRNA transcription and gene MLO1 was edited successfully in tomato. Sequence analysis revealed that all types of gene mutations are base substitution and the hotspot of mutation only exists in the target region of endogenous gene. 【Conclusion】4 kinds of SlU6 promoters with high transcription efficiency were obtained from tomato. The established CRISPR/Cas9 system based on SlU6-2 promoter could successfully achieve the editing of endogenous genes in tomato.

Key words: CRISPR/Cas9, SlU6 promoter, clone, tomato, gene editing

蒲艳，刘超，李继洋，阿尔祖古丽·塔什，胡燕，刘晓东. 番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立[J]. 中国农业科学, 2018, 51(2): 315-326.

PU Yan, LIU Chao, LI Ji-Yang, AERZU GULI·TaShi, HU Yan, LIU XiaoDong. Different SlU6 Promoters Cloning and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Tomato[J]. Scientia Agricultura Sinica, 2018, 51(2): 315-326.

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参考文献

[1] Sato S, Tabata S, Hirakawa H, Asamizu E, Shirasawa K, Isobe S, Kaneko T, Egholm M. The tomato genome sequence provides insights into fleshy fruit evolution. Nature, 2012, 485(7400): 635-641.

[2] Hisamatsu S, Sakaue M, Takizawa A, Kato T, Kamoshita M, Ito J, Kashiwazaki N. Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease. Animal Science Journal, 2015, 86(2): 132-137.

[3] Sun N, Zhao H. Transcription activator-like effector nucleases (TALENs): A highly efficient and versatile tool for genome editing. Biotechnology and Bioengineering, 2013, 110(7): 1811-1821.

[4] Chen K, Gao C. Targeted genome modification technologies and their applications in crop improvements. Plant Cell Reports, 2014, 33(4): 575-583.

[5] Chen X, Lu X, Shu N, Wang S, Wang J, Wang D, Guo L, Ye W. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Scientific Reports, 2017, 7: 44304.

[6] Li C, Unver T, Zhang B. A high-efficiency CRISPR/Cas9 system for targeted mutagenesis In Cotton (Gossypium hirsutum L.). Scientific Reports, 2017, 7: 43902.

[7] Liu X, Xie C, Si H, Yang J. CRISPR/Cas9-mediated genome editing in plants. Methods,2017, 3: 9.

[8] Friedland A E, Tzur Y B, Esvelt K M, Colaiácovo M P, Church G M, Calarco J A. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nature Methods, 2013, 10(8): 741-743.

[9] Wang M B, Helliwell C A, Wu L M, Waterhouse P M, Peacock W J, Dennis E S. Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants. RNA Biology, 2008, 14(5): 903-913.

[10] Domitrovich A M, Kunkel G R. Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies. Nucleic Acids Research, 2003, 31(9): 2344-2352.

[11] Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/sgRNA. Public Library of Science One, 2014, 9(4): e93806.

[12] 雷建峰, 伍娟, 陈晓俊, 於添平, 倪志勇, 李月, 张巨松, 刘晓东. 棉花花粉中高效转录U6启动子的克隆及功能分析. 中国农业科学, 2015, 48(19): 3794-3802.

Lei J F, Wu J, Chen X J, Yu T P, Ni Z Y, Li Y, Zhang J S, Liu X D. Cloning and functional analysis of cotton U6 promoter with high transcription activity in cotton pollen. Scientia Agricultura Sinica, 2015, 48(19):3794-3802. (in Chinese)

[13] Simmen K A, Mattaj I W. Complex requirements for RNA polymerase III transcription of the Xenopus U6 promoter. Nucleic Acids Research, 1990, 18(19): 5649-5657.

[14] 刘玟杉. 基于CRISPR/Cas9的拟南芥多基因编辑及其在基因功能研究中的应用[D]. 重庆: 重庆大学, 2015.

Liu W S. CRISPR-Cas9-mediated multiplex genome editing in Arabidopsis thaliana and the application in gene functions study [D]. Chongqing: Chongqing University, 2015. (in Chinese)

[15] Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. Plant Biology, 2014, 14(1): 327.

[16] 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. Nature Biotechnology, 2013, 31(8): 688-691.

[17] Erijman A, Shifman J M, Peleg Y. A single-tube assembly of DNA using the transfer-PCR (TPCR) platform. DNA Cloning and Assembly Methods, 2014, 1116: 89-101.

[18] Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature protocols, 2007, 2(7): 1565-1572.

[19] Lu Y M, Chen X, Wu Y X, Wang Y P, He Y Q, Wu Y. Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay. Public Library of Science One, 2013, 8(2): e57171.

[20] Janga M R, Campbell L M, Rathore K S. CRISPR/Cas9- mediated targeted mutagenesis in upland cotton (Gossypium hirsutum L.). Plant Molecular Biology, 2017, 94: 349-360.

[21] Liang Z, Zhang K, Chen K, Gao C. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. Journal of Genetics and Genomics, 2014, 41(2): 63-68.

[22] Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nature Protocols, 2014, 9(10): 2395-2410.

[23] Jiang W, Zhou H, Bi H, Fromm M, Yang B. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis，tobacco，sorghum and rice. Nucleic Acids Research, 2013, 41(20): e188.

[24] Jacobs T B，LaFayette P R，Schmitz R J，Parrott W A. Targeted genome modifications in soybean with CRISPR/Cas9. Bio Med Central Biotechnology, 2015, 15(1): 16.

[25] Cao H X, Wang W, Le H T T, Vu G T H. The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding. International Journal of Genomics, 2016.

[26] Cong L, Ran F A, Cox D, Lin S, Barretto R, Habi b, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823.

[27] Mali P, Yang L, Esvelt K M, Aach J, Guell M, DiCarlo J E, Church G M. RNA-guided human genome engineering via Cas9. Science, 2013, 339(6121): 823-826.

[28] Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V. Plant genome editing made easy: Targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods, 2013, 9(1): 39.

[29] Jacobs T B, LaFayette P R, Schmitz R J, Parrott W A. Targeted genome modifications in soybean with CRISPR/Cas9. Bio Med Central Biotechnology, 2015, 15(1): 16.

[30] Brooks C, Nekrasov V, Lippman Z B, Van Eck J. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 System . Plant Physiology, 2014, 166(3): 1292-1297.

[31] Zhang Z, Mao Y, Ha S, Liu W, Botella J R, Zhu J K. A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Report, 2016, 35(7): 1519-1533.

[32] Zhou H, Liu B, Weeks D P, Spalding M H, Yang B. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Research, 2014, 42(17): 10903.

[33] Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(39): 2579-2586.

[34] 雷建峰, 李月, 徐新霞, 阿尔祖古丽·塔什, 蒲艳, 张巨松, 刘晓东. 棉花不同GbU6启动子截短克隆及功能鉴定. 作物学报, 2016, 42(5): 675-683.

Lei J F, Li Y, Xu X X, Tashi A, Pu Y, Zhang J S, Liu X D. Cloning and functional analysis of different truncated GbU6 promoters in cotton. Acta Agronomica Sinica,2016, 42(5): 675-683.(in Chinese)

[35] Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V. Plant genome editing made easy: Targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods, 2013, 9(1): 39.

[36] Farboud B, Meyer B J. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design. Genetics, 2015, 199(4): 959-971.