作物学报 ›› 2016, Vol. 42 ›› Issue (08): 1160-1167.doi: 10.3724/SP.J.1006.2016.01160

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇



  1. 华南农业大学国家植物航天育种工程技术研究中心,广东广州 510642
  • 收稿日期:2015-12-17 修回日期:2016-05-09 出版日期:2016-08-12 网络出版日期:2016-05-23
  • 通讯作者: 郭涛,E-mail: guoguot@scau.edu.cn, Tel: 020-38604903; 陈志强,E-mail: chenlin@scau.edu.cn, Tel: 020-85283237
  • 基金资助:


Construction of tgw6 Mutants in Rice Based on CRISPR/Cas9 Technology

WANG Jia-Feng,ZHENG Cai-Min,LIU Wei,LUO Wen-Long,WANG Hui,CHEN Zhi-Qiang*,GUO Tao*   

  1. National Engineering Research Center of Plant Space Breeding, South China Agricultural University, Guangzhou 510642, China?
  • Received:2015-12-17 Revised:2016-05-09 Published:2016-08-12 Published online:2016-05-23
  • Contact: 郭涛,E-mail: guoguot@scau.edu.cn, Tel: 020-38604903; 陈志强,E-mail: chenlin@scau.edu.cn, Tel: 020-85283237
  • Supported by:

    This study was supported by Public Welfare Research and Capacity Building Transformation Funds in Guangdong (20150209), the National High Technology Research and Development Program of China (863 Program) (2011AA10A101) and Special Funds for the Construction of Modern Agricultural Industry Technology System (CARS-01-12).


利用CRISPR/Cas9技术对调控水稻产量千粒重基因TGW6定点编辑,获得了一套有重要育种价值的tgw6突变体。设计了分别由U3、U6a和U6b启动子驱动、长20 bp的guide RNA (gRNA)靶点以靶向编辑TGW6基因的外显子,首先将这3个靶点一起组装到pYLCRISPR/Cas9-MT(I)载体上,然后利用农杆菌介导侵染水稻材料H447 (R819/玉针香//R819的BC3F6);提取T0代转基因植株的基因组DNA并对编辑位点附近的DNA片段进行PCR检测及测序分析。结果表明,T0代材料中tgw6的突变频率高达90%,其中纯合缺失突变率约占51%。对T1代纯合缺失突变体的千粒重性状的调查分析结果表明,部分tgw6的缺失突变能显著提高千粒重(大于5%)。不同类型tgw6突变体的成功创建不仅丰富了tgw6的变异类型,为水稻的高产稳产奠定了重要的材料基础,还证实了CRISPR/Cas9技术在水稻基因工程育种中高效、易操作的特点,具有重要的理论与实践意义。

关键词: 水稻, 基因编辑, CRISPR/Cas9, TGW6, 千粒重


A set of tgw6 (Thousand grain weight 6) mutants were constructed using CRISPR/Cas9 technology in this study. Three sites of 20 nt guide RNA (gRNA) targeted to the exon of TGW6 were designed and transcribed from the U3, U6a and U6b promoters, respectively. The three target sites of gRNA were then ligated to the vector pYLCRISPR/Cas9-MT(I) based on golden gate cloning strategy. The recombinant plasmid was transferred to a rice cultivar, H447 (R819/Yuzhenxiang//R819 BC3F6) by Agrobacterium-mediated transformation. Sequencing for the genomic DNA of TGW6 locusinT0 rice showed the mutagenesis frequency for TGW6 was more than 90%, including 51% of the homozygous deletion mutations. Further analysis for the T1 mutants showed almost all the homozygous deletion mutants improved the thousand grain weight significantly (more than 5%). The successful tgw6 editing not only provided a series of tgw6 mutants for high and stable yield of rice but also proved that CRISPR/Cas9 is a facile and powerful means of rice genetic engineering for scientific and agricultural applications, which has important theoretical and practical significance for rice breeding.

Key words: Rice, Genome editing, CRISPR/Cas9, TGW6, Thousand grain weight

[1] You A Q, Lu X G, Jin H J, Ren X, Liu K, Yang G C, Yang H Y, Zhu L L, He G C. Identification of quantitative trait loci across recombinant inbred lines and testcross populations for traits of agronomic importance in rice. Genetics, 2006, 172: 1287–1300
[2] Shomura A, Izawa T, Ebana K, Ebitani T, Kanegae H, Konishi S, Yano M. Deletion in a gene associated with grain size increased yields during rice domestication. Nat Genet, 2008, 40: 1023–1028
[3] Weng J F, Gu S H, Wan X Y, Gao H, Guo T, Su N, Lei C, Zhang X, Cheng Z J, Guo X P, Wang J L, Jiang L, Zhai H Q, Wan J M. Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight. Cell Res, 2008, 18: 1199–1209
[4] Ishimaru K, Hirotsu N, Madoka Y, Murakami N, Hara N, Onodera H, Kashiwagi T, Ujiie K, Shimizu B, Onishi A, Miyagawa H, Katoh E. Loss of function of the IAA–glucose hydrolase gene TGW6 enhances rice grain weight and increases yield. Nat Genet, 2013, 45: 707–711
[5] Fan C C, Xing Y Z, Mao H L, Lu T T, Han B, Xu C G, Li X H, Zhang Q F. GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor Appl Genet, 2006, 112: 1164–1171
[6] Li Y B, Fan C C, Xing Y Z, Jiang Y H, Luo L J, Sun L, Shao D, Xu C J, Li X, Xiao J H, He Y Q, Zhang Q F. Natural variation in GS5 plays an important role in regulating grain size and yield in rice. Nat Genet, 2011, 43: 1266–1269
[7] Song X J, Huang W, Shi M, Zhu M Z, Lin H X. A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nat Genet, 2007, 39: 623–630
[8] Wang S K, Wu K, Yuan Q, Liu X, Liu Z, Lin X, Zeng R, Zhu H, Dong G, Qian Q, Zhang G Q, Fu X D. Control of grain size, shape and quality by OsSPL16 in rice. Nat Genet, 2012, 44: 950–954
[9] Hu Z J, He H H, Zhang S Y, Sun F, Xin X, Wang W, Qian X, Yang J S, Luo X J.A Kelch motif—containing serine/threonine protein phosphatase determines the large grain QTL trait in rice. J Integr Plant Biol, 2012, 54: 979–990
[10] Qi P, Lin Y S, Song X J, Shen J B, Huang W, Shan J X, Zhu M Z, Jiang L, Gao J P, Lin H X. The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1; 3. Cell Res, 2012, 22: 1666–1680
[11] Zhang X, Wang J, Huang J, Lan H, Wang C, Yin C, Wu Y, Tang H, Qian Q, Li J, Zhang H. Rare allele of OsPPKL1 associated with grain length causes extra-large grain and a significant yield increase in rice. Proc Natl Acad Sci USA, 2012, 109: 21534–21539
[12] Rath D, Amlinger L, Rath A, Lundgren M. The CRISPR-Cas immune system: Biology, mechanisms and applications. Biochimie, 2015, 117: 119–128
[13] Belhaj K, Chaparro-Garcia A, Kamoun S, Patron N J, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol, 2015, 32: 76–84
[14] Osakabe Y, Osakabe K. Genome editing with engineered nucleases in plants. Plant Cell Physiol, 2015, 56: 389–400
[15] Jiang W, Bikard D, Cox D, Zhang F, Marraffini L A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 2013, 31: 233–239
[16] Feng Z, Zhang B, Ding W, Liu X, Yang D L, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu J K. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res, 2013, 23: 1229–1232
[17] Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv, 2015, 33: 41–52
[18] 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. Mol Plant, 2015, 8: 1274–1284
[19] Xu, R F, Li H, Qin R Y, Li J, Qiu C H, Yang Y C, Ma H, Li L, Wei P C, Yang J B. Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci Rep, 2015, 11491. doi: 10.1038/srep11491
[20] Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6: 271–282
[21] Wang H, Chu Z, Ma X, Li R, Liu Y. A high through-Put protocol of plant genomic DNA preparation for PCR. Acta Agron Sin, 2013, 39: 1200–1205
[22] Bibikova M, Golic M, Golic K G, Carroll D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics, 2002, 161: 1169–1175
[23] Bibikova M, Beumer K, Trautman J K, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science, 2003, 300: 764
[24] Dreier B, Fuller R P, Segal D J, Lund C V, Blancafort P, Huber A, Koksch B, Barbas C F. Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem, 2005, 280: 35588–35597
[25] Hockemeyer D, Wang H, Kiani S, Lai C S, Gao Q, Cassady J P, Cost G J, Zhang L, Santiago Y, Miller J C, Zeitler B, Cherone J M, Meng X, Hinkley S J, Rebar E J, Gregory P D, Urnov F D, Jaenisch R. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol, 2011, 29: 731–734
[26] Tesson L, Usal C, Ménoret S, Leung E, Niles B J, Remy S, Santiago Y, Vincent A I, Meng X, Zhang L, Gregory P D, Anegon I, Cost G J. Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol, 2011, 29: 695–696
[27] Huang P, Xiao A, Zhou M G, Zhu Z Y, Lin S, Zhang B. Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol, 2011, 29: 699–700
[28] Endo M, Mikami M, Toki S. Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant Cell Physiol, 2015, 56: 41–47
[29] 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
[30] DiCarlo J E, Norville J E, Mali P, Rios X, Aach J, Church G M. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res, 2013, 41: 4336–4343
[31] Shen B, Zhang J, Wu H Y, Wang J, Ma K, Li Z, Zhang X G, Zhang P, Huang X. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res, 2013, 23: 720–723
[32] Gratz S J, Cummings A M, Nguyen J N, Hamm D C, Donohue L K, Harrison M M, Wildonger J, O'Connor-Giles K M. Genome engineering of Drosophila with the CRISPR RNA guided Cas9 nuclease. Genetics, 2013, 194: 1029–1035
[33] Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339: 819–823
[34] Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong J W, Xi J J. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res, 2013, 23: 465–472
[35] Dickinson D J, Ward J D, Reiner D J, Goldstein B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods, 2013, 10: 1028–1034
[36] ?ermák T, Baltes N J, ?egan R, Zhang Y, Voytas D F. High-frequency, precise modification of the tomato genome. Genome Biol, 2015, 16(1): 232. doi: 10.1186/s13059-015-0796-9
[37] Yin K, Han T, Liu G, Chen T, Wang Y, Yu A Y, Liu Y. A geminivirus-based guide RNA delivery system for CRISPR/ Cas9 mediated plant genome editing. Sci Rep, 2015, 14926. doi: 10.1038/ srep14926

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