欢迎访问作物学报,今天是

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

基于CRISPR/Cas9技术的水稻千粒重基因tgw6突变体的创建

王加峰,郑才敏,刘维,罗文龙,王慧,陈志强*,郭涛*   

  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:

    本研究由广东省公益研究与能力建设转型项目(20150209),国家高技术研究发展计划(863计划)项目(2011AA10A101)和国家现代农业产业技术体系建设专项(CARS-01-12)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).

摘要:

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

Abstract:

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

[1] 田甜, 陈丽娟, 何华勤. 基于Meta-QTL和RNA-seq的整合分析挖掘水稻抗稻瘟病候选基因[J]. 作物学报, 2022, 48(6): 1372-1388.
[2] 郑崇珂, 周冠华, 牛淑琳, 和亚男, 孙伟, 谢先芝. 水稻早衰突变体esl-H5的表型鉴定与基因定位[J]. 作物学报, 2022, 48(6): 1389-1400.
[3] 周文期, 强晓霞, 王森, 江静雯, 卫万荣. 水稻OsLPL2/PIR基因抗旱耐盐机制研究[J]. 作物学报, 2022, 48(6): 1401-1415.
[4] 郑小龙, 周菁清, 白杨, 邵雅芳, 章林平, 胡培松, 魏祥进. 粳稻不同穗部籽粒的淀粉与垩白品质差异及分子机制[J]. 作物学报, 2022, 48(6): 1425-1436.
[5] 颜佳倩, 顾逸彪, 薛张逸, 周天阳, 葛芊芊, 张耗, 刘立军, 王志琴, 顾骏飞, 杨建昌, 周振玲, 徐大勇. 耐盐性不同水稻品种对盐胁迫的响应差异及其机制[J]. 作物学报, 2022, 48(6): 1463-1475.
[6] 杨建昌, 李超卿, 江贻. 稻米氨基酸含量和组分及其调控[J]. 作物学报, 2022, 48(5): 1037-1050.
[7] 杨德卫, 王勋, 郑星星, 项信权, 崔海涛, 李生平, 唐定中. OsSAMS1在水稻稻瘟病抗性中的功能研究[J]. 作物学报, 2022, 48(5): 1119-1128.
[8] 朱峥, 王田幸子, 陈悦, 刘玉晴, 燕高伟, 徐珊, 马金姣, 窦世娟, 李莉云, 刘国振. 水稻转录因子WRKY68在Xa21介导的抗白叶枯病反应中发挥正调控作用[J]. 作物学报, 2022, 48(5): 1129-1140.
[9] 王小雷, 李炜星, 欧阳林娟, 徐杰, 陈小荣, 边建民, 胡丽芳, 彭小松, 贺晓鹏, 傅军如, 周大虎, 贺浩华, 孙晓棠, 朱昌兰. 基于染色体片段置换系群体检测水稻株型性状QTL[J]. 作物学报, 2022, 48(5): 1141-1151.
[10] 王泽, 周钦阳, 刘聪, 穆悦, 郭威, 丁艳锋, 二宫正士. 基于无人机和地面图像的田间水稻冠层参数估测与评价[J]. 作物学报, 2022, 48(5): 1248-1261.
[11] 陈悦, 孙明哲, 贾博为, 冷月, 孙晓丽. 水稻AP2/ERF转录因子参与逆境胁迫应答的分子机制研究进展[J]. 作物学报, 2022, 48(4): 781-790.
[12] 石育钦, 孙梦丹, 陈帆, 成洪涛, 胡学志, 付丽, 胡琼, 梅德圣, 李超. 通过CRISPR/Cas9技术突变BnMLO6基因提高甘蓝型油菜的抗病性[J]. 作物学报, 2022, 48(4): 801-811.
[13] 王吕, 崔月贞, 吴玉红, 郝兴顺, 张春辉, 王俊义, 刘怡欣, 李小刚, 秦宇航. 绿肥稻秆协同还田下氮肥减量的增产和培肥短期效应[J]. 作物学报, 2022, 48(4): 952-961.
[14] 巫燕飞, 胡琴, 周棋, 杜雪竹, 盛锋. 水稻延伸因子复合体家族基因鉴定及非生物胁迫诱导表达模式分析[J]. 作物学报, 2022, 48(3): 644-655.
[15] 陈云, 李思宇, 朱安, 刘昆, 张亚军, 张耗, 顾骏飞, 张伟杨, 刘立军, 杨建昌. 播种量和穗肥施氮量对优质食味直播水稻产量和品质的影响[J]. 作物学报, 2022, 48(3): 656-666.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!