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作物学报 ›› 2018, Vol. 44 ›› Issue (02): 227-235.doi: 10.3724/SP.J.1006.2018.00227

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基于棉花U6启动子的海岛棉CRISPR/Cas9基因组编辑体系的建立

李继洋, 雷建峰, 代培红, 姚瑞, 曲延英, 陈全家, 李月, 刘晓东*()   

  1. 新疆农业大学农学院 / 新疆农业大学农业生物技术重点实验室, 新疆乌鲁木齐 830052;
  • 收稿日期:2017-05-21 接受日期:2017-11-21 出版日期:2018-02-12 网络出版日期:2017-12-11
  • 通讯作者: 刘晓东
  • 作者简介:

    qinlixia11@163.com, Tel: 0359-2161515

  • 基金资助:
    本研究由国家自然科学基金项目(31660433)和新疆维吾尔自治区研究生科研创新项目(XJGRI2016055)资助

Establishment of CRISPR/Cas9 Genome Editing System Based on GbU6 Promoters in Cotton (Gossypium barbadense L.)

Ji-Yang LI, Jian-Feng LEI, Pei-Hong DAI, Rui YAO, Yan-Ying QU, Quan-Jia CHEN, Yue LI, Xiao-Dong LIU*()   

  1. College of Agronomy, Xinjiang Agricultural University / Key Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China;
  • Received:2017-05-21 Accepted:2017-11-21 Published:2018-02-12 Published online:2017-12-11
  • Contact: Xiao-Dong LIU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31660433) and the Innovation Research Program for Graduate Students in Xinjiang (XJGRI2016055).

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摘要：

CRISPR/Cas9基因组编辑技术是基因功能研究的一种强有力的工具, 目前已在许多生物体中成功实现内源靶向基因的突变。利用已克隆的海岛棉新海16的2个U6启动子, 分别构建带有新海16内源基因(GbGGB和GbERA1)靶位点DNA片段的CRISPR/Cas9基因编辑载体。以新海16的胚性愈伤组织为供试材料, 制备海岛棉的原生质体。通过PCR方法大量富集构建好的CRISPR/Cas9基因编辑载体的核心片段(包括GbU6::sgRNA和CAMV35S::Cas9两部分), 并利用PEG法转化海岛棉的原生质体。对原生质体基因组DNA进行酶切后PCR, 成功检测到内源靶基因的突变现象。对PCR产物进行克隆测序, 结果显示序列突变的类型主要以碱基替换为主, 少数为碱基缺失。结果表明基于海岛棉U6启动子的CRISPR/Cas9基因编辑系统能在海岛棉中实现靶向基因编辑的功能, 为棉花功能基因组学研究提供了重要的技术基础。

关键词: 棉花, 原生质体, CRISPR/Cas9, 基因组编辑

Abstract:

CRISPR/Cas9 genome editing is a powerful tool for genes functional analyses, and the mutation of endogenous genes has been successfully implemented in many organisms using the tool. Two cloned U6 promoter from sea island cotton Xinhai 16 were used to construct CRISPR/Cas9 gene editing vectors with target (GbGGB and GBERA1) DNA fragments from Xinhai 16 respectively. Through PEG method, the core fragments (including GbU6::sgRNA and CAMV35S::Cas9) of the CRISPR/Cas9 gene editing vectors enriched by PCR method were transformed into the cotton protoplast prepared from the embryo callus of Xinhai 16. The mutation of endogenous target genes was successfully detected by a restriction enzyme PCR (RE-PCR) assay of protoplast genome. The cloning and sequencing of the PCR product, showed that the two Cas9-GbU6-sgRNA vectors could both induce targeted mutagenesis. Sequence analysis revealed that most of the mutations were nucleotide substitutions and the few were nucleotide deletion. The results indicate that the CRISPR/Cas9 gene editing vector system based on GbU6 promoter can realize targeted mutagenesis in sea island cotton, which provides an important technical basis for cotton functional genomics research.

Key words: cotton, protoplast, CRISPR/Cas9, genome editing

图1

海岛棉基因组编辑载体结构示意图 A: 含Cas9I编辑载体; B: 含Cas9II编辑载体。"

表1

本研究使用的引物信息"

引物名称
Primer name		 序列
Sequence (5'-3')		 引物名称
Primer name		 序列
Sequence (5'-3')
GbU6-4PERA1-sg1F GATTGTTCGCAGAATGCATGACGG GbU6-5PGGB-sg2F AAGTGCTCTGTCGAAGTACTGAAG
GbU6-5PERA1-sg1F AAGTGTTCGCAGAATGCATGACGG GbU6GGB-sg2R AAACCTTCAGTACTTCGACAGAGC
GbU6ERA1-sg1R AAACCCGTCATGCATTCTGCGAAC Test GGB-sgF AAGTGGAAAGAGAATGGCGAC
GbU6-4PERA1-sg2F GATTGTCTTTCGCAGAATGCATGA Test GGB-sgR AGCTAATTCGCTATTGCAATCAATC
GbU6-5PERA1-sg2F AAGTGTCTTTCGCAGAATGCATGA Test ERA1-sgF GTATCCTCTTACCGCCTTTC
GbU6ERA1-sg2R AAACTCATGCATTCTGCGAAAGAC Test ERA1-sgF CATACTTACTGAGATGGCTGT
GbU6-4PGGB-sg1F GATTGTGTCGAAGTACTGAAGCGG FJ-1F GTAAAACGACGGCCAG
GbU6-5PGGB-sg1F AAGTGTGTCGAAGTACTGAAGCGG FJ-1R CAGGAAACAGCTATGAC
GbU6GGB-sg1R AAACCCGCTTCAGTACTTCGACAC FJ-2F TAAACTGAAGGCGGGAAACG
GbU6-4PGGB-sg2F GATTGCTCTGTCGAAGTACTGAAG FJ-2F CGGTTCTGTCAGTTCCAAACG

图2

基因编辑载体的酶切鉴定 M1, M2: 2K plus DNA marker. A: M-1 to M-4 were GbU6-5P::GGB-sgRNA1-Cas9IIGbU6-5P::GGB-sgRNA2-Cas9II, GbU6-5P::ERA1-sgRNA1-Cas9II, and GbU6-5P:: ERA1-sgRNA2-Cas9II, respectively. B: 35-1 to 35-4 were GbU6-5P::GGB-sgRNA1-Cas9I, GbU6-5P::GGB-SgRNA2-Cas9I, GbU6-5P::ERA1-sgRNA1-Cas9I, and GbU6-5P:: ERA1-sgRNA2-Cas9I."

图3

棉花原生质体转化前后形态图 A: 棉花原生质体酶解过夜镜检结果(×40); B: 转化GbU6-5-GGB-sgRNA1-Cas9I载体核心片段的棉花原生质体过夜孵育后镜检结果(×40)。Bar=100 μm。"

图4

酶切/PCR检测CRISPR/Cas9基因组编辑效应原理示意图红色区域为被编辑突变的酶切位点。"

图5

酶切/PCR检测CRISPR/Cas9基因组编辑效应 M: 2000 plus DNA marker。A: 1、2分别为转化GbU6-5P::ERA1-sgRNA1-Cas9I基因组酶切前、后PCR扩增结果, 3、4分别为转化GbU6-5P::GGB-sgRNA1-Cas9I基因组酶切前、后PCR扩增结果。B: 1、2分别为转化GbU6-5P::ERA1-sgRNA2-Cas9I基因组酶切前、后PCR扩增结果, 3、4分别为转化GbU6-5P::GGB-sgRNA2-Cas9I基因组酶切前、后PCR扩增结果, CK1、CK2为转化阴性对照载体基因组酶切前后PCR扩增结果。"

图6

GbGGB-sgRNA2靶位点编辑效应(碱基置换型)测序检测结果"

图7

GbGGB-sgRNA2靶位点编辑效应(碱基置换型)测序峰图红色方框指示碱基突变的区域; 黑色方框指示PAM位点区域。"

图8

GbGGB-sgRNA2靶位点编辑效应（碱基缺失型）测序峰图红色方框指示突变序列（B）碱基相比对照（A）测序峰图发生碱基缺失区域;C图显示碱基缺失区域。"

图9

GbGGB-sgRNA2靶位点区域(guide RNA)及其两侧位点的突变效率分析 L30、L60、L90分别表示guide DNA靶序列左侧30、60、90 bp区域; R30、R60、R90分别表示guide DNA靶序列右侧30、60、90 bp区域。"

[1] 李付广, 袁有禄, 棉花分子育种学. 北京: 中国农业大学出版社, 2013
Li F G, Yuan Y L.Molecular Breeding of Cotton. Beijing: China Agricultural University Press, 2013 (in Chinese)
[2] 张德超. 棉花叶片干旱胁迫蛋白的表达分析与鉴定. 中国农业科学院硕士学位论文, 北京, 2013
Zhang D C.Analysis and Identification of Drought Stress Proteins in Cotton Leaves. MS Thesis of Chinese Academy of Agricultural Sciences, Beijing, China, 2013 (in Chinese)
[3] 丁震乾, 陈天子, 刘廷利, 刘小双, 张保龙, 周兴根. 棉花干旱诱导MYB类转录因子GhRAX3的功能分析. 中国农业科学, 2015, 18: 3569-3579
Ding Z Q, Chen T Z, Liu T L, Liu X S, Zhang B L, Zhou G X.Functional analysis of MYB transcription factor GhRAX3 induced by Cotton Drought.Sci Agric Sin, 2015, 18: 3569-3579 (in Chinese with English abstract)
[4] 许宗弘. 棉花枯黄萎病研究现状及展望. 知识经济, 2010, (16): 132
Xu Z H.Research status and Prospect of cotton wilt disease.Knowledge Economy, 2010, (16): 132 (in Chinese)
[5] 任爱霞. 棉花枯黄萎病抗性遗传及生化机理研究. 浙江大学硕士学位论文, 浙江杭州, 2002
Ren A X.Genetic and Biochemical Mechanism of Resistance to Verticillium wilt of Cotton. MS Thesis of Zhejiang University, Hangzhou, China, 2002 (in Chinese)
[6] 戴敬, 徐俊兵, 杨举善, 吕丽兰. 棉花留叶枝栽培的研究现状与应用前景. 中国农业科技导报, 2003, (6): 19-23
Dai J, Xu J B, Yang J H, Lyu L L.Study on the cultivation of cotton retaining with status and application prospect.J Agric Sci Technol, 2003, (6): 19-23 (in Chinese with English abstract)
[7] 徐立华. 我国棉花高产、高效栽培技术研究现状与发展思路. 中国棉花, 2001, (3): 5-8
Xu L H.Research status and development of high yield and high efficiency cultivation techniques of cotton in China.China Cotton, 2001, (3): 5-8 (in Chinese)
[8] 孙学振, 施培, 周治国. 我国棉花高产栽培技术理论研究现状与展望. 中国棉花, 1999, (4): 2-7
Sun X Z, Shi P, Zhou Z G.Current situation and prospect of high yield cultivation techniques of cotton in China. China Cotton, 1999, (4): 2-7 (in Chinese)
[9] Sun Y, Li J, Xia L.Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement.Front Plant Sci, 2016, 7: 1928
[10] Cao H X, Wang W, Le H T T, Giang T H Vu. The power of CRISPR-Cas9-induced genome editing to speed up plant breeding.Int J Genom, 2016: 5078796
[11] Gilbert L A, Larson H M, Morsut L, Z R Liu, Brar G A, Torres S E, Ginossar N S, Brandman O, Whitehead E H, Doudna J A, Lim W A, Weissman J S, Qi L S. CRISPR-mediated modular RNA-guided regulation of transcription in Eukaryotes.Cell, 2013, 154: 442-451
[12] Hsu P D, Lander E S, Zhang F.Development and applications of CRISPR-Cas9 for genome engineering.Cell, 2014, 157: 1262-1278
[13] Bassett A R, C Tibbit, Ponting C P, Liu J L. Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system.Cell Rep, 2013, 4: 220-228
[14] Barrangou R, Marraffini L A.CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity.Mol Cell, 2014, 54: 234-244
[15] Mao Y F, Zhang Z G, Feng Z Y, Wei P L, Zhang H, Botella J R, Zhu J K.Development of germline specific CRISPR/Cas9 systems to improve the production of heritable gene modifications inArabidopsis. Plant Biotechnol J, 2016, 14: 519-532
[16] Kim H, Kim S T, Ryu J, Choi M K, Kweon J, Kang B C, Ahn S M, Bae S J, Kim J G, Kim J S, Kim S G.A simple flexible and high-throughput cloning system for plant genome editing via CRISPR/Cas system.J Integr Plant Biol, 2016, 58: 705-712
[17] Gao S L, Tong Y Y, Wen Z Q.Multiplex gene editing of theYarrowia lipolytica genome using the CRISPR/Cas9 system. Microbiol Biotech, 2016, 43: 1085-1093
[18] Johnson R A, Gurevich V, Filler S, Samach A, Levy A A.Comparative assessments of CRISPR-Cas nucleases cleavage efficiency in planta.Plant Mol Biol, 2015, 87: 143-156
[19] Xu R F, Li H, Qin R Y, Wang L, Li L, Wei P C, Yang J B.Gene targeting using theAgrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice, 2014, 7: 5
[20] Kumar V, Jain M.The CRISPR/Cas system for plant genome editing: advances and opportunities.J Exp Bot, 2015, 66: 47-57
[21] Sharma K, Hrle A, Kramer K, Sachsenberg T, Staals R H J, Randau L, Marchfelder A, van der Oost J, Kohlbacher O, Conti E, Urlaub H. Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry.Methods, 2015, 89: 138-148
[22] Basak J, Nithin C.Targeting non-coding RNAs in plants with the CRISPR-Cas technology is a challenge yet worth accepting.Front Plant Sci, doi: 10.3389/fpls.2015.01001
[23] Zlotorynski E.Plant cell biology: CRISP-Cas protection from plant viruses.Nat Rev Mol Cell Biol, 2015, 16: 642
[24] Liu L, Fan X D.CRISPR-Cas system: a powerful tool for genome engineering.Plant Mol Biol, 2014, 3: 209-218
[25] Chen X, Lu X, Shu N, Wang S, Wang J, Wang D, Guo L, Ye W W.Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Sci Rep, 2017, 7: 44304
[26] Li C, Unver T, Zhang B.A high efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton (Gossypium hirsutum L.). Sci Rep, 2017, 7: 43902
[27] Johnson C D, Chary S N, Chernoff E A, Zeng Q, Running M P, Crowell D N.Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling inArabidopsis. Plant Physiol, 2005, 139: 722-733
[28] Andrews M, Huizinga D H, Crowell D N.The CaaX specificities of Arabidopsis proteinprenyltransferases explainera1 and ggb phenotypes. BMC Plant Biol, 2010, 10: 118
[29] 雷建峰, 徐新霞, 李月, 代培红, 刘超, 刘晓东. CRISPR/Cas9介导靶向敲除拟南芥GGB基因突变体的鉴定. 西北植物学报, 2016, 36: 857-864
Lei J F, Xu X X, Li Y, Dai P H, Liu C, Liu X D.CRISPR/Cas9 mediated targeting to in identification ofGGB mutants of Arabidopsis. J Northwest Plants, 2016, 36: 857-864
[30] 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: 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 high efficient U6 promoter in cotton pollen.Chin J Agric Sci, 2015, 48: 3794-3802
[31] 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.PLoS One, 2013, 8: e57171
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