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应用CRISPR/Cas9基因编辑技术获得高直链淀粉水稻种质

吴敏 黄娟 石桃雄 朱丽伟 邓娇 梁成刚 汪燕 刘飞 李荣 蔡芳 陈庆富

吴敏,黄娟,石桃雄,等. 应用CRISPR/Cas9基因编辑技术获得高直链淀粉水稻种质 [J]. 福建农业学报,2024,39(1):17−24 doi: 10.19303/j.issn.1008-0384.2024.01.003
引用本文: 吴敏,黄娟,石桃雄,等. 应用CRISPR/Cas9基因编辑技术获得高直链淀粉水稻种质 [J]. 福建农业学报,2024,39(1):17−24 doi: 10.19303/j.issn.1008-0384.2024.01.003
WU M, HUANG J, SHI T X, et al. CRISPR/Cas9 Technology-generated High-amylose Rice Varieties [J]. Fujian Journal of Agricultural Sciences,2024,39(1):17−24 doi: 10.19303/j.issn.1008-0384.2024.01.003
Citation: WU M, HUANG J, SHI T X, et al. CRISPR/Cas9 Technology-generated High-amylose Rice Varieties [J]. Fujian Journal of Agricultural Sciences,2024,39(1):17−24 doi: 10.19303/j.issn.1008-0384.2024.01.003

应用CRISPR/Cas9基因编辑技术获得高直链淀粉水稻种质

doi: 10.19303/j.issn.1008-0384.2024.01.003
基金项目: 国家自然科学基金项目(32060508);贵州省科技计划项目(黔科合基础-ZK〔2021〕一般 109、黔科合基础-ZK〔2023〕 一般278);贵州师范大学学术新苗基金项目(黔师新苗〔2021〕A16号);云南省重大科技专项与重点研发计划项目(202202AE090020)
详细信息
    作者简介:

    吴敏(1999 —),女,硕士研究生,主要从事苦荞育种研究,E-mail:1930541367@qq.com

    通讯作者:

    黄娟(1988 —),女,博士,副教授,主要从事荞麦品质性状重要基因功能研究 ,E-mail:huang200699@163.com

  • 中图分类号: S511

CRISPR/Cas9 Technology-generated High-amylose Rice Varieties

  • 摘要:   目的  通过CRISPR/Cas9基因编辑技术遗传改良水稻种质,创制高直链淀粉新材料。  方法  以水稻品种中花11为试验材料,利用CRISPR/Cas9编辑系统对水稻淀粉分支酶(Starch branching enzymes, SBE)基因OsSBE 3进行靶向编辑,利用PCR技术鉴定无标记纯合突变体,并测定其淀粉含量。  结果  T0代获得10株突变体株系,T1代获得5个无标记纯合突变株系,其中4个株系(sbe3-22-6sbe3-25-3sbe3-25-4sbe3-25-6)的直链淀粉含量和淀粉直支比显著高于野生型。  结论  本研究创制了高直链淀粉含量的水稻新种质,为水稻品质改良提供了参考。
  • 图  1  YL-Hu-SBE3基因编辑载体信息

    红色方框表示gRNA插入位置。

    Figure  1.  Information on YL-Hu-SBE3 knockout vector

    Red box: gRNA insertion position.

    图  2  基因编辑T0代水稻突变体的检测

    WT表示野生型;黑色下划线表示靶点2序列;红色字体表示碱基插入;黑色虚线表示碱基缺失;||表示染色体正负链。

    Figure  2.  Detection of mutant lines in T0 generation rice

    WT: Wild type; black underline: sequence of Target 2; red font: base insertion; black dotted line: base deletion; ||: plus and minus chains of chromosome.

    图  3  T1代突变体无T-DNA插入原件筛选

    M:DL2000 DNA 标记;WT:野生型。泳道1~55 为sbe3 T1代突变体植株,其中5、6、10、11、18、23、24、26、27、31、37、39和54分别是sbe3-6-1sbe3-26-4sbe3-12-4sbe3-22-6sbe3-26-7sbe3-25-2sbe3-33-1sbe3-25-3sbe3-25-7sbe3-31-2sbe-25-5sbe3-25-4sbe3-25-6

    Figure  3.  Selection of T-DNA-free mutants in T1 generation

    M: DL2000 DNA marker; WT: wild type. Lanes 1–55: sbe3 mutants in T1 generation, of which 5, 6, 10, 11, 18, 23, 24, 26, 27, 31, 37, 39, and 54 represent sbe3-6-1, sbe3-26-4, sbe3-12-4, sbe3-22-6, sbe3-26-7, sbe3-25-2, sbe3-33-1, sbe3-25-3, sbe3-25-7, sbe3-31-2, sbe-25-5, sbe3-25-4, and sbe3-25-6, respectively.

    图  4  OsSBE 3基因 T1代突变体测序峰图

    A:OsSBE3基因结构和靶点2示意图; B~F:sbe3-12-4sbe3-22-6sbe3-25-3sbe3-25-4sbe3-25-6测序峰图;黑色下划线:靶点2位置;箭头:突变位置。

    Figure  4.  Sequences and peak map of Ossbe3 mutants in T1 generation

    A: Schematic diagram of gene structure and Target 2 of OsSBE3; B–F: sequences and peak map of sbe3-12-4, sbe3-22-6, sbe3-25-3, sbe3-25-4, and sbe3-25-6, respectively; black underline: position of target 2; arrow: mutation position.

    图  5  直链淀粉标准曲线

    Figure  5.  Standard curve of amylose

    图  6  支链淀粉标准曲线

    Figure  6.  Standard curve of amylopectin

    图  7  OsSBE3基因编辑后代淀粉含量

    A:直链淀粉含量;B:支链淀粉含量;C:淀粉直支比;****:与WT相比,差异极显著(P<0.01);ns:与WT相比,没有显著差异(P>0.05)。

    Figure  7.  Starch content of progeny after genetic editing on OsSBE3

    A: Amylose content; B: amylopectin content; C: amylose/amylopectin ratio; ****: extremely significant difference from WT (P<0.01); ns: not significantly differ from WT (P>0.05).

    表  1  引物信息

    Table  1.   Information on primers applied

    引物名称
    Primer name
    引物序列(5′-3′)
    Primer sequence (5′- 3′)
    用途
    Application
    YL-Hu-SBE3-Y1+cagtGGTCTCatgcaGAGAGCAGCGACCGCGACGT载体构建 Carrier construction
    YL-Hu-SBE3-Y1-cagtGGTCTCaaaacACGTCGCGGTCGCTGCTCTC载体构建 Carrier construction
    YL-Hu-SBE3-B1+cagtGGTCTCatgcaTTGCTCATGCGGTCTGCATTt载体构建 Carrier construction
    YL-Hu-SBE3-B1-cagtGGTCTCaaaacAATGCAGACCGCATGAGCAA载体构建 Carrier construction
    Pyl-ACCGGTAAGGCGCGCCGTAGT鉴定引物 Identification primer
    Pbw2-GCGATTAAGTTGGGTAACGCCAGGG鉴定引物 Identification primer
    Cas9-CL-FGAACGGTCGTAAGAGGATGC无标记检测 Unmarked detection
    Cas9-CL-RGGTGATGGACTGGTGGATGAG无标记检测 Unmarked detection
    SBE3-C-FTGAAGGTGTCACTTATCGAGAA纯合突变检测 Homozygous mutation detection
    SBE3-C-RACCACTGCGCTATACATGCGTT纯合突变检测 Homozygous mutation detection
    HYG-FGGTGATGGACTGGTGGATGAG鉴定引物 Identification primer
    HYG-RGGAAGTGCTTGACATTGGGGAGTTT鉴定引物 Identification primer
    32660-BA1-FGCGCACACCCACACACCGACCA靶点1检测 Target 1 detection
    32660-BA1-441RgGCGAACGGCACCTGGACACGAGA靶点1检测 Target 1 detection
    OsBE3_BA2_RACCACTGCGCTATACATGCGTT靶点2检测 Target 2 detection
    OsBE3_BA2_FTGAAGGTGTCACTTATCGAGAA靶点2检测 Target 2 detection
    黑色下划线表示Eco31 I/Bsa I酶切位点,小写斜体字母为酶切位点的保护碱基。
    Black underline: Eco31 I/Bsa I cleaving site; lowercase italics: protective base of site.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2023-09-16
  • 修回日期:  2023-12-21
  • 网络出版日期:  2024-01-06
  • 刊出日期:  2024-01-28

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