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利用基因组编辑技术定点突变IPA1基因创制水稻新株型材料

刘华清 孙庆山 杨绍华 周淑芬 王锋

刘华清, 孙庆山, 杨绍华, 周淑芬, 王锋. 利用基因组编辑技术定点突变IPA1基因创制水稻新株型材料[J]. 福建农业学报, 2019, 34(8): 867-872. doi: 10.19303/j.issn.1008-0384.2019.08.001
引用本文: 刘华清, 孙庆山, 杨绍华, 周淑芬, 王锋. 利用基因组编辑技术定点突变IPA1基因创制水稻新株型材料[J]. 福建农业学报, 2019, 34(8): 867-872. doi: 10.19303/j.issn.1008-0384.2019.08.001
LIU Hua-qing, SUN Qing-shan, YANG Shao-hua, ZHOU Shu-fen, WANG Feng. Morphological Alternation of Rice Plant by Site-Directed Mutagenesis on IPA1 Gene[J]. Fujian Journal of Agricultural Sciences, 2019, 34(8): 867-872. doi: 10.19303/j.issn.1008-0384.2019.08.001
Citation: LIU Hua-qing, SUN Qing-shan, YANG Shao-hua, ZHOU Shu-fen, WANG Feng. Morphological Alternation of Rice Plant by Site-Directed Mutagenesis on IPA1 Gene[J]. Fujian Journal of Agricultural Sciences, 2019, 34(8): 867-872. doi: 10.19303/j.issn.1008-0384.2019.08.001

利用基因组编辑技术定点突变IPA1基因创制水稻新株型材料

doi: 10.19303/j.issn.1008-0384.2019.08.001
基金项目: 

福建省科技计划项目——省属公益类科研院所基本科研专项 2015R1019-1

福建省科技重大专项 2015NZ0002-3

详细信息
    作者简介:

    刘华清(1967—), 男, 博士, 副研究员, 主要从事水稻遗传育种研究(E-mail:lhq@fjage.org)

    通讯作者:

    王锋(1963—), 男, 博士, 研究员, 主要从事水稻分子育种研究(E-mail:wf@fjage.org)

  • 中图分类号: S511;S330;Q812

Morphological Alternation of Rice Plant by Site-Directed Mutagenesis on IPA1 Gene

  • 摘要:   目的  水稻株型改良是提高水稻产量的一个有效途径。水稻理想株型基因IPA1是一个调控水稻株型的关键基因,利用基因组编辑技术(TALENs技术)定点突变水稻IPA1基因,了解IPA1基因不同序列变异的株型效应,为进一步利用IPA1基因创制实用型水稻新株型材料奠定基础。  方法  利用TALENs技术定点突变优良恢复系明恢86的IPA1基因,通过测序鉴定突变体,种植于标准小区,调查分析其株型相关性状。  结果  利用TALENs技术获得了8种不同序列突变的水稻ipa1突变体,并通过转基因植株自交结合PCR分析筛选到去除了TALENs表达框,获得4种不含外源转基因成分的纯合突变体材料(IPA1基因表达区分别缺失2、4、16、23 bp)。表型分析发现,IPA1基因突变能够显著改变水稻的株高、有效穗数、穗长及穗粒数等性状。与野生型比较,缺失移码突变体株高降低7.9%~11.4%,有效穗数增加46.9%~68.4%,穗长短24.2%~29.3%,穗粒数减少31%~34%,结实率和千粒重差异不明显。  结论  利用TALENs技术定点突变水稻IPA1基因能够明显改变水稻株高、有效穗数、穗长及穗粒数等主要性状,产生水稻新株型。
  • 图  1  TALENs编辑水稻IPA1基因的靶位点

    注:空心框为基因IPA1的非翻译区,实心框为IPA1的编码区,实线为内含子;箭头所示为miRNA156结合序列区;TIPA1-1、TIPA1-2为基于TALENs设计的2个靶位点。

    Figure  1.  Target sites in rice IPA1 gene for TALENs editing

    Note: Empty frame encloses untranslated region; filled frame, coding region; and, solid line, intron. Arrow indicates binding sequence of microRNA156; TIPA1-1 and TIPA1-2 are two target sites designed based on TALENs.

    图  2  基因编辑T0代阳性转基因植株筛选

    注:CK+为阳性(质粒),CK-为阴性对照(野生型明恢86);1~11为部分转基因植株;M为DNA Ladder。

    Figure  2.  Screening of gene-edited T0 mutant transgenic plants

    Note: CK+: indicates positive control (plasmid); CK-:negative control (wild type Minghui 86); numbers 1-11:parts of transgenic plants; M: DNA ladder.

    图  3  TALENs编辑基因IPA1的序列突变类型

    注:下划线部分序列为TALENs识别序列,中间阴影部分为Spacer序列,缺失的碱基用“-”表示; 左边编号A起头的为未经过连续继代的抗性愈伤组织直接分化苗,编号B起头和C起头的分别为抗性愈伤组织连续继代2次和3次后的分化苗。右边数字为变化碱基数,缺失为“-”,插入为“+”;Homo为纯合突变,未标示者为杂合突变。

    Figure  3.  Mutation types of IPA1 produced by TALENs

    Note: Underscored sequences are TALENs recognition sequences; shaded part, spacer sequence; and, "-", missing base. On the left, numbers begin with A are directly differentiated from non-successive resistant callus; numbers with B, successively sub-cultured twice; and, numbers with C, successively sub-cultured three times. On the right, data are counts of variable base; and, "-" represents deletion; "+", insertion; "Homo", homozygous mutation; and, unmarked heterozygous mutation.

    图  4  不含转基因成分(TALENs载体成分)的T1植株筛选

    注:泳道1~10为靶基因有突变的一个转基因T1代株系,其中无扩增条带的为不含转基因成分植株;泳道11:野生型明恢86;泳道12:清水;M:DNA Ladder。

    Figure  4.  Selection of T1 plants without transgenic (TALENs vector) components

    Note: Lanes 1-10 are T1 transgenic plants mutated in target gene; and, those without amplified bands, non-transgenic. Lane 11 is wild-type Minghui 86; Lane 12, water; and, M, DNA ladder.

    图  5  不含转基因成分的突变体筛选

    注:A为测序检测野生型及缺失2 bp(-CT)纯合和杂合突变体靶序列; B为聚丙烯酰胺凝胶电泳结果,野生型(wt),缺失4 bp(-CTCT)的纯合(m)和杂合子(hm)。

    Figure  5.  Selection on mutants free of transgenic components

    Note: A represents detection of target sequences in wild type and mutants with 2 bp-deletion (-CT) homozygous or heterozygous by sequencing; and, B, marker analysis of target site in wild type (wt) and mutants with 4 bp-deletion (-CTCT), homozygous (m) or heterozygote (hm) by polyacrylamide gel electrophoresis.

    图  6  IPA1基因编辑突变体株型

    注:ipa1-CK为杂合突变体分离得到的野生型;ipa1-2、ipa1-4、ipa1-16分别为T2代缺失2、4、16 bp的纯合突变体。

    Figure  6.  Plant architectures of wild-type and IPA1 gene edited mutants

    Note:ipa1-CK is a wild-type from progeny of a heterozygous mutant; ipa1-2, ipa1-4, and ipa1-16 are T2 homozygous mutants with 2 bp, 4 bp, and 16 bp detection, respectively.

    表  1  基因编辑的靶位点序列

    Table  1.   Target sequence for gene-editing

    靶位点编号
    Target Num.
    靶位点序列
    Target sequences
    TIPA1-1 左臂Left arm:GCCGCCACCGACTCGAG
    右臂Right arm:TCCCATGGCTGGGTTGACA
    TIPA1-2 左臂Left arm:CGGTGCCGCCACCGACT
    右臂Right arm:GGGTTGACAGAAGAG
    下载: 导出CSV

    表  2  基因编辑再生植株阳性率及靶位点突变率

    Table  2.   Positive transgenic and mutation rates of target gene editing on plants

    载体名称
    Vector name
    再生克隆数 阳性克隆数 阳性率/% 突变克隆数 阳性克隆突变率/%
    TIPA1-1 38 36 94.7 2 5.6
    TIPA1-2 67 57 85.1 0 0.0
    注:再生克隆数Num. of regenerated clones,阳性克隆数Num. of positive clones,阳性率Positive percentage,突变克隆数Num. of mutation clones,阳性克隆突变率Mutation percentage of positive clones。
    下载: 导出CSV

    表  3  ipa1纯合突变体主要农艺性状

    Table  3.   Main agronomic traits of IPA1 mutants

    样品编号
    Sample NO.
    株高
    Plant height/cm
    有效穗数
    Effective panicle number
    穗长Average of panicle length/cm 穗粒数
    grains per panicle
    结实率
    SettingPercentage/%
    千粒重1000-grain weight/g
    ipa1-CK 102.2Aa 9.8A 25.6 A 178.2Aa 81.4a 28.3 a
    ipa1-2 90.6Bb 15.8C 19.3B 122.7Bb 79.20 a 28.1 a
    ipa1-4 90.7Bb 16.5C 19.4B 121.2Bb 83.70 a 28.6 a
    ipa1-16 94.1Bc 14.4B 18.1C 113.2Bc 82.50 a 28.8 a
    注:ipa1-CK为杂合突变体分离得到的野生型;ipa1-2、ipa1-4、ipa1-16分别为T2代缺失2、4、16 bp的纯合突变体。表中同列数据后无相同大、小写字母者分别表示同一性状不同样品材料之间差异达极显著水平(P < 0.01)和显著水平(P < 0.05)。
    Note:ipa1-CK is a wild-type from progeny of a heterozygous mutant; ipa1-2, ipa1-4, and ipa1-16 are T2 homozygous mutants with 2 bp, 4 bp, and 16 bp detection, respectively. Data in a same column with different capital letters indicate extremely significant differences (P < 0.01) and with different lowercase letters, significant differences (P < 0.05).
    下载: 导出CSV
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  • 收稿日期:  2019-04-12
  • 修回日期:  2019-07-14
  • 刊出日期:  2019-08-01

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