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不同水稻tms5突变位点对雄性不育起点温度的影响

吴明基 刘华清 陈建民 付艳萍 杨绍华 宋亚娜 林艳 王锋

吴明基,刘华清,陈建民,等. 不同水稻tms5突变位点对雄性不育起点温度的影响 [J]. 福建农业学报,2023,38(7):761−771 doi: 10.19303/j.issn.1008-0384.2023.07.001
引用本文: 吴明基,刘华清,陈建民,等. 不同水稻tms5突变位点对雄性不育起点温度的影响 [J]. 福建农业学报,2023,38(7):761−771 doi: 10.19303/j.issn.1008-0384.2023.07.001
WU M J, LIU H Q, CHEN J M, et al. Critical Sterility-inducing Temperature of Rice Affected by tms5 Mutation Site [J]. Fujian Journal of Agricultural Sciences,2023,38(7):761−771 doi: 10.19303/j.issn.1008-0384.2023.07.001
Citation: WU M J, LIU H Q, CHEN J M, et al. Critical Sterility-inducing Temperature of Rice Affected by tms5 Mutation Site [J]. Fujian Journal of Agricultural Sciences,2023,38(7):761−771 doi: 10.19303/j.issn.1008-0384.2023.07.001

不同水稻tms5突变位点对雄性不育起点温度的影响

doi: 10.19303/j.issn.1008-0384.2023.07.001
基金项目: 福建省自然科学基金项目(2020J011359);福建省农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021002);福建省科技重大专项(2020NZ08017)
详细信息
    作者简介:

    吴明基(1976 —),男,博士,助理研究员,主要从事水稻遗传育种研究,E-mail:839845032@qq.com

    通讯作者:

    王锋(1963 —),男,博士,研究员,主要从事水稻基因工程与遗传育种研究,E-mail:wf@fjage.org

  • 中图分类号: S511

Critical Sterility-inducing Temperature of Rice Affected by tms5 Mutation Site

  • 摘要:   目的  研究不同水稻tms5突变位点对雄性不育起点温度的影响,探讨不育起点温度遗传调控途径。  方法  在水稻TMS5的6个外显子上设计11个CRISPR/Cas9基因编辑靶点,依次命名为T501 — T511,构建相应载体转化粳稻品种日本晴和籼稻品种明恢86,获得各靶点的tms5移码突变体。田间自然高温及人工气候箱(设日平均22 、24和28 ℃ 3种温度)条件下分析tms5突变体的花粉碘染及自交结实率,鉴定不育起点温度。  结果  粳稻日本晴tms5突变体的不育起点温度高于28 ℃,籼稻明恢86的不同tms5突变体不育起点温度为22~28 ℃。此外,同一遗传背景下,通过T501靶点编辑产生的tms5-1移码突变体不育起点温度均显著高于T502靶点的tm5-2突变体,其他位点上的tms5突变体育性特征与tm5-2突变体并无差异。基因表达量分析表明,tms5-1突变体幼穗UbL40 基因表达量显著低于tm5-2突变体的表达量。  结论  水稻tms5突变体不育起点温度不仅受遗传背景的影响,tms5基因突变位点不同也会影响不育起点温度,特别是T501位点与其余位点突变体间不育起点温度差异显著,为研究水稻tms5两系不育起点温度的分子机理及遗传调控网络提供了新思路。
  • 图  1  CRISPR/Cas9系统介导的TMS5基因特异性突变

    A:TMS5基因结构的示意图,黑色矩形代表TMS5的6个外显子,图中短竖线所指位置为各靶点在TMS5基因上的位置。B:CRISPR/Cas9介导的TMS5突变实例,每个靶点只列出了一种突变类型, T505、T508、T510靶点列出的是杂合型突变,T511靶点是双突变,其余位点列出的均为纯合型突变;图上三角形处为缺失或插入突变发生的位置。

    Figure  1.  CRISPR/Cas9-mediated genome editing of tms5

    A.Schematic tms5 structure; black rectangles represent 6 exons of tms5; short vertical line refers to position of each target. B. CRISPR/Cas9-mediated tms5 mutants; only one mutation type is listed for each target; T505, T508, and T510 are listed as heterozygous mutations; T511 as biallelic mutation; and remainders as homozygous mutations; triangles point at deletion and insertion sites.

    图  2  T1tms5突变体在田间高温下的花粉育性

    ①A和B:明恢86(A)及日本晴(B)遗传背景的tms5突变体T1代材料花粉育性;两种背景的tms5突变体(依次为T501至T511靶点突变)育性敏感期处于福州8月初的自然长日高温条件;MH86为明恢86,NIP为日本晴;M-#和N-#分别代表明恢86和日本晴遗传背景的突变体。C:明恢86和日本晴两种遗传背景的野生型、tms5-1突变体及tms5-2突变体穗部开花特征。D: M-T501-#1同一穗上出现可育与不育的颖花。②标尺长度:80 µm(A、B), 0.5 cm(C、D)。

    Figure  2.  Pollen fertility of T1 tms5 mutants under NHT

    ①Aand B: Pollen fertility of tms5 mutants in MH86 (A) and NIP (B) induced at 11 targets from T501 to T511; NHT was in early August in Fuzhou; M-# and N-# are mutant plants of MH86 and NIP, respectively. C: Flowering characteristics of WT, tms5-1, and tms5-2 mutants of NIP and MH86 backgrounds. D: Both fertile and sterile spikelets were set on same panicle of M-T501-#1. ②Scale bars represent 80 μm for A and B, 0.5 cm for C and D.

    图  3  T2tms5突变体在不同温度条件下的花粉育性

    ① 22℃ 、24 ℃、28 ℃、NHT分别表示明恢86及日本晴遗传背景的tms5-1tms5-2突变体在人工气候箱22 ℃、24 ℃、28 ℃及福州8月初自然高温条件下(NHT)的花粉育性。MH86为明恢86,NIP为日本晴,M-#和N-#分别代表明恢86和日本晴遗传背景的突变体。②标尺长度:80 µm。

    Figure  3.  Pollen fertility of T2 tms5 mutants under varied temperatures

    ① Pollen fertility of tms5-1 and tms5-2 mutants of MH86 and NIP backgrounds cultivated at 22 ℃, 24 ℃ or 28 ℃ in phytotrons, or under NHT, respectively; M-# and N-# represent mutant plants of MH86 and NIP backgrounds, respectively. ② Scale bars are 80 μm in length.

    图  4  Ub L40基因的表达量差异分析

    A、B和C分别表示明恢86及日本晴tms5突变体UbL401、UbL402UbL404的表达特征,数据为平均数±标准差,n=3。

    Figure  4.  Differential expressions of UbL40

    A, B and C: expressions of UbL401, UbL402, and UbL404 in tms5 mutants of MH86 and NIP, respectively. Data are mean ± standard deviation calculated from 3 replicates.

    表  1  本试验所用sgRNA序列

    Table  1.   sgRNA applied

    靶位点
    Target
    序列(5′- 3′)
    Sequence
    试验目的
    Purpose
    T501S CAGGAACAGCGGCAAGTCATCGC sgRNA构建/
    sgRNA Construction
    T501A AACGCGATGACTTGCCGCTGTTC
    T502S CAGCCACCGCGCCGCCACCGGGT sgRNA构建/
    sgRNA Construction
    T502A AACACCCGGTGGCGGCGCGGTGG
    T503S CAGCACCGTCGAGGGCTACCCCG sgRNA构建/
    sgRNA Construction
    T503A AACCGGGGTAGCCCTCGACGGTG
    T504S CAGCCCATGTACGTCGCCACCCG sgRNA构建/
    sgRNA Construction
    T504A AACCGGGTGGCGACGTACATGGG
    T505S CAGAACCTCGTCCCCCTCGAGAT sgRNA构建/
    sgRNA Construction
    T505A AACATCTCGAGGGGGACGAGGTT
    T506S CAGGTAGGGGTATGTGATATACA sgRNA构建/
    sgRNA Construction
    T506A AACTGTATATCACATACCCCTAC
    T507S CAGATCAAGCAGCTGAAGCTGTC sgRNA构建/
    sgRNA Construction
    T507A AACGACAGCTTCAGCTGCTTGAT
    T508S CAGTGCTTTTACCGGAGATACGA sgRNA构建/
    sgRNA Construction
    T508A AACTCGTATCTCCGGTAAAAGCA
    T509S CAGTGGGCACACCCATCTGTTTG sgRNA构建/
    sgRNA Construction
    T509A AACCAAACAGATGGGTGTGCCCA
    T510S CAGTTCTGCTCGTTATACCGCAG sgRNA构建/
    sgRNA Construction
    T510A AACCTGCGGTATAACGAGCAGAA
    T511S CAGAAGTAGAGTTCATGCATTGA sgRNA构建/
    sgRNA Construction
    T511A AACTCAATGCATGAACTCTACTT
    下载: 导出CSV

    表  2  供试引物

    Table  2.   Primers selected

    引物名
    Primer name
    序列
    Sequence (5′-3′)
    试验目的
    Purpose
    HptFAGGTCAGGCTCTCGCTAAAC转基因阳性株检测/
    Identify transgenic plants
    Hpt-RACGTAAGGGATGACGCACAAT
    TMS5FCCATCGTGCTTCGTGCCATMS5基因扩增、测序
    PCR and Sanger sequence for TMS5
    TMS5RGAGTTCTTGGTACATGAGTGC
    UBL401FCTACCCCAAGGGGATCGAGReal-Time PCR
    UBL401RGCAAGGCGGTCGATTGAACT
    UBL402FTCGCTCAGGGTCCTCGCCTAReal-Time PCR
    UBL402RCTAGATTCGGCATCCAGTAG
    UBL404FTACACCATCCAGGAGCCCAReal-Time PCR
    UBL404RGGTAGCTGGGCATACGAAG
    ActinFCGTCTGCGATAATGGAACTGReal-Time PCR
    ActinRTCTGGGTCATCTTCTCACGA
    下载: 导出CSV

    表  3  各载体转化T0代植株突变分析

    Table  3.   Mutation of T0 transgenic plants

    载体
    Construct
    靶点位置
    Target site
    品种
    Variety
    转基因植株数
    Numbers of transgenic plants
    含突变植株数
    Numbers of mutation plants
    突变率
    Mutation rates/%
    TMS501 TMS5第一外显子
    First exon of TMS5
    Nipponbare 21 15 71.4
    MH86 18 16 88.9
    TMS502 Nipponbare 22 21 95.5
    MH86 11 11 100.0
    TMS503 Nipponbare 29 11 37.9
    MH86 9 6 33.3
    TMS504 Nipponbare 21 11 52.4
    MH86 15 7 46.7
    TMS505 Nipponbare 21 6 28.6
    MH86 21 7 33.3
    TMS506 TMS5第二外显子
    2nd exon of TMS5
    Nipponbare 19 13 68.4
    MH86 24 21 87.5
    TMS507 Nipponbare 10 2 20.0
    MH86 11 1 9.1
    TMS508 TMS5第三外显子
    3rd exon of TMS5
    Nipponbare 10 1 10.0
    MH86 18 2 11.1
    TMS509 TMS5第四外显子
    4th exon of TMS5
    Nipponbare 21 14 66.6
    MH86 18 13 72.2
    TMS510 TMS5第五外显子
    5th exon of TMS5
    Nipponbare 10 1 10.0
    MH86 15 2 13.3
    TMS511 TMS5第六外显子
    6th exon of TMS5
    Nipponbare 14 12 85.7
    MH86 11 9 81.8
    下载: 导出CSV

    表  4  T1tms5材料结实率

    Table  4.   Seed setting rates of T1 generation of tms5 mutants

    背景
    Background
    材料
    Lines
    突变序列
    § Mutation sequences
    平均结实率
    †Average seed setting rates/%
    明恢86
    MH86
    MH8687.5±4.3 a
    M-T501-#1T501gaacagcggcaagtcatAcgccgg6.9±9.6 b
    M-T501-#2T501gaacagcggcaagtcatTcgccgg4.0±5.6 b
    M-T501-#3T501gaacagcggcaagtcatCcgccgg5.4±6.9 b
    M-T502-#1T502ccaccgcgcc-------ggtc0 c
    M-T502-#2T502ccaccgcgccgccaccgCggtcgg0 c
    M-T502-#3T502ccaccgcgccgccaccgTggtcgg0 c
    M-T511-#1T511gtagagttcatgcat-gaaggaa0 c
    M-T511-#2T511gtagagttcatgcatTtgaaggaa0 c
    M-T511-#3T511gtagagttcatgcatAtgaaggaa0 c
    Nipponbare89.5±2.0 a
    日本晴
    Nipponbare
    N-T501-#1T501gc-----------------gacc48.9±22.1 c
    N-T501-#2T501gaacagcggcaagtca--gccgg54.2±15.8 b
    N-T501-#3T501gaacagcggcaagtcatTcgccgg44.1±12.2 c
    N-T502-#1T502ccaccgcgccg------Cggtcggc10.1±10.2 d
    N-T502-#2T502ccaccgcgccgccaccgAggtcggc8.8±10.1 d
    N-T502-#3T502ccaccgcgccgccaccgTggtcgg9.4±9.6 d
    N-T511-#1T511gtagagttcatgca--gaaggaa9.6±8.4 d
    N-T511-#2T511gtagagttcatgcatCtgaaggaa10.6±10.0 d
    N-T511-#3T511gtagagttcatgcatAtgaaggaa10.4±8.9 d
    ①§靶点位置的大写字母和短划线分别表示插入与缺失突变类型。 ②†结实率为10穗结实率的平均值±标准差。③同列数据后不同小写字母表示依据LSD多重比较,同一遗传背景材料间的差异达显著水平(P<0.05),下同。
    ①§ Capital letters and dashes at target sites indicate type of insertion and deletion mutations, respectively. ②† Average seed setting rates are mean ± standard deviation calculated from 10 panicles. ③ Data with different lowercase letters on same column indicate significant difference according to LSD multiple comparison (P<0.05), Same for below.
    下载: 导出CSV

    表  5  T2tms5 突变体花粉育性及自交结实率

    Table  5.   Pollen fertility and seed setting rates of T2 tms5 mutants

    植株编号
    Plant ID
    花粉黑染率
    Stained pollens rate/%
    自交结实率
    Seed setting rate/%
    22 ℃24 ℃28 ℃自然条件
    Natural condition
    22 ℃24 ℃28 ℃自然条件
    Natural condition
    MH86 86.7±7.8 a 87.1±6.9 a 87.2±5.8 a 88.3±7.1 a 83.4±8.7 a 83.1±6.4 a 85.2±7.2 a 85.9±6.5 a
    M-T501-#1 87.5±10.3 a 61.6±13.5 c 0 b 20.5±11.5 c 67.8±20.3 b 30.7±12.3 c 0 b 8.5±10.6 c
    M-T501-#2 80.1±12.0 b 62.7±10.4 c 0 b 28.2±16.3 b 65.3±16.5 bc 40.2±20.5 b 0 b 21.3±16.3 b
    M-T501-#3 83.3±8.7 ab 71.2±20.2 b 0 b 25.6±15.6 bc 62.2±8.5 c 41.4±17.4 b 0 b 18.7±21.5 b
    M-T502-#1 12.1±10.6cd 0 d 0 b 0 d 1.5±0.3 d 0 d 0 b 0 d
    M-T502-#2 9.3±5.8d 0 d 0 b 0 d 1.8±0.6 d 0 d 0 b 0 d
    M-T502-#3 13.9±10.1 c 0 d 0 b 0 d 1.9±1.3 d 0 d 0 b 0 d
    Nipponbare 92.5±5.9a 90.6±7.3 a 91.1±6.3 a 92.5±6.5 a 87.6±8.2 a 88.1±9.3 a 87.8±6.6 a 90.5±2.3 a
    N-T501-#1 84.2±5.7 b 78.2±20.4 b 35.1±11.3 c 67.4±6.9 c 84.1±8.3 a 66.4±10.6 b 13.1±7.2 c 45.2±33.1 c
    N-T501-#2 86.2±19.5 b 80.2±12.9 b 37.1±8.8 bc 75.7±15.2 b 83.2±7.8 a 68.5±10.3 b 14.8±5.1 bc 52.1±35.8b
    N-T501-#3 82.3±21.2 b 80.1±18.1 b 40.1±10.5 b 69.1±9.6 c 83.1±10.9 a 50.5±12.2 c 18.6±9.5 b 50.7±28.3 bc
    N-T502-#1 82.2±6.8 b 70.0±20.2 c 6.1±2.5 d 25.2±19.0 e 72.3±9.2 bc 35.3±28.1 d 3.5±1.6 c 14.1±11.2 d
    N-T502-#2 85.5±6.3 b 72.2±18.5 c 7.6±6.6 d 31.1±16.9d 75.8±12.3 b 33.4±16.9 d 7.5±8.1 c 18.6±10.3 d
    N-T502-#3 84.1±9.2 b 70.5±16.7 c 9.2±8.1 d 26.3±27.7 e 69.6±9.2 c 25.4±20.3 e 4.2±2.5 c 17.6±16.3 d
    花粉黑染率及自交结实率均为5株的平均值,自然条件指福州8月份的长日高温条件。
    Data are means on 5 plants; NHT was defined as long day and high temperature in August in Fuzhou.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2023-05-12
  • 修回日期:  2023-05-22
  • 网络出版日期:  2023-08-16
  • 刊出日期:  2023-07-28

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