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AtJAR1基因在拟南芥耐盐性中的功能分析

李丹丹 林蓉 李新国 郑月萍

李丹丹, 林蓉, 李新国, 郑月萍. AtJAR1基因在拟南芥耐盐性中的功能分析[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210742
引用本文: 李丹丹, 林蓉, 李新国, 郑月萍. AtJAR1基因在拟南芥耐盐性中的功能分析[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210742
LI Dandan, LIN Rong, LI Xinguo, ZHENG Yueping. Functional analysis of AtJAR1 gene in salt tolerance of Arabidopsis thaliana[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210742
Citation: LI Dandan, LIN Rong, LI Xinguo, ZHENG Yueping. Functional analysis of AtJAR1 gene in salt tolerance of Arabidopsis thaliana[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210742

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AtJAR1基因在拟南芥耐盐性中的功能分析

doi: 10.11833/j.issn.2095-0756.20210742
基金项目: 国家重点研发计划项目(2018YFD1000906-3);浙江省自然科学基金青年基金项目(Q21C020003)
详细信息
    作者简介: 李丹丹(ORCID: 0000-0003-3501-1220),从事植物生物技术研究。E-mail: 2551448078@qq.com
    通信作者: 郑月萍(ORCID: 0000-0002-8702-9556),讲师,博士,从事植物生化和代谢研究。E-mail: 630713861@qq.com
  • 中图分类号: S722.3; Q781

Functional analysis of AtJAR1 gene in salt tolerance of Arabidopsis thaliana

  • 摘要:   目的  茉莉酰氨基酸结合物合成酶(jasmonoyl amino acid conjugate synthase,JAR1)可以催化茉莉酸(jasmonic acid,JA)形成茉莉酸的活性形式茉莉酸异亮氨基酸复合体(jasmonic acid-isoleucine,JA-Ile),从而激活JA信号途径。JA信号途径在介导植物盐胁迫的响应中发挥重要作用,因此,探究AtJAR1在植物耐盐性中的功能对于研究JA信号途径影响植物耐盐性的机制具有重要作用。  方法  运用CRISPR/Cas9基因编辑技术,创建了2个不同的拟南芥Arabidopsis thaliana AtJAR1基因突变体,并对这2个突变体进行地上部生物量的统计分析和JA信号标记基因的表达分析,以确定AtJAR1基因功能缺失。之后,观察分析不同浓度氯化钠和脱落酸(ABA)处理对jar1突变体的种子萌发和幼苗建成的影响,明确AtJAR1基因对拟南芥耐盐性的影响。最后,通过比较分析盐处理前后野生型和突变体的钾离子(K+)和钠离子(Na+)质量摩尔浓度,以及高亲和力K+转运蛋白基因AtHAK5的表达变化情况,初步探究AtJAR1基因在拟南芥耐盐性中的功能。  结果  JA信号标记基因AtVSP1和AtVSP2的表达量大幅下调,表明AtJAR1基因功能丧失。与点突变产生的jar1#1突变体不同的是,这2个突变体表现为前3周生长加快,之后逐渐减缓并出现叶片萎蔫的表型。同时,AtJAR1突变可以缓解盐胁迫和ABA对种子萌发和根系生长产生的抑制作用。此外,盐胁迫下AtJAR1突变可以促进AtHAK5的表达和根系对K+的吸收转运。  结论  JA信号途径可能通过与ABA交互作用影响AtHAK5的表达量,以调节植物根系对K+的吸收转运,进而改变细胞内K+/Na+平衡,最终影响植物耐盐性。图8表2参52
  • 图  1  jar1#1和jar1#2突变序列信息

    Figure  1  Sequence information of jar1#1 and jar1#2 mutants

    图  2  AtJAR1基因突变体与野生型的表型差异

    Figure  2  Phenotypic differences between AtJAR1 gene mutants and wild type

    图  3  不同盐浓度下拟南芥种子的发芽率

    Figure  3  Germination rate of A. thaliana seeds under different salt concentrations

    图  4  atjar1突变体主根长对盐胁迫响应

    Figure  4  Effect of salt stress on the main root length of atjar1 mutant

    图  5  不同ABA浓度下拟南芥种子的萌发率

    Figure  5  Germination rate of A. thaliana seeds under different ABA concentrations

    图  6  atjar1突变体主根长对ABA的响应

    Figure  6  Effect of ABA on the main root length of atjar1 mutant

    图  7  野生型和AtJAR1基因突变体根K+和Na+变化

    Figure  7  Determination of element contents in wild-type and mutants of AtJAR1 gene

    图  8  盐胁迫下野生型和突变体中AtHAK5基因的相对表达量

    Figure  8  Relative expression of AtHAK5 gene in wild-type and mutants under salt stress

    表  1  1/5 Hoagland水培培养液配方

    Table  1.   1/5 Hoagland formula for hydroponic culture solution

    组成母液浓度/
    (mol·L−1)
    母液添加体
    积/(mL·L−1)
    水培液中浓
    度/(mmol·L−1)
    组成母液浓度/
    (mol·L−1)
    母液添加体
    积(mL·L−1)
    水培液中浓
    度(mmol·L−1)
    KNO3 1.0 1.25 1.25 KH2PO4 0.5 1.00 0.50
    Ca(NO3)·4H2O 1.0 1.00 1.00 Fe盐 1.00
    MgSO4·7H2O 0.4 1.00 0.40 微量元素 0.10
      说明:①1 000倍铁盐母液的配置:称取5.56 g七水合硫酸亚铁(FeSO4·7H2O)放入100 mL烧杯中,边加水边搅拌;将7.5 g 二水合乙     二胺四乙酸二钠(EDTA·2Na·2H2O)放入1 L烧杯中加水煮沸,缓慢加入FeSO4溶液,于微波炉中煮沸2 min;避光放入60 ℃烘     箱烘2 h以上;室温静置,冷却后定容成1 L,即为浓度为20 mmol·L−1的母液。使用时稀释1000倍。②10 000倍微量元素母液的     配置:称取H3BO3 6.18 g;MnCl2·4H2O 0.99 g;CuSO4·5H2O 1.25 g;ZnSO4·7H2O 1.44 g;H2MoO4 0.08 g;NaCl 0.20 g溶解     于水中,冷却后定容至1 L。使用时稀释10 000倍
    下载: 导出CSV

    表  2  相关引物序列

    Table  2.   Sequence of related primers

    基因上游引物(5′→3′)下游引物(5′→3′)
    AtActin2 GTCGTACAACCGGTATTGTGCT TGTCTCTTACAATTTCCCGCTCT
    AtVSP1 TGGATCTTTGACCTAGACGACA GAGTTCCAAGAGGTTTTCGTA
    AtVSP2 TGACCTAGATGATACCCTCCTCTC CAATCCCGAGCTCTATGATGTT
    AtHAK5 TCTGCATCACTGGGACGGAG CAGTATAACGGATCAGGGATTGA
    下载: 导出CSV
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  • 收稿日期:  2021-11-11
  • 修回日期:  2022-04-24

AtJAR1基因在拟南芥耐盐性中的功能分析

doi: 10.11833/j.issn.2095-0756.20210742
    基金项目:  国家重点研发计划项目(2018YFD1000906-3);浙江省自然科学基金青年基金项目(Q21C020003)
    作者简介:

    李丹丹(ORCID: 0000-0003-3501-1220),从事植物生物技术研究。E-mail: 2551448078@qq.com

    通信作者: 郑月萍(ORCID: 0000-0002-8702-9556),讲师,博士,从事植物生化和代谢研究。E-mail: 630713861@qq.com
  • 中图分类号: S722.3; Q781

摘要:   目的  茉莉酰氨基酸结合物合成酶(jasmonoyl amino acid conjugate synthase,JAR1)可以催化茉莉酸(jasmonic acid,JA)形成茉莉酸的活性形式茉莉酸异亮氨基酸复合体(jasmonic acid-isoleucine,JA-Ile),从而激活JA信号途径。JA信号途径在介导植物盐胁迫的响应中发挥重要作用,因此,探究AtJAR1在植物耐盐性中的功能对于研究JA信号途径影响植物耐盐性的机制具有重要作用。  方法  运用CRISPR/Cas9基因编辑技术,创建了2个不同的拟南芥Arabidopsis thaliana AtJAR1基因突变体,并对这2个突变体进行地上部生物量的统计分析和JA信号标记基因的表达分析,以确定AtJAR1基因功能缺失。之后,观察分析不同浓度氯化钠和脱落酸(ABA)处理对jar1突变体的种子萌发和幼苗建成的影响,明确AtJAR1基因对拟南芥耐盐性的影响。最后,通过比较分析盐处理前后野生型和突变体的钾离子(K+)和钠离子(Na+)质量摩尔浓度,以及高亲和力K+转运蛋白基因AtHAK5的表达变化情况,初步探究AtJAR1基因在拟南芥耐盐性中的功能。  结果  JA信号标记基因AtVSP1和AtVSP2的表达量大幅下调,表明AtJAR1基因功能丧失。与点突变产生的jar1#1突变体不同的是,这2个突变体表现为前3周生长加快,之后逐渐减缓并出现叶片萎蔫的表型。同时,AtJAR1突变可以缓解盐胁迫和ABA对种子萌发和根系生长产生的抑制作用。此外,盐胁迫下AtJAR1突变可以促进AtHAK5的表达和根系对K+的吸收转运。  结论  JA信号途径可能通过与ABA交互作用影响AtHAK5的表达量,以调节植物根系对K+的吸收转运,进而改变细胞内K+/Na+平衡,最终影响植物耐盐性。图8表2参52

English Abstract

李丹丹, 林蓉, 李新国, 郑月萍. AtJAR1基因在拟南芥耐盐性中的功能分析[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210742
引用本文: 李丹丹, 林蓉, 李新国, 郑月萍. AtJAR1基因在拟南芥耐盐性中的功能分析[J]. 浙江农林大学学报. doi: 10.11833/j.issn.2095-0756.20210742
LI Dandan, LIN Rong, LI Xinguo, ZHENG Yueping. Functional analysis of AtJAR1 gene in salt tolerance of Arabidopsis thaliana[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210742
Citation: LI Dandan, LIN Rong, LI Xinguo, ZHENG Yueping. Functional analysis of AtJAR1 gene in salt tolerance of Arabidopsis thaliana[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20210742

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