Functional analysis of AtJAR1 gene in salt tolerance of Arabidopsis thaliana
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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 -
关键词:
- 拟南芥 /
- CRISPR/Cas9 /
- 茉莉酰氨基酸结合物合成酶 /
- 耐盐性 /
- K+/Na+平衡
Abstract:Objective Jasmonal amino acid conjugate synthase (JAR1) can catalyze jasmonic acid (JA) to form jasmonic acid-soleucine (JA-Ile), an active form of jasmonic acid, and activate the JA signal pathway. JA signaling pathway plays an important role in mediating the response of plants to salt stress. This study aims to explore the function of AtJAR1 in plant salt tolerance, which plays an important role in studying the mechanism of JA signal pathway affecting plant salt tolerance. Method CRISPR/cas9 gene editing technology was used to create two different Arabidopsis thaliana AtJAR1 gene mutants. The aboveground biomass of the two mutants and the expression of JA signal marker gene were analyzed to determine the loss of AtJAR1 gene function. Then, the effects of different concentrations of NaCl and ABA treatment on seed germination and seedling establishment of jar1 mutants were observed and analyzed to clarify the effect of AtJAR1 gene on salt tolerance of A. thaliana. Finally, the role of AtJAR1 gene in salt tolerance of A. thaliana was investigated by comparing the content of potassium and sodium ions in wild type and mutants before and after salt treatment, as well as the expression of AtHAK5, a high affinity potassium ion transporter gene. Result The expression of JA signal marker genes AtVSP1 and AtVSP2 decreased significantly, indicating the loss of AtJAR1 gene function. Different from the jar1#1 mutant produced by point mutation, the growth of the two mutants accelerated in the first three weeks, then gradually slowed down and the leaves wilted. At the same time, AtJAR1 mutation can alleviate the inhibition of salt stress and ABA on seed germination and root growth. In addition, AtJAR1 mutation can promote the expression of AtHAK5 and the absorption and transport of potassium ions in roots under salt stress. Conclusion JA signaling pathway may affect the expression of AtHAK5 through interaction with ABA, so as to regulate the absorption and transport of K+ by plant roots, change the intracellular K+/Na+ balance, and finally affect plant salt tolerance. [Ch, 8 fig. 2 tab. 52 ref.] -
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 1.00 MgSO4·7H2O 0.4 1.00 0.40 微量元素② 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 
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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
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