一个调控拟南芥盐/干旱胁迫响应的基因功能研究
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摘要
借助已有的基因表达谱分析,发现拟南芥中一个功能未知的基因DRS1被盐和干旱诱导表达。然而,DSR1基因是否在盐和干旱胁迫响应中起重要的作用尚不清楚。
为阐明DSR1基因在耐盐和抗旱中作用,获得了两个T-DNA插入DSR1基因的功能缺失型等位系dsr1-1和dsr1-2,本文研究了拟南芥野生型WT和突变体dsr1-1、dsr1-2对盐和干旱胁迫响应的特征。研究结果如下：
1.在不同浓度的NaCl胁迫条件下,dsr1-1突变体的存活率都明显高于野生型：在175mM NaCl条件下,dsr1-1的存活率比野生型高出40%；在125mM的NaCl条件下,突变体dsr1-1、dsr1-2比野生型WT的发芽率分别高出21.62%和27.03%；竖培实验中在100mM的NaCl条件下,突变体dsr1-1、dsr1-2比野生型WT的根长长1.5cm,突变体鲜重比野生型高4.5mg／5株。表明突变体dsr1-1、dsr1-2的耐盐能力显著高于野生型。
2.盐胁迫相关基因表达的QRT-PCR分析结果也表明,这种耐受性的提高可能与在盐胁迫下突变体dsr1-1、dsr1-2中MYB2/MYC2及其下游靶基因的表达水平提高有关,表明DSR1基因可能通过调控MYB2/MYC2介导的信号转导途径来调节盐胁迫响应。
3.在干旱胁迫条件下,突变体dsr1-1、dsr1-2的存活率显著高于野生型;突变体dsr1-1、dsr1-2对渗透胁迫的耐受性比野生型更强,且突变体体内可溶性总糖和脯氨酸的积累均比野生型高,这表明突变体dsr1-1、dsr1-2比野生型更加耐受干旱胁迫。
4.干旱胁迫相关基因表达的QRT-PCR分析结果表明,突变体dsr1-1、dsrl-2对干旱胁迫的耐受性可能与在干旱胁迫下突变体中脯氨酸合成基因P5CS及一些下游胁迫相关基因的表达水平升高有关。
5.ABA发芽率实验中,在5mM的ABA条件下,突变体dsr1-1、dsr1-2比野生型发芽率分别高出29.73%和32.43%,差异显著；而在ABA竖培实验中根长鲜重表型没有差异,相关胁迫基因的表达也几乎没有差异,说明突变体dsr1-1、dsr1-2的耐盐抗旱性可能与ABA依赖途径有关。
6.在KCl、LiCl胁迫实验中进一步证实了突变体dsr1-1、dsr1-2耐盐性可能与离子平衡机制相关,H2O2耐受又说明DSR1基因也参与了活性氧清除机制的调节,而甘露醇胁迫下的相关基因表达则说明了突变体dsr1-1、dsr1-2高盐及干旱胁迫的主要调节方式是通过渗透调节。
总之,所有这些结果表明,DSR1基因在调控盐和干旱胁迫响应过程中起着重要的作用。
Expression of the DSR1 gene encoding a unkown protein was found to be induced by salt and drought stresses by using gene expression microarray analysis data. However, it is unclear whether the DSR1 gene plays important roles in the regulation of the responses of Arabidopsis plants to salt and drought stresses.
To clarify the role of the DSR1 gene in salt/drought-stress responses, we studied the characteristics of responses of wild-type Arabidopsis and mutants dsr1-1, dsr1-2 to salt and drought stresses. The results are as follows:
1. Under different concentrations of NaCl stress conditions, the survival rate of dsr1-1 mutant is significantly higher than that of wild type. In 175mM NaCl conditions, survival rate of the mutant dsr1-1 was 40% higher than that of the wild type; in germination rate experiment, in 125mM NaCl conditions, the germination rates of mutants dsr1-1, dsr1-2 were higher than the wild type 21.62% and 27.03%, respectively; under vertical culture experiment conditions of 100mM of NaCl, the root length of mutant dsr1-1, dsr1-2 was about 1.5cm longer than that of the wild type, Fresh weight of mutant was approximately 4.5mg/5plants higher than that of the wild type. suggesting that the mutants dsr1-1, dsr1-2 are more resistant to salt stress than the wild type.
2. QRT-PCR analysis of salt stress-related gene expression also showed that enhanced resisitance of mutants dsr1-1, dsr1-2 to salt stress may be related to the increases in expression levels of MYB2/MYC2 and their downstream targeting genes, indicating that DSR1 gene could regulate MYB2/MYC2-mediated signal transduction pathway to regulate salt stress responses.
3. Under drought stress, the survival rate of mutants dsr1-1, dsr1-2 wereas significantly higher than that of wild type; and the tolerance of mutants dsr1-1, dsr1-2 to osmotic stress was more resistant to osmotic stress than the wild type, and. InUnder drought stress conditions, the levels of mutants soluble sugar and proline accumulation in mutants were significantly higher than those of the wild type, indicating that enhanced resistance of mutants dsr1-1 and dsr1-2 is assiacted with increases in the levels of soluble sugar and proline accumulationmore tolerant than wild-type to drought stress.
4. QRT-PCR analysis of drought stress-related gene expression showed that enhanced tolerance of mutants dsr1-1 and dsr1-2 to drought stress may be related to increases in the expression levels of the proline synthesis gene P5CS and some stress downstream genes under drought stress.
5. In the ABA germination experiments, in 5mM ABA condition, the germination rates of mutants dsr1-1 and dsr1-2 were significantly higher than the wild-type 29.73% and 32.43%, respectively, indicating responses of mutants dsr1-1and dsr1-2 to salt and drought stresses may be ABA dependent.
6.In KC1, and LiCl stress experiments further confirmed enhanced salt tolerance of mutants dsr1-1, and dsr1-2 are was associated with the mechanism of ion balance to salt tolerance. In addition, mutants also showed, there is another explanation of enhanced H2O2 resistance compared with the wild type,mutants suggesting that enhanced salt/drought resistance of mutants is also assicated with oxygen scavenging mechanism.
In sum, all these results show that DSR1 gene in the regulation of salt and drought stress plays an important role in the regulation of salt and drought stress responses.
为阐明DSR1基因在耐盐和抗旱中作用,获得了两个T-DNA插入DSR1基因的功能缺失型等位系dsr1-1和dsr1-2,本文研究了拟南芥野生型WT和突变体dsr1-1、dsr1-2对盐和干旱胁迫响应的特征。研究结果如下：
1.在不同浓度的NaCl胁迫条件下,dsr1-1突变体的存活率都明显高于野生型：在175mM NaCl条件下,dsr1-1的存活率比野生型高出40%；在125mM的NaCl条件下,突变体dsr1-1、dsr1-2比野生型WT的发芽率分别高出21.62%和27.03%；竖培实验中在100mM的NaCl条件下,突变体dsr1-1、dsr1-2比野生型WT的根长长1.5cm,突变体鲜重比野生型高4.5mg／5株。表明突变体dsr1-1、dsr1-2的耐盐能力显著高于野生型。
2.盐胁迫相关基因表达的QRT-PCR分析结果也表明,这种耐受性的提高可能与在盐胁迫下突变体dsr1-1、dsr1-2中MYB2/MYC2及其下游靶基因的表达水平提高有关,表明DSR1基因可能通过调控MYB2/MYC2介导的信号转导途径来调节盐胁迫响应。
3.在干旱胁迫条件下,突变体dsr1-1、dsr1-2的存活率显著高于野生型;突变体dsr1-1、dsr1-2对渗透胁迫的耐受性比野生型更强,且突变体体内可溶性总糖和脯氨酸的积累均比野生型高,这表明突变体dsr1-1、dsr1-2比野生型更加耐受干旱胁迫。
4.干旱胁迫相关基因表达的QRT-PCR分析结果表明,突变体dsr1-1、dsrl-2对干旱胁迫的耐受性可能与在干旱胁迫下突变体中脯氨酸合成基因P5CS及一些下游胁迫相关基因的表达水平升高有关。
5.ABA发芽率实验中,在5mM的ABA条件下,突变体dsr1-1、dsr1-2比野生型发芽率分别高出29.73%和32.43%,差异显著；而在ABA竖培实验中根长鲜重表型没有差异,相关胁迫基因的表达也几乎没有差异,说明突变体dsr1-1、dsr1-2的耐盐抗旱性可能与ABA依赖途径有关。
6.在KCl、LiCl胁迫实验中进一步证实了突变体dsr1-1、dsr1-2耐盐性可能与离子平衡机制相关,H2O2耐受又说明DSR1基因也参与了活性氧清除机制的调节,而甘露醇胁迫下的相关基因表达则说明了突变体dsr1-1、dsr1-2高盐及干旱胁迫的主要调节方式是通过渗透调节。
总之,所有这些结果表明,DSR1基因在调控盐和干旱胁迫响应过程中起着重要的作用。
Expression of the DSR1 gene encoding a unkown protein was found to be induced by salt and drought stresses by using gene expression microarray analysis data. However, it is unclear whether the DSR1 gene plays important roles in the regulation of the responses of Arabidopsis plants to salt and drought stresses.
To clarify the role of the DSR1 gene in salt/drought-stress responses, we studied the characteristics of responses of wild-type Arabidopsis and mutants dsr1-1, dsr1-2 to salt and drought stresses. The results are as follows:
1. Under different concentrations of NaCl stress conditions, the survival rate of dsr1-1 mutant is significantly higher than that of wild type. In 175mM NaCl conditions, survival rate of the mutant dsr1-1 was 40% higher than that of the wild type; in germination rate experiment, in 125mM NaCl conditions, the germination rates of mutants dsr1-1, dsr1-2 were higher than the wild type 21.62% and 27.03%, respectively; under vertical culture experiment conditions of 100mM of NaCl, the root length of mutant dsr1-1, dsr1-2 was about 1.5cm longer than that of the wild type, Fresh weight of mutant was approximately 4.5mg/5plants higher than that of the wild type. suggesting that the mutants dsr1-1, dsr1-2 are more resistant to salt stress than the wild type.
2. QRT-PCR analysis of salt stress-related gene expression also showed that enhanced resisitance of mutants dsr1-1, dsr1-2 to salt stress may be related to the increases in expression levels of MYB2/MYC2 and their downstream targeting genes, indicating that DSR1 gene could regulate MYB2/MYC2-mediated signal transduction pathway to regulate salt stress responses.
3. Under drought stress, the survival rate of mutants dsr1-1, dsr1-2 wereas significantly higher than that of wild type; and the tolerance of mutants dsr1-1, dsr1-2 to osmotic stress was more resistant to osmotic stress than the wild type, and. InUnder drought stress conditions, the levels of mutants soluble sugar and proline accumulation in mutants were significantly higher than those of the wild type, indicating that enhanced resistance of mutants dsr1-1 and dsr1-2 is assiacted with increases in the levels of soluble sugar and proline accumulationmore tolerant than wild-type to drought stress.
4. QRT-PCR analysis of drought stress-related gene expression showed that enhanced tolerance of mutants dsr1-1 and dsr1-2 to drought stress may be related to increases in the expression levels of the proline synthesis gene P5CS and some stress downstream genes under drought stress.
5. In the ABA germination experiments, in 5mM ABA condition, the germination rates of mutants dsr1-1 and dsr1-2 were significantly higher than the wild-type 29.73% and 32.43%, respectively, indicating responses of mutants dsr1-1and dsr1-2 to salt and drought stresses may be ABA dependent.
6.In KC1, and LiCl stress experiments further confirmed enhanced salt tolerance of mutants dsr1-1, and dsr1-2 are was associated with the mechanism of ion balance to salt tolerance. In addition, mutants also showed, there is another explanation of enhanced H2O2 resistance compared with the wild type,mutants suggesting that enhanced salt/drought resistance of mutants is also assicated with oxygen scavenging mechanism.
In sum, all these results show that DSR1 gene in the regulation of salt and drought stress plays an important role in the regulation of salt and drought stress responses.
引文
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