一株拟南芥敏感突变体的筛选及表型分析
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摘要
重金属镉(Cd)毒性高,难降解,是对生物体有较强遗传毒性的物质。近年来,有关植物对重金属Cd胁迫响应的基因已有很多报道,这些基因主要有ZIP家族,HMA家族等。通过对这些基因的研究了解了一些植物应答重金属Cd胁迫过程中的自我保护机制,例如HMA家族基因对区室化的作用等。但是,植物应对Cd胁迫是一个复杂的过程,这其中涉及的应答机制相关研究尚不深入。
我们通过优化拟南芥Cd敏感突变体筛选条件,从化学诱导激活型拟南芥T-DNA插入突变体库中筛选出一些具有Cd胁迫敏感表型的株系；将其中表型最明显的植株为命名为atcsr-2,作为本研究的材料。通过Tail-PCR鉴定表明该突变体的T-DNA插入位点为At2g36130(相应地称作AtCSR).该基因能够编码一种肽酰-脯氨酰顺反式异构酶(Peptidyl-Prolyl cis/trans Isomerase),此酶能够降低蛋白质折叠过程中所需的能量,有助于蛋白质正确折叠。
为了探究CSR基因位点突变在生理水平上对拟南芥应答Cd胁迫的影响,我们对根长、Cd含量累积量、叶绿素含量、MDA、H202、脯氨酸、可溶性糖、可溶性蛋白等植物胁迫损伤重要指标进行了检测。通过对比Col-0和atcsr-2在Cd胁迫处理条件下这些指标的差异,发现atcsr-2突变体体内积累了大量的Cd2+、MDA、H2O2,受到了更加严重的自由基损伤。在确定atcsr-2突变体受到的氧化胁迫较野生型更为明显后,为进一步了解其相关的抗氧化机制,我们对比了两者在Cd胁迫条件下抗氧化酶活性和相关基因表达量变化以及Cd转运蛋白编码基因的表达情况。结果表明atcsr-2突变体中CAT、SOD、POD、APX等抗氧化酶活性均低野生型植株,其编码基因表达水平变化与酶活性变化趋势一致。而Cd的内转运蛋白编码基因IRT1表达量增高,Cd离子区室化转运蛋白MRP3的表达量降低,这导致细胞质中Cd离子累积,毒害增强。另外,我们将CSR构建到原核表达载体在大肠杆菌中进行诱导表达,观察大肠杆菌在Cd胁迫条件下的耐受性变化,并且测定了SOD、CAT酶活性。结果表明诱导表达AtCSR的细菌对Cd的耐受性明显增强,抗氧化酶活性相应升高。
本研究分别从表型水平、生理生化水平、分子水平等多个方面分析了atcsr-2突变体Cd敏感表型的原因,为进一步了解AtCSR响应重金属Cd胁迫的机制奠定基础。
Cd is considered to be highly toxic, difficult to degrade and genetically toxic substance. In recent years, it has been reported that a large number of genes in plants are related to cadmium stress response. These genes mainly include ZIP family, HMA family and so on. Through studying those genes in response to cadmium stress, some self-protection mechanisms including chelation, sequestration and etc have been found. However, it is a complex process that how plants respond to cadmium stress, during which some relevant mechanisms are still not deeply understood.
We optimized screening conditions for Arabidopsis cadmium sensitive mutant, and have founded some plants that are sensitive to cadmium stress in the T-DNA insertion Arabidopsis mutant library. After that, we chose the mutant with the most obvious phenotype and named it atcsr-2, which was used for the following experiment materials. According to Tail-PCR, we knew that the insertional locus of atcsr-2is in At2g36130(correspondingly called AtCSR). This gene encodes peptidyl-prolyl cis/trans isomerases that could reduce energy in the process of protein folding, thus helping protein fold exactly.
In order to explore the influence of the AtCSR gene locus mutation on Arabidopsis responding to cadmium stress at the physiological level, we measured some important physiological indexes, such as root length, cadmium cumulant、chlorophyll content、MDA content、H2O2level、proline content、soluble sugar and soluble protein. Therefore, after contracting these indexes between wide type and mutant with cadmium stress, we found that cadmium caused more damage to atcsr-2. It accumulated higher level of Cd2+, MDA, H2O2, and suffered more free radical damage. After identifying (confirming) atcsr-2suffering more significant oxidativestress than Col-O, we compared antioxidase activity and the expression of genes that encode those enzymes as well as gene expressions related to cadmium transportation between Col-0and atcsr-2with or without cadmium stress, all of which is in order to learn more correlative oxidation mechanisms. The results showed that oxidativestress activities such as CAT, SOD, POD, APX were lower in atcsr-2than those in wide type, and the results of gene expression matched well with those of oxidativestress activities. The expression of Cd intracellular transporter activity IRT1was elevated, while the expression of MRP3was downregulated, which led to Cd accumulation and enhancement of toxicity. In addition, through prokaryotic expression of AtCSR, we studied E. coli growth with cadmium stress, and assayed SOD, CAT activity. The findings revealed that inducible E. coli was less restrained contrast to not inducible E. coli, and oxidativestress activities were higher in the former.
In conclusion, we studied sensitive phenotype of atcsr-2from physiological and biochemical index as well as molecular level, thus laying the foundation for further research in cadmium sensitive mechanism of atcsr-2.
我们通过优化拟南芥Cd敏感突变体筛选条件,从化学诱导激活型拟南芥T-DNA插入突变体库中筛选出一些具有Cd胁迫敏感表型的株系；将其中表型最明显的植株为命名为atcsr-2,作为本研究的材料。通过Tail-PCR鉴定表明该突变体的T-DNA插入位点为At2g36130(相应地称作AtCSR).该基因能够编码一种肽酰-脯氨酰顺反式异构酶(Peptidyl-Prolyl cis/trans Isomerase),此酶能够降低蛋白质折叠过程中所需的能量,有助于蛋白质正确折叠。
为了探究CSR基因位点突变在生理水平上对拟南芥应答Cd胁迫的影响,我们对根长、Cd含量累积量、叶绿素含量、MDA、H202、脯氨酸、可溶性糖、可溶性蛋白等植物胁迫损伤重要指标进行了检测。通过对比Col-0和atcsr-2在Cd胁迫处理条件下这些指标的差异,发现atcsr-2突变体体内积累了大量的Cd2+、MDA、H2O2,受到了更加严重的自由基损伤。在确定atcsr-2突变体受到的氧化胁迫较野生型更为明显后,为进一步了解其相关的抗氧化机制,我们对比了两者在Cd胁迫条件下抗氧化酶活性和相关基因表达量变化以及Cd转运蛋白编码基因的表达情况。结果表明atcsr-2突变体中CAT、SOD、POD、APX等抗氧化酶活性均低野生型植株,其编码基因表达水平变化与酶活性变化趋势一致。而Cd的内转运蛋白编码基因IRT1表达量增高,Cd离子区室化转运蛋白MRP3的表达量降低,这导致细胞质中Cd离子累积,毒害增强。另外,我们将CSR构建到原核表达载体在大肠杆菌中进行诱导表达,观察大肠杆菌在Cd胁迫条件下的耐受性变化,并且测定了SOD、CAT酶活性。结果表明诱导表达AtCSR的细菌对Cd的耐受性明显增强,抗氧化酶活性相应升高。
本研究分别从表型水平、生理生化水平、分子水平等多个方面分析了atcsr-2突变体Cd敏感表型的原因,为进一步了解AtCSR响应重金属Cd胁迫的机制奠定基础。
Cd is considered to be highly toxic, difficult to degrade and genetically toxic substance. In recent years, it has been reported that a large number of genes in plants are related to cadmium stress response. These genes mainly include ZIP family, HMA family and so on. Through studying those genes in response to cadmium stress, some self-protection mechanisms including chelation, sequestration and etc have been found. However, it is a complex process that how plants respond to cadmium stress, during which some relevant mechanisms are still not deeply understood.
We optimized screening conditions for Arabidopsis cadmium sensitive mutant, and have founded some plants that are sensitive to cadmium stress in the T-DNA insertion Arabidopsis mutant library. After that, we chose the mutant with the most obvious phenotype and named it atcsr-2, which was used for the following experiment materials. According to Tail-PCR, we knew that the insertional locus of atcsr-2is in At2g36130(correspondingly called AtCSR). This gene encodes peptidyl-prolyl cis/trans isomerases that could reduce energy in the process of protein folding, thus helping protein fold exactly.
In order to explore the influence of the AtCSR gene locus mutation on Arabidopsis responding to cadmium stress at the physiological level, we measured some important physiological indexes, such as root length, cadmium cumulant、chlorophyll content、MDA content、H2O2level、proline content、soluble sugar and soluble protein. Therefore, after contracting these indexes between wide type and mutant with cadmium stress, we found that cadmium caused more damage to atcsr-2. It accumulated higher level of Cd2+, MDA, H2O2, and suffered more free radical damage. After identifying (confirming) atcsr-2suffering more significant oxidativestress than Col-O, we compared antioxidase activity and the expression of genes that encode those enzymes as well as gene expressions related to cadmium transportation between Col-0and atcsr-2with or without cadmium stress, all of which is in order to learn more correlative oxidation mechanisms. The results showed that oxidativestress activities such as CAT, SOD, POD, APX were lower in atcsr-2than those in wide type, and the results of gene expression matched well with those of oxidativestress activities. The expression of Cd intracellular transporter activity IRT1was elevated, while the expression of MRP3was downregulated, which led to Cd accumulation and enhancement of toxicity. In addition, through prokaryotic expression of AtCSR, we studied E. coli growth with cadmium stress, and assayed SOD, CAT activity. The findings revealed that inducible E. coli was less restrained contrast to not inducible E. coli, and oxidativestress activities were higher in the former.
In conclusion, we studied sensitive phenotype of atcsr-2from physiological and biochemical index as well as molecular level, thus laying the foundation for further research in cadmium sensitive mechanism of atcsr-2.
引文
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