兰州大学机构库 >生命科学学院
高山离子芥铁蛋白基因CbFer的克隆、表达及其抗氧化功能分析
Alternative TitleMolecular Cloning, Expression and Antioxidant Effect Analysis of a Novel Ferritin Gene CbFer in Chorispora bungeana
张丽静
Thesis Advisor安黎哲
2006-05-04
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword高山离子芥 冷胁迫 铁蛋白 CbFer基因克隆 表达分析 抗氧化
Abstract

高山离子芥(Chorispora bungeana Fisch. & C.A. Mey)是一种稀有的高山冰缘植物,具有很强抗寒能力,它不具备特殊的形态结构,但在生理和分子水平上是研究植物适应冷冻环境机制的理想材料。本文以高山离子芥为实验材料,克隆到一个全新的铁蛋白基因(CbFer),在不同温度下(4℃、0℃和-4℃)对其进行低温胁迫后,系统性的检测了AsA-GSH系统和CbFer基因对低温的响应表达,重点对诱导CbFer基因表达的信号途径进行了初步研究。结果认为高山离子芥作为高山冰缘植物所特有的抗寒机制主要表现在以下几个方面: 1、高山离子芥铁蛋白基因(CbFer)全长975bp, 有一个编码260个氨基酸的开放阅读框(ORF)。对应编码的蛋白分子量为29.17 kDa,等电点是5.44。在氨基酸结构域分析中,揭示了高山离子芥铁蛋白有类似铁蛋白双铁结构域并且具有铁蛋白铁结合区域的特征。 2、CbFer cDNA编码的铁蛋白亚基含有一段定位于叶绿体蛋白的转运肽。RT-PCR分析表明这个基因是组织特异性表达的基因,只能在叶片中检测到它的表达。这表明CbFer基因编码的铁蛋白功能在叶片中发挥。 3、检测这个基因在冷冻胁迫(4℃、0℃和 -4℃)下的表达情况,发现它的转录物在4℃逐渐积累,而在0℃和-4℃的积累反而直接和迅速。这表明CbFer基因的表达与温度密切相关、对温度反应敏感,所以CbFer基因的表达是高山离子芥传递抵御低温胁迫信号的一个重要保护机制。随着胁迫强度的增大,植物启动这个保护机制就越迅速。这恰恰就反映了高山离子芥具有适应温度剧烈变化的能力。 4、铁鳌合剂(Ferrozine)处理明显的抑制了CbFer基因的表达,表明铁是调控CbFer基因表达的一个重要限制因子。但值得注意的是经铁鳌合剂处理后再进行冷冻胁迫又可以重新诱导CbFer基因的表达。这说明在冷冻情况下诱导CbFer表达的信号除了铁这个因素以外还可能包含其他因素。 5、用外源信号处理发现:过量铁和H2O2正调控CbFer基因的表达;ABA负调控CbFer基因的表达;SNP处理不能引起CbFer基因表达的变化。铁鳌合剂处理后引起的CbFer基因的表达抑制不能被外源ABA、Fe、SNP处理所恢复。但在同样情况下,用H2O2处理在一定程度上诱导了CbFer的表达。这表明CbFer 基因表达不受ABA、NO作用,但受H2O2氧化,部分解除抑制使基因重新表达。 6、对高山离子芥蛋白水平的分析表明,铁蛋白含量随着温度的降低升高,增强了植物在冷冻胁迫下加速转换Fe2+ Fe3+的能力,限制了OH.产生。 7、在低温处理过程中我们还发现高山离子芥抗寒性体现在AsA-GSH系统的几种酶活性上:其中SOD和CAT 这两种直接清除活性氧的酶表现相同的变化趋势,随着温度的降低,酶活性反而升高;AsA-POD是叶绿体中清除H2O2的主要酶类, 4℃和0℃处理时酶活性升高,0℃处理时酶活性明显高于4℃,但在-4℃时酶活性的变化近似于对照水平;DHAR酶活性一直保持在较平稳的、高于对照水平上;MDHAR和GR作为AsA-GSH系统的动力酶在低温胁迫过程中保持了较高活性,为其他酶提供了足够的动力。高山离子芥存在的这种对温度敏感的快速清除活性氧系统,与CbFer基因对温度反应敏感结果一致,一旦低于正常生长温度,酶保护系统与CbFer基因同步运作,这正是酶系统与CbFer基因表达的同步协调表达的结果。使高山离子芥在低温胁迫过程中的活性氧水平一直处于较低水平,保护植物免受膜脂过氧化的伤害。

我们认为高山离子芥能够在低温环境中正常生长,是通过CbFer基因的表达、快速启动AsA-GSH系统二者协同作用来实现的。酶活性提高加速抗氧化系统的运行清除活性氧;铁蛋白的增加,吸收Fe 2+ ,减少羟自由基的生成而起到减轻膜损伤的保护作用。AsA-GSH抗氧化系统和铁蛋白协同作用,减少了Fenton反应所需的两个必需的底物(H2O2 和Fe2+ ),从而减少了羟自由基的产生,减少了膜脂过氧化产物MDA的产生,由此保护高山离子芥抵御了冷冻胁迫的伤害。

Other Abstract

Chorispora bungeana Fisch. & C.A. Mey (C. bungeana) is a rare alpine subnival plant species that is highly capable of resisting freezing environment. Lacking special morphological structure, it is an ideal plant in studying physiological and molecular mechanism in resisting freezing stress. Ferritin cDNA, CbFer, was cloned from C. bungeana. The responses of AsA-GSH system and CbFer to temperature change were examined during the course of different temperature (4℃、0℃ and -4℃) stresses, the signal transduction way of the expression of CbFer was also investigated in this paper. The special cold resistance ability of C. bungeana shows in the following aspects: 1. CbFer gene is 975 bp in length with an open reading frame (ORF) of 260 amino acids, corresponding to a protein of predicted molecular mass 29.17 kDa and an isoelectric point of 5.44. The deduced amino acid sequence possesses ferritin-like diiron domain and the ferritin iron-binding regions signature. 2. CbFer cDNA codes for a ferritin subunit plus a chloroplast-targeting transit peptide. RT-PCR analysis revealed that CbFer was a tissue-specfic gene, the expression could only be detected in leaves. These results suggest that the protein coded by CbFer plays function only in leaves. 3. The expression patterns of CbFer gene were investigated in relation to cold stress. The CbFer gene transcript progressively increased under 4℃ and increased much more distinctly and quickly under 0℃ and –4℃. This result suggests there is a close relationship between the expression of CbFer and cold stress, CbFer expression is sensitive to low temperature. CbFer plays an important protective mechanism in signal transduction to resist cold stress in C. bungeana. Lower temperature could trigger the expression more quickly exactly,this phenomenon reflects fitly that C. bungeana has the ability to adapt to acute temperature change in the growth environment. 4. The expression of CbFer was strongly inhibited by treatment with iron chelating agent Ferrozine. This result indicates that iron is a main factor in the regulating of CbFer gene expression. It was remarkable that we found cold stress could induce the expression of CbFer after Ferrozine treatment, which means there is another signal except iron induces the expression of the gene during cold stress. 5. Two tested exogenous signals, excessive iron and H2O2, up-regulated the expression of CbFer gene. However, the expression of the gene was down-regulated by ABA. SNP treatment didn’t influence the expression of CbFer. We also found that plant cells pretreated with Ferrozine showed no response to excessive iron, abscisic acid (ABA) and SNP, however, H 2 O 2 induced the expression of CbFer to some extent in the same status. These results indicates the activated transduction way of the cold induced expression of CbFer gene is independent from ABA and NO, and suggested that H 2 O 2 would be involved in the regulation of CbFer gene expression. 6. The SDS-PAGE of soluble protein showed that the content of ferritin increased with the descending temperature. This result suggests that C. bungeana improves the capability of conversion Fe 2+ to Fe 3+ during cold stress, decreases the production of OH 7. The enzymes activities in AsA-GSH system of C. bungeana showed different changing current during cold stress. The activities of two major ROS scavenging enzymes, SOD and CAT, increased quickly during the cold stress, lower temperature caused higher enzyme activity at the same time point; As the major enzyme scavenging H2O2 in chloroplast, the AsA-POD activities were promoted by 4℃and 0℃ treatment, however, the enzyme activities didn’t change obviously during -4℃ treatment; DHAR activity showed high level throughout cold stress; the activities of two energy-providing enzymes, MDHAR and GR, showed high level and provided enough energy to support the normal running of AsA-GSH system. These results show that C. bungeana possesses a temperature-sensitive ROS scavenging system, which is consistent with the expression pattern of CbFer gene in cold stress. The enzyme in AsA-GSH system and CbFer cooperated when plant suffered cold stress, and then the ROS level was kept at a relative low level. The plant was protected from injuring induced by cold stress.

In conclusion, C. bungeana possesses a ability to trigger the operation of antioxidant system rapidly during cold stress. When C. bungeana suffered from cold stress, a high level operation of AsA-GSH system and increased content of ferritin cooperate to hold back the production of OH . in Fenton reaction, and then the membrane damage was weakened. In this way, plant survived during cold stress.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221362
Collection生命科学学院
Recommended Citation
GB/T 7714
张丽静. 高山离子芥铁蛋白基因CbFer的克隆、表达及其抗氧化功能分析[D]. 兰州. 兰州大学,2006.
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