Heat, acid and cold stresses enhance the expression of DnaK gene in Alicyclobacillus acidoterrestris
Heat, acid and cold stresses enhance the expression of DnaK gene in Alicyclobacillus acidoterrestris
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热、酸和冷胁迫增强酸土脂环酸芽孢杆菌 DnaK 基因的表达
DOI：
10.1016/j.foodres.2014.11.023
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发表时间：
2015-01-01
影响因子：
8.1
通讯作者：
Wang Junjie
中科院分区：
文献类型：
--
作者：
Jiao Lingxia;Ran Junjian;Wang Junjie
编码70 kda热休克蛋白（Hsp70）的DnaK基因在保护微生物免受恶劣环境和食品保存条件（如加热、酸性、碱性和冷却）的影响中起着至关重要的作用，并被认为与嗜酸地酸杆菌（a . acidoterrestris）的嗜热嗜酸性有关。本研究采用基因组行走技术克隆DnaK基因，并利用在线软件预测DnaK的生物信息学特征。采用实时荧光定量PCR （qRT-PCR）技术，研究了酸地草在高温、酸性和冷胁迫下的生长和产孢情况，并检测了DnaK基因在酸地草中的表达水平。DnaK基因(GenBank登录号：A. acidoterrestris DSM 3922(T)的HQ893543)包含一个1854 bp的开放阅读框，编码617个氨基酸，推断出的氨基酸序列与acidocalarius Alicyclobacillus DnaK伴侣蛋白同源。根据氨基酸序列、折叠多肽和卷曲多肽功能的一级和二级结构分析，鉴定出该蛋白为耐热蛋白。采用70℃热胁迫、1.0 pH酸胁迫和0℃冷胁迫3种环境胁迫对酸地草进行DnaK的表达研究。热胁迫下DnaK基因的转录水平在5 min内迅速上升至峰值，15 min后下降至最低水平；酸胁迫下DnaK基因转录量在0.5 h内显著升高，在1 h内达到峰值，在3 h内下降至最低水平，且热胁迫与酸胁迫的峰值mRNA表达量存在差异。45℃条件下，热胁迫5 min和酸胁迫1 h时DnaK表达量分别是非胁迫条件下的1.4倍和4.3倍。在0℃条件下，冷处理1 h后，DnaK表达量急剧下降，为对照的0.48倍。热胁迫40 min、酸胁迫5 h、冷胁迫6 h后，酸地草产生孢子，表明DnaK基因调控酸地草对突发性非生物刺激的抗逆性不同于产孢。然而，胁迫条件下DnaK的表达随着诱导胁迫的持续而呈现出不同的变化模式，特别是在酸胁迫的情况下，这表明DnaK的调控机制参与了酸地草的胁迫响应。(C) 2014 Elsevier Ltd.版权所有。
DnaK gene coding for the 70-kDa heat shock protein (Hsp70) plays an essential role in protecting microorganisms from a hostile environmental and food preservation conditions such as heating, acidity, alkalinity and chilling and is believed to be associated with the thermo-acidophilic resistance of Alicyclobacillus acidoterrestris (A. acidoterrestris). In this study, the genome walking technique was used to clone DnaK gene and the bioinformatics characteristics of DnaK was predicted using online software. The growth and sporulation of A. acidoterrestris were explored and the expression levels of DnaK gene in A. acidoterrestris were determined by quantitative real time PCR (qRT-PCR) under heat, acid and cold stresses. DnaK gene (GenBank Accession No. HQ893543) from A. acidoterrestris DSM 3922(T) contains an open reading frame of 1854 bp coding for 617 amino acids and the deduced amino acid sequence is homologous to the DnaK chaperone protein of Alicyclobacillus acidocaldarius. The protein translated from DnaK gene of A. acidoterrestris was characterized as a thermostable protein based on the primary and secondary structure analyses which predict the functions of amino acid sequence, and folding and coiling polypeptides, respectively. Three environmental stresses including heat stress at 70 degrees C, acid stress at the pH of 1.0, and cold stress at 0 degrees C were imposed on A. acidoterrestris to investigate the expression of DnaK. The transcriptional level of DnaK gene under heat stress increased quickly to peak level in 5 min and then dropped to the minimum level in 15 min; the transcriptional levels of DnaK gene under acid stress increased markedly in 0.5 h to peak level in 1 h and then decreased to the minimum level in 3 h, and the peak mRNA expression levels differed between heat and acid stress. DnaK expression levels of 5 min heat stress and 1 h acid stress were 1.4 times and 4.3 times higher than those of the control under non-stress condition at 45 degrees C, respectively. In response to told stress at 0 degrees C, the DnaK expression level decreased drastically to 0.48 times of the control after 1 h cold treatment. Spores of A. acidoterrestris were produced after the bacteria received 40 min heat stress, 5 h acid stress or 6 h cold stress, which suggested that DnaK gene regulates the stress resistance of A. acidoterrestris against abrupt abiotic stimuli differently from sporulation. However, the expression of DnaK under stress conditions demonstrated the different pattern of changes as the induced stresses continued, especially in the case of acid stress, which implied that a more complicated mechanism of DnaK regulation is involved in the stress response of A. acidoterrestris. (C) 2014 Elsevier Ltd. All rights reserved.