耐辐射异常球菌σ因子Sig1和Sig2的功能鉴定及其热激胁迫反应的转录分析
发布时间：2019-06-19 09:38
【摘要】：σ因子(Sigma Factors)是原核生物RNA聚合酶的一个亚基,是转录起始所必需的因子,它能可逆地与RNA聚合酶核心酶的活性催化位点结合来激活起始转录,影响RNA聚合酶对转录起始位点的正确识别,特异的起始相应基因的表达。耐辐射异常球菌(Deinooccus radiodurans)具有超强的极端辐射氧化抗性及环境胁迫适应性,该菌含有3个σ因子(分别为Sig1(DR_0180)、Sig2(DR_0804)和SigA(DR_0916))。有关Sig1特别是Sig2的功能目前不是太清楚,相关报道甚少。本研究工作通过单缺失突变株和双缺失突变株的构建,利用逆境胁迫冲击实验、荧光实时定量技术以及转录组学数据分析,对sig1、sig2两个基因的功能及其调节作用进行了研究,取得如下研究进展：(1)生物信息学分析预测1)einococcus radiodurans R1 Sig1为ECF亚家族σ因子,隶属于σ70家族4,sig1(DR_0180)基因全长690bp,编码蛋白含有229个氨基酸,蛋白分子量约为2589 kDa；Sig2(DR_0804)也属于σ70家族的RNA聚合酶。因子,其生理功能未知,sig2基因全长849bp,编码蛋白包含282个氨基酸,分子量约为29.91kDa。氨基酸序列比对发现,Sigl蛋白与同属中的σ因子具有一定的相似性,序列相似性达到64%以上,且ECF结构域在Deinococcus属中高度保守；Sig2与同属1Deinococcus sp.YIM 77859的RNA聚合酶亚基σ24的序列一致性最高,但仅为47%。Sig1和Sig2都具有σ70蛋白中最保守的区域2,该区域是σ因子识别核心酶的关键区域。(2)为了研究Sig1和Sig2的生物学功能,我们利用融合PCR技术构建了sig1缺失突变株(Δsig1),sig2缺失突变株(Δsig2)以及sig1和sig2双缺失突变株(Δsigl sig2)。表型分析表明sig1基因的突变,sig2基因的突变以及sig1和sig2基因的双缺失突变均不影响该菌的生长。但在酸和5M NaCl冲击下,Δsigl比野生型菌株的存活能力显著下降,表明sig1突变导致了菌株对酸和盐胁迫敏感,sigl和sig2的单突变或双突变引起菌株对热(48℃)和氧化(100 mM过氧化氢)胁迫敏感。(3)为了进一步研究σ因子对细胞代谢的调控作用,本论文开展了正常生长和48℃高温胁迫条件下野生型和Δsig1、Δsig2突变株的转录组学分析。结果表明,耐辐射异常球菌野生型受到热激胁迫后有656个基因发生显著变化,其中329个基因上调,327个基因下调。表达差异基因中23%属于调控细胞信息存储和处理的基因；28%属于调控细胞新陈代谢的基因；49%是功能未知的基因；表达差异基因涉及多个代谢途径。sig1的突变导致265个基因发生显著变化,其中59个基因上调,206个基因下调；在热激胁迫处理2h后有293个基因发生显著变化,其中92个基因上调,201个基因下调；其中,有12个与核糖体蛋白结构相关的基因表达增强,分别为rpsJ、rplC、rplW、rplB、rpsS、 rplV、rplP、rplN、rplF、rplO、rpsM 和rpsD。sig2的突变导致317个基因发生显著变化,其中130个基因上调,187个基因下调；在热激胁迫处理2h后有376个基因发生显著变化,其中279基因上调,97个基因下调。表达差异基因产物与细胞壁/细胞膜/细胞包膜生物合成、翻译/核糖体结构和生物合成、复制重组和修复、信号转导机制、能量产生和转换、氨基酸运输和代谢、脂类运输和代谢以及次级代谢产物的合成运输和分解代谢等有关。其中,热激胁迫导致分子伴侣dnaK在Δsig1中上调1.3倍,在Sig2缺失突变株中下调1.5倍。实时定量结果显示,分子伴侣groES、groEL、dnaK、dnaJ以及热激保护基因DR_1314、DR_0972和DR 0326在热激胁迫1h和3h均发生显著变化。结合转录组数据和实时定量实验结果,表明sig1、sig2基因可能参与了上述热激胁迫相关基因的转录。将热激胁迫后Sigl和Sig2转录组数据进行比较,发现变化显著的基因中有28个相同,其中DR_0179、DR_1783和DR_A0248在Sigl和Sig2的单缺失突变株中均发生上调。DR_A0248调控MarR家族蛋白的转录,表明Sigl和Sig2可以直接调控MarR家族蛋白在热激胁迫时的转录表达。本研究表明,耐辐射异常球菌σ因子Sigl和Sig2通过直接或间接的调控作用,影响了细胞的多种生理胁迫反应,包括细胞的热激胁迫保护、抗氧化保护及核糖体蛋白合成等过程,为进一步揭示耐辐射异常球菌极端环境的适应机制奠定了理论基础。
[Abstract]:The transcription factor (Sigma Factors) is a subunit of a prokaryote RNA polymerase, a factor necessary for transcription initiation, which can reversibly bind to the active catalytic site of the RNA polymerase core enzyme to activate the initial transcription, affecting the correct recognition of the transcription initiation site by the RNA polymerase, Specific initiation of the expression of the corresponding gene. Deinococcus radiodurans has super-strong extreme radiation oxidation resistance and environmental stress adaptability, which contains 3 risk factors (Si1 (DR _ 0180), Si2 (DR _ 0804) and SigA (DR _ 0916)). The function of Si1, in particular the Sig2, is not so clear at this time, and the relevant reports are very little. The study on the function and regulation of the two genes of sig1 and sig2 was studied by the construction of single-deletion mutant and double-deletion mutant, and the function and regulation of the two genes of sig1 and sig2 were studied by using stress-stress-shock test, fluorescence real-time quantitative technique and the data analysis of the transcriptome. (1) Bioinformatics analysis predicts that 1) einococcus radiodurans R1 Si1 is an ECF subfamily, and the total length of the sig1 (DR _ 0180) gene is 690bp, the coding protein contains 229 amino acids, the molecular weight of the protein is about 2589 kDa, and Si2 (DR _ 0804) also belongs to the RNA polymerase of the family of A70. Its physiological function is unknown, the total length of the sig2 gene is 849 bp, the encoded protein contains 282 amino acids, and the molecular weight is about 29.91 kDa. The sequence of the amino acid sequence is more than 64% and the ECF domain is highly conserved in the genus Deinococcus, and the sequence consistency of the Si2 with the RNA polymerase subunit of the same 1 Deinococcus sp. YIM 77859 is the highest, However, only 47%. Si1 and Si2 have the most conserved region 2 in the p70 protein, which is the key area of the transcription factor identifying the core enzyme. (2) In order to study the biological function of Si1 and Si2, we constructed the sig1 deletion mutant strain (visi1), the sig2 deletion mutant (bsigma2) and the sig1 and sig2 double-deletion mutant strains using the fusion PCR technique. The phenotype analysis indicated that the mutation of the sig1 gene, the mutation of the sig2 gene, and the double deletion mutation of the sig1 and sig2 genes did not affect the growth of the bacterium. However, in the presence of acid and 5M NaCl, the presence of sigl was significantly lower than that of the wild-type strain, indicating that the sig1 mutation resulted in a strain sensitive to acid and salt stress, a single mutation or a double mutation of sigl and sig2 causing the strain to be sensitive to heat (48. degree. C.) and oxidation (100 mM hydrogen peroxide). (3) In order to further study the effect of the transcription factor on the cell metabolism, this paper carried out the metabolomics analysis of wild-type and sigma1 and sigma-2 mutants under the conditions of normal growth and high temperature stress of 48 鈩，
本文编号：2502255
[Abstract]:The transcription factor (Sigma Factors) is a subunit of a prokaryote RNA polymerase, a factor necessary for transcription initiation, which can reversibly bind to the active catalytic site of the RNA polymerase core enzyme to activate the initial transcription, affecting the correct recognition of the transcription initiation site by the RNA polymerase, Specific initiation of the expression of the corresponding gene. Deinococcus radiodurans has super-strong extreme radiation oxidation resistance and environmental stress adaptability, which contains 3 risk factors (Si1 (DR _ 0180), Si2 (DR _ 0804) and SigA (DR _ 0916)). The function of Si1, in particular the Sig2, is not so clear at this time, and the relevant reports are very little. The study on the function and regulation of the two genes of sig1 and sig2 was studied by the construction of single-deletion mutant and double-deletion mutant, and the function and regulation of the two genes of sig1 and sig2 were studied by using stress-stress-shock test, fluorescence real-time quantitative technique and the data analysis of the transcriptome. (1) Bioinformatics analysis predicts that 1) einococcus radiodurans R1 Si1 is an ECF subfamily, and the total length of the sig1 (DR _ 0180) gene is 690bp, the coding protein contains 229 amino acids, the molecular weight of the protein is about 2589 kDa, and Si2 (DR _ 0804) also belongs to the RNA polymerase of the family of A70. Its physiological function is unknown, the total length of the sig2 gene is 849 bp, the encoded protein contains 282 amino acids, and the molecular weight is about 29.91 kDa. The sequence of the amino acid sequence is more than 64% and the ECF domain is highly conserved in the genus Deinococcus, and the sequence consistency of the Si2 with the RNA polymerase subunit of the same 1 Deinococcus sp. YIM 77859 is the highest, However, only 47%. Si1 and Si2 have the most conserved region 2 in the p70 protein, which is the key area of the transcription factor identifying the core enzyme. (2) In order to study the biological function of Si1 and Si2, we constructed the sig1 deletion mutant strain (visi1), the sig2 deletion mutant (bsigma2) and the sig1 and sig2 double-deletion mutant strains using the fusion PCR technique. The phenotype analysis indicated that the mutation of the sig1 gene, the mutation of the sig2 gene, and the double deletion mutation of the sig1 and sig2 genes did not affect the growth of the bacterium. However, in the presence of acid and 5M NaCl, the presence of sigl was significantly lower than that of the wild-type strain, indicating that the sig1 mutation resulted in a strain sensitive to acid and salt stress, a single mutation or a double mutation of sigl and sig2 causing the strain to be sensitive to heat (48. degree. C.) and oxidation (100 mM hydrogen peroxide). (3) In order to further study the effect of the transcription factor on the cell metabolism, this paper carried out the metabolomics analysis of wild-type and sigma1 and sigma-2 mutants under the conditions of normal growth and high temperature stress of 48 鈩，
本文编号：2502255
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