【摘要】：σ因子(Sigma Factors)是原核生物RNA聚合酶的一個(gè)亞基,是轉(zhuǎn)錄起始所必需的因子,它能可逆地與RNA聚合酶核心酶的活性催化位點(diǎn)結(jié)合來(lái)激活起始轉(zhuǎn)錄,影響RNA聚合酶對(duì)轉(zhuǎn)錄起始位點(diǎn)的正確識(shí)別,特異的起始相應(yīng)基因的表達(dá)。耐輻射異常球菌(Deinooccus radiodurans)具有超強(qiáng)的極端輻射氧化抗性及環(huán)境脅迫適應(yīng)性,該菌含有3個(gè)σ因子(分別為Sig1(DR_0180)、Sig2(DR_0804)和SigA(DR_0916))。有關(guān)Sig1特別是Sig2的功能目前不是太清楚,相關(guān)報(bào)道甚少。本研究工作通過(guò)單缺失突變株和雙缺失突變株的構(gòu)建,利用逆境脅迫沖擊實(shí)驗(yàn)、熒光實(shí)時(shí)定量技術(shù)以及轉(zhuǎn)錄組學(xué)數(shù)據(jù)分析,對(duì)sig1、sig2兩個(gè)基因的功能及其調(diào)節(jié)作用進(jìn)行了研究,取得如下研究進(jìn)展：(1)生物信息學(xué)分析預(yù)測(cè)1)einococcus radiodurans R1 Sig1為ECF亞家族σ因子,隸屬于σ70家族4,sig1(DR_0180)基因全長(zhǎng)690bp,編碼蛋白含有229個(gè)氨基酸,蛋白分子量約為2589 kDa；Sig2(DR_0804)也屬于σ70家族的RNA聚合酶。因子,其生理功能未知,sig2基因全長(zhǎng)849bp,編碼蛋白包含282個(gè)氨基酸,分子量約為29.91kDa。氨基酸序列比對(duì)發(fā)現(xiàn),Sigl蛋白與同屬中的σ因子具有一定的相似性,序列相似性達(dá)到64%以上,且ECF結(jié)構(gòu)域在Deinococcus屬中高度保守；Sig2與同屬1Deinococcus sp.YIM 77859的RNA聚合酶亞基σ24的序列一致性最高,但僅為47%。Sig1和Sig2都具有σ70蛋白中最保守的區(qū)域2,該區(qū)域是σ因子識(shí)別核心酶的關(guān)鍵區(qū)域。(2)為了研究Sig1和Sig2的生物學(xué)功能,我們利用融合PCR技術(shù)構(gòu)建了sig1缺失突變株(Δsig1),sig2缺失突變株(Δsig2)以及sig1和sig2雙缺失突變株(Δsigl sig2)。表型分析表明sig1基因的突變,sig2基因的突變以及sig1和sig2基因的雙缺失突變均不影響該菌的生長(zhǎng)。但在酸和5M NaCl沖擊下,Δsigl比野生型菌株的存活能力顯著下降,表明sig1突變導(dǎo)致了菌株對(duì)酸和鹽脅迫敏感,sigl和sig2的單突變或雙突變引起菌株對(duì)熱(48℃)和氧化(100 mM過(guò)氧化氫)脅迫敏感。(3)為了進(jìn)一步研究σ因子對(duì)細(xì)胞代謝的調(diào)控作用,本論文開(kāi)展了正常生長(zhǎng)和48℃高溫脅迫條件下野生型和Δsig1、Δsig2突變株的轉(zhuǎn)錄組學(xué)分析。結(jié)果表明,耐輻射異常球菌野生型受到熱激脅迫后有656個(gè)基因發(fā)生顯著變化,其中329個(gè)基因上調(diào),327個(gè)基因下調(diào)。表達(dá)差異基因中23%屬于調(diào)控細(xì)胞信息存儲(chǔ)和處理的基因；28%屬于調(diào)控細(xì)胞新陳代謝的基因；49%是功能未知的基因；表達(dá)差異基因涉及多個(gè)代謝途徑。sig1的突變導(dǎo)致265個(gè)基因發(fā)生顯著變化,其中59個(gè)基因上調(diào),206個(gè)基因下調(diào)；在熱激脅迫處理2h后有293個(gè)基因發(fā)生顯著變化,其中92個(gè)基因上調(diào),201個(gè)基因下調(diào)；其中,有12個(gè)與核糖體蛋白結(jié)構(gòu)相關(guān)的基因表達(dá)增強(qiáng),分別為rpsJ、rplC、rplW、rplB、rpsS、 rplV、rplP、rplN、rplF、rplO、rpsM 和rpsD。sig2的突變導(dǎo)致317個(gè)基因發(fā)生顯著變化,其中130個(gè)基因上調(diào),187個(gè)基因下調(diào)；在熱激脅迫處理2h后有376個(gè)基因發(fā)生顯著變化,其中279基因上調(diào),97個(gè)基因下調(diào)。表達(dá)差異基因產(chǎn)物與細(xì)胞壁/細(xì)胞膜/細(xì)胞包膜生物合成、翻譯/核糖體結(jié)構(gòu)和生物合成、復(fù)制重組和修復(fù)、信號(hào)轉(zhuǎn)導(dǎo)機(jī)制、能量產(chǎn)生和轉(zhuǎn)換、氨基酸運(yùn)輸和代謝、脂類運(yùn)輸和代謝以及次級(jí)代謝產(chǎn)物的合成運(yùn)輸和分解代謝等有關(guān)。其中,熱激脅迫導(dǎo)致分子伴侶dnaK在Δsig1中上調(diào)1.3倍,在Sig2缺失突變株中下調(diào)1.5倍。實(shí)時(shí)定量結(jié)果顯示,分子伴侶groES、groEL、dnaK、dnaJ以及熱激保護(hù)基因DR_1314、DR_0972和DR 0326在熱激脅迫1h和3h均發(fā)生顯著變化。結(jié)合轉(zhuǎn)錄組數(shù)據(jù)和實(shí)時(shí)定量實(shí)驗(yàn)結(jié)果,表明sig1、sig2基因可能參與了上述熱激脅迫相關(guān)基因的轉(zhuǎn)錄。將熱激脅迫后Sigl和Sig2轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行比較,發(fā)現(xiàn)變化顯著的基因中有28個(gè)相同,其中DR_0179、DR_1783和DR_A0248在Sigl和Sig2的單缺失突變株中均發(fā)生上調(diào)。DR_A0248調(diào)控MarR家族蛋白的轉(zhuǎn)錄,表明Sigl和Sig2可以直接調(diào)控MarR家族蛋白在熱激脅迫時(shí)的轉(zhuǎn)錄表達(dá)。本研究表明,耐輻射異常球菌σ因子Sigl和Sig2通過(guò)直接或間接的調(diào)控作用,影響了細(xì)胞的多種生理脅迫反應(yīng),包括細(xì)胞的熱激脅迫保護(hù)、抗氧化保護(hù)及核糖體蛋白合成等過(guò)程,為進(jìn)一步揭示耐輻射異常球菌極端環(huán)境的適應(yīng)機(jī)制奠定了理論基礎(chǔ)。
[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，

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