本文關(guān)鍵詞：阿維鏈霉菌中γ-丁酸內(nèi)酯受體同源蛋白AvaR2和AvaR1的調(diào)控機(jī)制　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 阿維鏈霉菌 阿維菌素 γ-丁酸內(nèi)酯受體同源蛋白 AvaR2 AvaR1

【摘要】：阿維鏈霉菌(Streptomyces avermitilis)是重要的工業(yè)微生物,其產(chǎn)生的阿維菌素(Avermectins)由于具有高效、廣譜和低毒的殺蟲活性被廣泛應(yīng)用于農(nóng)業(yè)、畜牧業(yè)和醫(yī)藥領(lǐng)域。在阿維鏈霉菌中,誘導(dǎo)阿維菌素合成的自調(diào)節(jié)因子信號-avenolide是一種新型的γ-丁烯酰類的小分子,而非常見的γ-丁酸內(nèi)酯(GBL)類自調(diào)節(jié)因子。為了闡明阿維菌素生物合成的調(diào)控機(jī)制,本論文針對阿維鏈霉菌中兩個(gè)GBL受體同源蛋白AvaR2和AvaR1的調(diào)控功能和作用機(jī)制進(jìn)行了研究。阿維鏈霉菌中存在三個(gè)屬于TetR家族轉(zhuǎn)錄調(diào)控因子的GBL受體同源蛋白:AvaR1(SAV3705)、AvaR2(SAV3702)和AvaR3(SAV3703),其中AvaR2與假GBL受體同源性最高。對avaR2進(jìn)行缺失、回補(bǔ)和過表達(dá),通過搖瓶發(fā)酵和形態(tài)觀察實(shí)驗(yàn),初步證實(shí)AvaR2負(fù)調(diào)控阿維菌素的生物合成和阿維鏈霉菌的生長,但不影響形態(tài)分化。進(jìn)一步通過qRT-PCR、EMSA、DNase I footprinting、5' RACE和ChIP-qPCR等實(shí)驗(yàn),證實(shí)AvaR2能直接負(fù)調(diào)控aveR(阿維菌素生物合成的途徑特異性正調(diào)控基因)、aco(avenolide關(guān)鍵合成酶基因)、自身基因以及其它兩個(gè)avaR基因和avaR1和avaR3)的轉(zhuǎn)錄,并且與aveRp的親和力最高。通過分析AvaR2在這5個(gè)啟動(dòng)子區(qū)的結(jié)合序列,得出其結(jié)合的一段保守的 18 bp不完全回文序列(AWWCCRBBHDDNMSGTWT.W:A或T:R:A或G:B:G、C或T;H:A、C或T;D:A、G或T;M:A或C;S:G或C;N:A、G、C或T)。利用該保守序列預(yù)測了一些新的AvaR2靶基因,并通過EMSA和qRT-PCR鑒定了 11個(gè)新的靶基因,它們分別參與初級代謝、核糖體蛋白合成、脅迫響應(yīng)等生理過程,表明AvaR2是一個(gè)多效調(diào)控因子。AvaR2不僅能以內(nèi)源的avenolide為配體,還能以外源的杰多霉素B(JadB)、安普霉素(Apr)和潮霉素B(HygB)作為配體調(diào)節(jié)其結(jié)合DNA的能力,表明AvaR2介導(dǎo)的信號傳導(dǎo)系統(tǒng)不僅在種內(nèi)而且在種間信息交流中發(fā)揮了重要作用。AvaR1與真GBL受體同源性最高。對avaR1進(jìn)行缺失、回補(bǔ)和過表達(dá),通過搖瓶發(fā)酵和形態(tài)觀察實(shí)驗(yàn),初步證實(shí)AvaR1負(fù)調(diào)控阿維菌素的生物合成,但不影響阿維鏈霉菌的生長和形態(tài)分化。進(jìn)一步通過qRT-PCR、EMSA、DNase I footprinting和ChIP-qPCR等實(shí)驗(yàn),證實(shí)AvaR1能直接負(fù)調(diào)控aveR、aco、自身基因以及其它兩個(gè)avaR基因(avaR2和avaR3)的轉(zhuǎn)錄,并且與avaR3p的親和力稍高。AvaR1與AvaR2在aveR、aco、avaR1、vaaR2和avaR3啟動(dòng)子區(qū)的保護(hù)位點(diǎn)相同,據(jù)此得出AvaR1和AvaR2結(jié)合的保守序列相同。通過EMSA和qRT-PCR鑒定了10個(gè)新的AvaR1靶基因,這些基因分別參與初級代謝、核糖體蛋白合成、脅迫響應(yīng)、核酸代謝等生理過程,表明AvaR1也是一個(gè)多效調(diào)控因子。AvaR1和AvaR2既有共同的靶基因,也有各自不同的靶基因,表明它們可交叉調(diào)控不同的生理過程。
[Abstract]:Streptomyces avermectinis is an important industrial microorganism which produces avermectins (Avermectins) because of its high efficiency. Broad spectrum and low toxicity insecticidal activities are widely used in agriculture, animal husbandry and medicine. Self-regulating factor signal -avenolide, which induces the synthesis of avermectin, is a new type of small 緯 -butenyl molecule. In order to elucidate the regulation mechanism of avermectin biosynthesis, the unusual self-regulating factors of 緯 -butyrolactone (GBL) were used to elucidate the regulation mechanism of avermectin biosynthesis. The regulatory function and mechanism of two GBL receptor homologous proteins AvaR2 and AvaR1 in Streptomyces avelicus were studied in this paper. There are three transcriptional modulations belonging to the TetR family in Streptomyces avelicus. The GBL receptor homologous protein of the control factor:. Ava R1 (. SAV3705). Ava R2 (SAV3702) and AvaR3 (SAV3703) had the highest homology with pseudo GBL receptor. AvaR2 was deleted, compensated and overexpressed. By shaking flask fermentation and morphological observation, it was preliminarily confirmed that AvaR2 negatively regulated the biosynthesis of avermectin and the growth of Streptomyces avermectin, but had no effect on morphological differentiation. Emsa DNase I footprinting 5'#en0# and ChIP-qPCR. It is confirmed that AvaR2 can directly and negatively regulate aveR( the specific positive regulation gene of avermectin biosynthesis). Transcription of autogenes and two other avaR genes and avaR1 and Ava R3. The binding sequence of AvaR2 in the five promoter regions was analyzed. A conservative 18bp incomplete palindromes sequence was obtained, which consisted of AWWCCRBBHDDNMSGTWT.W: a or T: R: a or G: B: GfU C or T; H: Ac or T; D: Agna G or T; M: a or C; S: G or C; Using the conserved sequence, we predicted some new AvaR2 target genes and identified 11 new target genes by EMSA and qRT-PCR. They are involved in primary metabolism, ribosomal protein synthesis, stress response and other physiological processes. The results showed that AvaR2 was a multieffectual regulator. Ava R2 could not only use endogenous avenolide as ligand, but also exogenous JadB. Apramycin (April) and hygromycin B (HygB) act as ligands to regulate their ability to bind to DNA. The results suggest that AvaR2 mediated signal transduction system plays an important role in the communication of information between species and species. AvaR1 has the highest homology with true GBL receptor and avaR1 is missing. Through flask fermentation and morphological observation, it was proved that AvaR1 negatively regulated the biosynthesis of avermectin. But it did not affect the growth and morphological differentiation of Streptomyces aviriformis. Further experiments were carried out by using qRT-PCR- EMSADNase I footprinting and ChIP-qPCR. It is confirmed that AvaR1 can directly and negatively regulate the transcription of aveRnaco, its own gene and two other avaR genes, Ava R2 and Ava R3. The protective sites of AvaR1 and AvaR2 in aveRacoavaR1nvaaR2 and avaR3 promoter region were similar to those of avaR3p. It was concluded that the conserved sequence of AvaR1 and AvaR2 binding was the same. Ten new AvaR1 target genes were identified by EMSA and qRT-PCR, which were involved in primary metabolism respectively. Ribosomal protein synthesis, stress response, nucleic acid metabolism and other physiological processes indicate that AvaR1 is also a multi-effect regulatory factor. AvaR1 and AvaR2 have common target genes. They also have different target genes, indicating that they can cross regulate different physiological processes.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q936

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