阿维链霉菌中γ-丁酸内酯受体同源蛋白AvaR2和AvaR1的调控机制

发布时间：2018-01-15 09:01

  本文关键词：阿维链霉菌中γ-丁酸内酯受体同源蛋白AvaR2和AvaR1的调控机制　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 阿维链霉菌 阿维菌素 γ-丁酸内酯受体同源蛋白 AvaR2 AvaR1

【摘要】：阿维链霉菌(Streptomyces avermitilis)是重要的工业微生物,其产生的阿维菌素(Avermectins)由于具有高效、广谱和低毒的杀虫活性被广泛应用于农业、畜牧业和医药领域。在阿维链霉菌中,诱导阿维菌素合成的自调节因子信号-avenolide是一种新型的γ-丁烯酰类的小分子,而非常见的γ-丁酸内酯(GBL)类自调节因子。为了阐明阿维菌素生物合成的调控机制,本论文针对阿维链霉菌中两个GBL受体同源蛋白AvaR2和AvaR1的调控功能和作用机制进行了研究。阿维链霉菌中存在三个属于TetR家族转录调控因子的GBL受体同源蛋白:AvaR1(SAV3705)、AvaR2(SAV3702)和AvaR3(SAV3703),其中AvaR2与假GBL受体同源性最高。对avaR2进行缺失、回补和过表达,通过摇瓶发酵和形态观察实验,初步证实AvaR2负调控阿维菌素的生物合成和阿维链霉菌的生长,但不影响形态分化。进一步通过qRT-PCR、EMSA、DNase I footprinting、5' RACE和ChIP-qPCR等实验,证实AvaR2能直接负调控aveR(阿维菌素生物合成的途径特异性正调控基因)、aco(avenolide关键合成酶基因)、自身基因以及其它两个avaR基因和avaR1和avaR3)的转录,并且与aveRp的亲和力最高。通过分析AvaR2在这5个启动子区的结合序列,得出其结合的一段保守的 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)。利用该保守序列预测了一些新的AvaR2靶基因,并通过EMSA和qRT-PCR鉴定了 11个新的靶基因,它们分别参与初级代谢、核糖体蛋白合成、胁迫响应等生理过程,表明AvaR2是一个多效调控因子。AvaR2不仅能以内源的avenolide为配体,还能以外源的杰多霉素B(JadB)、安普霉素(Apr)和潮霉素B(HygB)作为配体调节其结合DNA的能力,表明AvaR2介导的信号传导系统不仅在种内而且在种间信息交流中发挥了重要作用。AvaR1与真GBL受体同源性最高。对avaR1进行缺失、回补和过表达,通过摇瓶发酵和形态观察实验,初步证实AvaR1负调控阿维菌素的生物合成,但不影响阿维链霉菌的生长和形态分化。进一步通过qRT-PCR、EMSA、DNase I footprinting和ChIP-qPCR等实验,证实AvaR1能直接负调控aveR、aco、自身基因以及其它两个avaR基因(avaR2和avaR3)的转录,并且与avaR3p的亲和力稍高。AvaR1与AvaR2在aveR、aco、avaR1、vaaR2和avaR3启动子区的保护位点相同,据此得出AvaR1和AvaR2结合的保守序列相同。通过EMSA和qRT-PCR鉴定了10个新的AvaR1靶基因,这些基因分别参与初级代谢、核糖体蛋白合成、胁迫响应、核酸代谢等生理过程,表明AvaR1也是一个多效调控因子。AvaR1和AvaR2既有共同的靶基因,也有各自不同的靶基因,表明它们可交叉调控不同的生理过程。
[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 纬 -butenyl molecule. In order to elucidate the regulation mechanism of avermectin biosynthesis, the unusual self-regulating factors of 纬 -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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q936

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