桔霉素降解菌的筛选及其降解机制研究
发布时间：2018-10-22 18:41
【摘要】：桔霉素(citrinin,CIT)是一种主要由青霉,曲霉和红曲霉属产生的次级代谢产物。研究证实CIT具有肾毒性,现已明确的CIT毒性机制是CIT会造成细胞内氧化还原系统和线粒体膜渗透功能障碍。CIT在食品和饲料中的污染范围十分广泛,而且往往与其他真菌毒素共存。研究发现,CIT可以与赭曲霉毒素A(OTA),展青霉素(PAT)发生协同增强作用。因此CIT是一种危险性很高的真菌毒素,严重危害人类的健康。当前的关键是要寻找控制CIT的方法,降低人类接触的风险,提高食品的安全性。论文筛选出了一株高效降解CIT的酵母菌,研究了此酵母菌降解CIT的效果及分子机制,主要研究成果如下:(1)研究了7株酵母菌对CIT的降解效果,发现Y3菌株可以高效降解CIT。通过5.8S rDNA-ITS区基因序列分析及形态学鉴定,确定这株酵母菌株为Cryptococcus podzolicus。通过动物实验对其进行安全性检验,发现此酵母菌属于实际无毒级,可以用于食品中CIT的控制与降解。(2)研究了不同酵母菌初始浓度、温度、pH值、CIT初始浓度、培养基等因素对C.podzolicus Y3降解CIT效果的影响,结果表明,C.podzolicus Y3初始浓度越高,降解效率越高;温度为28?C时,CIT降解效率最高;碱性条件不利于CIT的降解;CIT初始浓度越高越能刺激C.podzolicus Y3对CIT的降解;C.podzolicus Y3在NYDB培养基中可以降解CIT,但在PDB培养基中不能降解CIT。(3)研究了C.podzolicus Y3细胞壁,C.podzolicus Y3的细胞外代谢物降解CIT的生理机制。结果显示,C.podzolicus Y3活细胞的细胞壁与死细胞的细胞壁对CIT没有降解作用;C.podzolicus Y3细胞对CIT没有吸收作用;NYDB正常培养的C.podzolicus Y3细胞外代谢物与CIT刺激后C.podzolicus Y3的细胞外代谢对CIT没有降解作用。(4)通过蛋白质组学技术,对C.podzolicus Y3在含有和不含有CIT的两种NYDB培养基上培养24 h后的差异表达蛋白进行分析,结果显示,糖基转移酶家族2(glycosyl transferase family 2),苹果酸脱氢酶DNA依赖型(malate dehydrogenase,NAD-dependent),铜锌超氧化物歧化酶(superoxide dismutase[Cu-Zn]),半胱氨酸过氧化物(cysteine peroxiredoxin),双链断裂修复Rad50腺苷三磷酸酶(DNA double-strand break repair Rad50 ATPase),细胞色素c(cytochrome c)等蛋白具有显著差异表达。(5)通过转录组技术,分析C.podzolicus Y3在含有和不含有CIT的两种NYDB培养基上的表达基因,并对9个相关基因进行RT-qPCR技术验证。结果表明,一共鉴定出C.podzolicus Y3的14551个基因。差异显著的基因(|log2(FoldChange)|2)有1208个,包括上调差异基因551个,占总差异基因43.05%,下调差异基因657个,占差异总基因56.95%。选取与C.podzolicus Y3酵母细胞降解CIT相关的8个上调基因,1个下调基因,经RT-PCR验证,结果与转录组数据基本一致。验证的9个基因中6个基因与C.podzolicus Y3降解CIT相关,分别为黄素单加氧酶(Flavin-binding monooxygenase,FMO),乙醇脱氢酶(Alcohol dehydrogenase,ADH),FAD(黄素腺嘌呤二核苷酸)依赖的氧化还原酶(FAD dependent oxidoreductase),谷胱甘肽-S-转移酶(Glutathione S-transferase,GST),乙酰基转移酶(Acetyltransferase(GNAT)),葡萄糖醛酸酶(beta-D-glucuronidase),均为正调控基因。3个基因与CIT对C.podzolicus Y3造成的损伤相关,分别为药物应答反应蛋白(Multidrug resistance regulator 1),过氧化物酶体膜蛋白(Peroxisomal membrane protein),与染色体合成相关的DNA聚合酶(DNA polymerase family A),前两个为上调基因,最后一个为下调基因。
[Abstract]:Citrinin (CIT) is a secondary metabolite produced mainly from Penicillium, Aspergillus and Aspergillus. Studies have shown that CIT has nephrotoxicity, and the now-defined CIT toxicity mechanism is that CIT can cause intracellular redox system and mitochondrial membrane penetration dysfunction. CIT has a wide range of contamination in food and feed and tends to co-exist with other mycotoxins. It has been found that CIT can play a role in synergistic potentiation with Aspergillus fumigatus toxin A (OTA) and Exhibition penicillin (PAT). So CIT is a highly dangerous mycotoxin that harms human health. The key is to find ways to control CIT, reduce the risk of human exposure and improve food safety. The results of this study were as follows: (1) The degradation effect of 7 strains of yeast on CIT was studied, and it was found that Y3 strain could degrade CIT efficiently. Based on the analysis and morphological identification of the 5. 8S rDNA-ITS region gene sequence, it was determined that the strain of this strain was Cryptococcus plutozolamide. The safety test was carried out by animal experiment. It was found that the yeast belongs to the actual non-toxic level and can be used in the control and degradation of CIT in food. (2) The effects of initial concentration, temperature, pH value, CIT initial concentration, culture medium and other factors on the degradation of CIT effect were studied. The results showed that the higher the initial concentration of C. podzolamide Y3, the higher the degradation efficiency and the highest degradation efficiency of CIT when the temperature was 28 掳 C. The higher the initial concentration of CIT can stimulate the degradation of CIT. The higher the initial concentration of CIT can stimulate the degradation of CIT; C. podzolizumab Y3 can degrade CIT in NYDB culture medium, but it is not able to degrade CIT in culture medium. (3) The extracellular metabolites of C. podzolicum Y3 cell walls, C. podzolicum Y3 were studied to degrade CIT. The results showed that the cell wall of the viable cells and the cell wall of the dead cells did not degrade CIT; C. podzolizumab Y3 cells did not absorb CIT; and the extracellular metabolites of C. podzolizumab Y3 cells cultured normally by NYDB did not degrade CIT after CIT stimulation. (4) After 24 hours of culture on two NYDB medium containing and without CIT, the differentially expressed proteins were analyzed by proteomic techniques. The results showed that the glycosyltransferases family 2, malate dehydrogenase DNA-dependent (NAD-dependent), Copper-zinc superoxide dismutase (Cu-Zn), cysteine peroxide, double-strand break repair Rad50-triphosphatase (DNA double-strand break repair Rad50 ATPase), cytochrome c (cytochrome c) and other proteins have significant differences in expression. (5) Through the transcription group technique, the expression genes of C. podzolicum Y3 in two NYDB medium containing and without CIT were analyzed, and 9 related genes were verified by RT-qPCR. The results showed that 14551 genes of C. podzolicum Y3 were identified in total. Among them, there were 1208 genes (| log2 (FoldChange) | 2), including 551 genes up-regulated differential genes, 43. 05% of total differential gene and 657 genes down-regulated genes, accounting for 56. 95% of the total genes. Eight up-regulated genes, one down-regulated gene and RT-PCR were selected to degrade CIT, and the results were consistent with the data of transcriptome. Six of the nine genes were related to the degradation of CIT by C. podzolicum Y3, which was the ketoreductase (FAD)-dependent redox enzyme (FMO), the glutathione-S-transferase (Glutathione S-transfectin), which was dependent on the Emodin monooxygenase (FMO), the alcohol dehydrogenase (Alcohol dew), the FAAD (Emodin-2), respectively. GST, Acetylene transfectin (GNAT), beta-D-glucuronidase, all of which are positive regulatory genes. Three genes and CIT are related to the damage caused by C. podzolizumab Y3, respectively, are drug response protein (Multidrug resistance regator 1), peroxidase body membrane protein (PPO), DNA polymerase (DNA polymerase family A) associated with chromosome synthesis, the first two were up-regulated genes, and the last one was the down-regulation gene.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS201.3
本文编号：2287993
[Abstract]:Citrinin (CIT) is a secondary metabolite produced mainly from Penicillium, Aspergillus and Aspergillus. Studies have shown that CIT has nephrotoxicity, and the now-defined CIT toxicity mechanism is that CIT can cause intracellular redox system and mitochondrial membrane penetration dysfunction. CIT has a wide range of contamination in food and feed and tends to co-exist with other mycotoxins. It has been found that CIT can play a role in synergistic potentiation with Aspergillus fumigatus toxin A (OTA) and Exhibition penicillin (PAT). So CIT is a highly dangerous mycotoxin that harms human health. The key is to find ways to control CIT, reduce the risk of human exposure and improve food safety. The results of this study were as follows: (1) The degradation effect of 7 strains of yeast on CIT was studied, and it was found that Y3 strain could degrade CIT efficiently. Based on the analysis and morphological identification of the 5. 8S rDNA-ITS region gene sequence, it was determined that the strain of this strain was Cryptococcus plutozolamide. The safety test was carried out by animal experiment. It was found that the yeast belongs to the actual non-toxic level and can be used in the control and degradation of CIT in food. (2) The effects of initial concentration, temperature, pH value, CIT initial concentration, culture medium and other factors on the degradation of CIT effect were studied. The results showed that the higher the initial concentration of C. podzolamide Y3, the higher the degradation efficiency and the highest degradation efficiency of CIT when the temperature was 28 掳 C. The higher the initial concentration of CIT can stimulate the degradation of CIT. The higher the initial concentration of CIT can stimulate the degradation of CIT; C. podzolizumab Y3 can degrade CIT in NYDB culture medium, but it is not able to degrade CIT in culture medium. (3) The extracellular metabolites of C. podzolicum Y3 cell walls, C. podzolicum Y3 were studied to degrade CIT. The results showed that the cell wall of the viable cells and the cell wall of the dead cells did not degrade CIT; C. podzolizumab Y3 cells did not absorb CIT; and the extracellular metabolites of C. podzolizumab Y3 cells cultured normally by NYDB did not degrade CIT after CIT stimulation. (4) After 24 hours of culture on two NYDB medium containing and without CIT, the differentially expressed proteins were analyzed by proteomic techniques. The results showed that the glycosyltransferases family 2, malate dehydrogenase DNA-dependent (NAD-dependent), Copper-zinc superoxide dismutase (Cu-Zn), cysteine peroxide, double-strand break repair Rad50-triphosphatase (DNA double-strand break repair Rad50 ATPase), cytochrome c (cytochrome c) and other proteins have significant differences in expression. (5) Through the transcription group technique, the expression genes of C. podzolicum Y3 in two NYDB medium containing and without CIT were analyzed, and 9 related genes were verified by RT-qPCR. The results showed that 14551 genes of C. podzolicum Y3 were identified in total. Among them, there were 1208 genes (| log2 (FoldChange) | 2), including 551 genes up-regulated differential genes, 43. 05% of total differential gene and 657 genes down-regulated genes, accounting for 56. 95% of the total genes. Eight up-regulated genes, one down-regulated gene and RT-PCR were selected to degrade CIT, and the results were consistent with the data of transcriptome. Six of the nine genes were related to the degradation of CIT by C. podzolicum Y3, which was the ketoreductase (FAD)-dependent redox enzyme (FMO), the glutathione-S-transferase (Glutathione S-transfectin), which was dependent on the Emodin monooxygenase (FMO), the alcohol dehydrogenase (Alcohol dew), the FAAD (Emodin-2), respectively. GST, Acetylene transfectin (GNAT), beta-D-glucuronidase, all of which are positive regulatory genes. Three genes and CIT are related to the damage caused by C. podzolizumab Y3, respectively, are drug response protein (Multidrug resistance regator 1), peroxidase body membrane protein (PPO), DNA polymerase (DNA polymerase family A) associated with chromosome synthesis, the first two were up-regulated genes, and the last one was the down-regulation gene.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS201.3
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