【摘要】：桔霉素(citrinin,CIT)是一種主要由青霉,曲霉和紅曲霉屬產(chǎn)生的次級代謝產(chǎn)物。研究證實CIT具有腎毒性,現(xiàn)已明確的CIT毒性機制是CIT會造成細胞內(nèi)氧化還原系統(tǒng)和線粒體膜滲透功能障礙。CIT在食品和飼料中的污染范圍十分廣泛,而且往往與其他真菌毒素共存。研究發(fā)現(xiàn),CIT可以與赭曲霉毒素A(OTA),展青霉素(PAT)發(fā)生協(xié)同增強作用。因此CIT是一種危險性很高的真菌毒素,嚴重危害人類的健康。當前的關鍵是要尋找控制CIT的方法,降低人類接觸的風險,提高食品的安全性。論文篩選出了一株高效降解CIT的酵母菌,研究了此酵母菌降解CIT的效果及分子機制,主要研究成果如下:(1)研究了7株酵母菌對CIT的降解效果,發(fā)現(xiàn)Y3菌株可以高效降解CIT。通過5.8S rDNA-ITS區(qū)基因序列分析及形態(tài)學鑒定,確定這株酵母菌株為Cryptococcus podzolicus。通過動物實驗對其進行安全性檢驗,發(fā)現(xiàn)此酵母菌屬于實際無毒級,可以用于食品中CIT的控制與降解。(2)研究了不同酵母菌初始濃度、溫度、pH值、CIT初始濃度、培養(yǎng)基等因素對C.podzolicus Y3降解CIT效果的影響,結(jié)果表明,C.podzolicus Y3初始濃度越高,降解效率越高;溫度為28?C時,CIT降解效率最高;堿性條件不利于CIT的降解;CIT初始濃度越高越能刺激C.podzolicus Y3對CIT的降解;C.podzolicus Y3在NYDB培養(yǎng)基中可以降解CIT,但在PDB培養(yǎng)基中不能降解CIT。(3)研究了C.podzolicus Y3細胞壁,C.podzolicus Y3的細胞外代謝物降解CIT的生理機制。結(jié)果顯示,C.podzolicus Y3活細胞的細胞壁與死細胞的細胞壁對CIT沒有降解作用;C.podzolicus Y3細胞對CIT沒有吸收作用;NYDB正常培養(yǎng)的C.podzolicus Y3細胞外代謝物與CIT刺激后C.podzolicus Y3的細胞外代謝對CIT沒有降解作用。(4)通過蛋白質(zhì)組學技術,對C.podzolicus Y3在含有和不含有CIT的兩種NYDB培養(yǎng)基上培養(yǎng)24 h后的差異表達蛋白進行分析,結(jié)果顯示,糖基轉(zhuǎn)移酶家族2(glycosyl transferase family 2),蘋果酸脫氫酶DNA依賴型(malate dehydrogenase,NAD-dependent),銅鋅超氧化物歧化酶(superoxide dismutase[Cu-Zn]),半胱氨酸過氧化物(cysteine peroxiredoxin),雙鏈斷裂修復Rad50腺苷三磷酸酶(DNA double-strand break repair Rad50 ATPase),細胞色素c(cytochrome c)等蛋白具有顯著差異表達。(5)通過轉(zhuǎn)錄組技術,分析C.podzolicus Y3在含有和不含有CIT的兩種NYDB培養(yǎng)基上的表達基因,并對9個相關基因進行RT-qPCR技術驗證。結(jié)果表明,一共鑒定出C.podzolicus Y3的14551個基因。差異顯著的基因(|log2(FoldChange)|2)有1208個,包括上調(diào)差異基因551個,占總差異基因43.05%,下調(diào)差異基因657個,占差異總基因56.95%。選取與C.podzolicus Y3酵母細胞降解CIT相關的8個上調(diào)基因,1個下調(diào)基因,經(jīng)RT-PCR驗證,結(jié)果與轉(zhuǎn)錄組數(shù)據(jù)基本一致。驗證的9個基因中6個基因與C.podzolicus Y3降解CIT相關,分別為黃素單加氧酶(Flavin-binding monooxygenase,FMO),乙醇脫氫酶(Alcohol dehydrogenase,ADH),FAD(黃素腺嘌呤二核苷酸)依賴的氧化還原酶(FAD dependent oxidoreductase),谷胱甘肽-S-轉(zhuǎn)移酶(Glutathione S-transferase,GST),乙�；D(zhuǎn)移酶(Acetyltransferase(GNAT)),葡萄糖醛酸酶(beta-D-glucuronidase),均為正調(diào)控基因。3個基因與CIT對C.podzolicus Y3造成的損傷相關,分別為藥物應答反應蛋白(Multidrug resistance regulator 1),過氧化物酶體膜蛋白(Peroxisomal membrane protein),與染色體合成相關的DNA聚合酶(DNA polymerase family A),前兩個為上調(diào)基因,最后一個為下調(diào)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 擄 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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TS201.3

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