發(fā)布時間：2018-05-21 11:16

  本文選題：紅曲霉 + 桔霉素��； 參考：《天津科技大學》2017年碩士論文

【摘要】：紅曲霉是天然的色素和功能性食品添加劑的發(fā)酵菌株,在亞洲的許多國家應用廣泛。紅曲霉發(fā)酵產生的色素具有例如消炎、抗氧化、抗動脈粥樣硬化的有益功效,另外,紅曲霉的其他次級代謝產物也具有多種機體調節(jié)功能。但是,隨之產生的桔霉素會對人體腎臟、肝臟產生毒害作用,使其成為紅曲霉產業(yè)的瓶頸,嚴重影響著產品的生產和出口。目前為止,紅曲霉桔霉素產生及調控機制尚不清晰,本課題的研究為桔霉素產生機制提供一定的理論依據(jù)。本試驗選取了三株在生長速度、產色素及桔霉素能力上明顯不同的紅曲霉菌種M2、FM46、FJ-1,利用7對引物擴增出三者的桔霉素合成基因簇,進行DNA測序,結果表明,三種紅曲霉該基因簇序列相似性在99.9 %以上,與NCBI發(fā)布的序列幾乎完全一致,證明該基因簇為紅曲霉基因組的保守區(qū)域。通過實時熒光定量PCR比較分析三種紅曲霉基因簇中相關基因表達水平,結合檢索到的基因的生物學信息,確定ctnI、orf6、ctnR1為目標基因。試驗通過根瘤農桿菌介導的遺傳轉化體系(ATMT)對紅曲霉的桔霉素合成有關基因敲除進行嘗試,并在ATMT體系基礎上成功實現(xiàn)紅曲霉orf6、ctnR1基因的敲除。試驗首先對轉化載體pAG1-H3進行了改造,獲得了紅曲霉ctnI、orf6、ctnR1基因敲除的載體以及orf6、ctnR1基因回復突變載體。ctnR1基因的同源重組概率可高達10.5%, orf6基因的同源重組概率為3 %。在基因功能鑒定方面,試驗所獲得的orf6、ctnR1突變株在營養(yǎng)生長速度略微減慢,orf6突變體色素合成稍微降低,ctnR1突變株色素的合成未受影響,但兩者的產桔霉素能力明顯升高,最高可比野生株高4.5倍,存在顯著差異。根據(jù)這一結果預測,orf6、ctnR1基因為桔霉素合成途徑中的調節(jié)因子,反饋抑制了桔霉素的合成,后續(xù)研究可通過基因過表達驗證這一構想。
[Abstract]:Monascus is a fermentative strain of natural pigments and functional food additives and is widely used in many countries in Asia. The pigment produced by Monascus fermentation has beneficial effects such as anti-inflammation, anti-oxidation and anti-atherosclerosis. In addition, the other secondary metabolites of Monascus also have a variety of organism regulation functions. However, the resulting citrinin will have toxic effects on human kidney and liver, which will become the bottleneck of Monascus industry and seriously affect the production and export of products. Up to now, the mechanism of production and regulation of citrinin is not clear. The research in this paper provides some theoretical basis for the mechanism of citrinin production. In this experiment, three Monascus species, M2FFM46 FJ-1, which have different growth rate, pigment production and citrinin-producing ability, were selected and their citrinin synthetic gene clusters were amplified by 7 pairs of primers and sequenced by DNA. The sequence similarity of the three species of Monascus gene cluster is over 99.9%, which is almost identical with the sequence released by NCBI, which indicates that the gene cluster is a conserved region of Monascus genome. The expression level of related genes in three species of Monascus gene clusters was compared by real-time fluorescence quantitative PCR, and the target gene ctnIorf6 ctnR1 was identified by combining the biological information of the genes retrieved. In this experiment, Agrobacterium tumefaciens-mediated genetic transformation system (ATMTT) was used to knockout orf6ctnR1 gene of Monascus, and the knockout of orf6ctnR1 gene was successfully realized on the basis of ATMT system. Firstly, the transformed vector pAG1-H3 was modified, and the vector of ctnIorf6ctnR1 gene knockout and orf6ctnR1 gene reverse-mutation vector. The probability of homologous recombination of the vector. The probability of homologous recombination of orf6 gene was as high as 10. 5%, and the probability of homologous recombination of orf6 gene was 3. In the aspect of gene function identification, the orf6 ctnR1 mutant decreased the pigment synthesis of orf6 mutant slightly, but the ability of producing citrinin was significantly increased. The highest height was 4.5 times higher than that of wild plants, and there was significant difference. According to this result, it is predicted that orf6ctnR1 can inhibit the synthesis of citrinin by feedback because of the regulatory factor in the citrinin synthesis pathway, which can be verified by gene overexpression.
【學位授予單位】：天津科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TS201.3

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本文編號：1918978