红曲霉桔霉素合成相关基因的功能验证

发布时间：2018-05-21 11:16

本文选题：红曲霉 + 桔霉素　；参考：《天津科技大学》2017年硕士论文

【摘要】：红曲霉是天然的色素和功能性食品添加剂的发酵菌株,在亚洲的许多国家应用广泛。红曲霉发酵产生的色素具有例如消炎、抗氧化、抗动脉粥样硬化的有益功效,另外,红曲霉的其他次级代谢产物也具有多种机体调节功能。但是,随之产生的桔霉素会对人体肾脏、肝脏产生毒害作用,使其成为红曲霉产业的瓶颈,严重影响着产品的生产和出口。目前为止,红曲霉桔霉素产生及调控机制尚不清晰,本课题的研究为桔霉素产生机制提供一定的理论依据。本试验选取了三株在生长速度、产色素及桔霉素能力上明显不同的红曲霉菌种M2、FM46、FJ-1,利用7对引物扩增出三者的桔霉素合成基因簇,进行DNA测序,结果表明,三种红曲霉该基因簇序列相似性在99.9 %以上,与NCBI发布的序列几乎完全一致,证明该基因簇为红曲霉基因组的保守区域。通过实时荧光定量PCR比较分析三种红曲霉基因簇中相关基因表达水平,结合检索到的基因的生物学信息,确定ctnI、orf6、ctnR1为目标基因。试验通过根瘤农杆菌介导的遗传转化体系(ATMT)对红曲霉的桔霉素合成有关基因敲除进行尝试,并在ATMT体系基础上成功实现红曲霉orf6、ctnR1基因的敲除。试验首先对转化载体pAG1-H3进行了改造,获得了红曲霉ctnI、orf6、ctnR1基因敲除的载体以及orf6、ctnR1基因回复突变载体。ctnR1基因的同源重组概率可高达10.5%, orf6基因的同源重组概率为3 %。在基因功能鉴定方面,试验所获得的orf6、ctnR1突变株在营养生长速度略微减慢,orf6突变体色素合成稍微降低,ctnR1突变株色素的合成未受影响,但两者的产桔霉素能力明显升高,最高可比野生株高4.5倍,存在显著差异。根据这一结果预测,orf6、ctnR1基因为桔霉素合成途径中的调节因子,反馈抑制了桔霉素的合成,后续研究可通过基因过表达验证这一构想。
[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.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS201.3

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