本文關(guān)鍵詞：溫度和藍(lán)光對紅曲霉莫納可林K代謝調(diào)控的影響　出處：《天津科技大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 紅曲霉 溫度 藍(lán)光 莫納可林K 代謝調(diào)控 桔霉素

【摘要】：紅曲霉次生代謝產(chǎn)物中的莫納可林K(Monacolin K)是有效的膽固醇合成抑制劑,而其產(chǎn)率普遍不高和紅曲霉代謝產(chǎn)物中桔霉素的存在,限制了紅曲產(chǎn)品的開發(fā)與應(yīng)用。紅曲霉的次生代謝產(chǎn)物受溫度、光照、pH、溶氧量等環(huán)境因子的影響。本文主要研究了溫度和藍(lán)光對紅曲霉發(fā)酵過程中莫納可林K產(chǎn)量的影響,并采用RT-qPCR技術(shù)對莫納可林K相關(guān)合成基因的表達(dá)情況進(jìn)行分析,探討莫納可林K代謝調(diào)控的機(jī)理；對功能紅曲菌株的產(chǎn)桔霉素特性進(jìn)行安全性評價(jià),為構(gòu)建高產(chǎn)莫納可林K不產(chǎn)桔霉素的基因工程菌提供依據(jù)。采用固態(tài)發(fā)酵方式,研究不同溫度對高產(chǎn)莫納可林K的紅曲霉MC2菌株發(fā)酵過程中莫納可林K產(chǎn)量的影響。結(jié)果顯示,在30-C條件下培養(yǎng)到第25 d時(shí)莫納可林K產(chǎn)量達(dá)到21.2 μg/g干重；在20-C條件下培養(yǎng)到第28 d時(shí)莫納可林K產(chǎn)量達(dá)到2917.7μg/g干重,兩者相差137倍。采用液態(tài)靜置發(fā)酵方式,研究藍(lán)光對莫納可林K產(chǎn)量的影響,結(jié)果發(fā)現(xiàn),藍(lán)光和黑暗條件均在第28 d產(chǎn)量達(dá)到最高,分別為519.5 μg/g干重和1213.4 μg/g干重。藍(lán)光照射培養(yǎng)條件下,莫納可林K的最高產(chǎn)量僅為黑暗培養(yǎng)條件下的43%。設(shè)計(jì)引物,克隆紅曲霉MC2基因組DNA中與莫納可林K合成相關(guān)的10個(gè)基因的部分片段,將PCR產(chǎn)物測序結(jié)果與NCBI上報(bào)道的桔霉素合成相關(guān)基因序列進(jìn)行BLAST比對,結(jié)果顯示,同源性均在99%以上。采用RT-qPCR方法,比較30℃與20℃條件下,以糯糙米為基質(zhì),在發(fā)酵過程中紅曲霉MC2菌株的莫納可林K合成相關(guān)基因表達(dá)量的差異,結(jié)果表明：低溫條件提高了mokA、mokC、mokD、mokE、mokF、mokG和MpLaeA的表達(dá)量,降低了mokB、mokH和mokI基因的表達(dá)量。采用RT-qPCR方法,比較藍(lán)光和黑暗培養(yǎng)條件下,莫納可林K合成相關(guān)基因表達(dá)量的差異,發(fā)現(xiàn)藍(lán)光照射培養(yǎng)條件下,除mokB外,所有與紅曲霉MC2菌株莫納可林K合成相關(guān)基因的表達(dá)量均低于黑暗培養(yǎng)條件,從而導(dǎo)致藍(lán)光下莫納可林K產(chǎn)量降低,其中mokA、mokC、mokD、mokE、mokG是莫納可林K合成過程中九酮化合物合成途徑中的相關(guān)基因,溫度和藍(lán)光都是通過影響莫納可林K九酮化合物的合成來影響莫納可林K的最終產(chǎn)量。分析了紅曲霉MC2、C100和M9菌株在不同培養(yǎng)基中的產(chǎn)桔霉素情況,發(fā)現(xiàn)紅曲霉MC2是一株不產(chǎn)桔霉素的菌株,該菌株缺少桔霉素合成的關(guān)鍵基因ctnA、pksCT和orf3。對紅曲霉C100和M9兩株正常產(chǎn)桔霉素菌株進(jìn)行培養(yǎng),發(fā)現(xiàn)在添加一定量NH4NO3的大米粉培養(yǎng)基中不產(chǎn)桔霉素,利用RT-PCR方法分析不產(chǎn)桔霉素時(shí)相關(guān)基因的表達(dá)情況,發(fā)現(xiàn)桔霉素合成基因簇中相關(guān)基因均有表達(dá)。
[Abstract]:Monacolin (Monacolin K), a secondary metabolite of Monascus, is an effective inhibitor of cholesterol synthesis, but its yield is generally low and citrinin is present in the metabolites of Monascus. The development and application of Monascus products were limited. The secondary metabolites of Monascus were subjected to temperature, light and pH. The effects of temperature and blue light on the yield of Monacolin K during Monascus fermentation were studied. RT-qPCR technique was used to analyze the expression of monacolin K related synthetic gene, and the mechanism of the regulation of monacolin K metabolism was discussed. The safety evaluation of citrinin-producing strain was carried out to provide the basis for the construction of genetically engineered bacteria with high yield of Monacolin K and no citrinin production. Solid state fermentation was adopted. The effects of different temperatures on the yield of Monacolin K during the fermentation of Monascus MC2 strain with high yield of Monacolin K were studied. The yield of Monacolin K reached 21.2 渭 g / g dry weight at the 25th day under 30-C condition. The yield of Monacolin K reached 2917.7 渭 g / g dry weight at the 28th day under 20-C condition, and the difference was 137 times. The effect of blue light on the yield of Monacolin K was studied. The results showed that both blue light and dark conditions reached the highest yield on the 28th day. Dry weight of 519.5 渭 g / g and 1213.4 渭 g / g of dry weight respectively. Under the condition of blue light irradiation, the maximum yield of Monacolin K was only 43 under dark culture. The partial fragments of 10 genes related to the synthesis of Monacolin K were cloned from the genomic DNA of Monascus MC2. The sequencing results of PCR products were compared with the related gene sequences of citrinin synthesis reported on NCBI. The results showed that the homology was above 99%. RT-qPCR method was used. The relative gene expression of Monacolin K biosynthesis of Monascus MC2 strain was compared at 30 鈩，

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