本文關(guān)鍵詞：兩種調(diào)控因子在嗜熱毀絲霉纖維素酶基因表達(dá)調(diào)控中的功能研究　出處：《深圳大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：木質(zhì)纖維素是植物細(xì)胞壁的主要成分之一,是地球上最為豐富的可再生碳源。利用纖維素酶降解木質(zhì)纖維素,在食品工業(yè)、造紙工業(yè)、畜牧業(yè)以及生物乙醇的研發(fā)等方面均有大量的研究應(yīng)用。如何獲得大量、高效、熱穩(wěn)定性好的纖維素酶已成為當(dāng)今科學(xué)界重要的研究課題。許多微生物所產(chǎn)生的纖維素酶都能有效的降解木質(zhì)纖維素,目前工業(yè)上主要用木霉、青霉等中溫真菌來(lái)生產(chǎn)纖維素酶。與中溫真菌相比,嗜熱真菌可耐高溫環(huán)境,培養(yǎng)過(guò)程中不易受中溫真菌污染,所產(chǎn)纖維素酶具有更好的熱穩(wěn)定性,可加快降解速度,有廣闊的應(yīng)用前景。嗜熱毀絲霉(Myceliophthora thermophile ATCC42464)是一種絲狀子囊真菌,研究表明該菌能產(chǎn)生熱穩(wěn)定性良好的纖維素酶類(lèi),具有水解生物質(zhì)大部分多糖的能力,是潛在的高效中高溫酶庫(kù)。本研究利用RNA-Seq技術(shù),篩選到嗜熱毀絲霉中兩個(gè)潛在的與纖維素酶表達(dá)調(diào)控相關(guān)的轉(zhuǎn)錄因子MHR1和MHR2,并分別運(yùn)用RNAi和同源過(guò)表達(dá)技術(shù)對(duì)上述兩因子進(jìn)行了研究。本論文的主要內(nèi)容如下:(1)本實(shí)驗(yàn)設(shè)計(jì)了mhr1基因的RNA干擾序列,利用pUC19-M質(zhì)粒構(gòu)建mhr1基因的沉默表達(dá)盒,經(jīng)原生質(zhì)體轉(zhuǎn)化、潮霉素平板篩選、PCR鑒定和測(cè)序,成功獲得五株轉(zhuǎn)化子,再經(jīng)RT-qPCR選出干擾率最高的一株轉(zhuǎn)化子MtR5,其mhr1基因的表達(dá)量?jī)H為原菌WT中表達(dá)量的1%。分別在誘導(dǎo)條件和非誘導(dǎo)條件下,同時(shí)培養(yǎng)轉(zhuǎn)化子MtR5和原菌WT,測(cè)定其胞外蛋白濃度、纖維素酶酶活及主要纖維素酶基因表達(dá)量。發(fā)現(xiàn)在非誘導(dǎo)培養(yǎng)144 h時(shí),轉(zhuǎn)化子MtR5的胞外蛋白濃度、濾紙酶活、內(nèi)切葡聚糖酶酶活和木聚糖酶酶活分別是原菌的1.94倍、1.52倍、1.47倍和1.20倍;經(jīng)qPCR檢測(cè)發(fā)現(xiàn),此時(shí)轉(zhuǎn)化子MtR5的主要纖維素酶基因中cbh1、egl3和xyr1比原菌中的表達(dá)量高出6-10倍。在誘導(dǎo)培養(yǎng)72 h時(shí),轉(zhuǎn)化子MtR5的胞外蛋白濃度和纖維素酶酶活均比原菌有所提高;qPCR檢測(cè)結(jié)果表明,此時(shí)轉(zhuǎn)化子MtR5的主要纖維素酶基因中cbh2、egl3和調(diào)控因子xyr1的表達(dá)量比原菌中高出28-56倍。研究表明,干擾mhr1基因的表達(dá),可提高嗜熱毀絲霉中部分纖維素酶基因的表達(dá)量,增強(qiáng)纖維素酶活性。從上述結(jié)果可知,該轉(zhuǎn)錄因子MHR1是作為阻遏因子發(fā)揮作用的。(2)本實(shí)驗(yàn)通過(guò)查詢(xún)NCBI數(shù)據(jù)庫(kù)獲得MHR2的基因全長(zhǎng)序列,構(gòu)建其過(guò)表達(dá)載體,經(jīng)原生質(zhì)體轉(zhuǎn)化、潮霉素平板篩選、PCR鑒定獲得五株陽(yáng)性過(guò)表達(dá)轉(zhuǎn)化子,再經(jīng)RT-qPCR選出過(guò)表達(dá)量最高的一株轉(zhuǎn)化子MtO24,研究了mhr2基因過(guò)表達(dá)對(duì)嗜熱毀絲霉胞外蛋白含量、纖維素酶活性及纖維素酶基因表達(dá)的影響。實(shí)驗(yàn)發(fā)現(xiàn),在誘導(dǎo)培養(yǎng)72h時(shí),轉(zhuǎn)化子MtO24和原菌WT的胞外蛋白濃度和纖維素酶酶活均達(dá)到峰值,且轉(zhuǎn)化子MtO24的胞外蛋白濃度是原菌WT的1.58倍,濾紙酶活與內(nèi)切葡聚糖酶酶活分別為原菌的1.30和1.24倍。qPCR檢測(cè)發(fā)現(xiàn),誘導(dǎo)培養(yǎng)72 h時(shí),轉(zhuǎn)化子MtO24的幾種主要纖維素酶基因的表達(dá)量均比原菌WT提高了3-9倍不等,這與纖維素酶活性的提高基本一致。本研究結(jié)果表明,MHR2是一種與嗜熱毀絲霉纖維素酶基因的表達(dá)調(diào)控相關(guān)的轉(zhuǎn)錄因子,且該因子對(duì)纖維素酶基因的表達(dá)具有激活作用。
[Abstract]:Lignocellulose is one of the main components of plant cell wall, is renewable carbon rich earth. The utilization of lignocellulose degradation of cellulase, in the food industry, paper industry, animal husbandry and the research and application of bio ethanol and other aspects of development. There are a lot of how to get a lot of high efficiency, good thermal stability, cellulase has become an important the research topic in the field of science. Generated by many microorganisms can degrade lignocellulose effectively, the industry is currently mainly used Trichoderma, Penicillium and other mesophilic fungi to produce cellulase. Compared with the mesophilic fungi and thermophilic fungi resistant to high temperature environment, the training process is not affected by the temperature of fungal contamination, thermal stability cellulase has better, can accelerate the degradation rate, and has a wide application prospect. Myceliophthora thermophila (Myceliophthora thermophile ATCC42464) is a filamentous The research shows that the fungi, bacteria can produce cellulases has good thermal stability, ability of hydrolysis of biomass polysaccharides is most efficient in high temperature, enzyme base potential. In this study, the use of RNA-Seq technology, screened by Myceliophthora thermophila in two potential cellulase and expression regulation of transcription factors related to MHR1 and MHR2, and then use RNAi and homologous overexpression was investigated based on the two factors mentioned above. The main contents of this thesis are as follows: (1) the experimental design of RNA interference sequence of mhr1 gene, mhr1 gene was constructed by pUC19-M gene silencing expression cassette by protoplast transformation, hygromycin screening, identification and PCR sequencing, successfully obtained five transformants were selected by RT-qPCR, then interference transformants were the highest rate of MtR5, the expression of mhr1 gene expression is only original bacteria amount of WT 1%. in induced and non induced conditions, At the same time culturing a transformant MtR5 and original strain WT, the determination of protein concentration, the expression of cellulase activity and cellulase gene. Found in the non induced 144 h, the concentration of extracellular protein MtR5 transformants, filter paper enzyme activity, enzyme activity of endoglucanase and xylanase activity were 1.94 times the original, bacteria 1.52 times, 1.47 times and 1.20 times; the results of qPCR showed that the cbh1 mainly cellulase transformant MtR5 genes, egl3 and xyr1 is 6-10 times higher than the original strain. The amount of expression in the induction of 72 h transformants, MtR5 protein concentration and cellulose enzyme the activity was compared with the original strain increased; the results of qPCR showed that the main cbh2 cellulase transformant MtR5 gene, expression and regulation of egl3 factor xyr1 is 28-56 times higher than the original strain. The research shows that interfering the expression of mhr1 gene can improve the thermophilic cellulase destroyed part of silk in the mould The amount of gene expression, enhanced cellulase activity. It can be seen from the above results, the transcription factor MHR1 is play a role as a repressor. (2) obtained the full-length sequence of MHR2 gene in this experiment by querying the NCBI database, the construction of its expression vector, by protoplast transformation, hygromycin screening, identification of five strains of PCR the positive expression of transformants by RT-qPCR had the highest expression level of selected transformants were MtO24, the overexpression of mhr2 gene on Myceliophthora thermophila extracellular protein content, the effect of cellulase activity and cellulase gene expression. The experimental results showed that, in the induction of 72h, extracellular protein concentration and cellulase enzyme conversion MtO24 and the original strain WT activities reached peak, the concentration of extracellular proteins and the transformant MtO24 was 1.58 times higher than the original strain WT, and endoglucanase enzyme activity were 1.30 and 1.24 times of.QPCR detection of pathogenic bacteria of filter paper activity Found that induction of 72 h, the expression of several major cellulase genes transformed MtO24 were compared with the original strain WT was increased by 3-9 times. This increase and cellulase activity are basically the same. The results of this study show that MHR2 is a Myceliophthora thermophila cellulase gene expression regulation of related transcription factors activate the expression, and the factor of cellulase gene.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q78;Q55

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