發(fā)布時間：2018-11-27 15:55

【摘要】：本論文對粗糙脈孢菌(Neurospora crassa)進行了兩方面的研究工作。一部分是三個纖維二糖轉運蛋白基因的研究,另一部分是對產(chǎn)酶培養(yǎng)基優(yōu)化的研究。 微生物將生物質降解為各種糖類物質,這些糖類物質通過糖轉運蛋白運送到細胞中,并在細胞中進行進一步的代謝。在自然界中,糖轉運蛋白對于碳水化合物的代謝起著至關重要的作用。在非葡萄糖轉運蛋白的研究中,纖維二糖轉運蛋白的研究非常少見。粗糙脈胞菌具有纖維素降解,乙醇發(fā)酵,木糖應用等杰出特點,是一個非常有工業(yè)應用潛力的絲狀真菌。2010年,有科學家首次從粗糙脈孢菌中克隆鑒定了纖維二糖轉運蛋白基因CDT1、CDT2。但是對其生理生化功能、基因表達調控等分子機理還不很了解。 本論文以三個纖維二糖轉運蛋白為研究對象,分別為cDT1、CDT2和CDT3。實驗中構建并獲得了三個基因的單敲除體菌株,雙敲除體菌株及三敲除體菌株。并且在不同碳源培養(yǎng)基中生長這些菌株,通過纖維素內切酶酶活,木聚糖酶酶活等指標反映各個菌株之間的生長差異。結果表明,在以纖維二糖為碳源的培養(yǎng)基中生長的三敲除體菌株T123生長較雙突變菌株D12差,說明CDT3基因與纖維二糖轉運相關。并通過綠色熒光蛋白GFP對糖轉運蛋白進行了定位。定位結果顯示,糖轉運蛋白均在各種膜上進行表達。 本論文第二部分是以粗糙脈孢菌基因組測序菌株FGSC2,489為對象,采用響應面分析法對粗糙脈孢菌搖瓶發(fā)酵產(chǎn)纖維素酶的培養(yǎng)基進行優(yōu)化。在初始培養(yǎng)基的基礎上,采用該優(yōu)化培養(yǎng)基,最大纖維素酶活可達：1.27FPU/mL,是優(yōu)化前纖維素酶活的2.02倍；CMC酶活14.15IU/mL是優(yōu)化前的1.88倍,木聚糖酶活24,13IU/mL是優(yōu)化前的1.86倍,葡萄糖苷酶酶活1.22IU/mL是優(yōu)化前的2.08倍。
[Abstract]:In this paper, two aspects of research on (Neurospora crassa) were carried out. One is the study of three cellular-disaccharide transporter genes, the other is the optimization of enzyme production medium. Microorganisms degrade biomass into various carbohydrates which are transported to cells by sugar transporters and further metabolized in cells. Sugar transporters play an important role in carbohydrate metabolism in nature. In the study of non-glucose transporter, the study of fibronectin is very rare. With outstanding characteristics of cellulose degradation, ethanol fermentation, xylose application and so on, C. crassica is a filamentous fungus with great potential for industrial application. Some scientists first cloned and identified the cellular-disaccharide transporter gene (CDT1,CDT2.) from C. crassa. However, its physiological and biochemical functions, gene expression regulation and other molecular mechanisms are not well understood. In this thesis, three cellular-disaccharide transporters (cDT1,CDT2 and CDT3.) were studied. Three single knockout strains, double knockout strains and triple knockout strains were constructed and obtained. These strains were grown in different carbon source media, and the growth differences were reflected by cellulose endonuclease activity and xylanase activity. The results showed that the growth of triple-knockout strain T123 was worse than that of double mutant strain D12 in the medium with cellulose disaccharide as carbon source, indicating that the CDT3 gene was related to fiber disaccharide transport. The sugar transporter was located by green fluorescent protein (GFP). The localization results showed that glucose transporters were expressed on various membranes. In the second part of this paper, the response surface analysis was used to optimize the medium for cellulase production by using response surface analysis (RSM), which was based on the genomic sequencing strain FGSC2489 of C. crassa. On the basis of the initial medium, the maximum cellulase activity was 1.27 FPU / mL, 2.02 times of that before optimization. The enzyme activity of CMC was 1.88 times of that before optimization, the activity of xylanase was 1.86 times of that before optimization, and the activity of glucosidase 1.22IU/mL was 2.08 times of that before optimization.
【學位授予單位】：天津科技大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R3411

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