發(fā)布時(shí)間：2018-11-27 15:55

【摘要】：本論文對(duì)粗糙脈孢菌(Neurospora crassa)進(jìn)行了兩方面的研究工作。一部分是三個(gè)纖維二糖轉(zhuǎn)運(yùn)蛋白基因的研究,另一部分是對(duì)產(chǎn)酶培養(yǎng)基優(yōu)化的研究。 微生物將生物質(zhì)降解為各種糖類物質(zhì),這些糖類物質(zhì)通過(guò)糖轉(zhuǎn)運(yùn)蛋白運(yùn)送到細(xì)胞中,并在細(xì)胞中進(jìn)行進(jìn)一步的代謝。在自然界中,糖轉(zhuǎn)運(yùn)蛋白對(duì)于碳水化合物的代謝起著至關(guān)重要的作用。在非葡萄糖轉(zhuǎn)運(yùn)蛋白的研究中,纖維二糖轉(zhuǎn)運(yùn)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(yīng)用等杰出特點(diǎn),是一個(gè)非常有工業(yè)應(yīng)用潛力的絲狀真菌。2010年,有科學(xué)家首次從粗糙脈孢菌中克隆鑒定了纖維二糖轉(zhuǎn)運(yùn)蛋白基因CDT1、CDT2。但是對(duì)其生理生化功能、基因表達(dá)調(diào)控等分子機(jī)理還不很了解。 本論文以三個(gè)纖維二糖轉(zhuǎn)運(yùn)蛋白為研究對(duì)象,分別為cDT1、CDT2和CDT3。實(shí)驗(yàn)中構(gòu)建并獲得了三個(gè)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(zhǎng)這些菌株,通過(guò)纖維素內(nèi)切酶酶活,木聚糖酶酶活等指標(biāo)反映各個(gè)菌株之間的生長(zhǎng)差異。結(jié)果表明,在以纖維二糖為碳源的培養(yǎng)基中生長(zhǎng)的三敲除體菌株T123生長(zhǎng)較雙突變菌株D12差,說(shuō)明CDT3基因與纖維二糖轉(zhuǎn)運(yùn)相關(guān)。并通過(guò)綠色熒光蛋白GFP對(duì)糖轉(zhuǎn)運(yùn)蛋白進(jìn)行了定位。定位結(jié)果顯示,糖轉(zhuǎn)運(yùn)蛋白均在各種膜上進(jìn)行表達(dá)。 本論文第二部分是以粗糙脈孢菌基因組測(cè)序菌株FGSC2,489為對(duì)象,采用響應(yīng)面分析法對(duì)粗糙脈孢菌搖瓶發(fā)酵產(chǎn)纖維素酶的培養(yǎng)基進(jìn)行優(yōu)化。在初始培養(yǎng)基的基礎(chǔ)上,采用該優(yōu)化培養(yǎng)基,最大纖維素酶活可達(dá)：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(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R3411

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李勇昊;周長(zhǎng)海;丁雷;孫元章;楊建明;;發(fā)酵培養(yǎng)基優(yōu)化策略[J];北京聯(lián)合大學(xué)學(xué)報(bào)(自然科學(xué)版);2011年02期

2 高蕾;肖文芳;李文燕;彭昌操;;擬南芥蔗糖轉(zhuǎn)運(yùn)蛋白(SUTs)的功能研究進(jìn)展[J];分子植物育種;2011年02期

3 ;中國(guó)新興能源產(chǎn)業(yè)最新數(shù)據(jù)公布(二)[J];節(jié)能與環(huán)保;2011年07期

4 宋安東;蘇麗娟;謝慧;曲音波;楊銘;;γ射線對(duì)斜臥青霉的誘變篩選及產(chǎn)酶條件優(yōu)化[J];核農(nóng)學(xué)報(bào);2008年03期

5 萬(wàn)根香;;生物質(zhì)成型燃料技術(shù)及其特性[J];能源研究與管理;2011年03期

6 于斐;吳雪昌;;己糖轉(zhuǎn)運(yùn)蛋白在釀酒酵母生長(zhǎng)與酒精發(fā)酵中的作用[J];科技通報(bào);2009年03期

7 張梁,石貴陽(yáng),洪劍輝,王正祥,章克昌;釀酒酵母纖維二糖代謝途徑的搭建[J];釀酒科技;2005年03期

8 李建波,宋欣,田敏,曲音波;粗糙脈孢菌產(chǎn)黑色素條件優(yōu)化及黑色素物理特性研究[J];山東大學(xué)學(xué)報(bào)(理學(xué)版);2004年04期

9 張妍妍;劉佩卉;劉新利;;粗糙脈孢菌及其在發(fā)酵工業(yè)中的應(yīng)用研究[J];山東科學(xué);2011年03期

10 鐘耀華;王曉利;汪天虹;;絲狀真菌高效表達(dá)異源蛋白研究進(jìn)展[J];生物工程學(xué)報(bào);2008年04期

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