本文選題：粘性絲孢酵母 + BG��； 參考：《西北民族大學(xué)》2016年碩士論文

【摘要】：粘性絲孢酵母作為一種油脂微生物,能夠?qū)⑻妓衔锱c普通的油脂轉(zhuǎn)化為微生物油脂儲(chǔ)存在體內(nèi),但不能直接利用木質(zhì)纖維素類的碳源合成微生物油脂。β-葡萄糖苷酶(β-glucosidase, BG)作為糖苷水解酶家族成員,屬于纖維素水解酶系,主要參與水解位于末端的糖基原子團(tuán)同芳基或烷基間的p-D-糖苷鍵生成葡萄糖,如p-葡萄糖苷和p-半乳糖苷,對(duì)于木糖及果糖苷也具有一定作用。水解纖維素產(chǎn)生的糖類能夠被粘性絲孢酵母利用合成微生物油脂。本試驗(yàn)旨在通過研究BG為粘性絲孢酵母高效利用木質(zhì)纖維素生產(chǎn)微生物油脂奠定一定的基礎(chǔ)。本試驗(yàn)選取粘性絲孢酵母BG基因作為研究對(duì)象,在NCBI上尋找己報(bào)道的不同菌屬BG基因序列設(shè)計(jì)引物,PCR擴(kuò)增得到BG基因部分CDs區(qū)并使用生物信息學(xué)軟件進(jìn)行分析,通過熒光定量PCR技術(shù)檢測(cè)其在不同培養(yǎng)時(shí)間、溫度、碳氮比以及pH表達(dá)量的變化,克隆得到的部分CDS區(qū)導(dǎo)入表達(dá)載體并在大腸桿菌(E.coil Top 10)中表達(dá)。研究結(jié)果如下：(1)經(jīng)過液氮研磨、反復(fù)凍融、超聲波破碎、鋼珠震蕩以及酶同鋼珠共同作用預(yù)處理方式,利用Trizol法提取粘性絲孢酵母總RNA。結(jié)果顯示液氮研磨和酶同鋼珠共同作用提取RNA相對(duì)完整無降解質(zhì)量較好,反復(fù)凍融方式次之,超聲波破碎出現(xiàn)明顯的降解,而鋼珠震蕩方式電泳未檢測(cè)出條帶,說明液氮研磨是破碎粘性絲孢酵母細(xì)胞壁提取總RNA有效的預(yù)處理方式。(2)通過TA克隆得到BG基因的部分CDs區(qū)序列。序列長(zhǎng)570bp,編碼189個(gè)氨基酸,生物信息學(xué)分析顯示該多肽屬于親水性蛋白,二級(jí)結(jié)構(gòu)中α-螺旋占25.9%,延伸鏈占22.89%,無規(guī)則卷曲占51.20%,無p-折疊等其他結(jié)構(gòu)。(3)通過熒光定量PCR技術(shù)結(jié)合SPSS軟件分析BG基因mRNA在不同培養(yǎng)時(shí)間、溫度、碳氮比以及pH表達(dá)量的變化,結(jié)果顯示培養(yǎng)溫度、pH、C/N對(duì)粘性絲孢酵母BG基因表達(dá)量無顯著性影響；培養(yǎng)時(shí)間對(duì)其具有顯著性影響,呈先上升后下降的趨勢(shì),在第3d時(shí)表達(dá)量最高且極顯著高于其他培養(yǎng)時(shí)期。(4)將pET28a質(zhì)粒表達(dá)載體與BG基因部分CDs區(qū)連接構(gòu)建原核表達(dá)載體,通過大腸桿菌表達(dá)其編碼的部分蛋白。經(jīng)菌液PCR與瓊脂糖凝膠電泳驗(yàn)證,表達(dá)載體在大腸桿菌中存在。SDS-PAGE凝膠電泳結(jié)果顯示,帶有HisTaq的目的蛋白分子量大小與預(yù)期相符合。Western blot驗(yàn)證結(jié)果顯示His標(biāo)簽有放射性反應(yīng),證明與HisTaq融合表達(dá)的目的蛋白在大腸桿菌內(nèi)也表達(dá),也進(jìn)一步證明SDS-PAGE凝膠電泳結(jié)果的準(zhǔn)確性,通過以上試驗(yàn)證明得到的蛋白為BG基因編碼的部分蛋白,為后續(xù)試驗(yàn)奠定一定的基礎(chǔ)。
[Abstract]:As a greasy microorganism, the yeast can convert carbohydrates and ordinary oils into microbial oils and store them in the body.However, microbial lipids could not be synthesized directly from the carbon source of lignocellulose. 尾 -glucosidase (BGase) is a member of the glycoside hydrolase family, which belongs to the cellulose hydrolase system.It is mainly involved in the hydrolysis of p-D- glucoside bonds between aryl and alkyl groups, such as p- glucoside and p- galactoside, which also play a certain role in xylose and fructoside.The carbohydrates produced by hydrolyzed cellulose can be used by Mycelia pastoris to synthesize microbial oils and fats.The purpose of this experiment was to establish a certain foundation for the production of microbial oil by using lignocellulose as BG yeast.In this experiment, BG gene was selected as the object of study, and some CDs regions of BG gene were amplified by primers designed on NCBI and analyzed by bioinformatics software.Fluorescence quantitative PCR technique was used to detect the changes of the expression amount in different culture time, temperature, C / N ratio and pH value. Some of the cloned CDS regions were introduced into the expression vector and expressed in E. coil Top 10 (E. coli).The results are as follows: (1) after liquid nitrogen grinding, repeated freezing and thawing, ultrasonic crushing, steel ball shaking and pretreatment of enzyme and steel ball interaction, the total RNAs were extracted by Trizol method.The results showed that the quality of RNA extraction by liquid nitrogen grinding and enzyme extraction with steel beads was relatively good, followed by repeated freezing and thawing, and obvious degradation appeared in ultrasonic crushing, while no band was detected in the electrophoresis of steel ball shock mode.The results showed that liquid nitrogen grinding was an effective pretreatment method for the extraction of total RNA from the cell wall of C. glutamicus. The partial CDs region of BG gene was obtained by TA cloning.The sequence was 570 BP and encoded 189 amino acids. Bioinformatics analysis showed that the peptide belonged to hydrophilic protein.The results showed that the culture temperature (pH = C / N) had no significant effect on the expression of BG gene, but the culture time had a significant effect on the expression of BG gene, which increased first and then decreased.On the 3rd day, the expression of pET28a plasmid was the highest and significantly higher than that of other culture period. (4) the pET28a plasmid was ligated with the CDs region of BG gene to construct the prokaryotic expression vector, and the part of protein was expressed by Escherichia coli.By PCR and agarose gel electrophoresis, the expression vector in Escherichia coli. SDS-PAGE gel electrophoresis results showed that the molecular weight of the target protein with HisTaq was in line with the expected. Western blot verification results showed that the His label had radioactivity.It was proved that the fusion protein expressed with HisTaq was also expressed in Escherichia coli, and the accuracy of SDS-PAGE gel electrophoresis was further proved. It was proved by the above experiments that the protein was part of the protein encoded by BG gene.To lay a certain foundation for the follow-up test.
【學(xué)位授予單位】：西北民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78;Q93

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