粘性丝孢酵母BG基因部分克隆与原核表达

发布时间：2018-04-10 23:04

本文选题：粘性丝孢酵母 + BG　；参考：《西北民族大学》2016年硕士论文

【摘要】：粘性丝孢酵母作为一种油脂微生物,能够将碳水化合物与普通的油脂转化为微生物油脂储存在体内,但不能直接利用木质纤维素类的碳源合成微生物油脂。β-葡萄糖苷酶(β-glucosidase, BG)作为糖苷水解酶家族成员,属于纤维素水解酶系,主要参与水解位于末端的糖基原子团同芳基或烷基间的p-D-糖苷键生成葡萄糖,如p-葡萄糖苷和p-半乳糖苷,对于木糖及果糖苷也具有一定作用。水解纤维素产生的糖类能够被粘性丝孢酵母利用合成微生物油脂。本试验旨在通过研究BG为粘性丝孢酵母高效利用木质纤维素生产微生物油脂奠定一定的基础。本试验选取粘性丝孢酵母BG基因作为研究对象,在NCBI上寻找己报道的不同菌属BG基因序列设计引物,PCR扩增得到BG基因部分CDs区并使用生物信息学软件进行分析,通过荧光定量PCR技术检测其在不同培养时间、温度、碳氮比以及pH表达量的变化,克隆得到的部分CDS区导入表达载体并在大肠杆菌(E.coil Top 10)中表达。研究结果如下：(1)经过液氮研磨、反复冻融、超声波破碎、钢珠震荡以及酶同钢珠共同作用预处理方式,利用Trizol法提取粘性丝孢酵母总RNA。结果显示液氮研磨和酶同钢珠共同作用提取RNA相对完整无降解质量较好,反复冻融方式次之,超声波破碎出现明显的降解,而钢珠震荡方式电泳未检测出条带,说明液氮研磨是破碎粘性丝孢酵母细胞壁提取总RNA有效的预处理方式。(2)通过TA克隆得到BG基因的部分CDs区序列。序列长570bp,编码189个氨基酸,生物信息学分析显示该多肽属于亲水性蛋白,二级结构中α-螺旋占25.9%,延伸链占22.89%,无规则卷曲占51.20%,无p-折叠等其他结构。(3)通过荧光定量PCR技术结合SPSS软件分析BG基因mRNA在不同培养时间、温度、碳氮比以及pH表达量的变化,结果显示培养温度、pH、C/N对粘性丝孢酵母BG基因表达量无显著性影响；培养时间对其具有显著性影响,呈先上升后下降的趋势,在第3d时表达量最高且极显著高于其他培养时期。(4)将pET28a质粒表达载体与BG基因部分CDs区连接构建原核表达载体,通过大肠杆菌表达其编码的部分蛋白。经菌液PCR与琼脂糖凝胶电泳验证,表达载体在大肠杆菌中存在。SDS-PAGE凝胶电泳结果显示,带有HisTaq的目的蛋白分子量大小与预期相符合。Western blot验证结果显示His标签有放射性反应,证明与HisTaq融合表达的目的蛋白在大肠杆菌内也表达,也进一步证明SDS-PAGE凝胶电泳结果的准确性,通过以上试验证明得到的蛋白为BG基因编码的部分蛋白,为后续试验奠定一定的基础。
[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.
【学位授予单位】：西北民族大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q93

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