异源耐热β-葡萄糖苷酶基因在黑曲霉中高效表达研究
发布时间：2018-09-13 13:44
【摘要】：β-葡萄糖苷酶(β-glucosidase;EC.3.2.1.21)全称β-D-葡萄糖苷水解酶,属于纤维素酶的一种,被认为是纤维素酶水解的限速酶。本论文以黑曲霉3.213为实验对象,对其产酶曲线、蛋白质标准曲线、葡萄糖标准曲线进行了测定;bgl基因的扩增和序列分析;同源重组敲除原bgl基因;构建异源耐热β-葡萄糖苷酶表达载体pPHT-bgl;表达质粒转化黑曲霉3.213和对转化的黑曲霉3.213进行产酶曲线的测定。具体结论如下:1.通过对其产酶标准曲线的绘制测得黑曲霉3.213最佳产酶时间为168 h,这点具有最高酶活1.704 U/mL。2.采用真菌基因组提取试剂盒法和CTAB法提取黑曲霉3.213的基因组DNA,以其为模板,设计引物,进行PCR扩增得到大小为2 083 bp的目的基因片段。将序列进行blastn比对,同源性达到100%,表明成功将黑曲霉3.213的bgl基因扩增。而且对其进行序列分析和多重序列比对。3.运用三段式PCR方法与同源重组对黑曲霉3.213原bgl基因进行了敲除,导入潮霉素抗性基因(PHT),通过潮霉素选择性筛选和PCR检测确定将原基因敲除,消除了原基因对目的基因的干扰。4.将TtrpC进行PCR,质粒pUC19与其产物连接方式为EcoRⅠ、HindⅢ双酶切,得到中间质粒;将中间质粒与PglaA双酶切连接;然后将其与PHT通过双酶切进行连接,得到的产物通过双酶切与bgl基因进行连接,最后构建出黑曲霉3.213的高效表达载体pPHT-bgl.5.用混合酶解液悬浮适量菌丝,振荡酶解3 h。过滤收集原生质体,通过一系列方法进行外源DNA的转化,将表达质粒通过原生质体转化黑曲霉3.213。对其进行PCR检测,目的条带大小约为2 300 bp,可初步认为异源耐热β-葡萄糖苷酶在黑曲霉3.213中进行表达。6.比较原菌株与转化后菌株的产酶能力的变化,可通过数据处理得出其产酶最高点比原菌株增加了33.5%,初步完成了异源耐热β-葡萄糖苷酶在黑曲霉3.213中的高效表达。
[Abstract]:尾 -glucosidase (EC.3.2.1.21) is a kind of cellulase, which is regarded as the rate-limiting enzyme of cellulase hydrolysis. In this paper, Aspergillus Niger 3.213 was used as the experimental object. The enzyme production curve, protein standard curve and glucose standard curve were used to determine the amplification and sequence analysis of BGL gene, homologous recombination knockout of the original bgl gene. The pPHT-bgl; expression vector of Heterothermic 尾 -glucosidase was constructed to transform Aspergillus Niger 3.213 and the transformed Aspergillus Niger 3.213 to determine the curve of enzyme production. The concrete conclusion is as follows: 1. The optimum enzyme production time of Aspergillus Niger 3.213 was 168h, which had the highest enzyme activity of 1.704 U / ml. 2. The genomic DNA, of Aspergillus Niger 3.213 was extracted by fungal genomic extraction kit method and CTAB method. Primers were designed and amplified by PCR to obtain the target gene fragment of 2083 bp. The sequence was compared with blastn, and the homology was 100%, which indicated that the bgl gene of Aspergillus Niger 3.213 was amplified successfully. And the sequence analysis and multiple sequence alignment. 3. The primordial bgl gene of Aspergillus Niger 3.213 was knocked out by three-segment PCR method and homologous recombination. The hygromycin selective screening and PCR detection were used to determine the knockout of the proto gene by introducing hygromycin resistant gene (PHT), which eliminated the interference of proto gene to the target gene. The PCR, plasmid pUC19 was ligated with its product by double digestion of EcoR 鈪，
本文编号：2241343
[Abstract]:尾 -glucosidase (EC.3.2.1.21) is a kind of cellulase, which is regarded as the rate-limiting enzyme of cellulase hydrolysis. In this paper, Aspergillus Niger 3.213 was used as the experimental object. The enzyme production curve, protein standard curve and glucose standard curve were used to determine the amplification and sequence analysis of BGL gene, homologous recombination knockout of the original bgl gene. The pPHT-bgl; expression vector of Heterothermic 尾 -glucosidase was constructed to transform Aspergillus Niger 3.213 and the transformed Aspergillus Niger 3.213 to determine the curve of enzyme production. The concrete conclusion is as follows: 1. The optimum enzyme production time of Aspergillus Niger 3.213 was 168h, which had the highest enzyme activity of 1.704 U / ml. 2. The genomic DNA, of Aspergillus Niger 3.213 was extracted by fungal genomic extraction kit method and CTAB method. Primers were designed and amplified by PCR to obtain the target gene fragment of 2083 bp. The sequence was compared with blastn, and the homology was 100%, which indicated that the bgl gene of Aspergillus Niger 3.213 was amplified successfully. And the sequence analysis and multiple sequence alignment. 3. The primordial bgl gene of Aspergillus Niger 3.213 was knocked out by three-segment PCR method and homologous recombination. The hygromycin selective screening and PCR detection were used to determine the knockout of the proto gene by introducing hygromycin resistant gene (PHT), which eliminated the interference of proto gene to the target gene. The PCR, plasmid pUC19 was ligated with its product by double digestion of EcoR 鈪，
本文编号：2241343
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