摘要：从本实验室保存的含目的基因的大肠杆菌DH5α菌株中提出质粒pMV-EG2，经过NotI和KpnI双酶切得到目的基因，并获得相同的粘性片段。再利用T4 DNA连接酶将目的基因与表达载体pKLACl连接，导入到感受态大肠杆菌DH5a中扩增。再次提出质粒，并用ScalI酶将重组质粒线性化以便导入受体细胞中。制备马克斯克鲁维酵母的感受态，将线性化质粒电转导入酵母中，利用乙酰胺为氮源筛选转化子。挑取阳性菌落，扩增培养工程菌，并使其表达。65509

毕业论文关键词：葡聚糖酶；马克斯克鲁维酵母；基因表达；  

Abstract: Proposed saved from this laboratory DH5a containing the gene plasmid pMV-EG2, through NotI and KpnI double digestion of the target gene, and get the same sticky fragments. Then using T4 DNA ligase target gene connected with the expression vector pKLACl, introduced into competent E. coli DH5a amplified. Plasmid proposed again, and with ScalI enzyme linearized plasmid into a recipient cell in order. Preparation marxianus competent, the linearized plasmid was transfected into yeast, using acetamide as nitrogen source transformants were selected. Positive colonies were picked, engineering bacteria were cultured and allowed to express.

Keywords：glucanase; Kluyveromyces marxianus; Gene expression;  

目录

1 前言 7

1.1 纤维素酶的概述 7

1.1.1纤维素酶的降解机制 7

1.1.2纤维素酶的性质 7

1.2马克斯克鲁维酵母的概述 8

2材料与方法 8

2.1材料 8

2.1.1菌株与质粒 8

2.1.2实验主要试剂 8

2.1.3主要仪器及设备 8

2.2主要试剂以及培养基的配制 9

2.3 实验方法 9

2.3.1重组克隆载体的提取 9

2.3.2目的基因片段的获取 10

2.3.3目的基因与表达载体的切胶回收 10

2.3.4酵母表达载体的构建 10

2.2.5重组质粒转化大肠杆菌DH5a感受态 11

2.2.6重组质粒的提取与验证 11

2.2.7重组质粒的线性化处理及回收 11

2.2.8马克斯克鲁维酵母感受态的制备 11

2.2.9马克斯克鲁维酵母的转化 12

2.2.10工程菌K.marxianus-pKLAC1-EG2的表达 12

3结果与分析 12

3.1 pMV-EG2质粒的提取 12

3.2表达载体pKLAC1质粒的提取 13

3.3 目的基因的获取 13

3.4 目的基因切胶回收并验证 14

3.5目的基因与表达载体的连接 14

3.6重组质粒的提取 14

3.7双酶切验证 15

3.8 ScaII酶线性化 15

3.9转化子筛选 16

3.10酶活测定