摘要：以里氏木霉Trichoderma reesei 基因组 DNA 为模板，采用外显子拼接的方法，克隆出内切β-1，4 -D-葡聚糖酶Ⅱ基因egl2 的全编码序列，通过PCR成功扩增出包含两段外显子的基因序列，平端连接到pHG-T simple载体上，通过一步法和CaCl2法两种方法转化大肠杆菌DH5a感受态，并进行两种方法的比较。转化成功的菌株提取出质粒提后送往深圳华大基因测序，并将序列与已有序列进行clustalx比对分析。为内切β-1，4-D-葡聚糖酶II的进一步研究奠定基础。57532

毕业论文关键词：里氏木霉；纤维素酶；基因克隆； clustalx比对 

Abstract:

In my study, the complete sequence encoding of the endo-1,4-glucanase II was amplified by the method of exons connecting, useing the Trichoderma reesei genomic DNA as a PCR template.

Two exons gene was connected to be the target gene by three times PCR amplifications which used to connected with the pHG -t simple vector. one step method and the method of CaCl2 was both used in transforming into competent E.coli DH5a.The two method was compared each other after the results.The plasmid extracted from successful strain sent to BGI to sequencing, and compare with the existing sequences by clustalx. 

Keywords：Trichoderma reesei，Cellulase, gene clone，clustalx alignment

目录

摘要 3

1前言 7

1.1纤维素酶的概述 7

1.1.1纤维素酶的分类及降解机制 7

1.1.2纤维素酶的结构 8

1.2里氏木霉的概述 8

1.2.1里氏木霉产纤维素酶的概况 8

1.2.2里氏木霉的研究动态 9

2材料与方法 9

2.1材料 9

2.1.1质粒和菌株 9

2.1.2主要仪器及设备 9

2.1.3酶和主要试剂 10

2.2试验方法 10

2.2.1主要试剂以及培养基的配制 10

2.2.2里氏木霉的活化培养 10

2.2.3里氏木霉菌体的分离 10

2.2.4里氏木霉总DNA的提取 11

2.2.5引物设计和pcr扩增特定片段基因 11

2.2.6基因片段切胶回收 13

2.2.7感受态大肠杆菌制作 14

2.2.8目的基因片段转与质粒连接 14

2.2.9转化大肠杆菌DH5a感受态。 14

2.2.10转化成功质粒提取酶切跑胶验证 15

2.2.11质粒测序并进行Blast比对分析 15

3结果与分析 15

3.1里氏木霉酶基因组的提取 15

3.2 PCR 扩增 15

3.2.1外显子E1扩增 15

3.2.2外显子E2扩增 16

3.2.3E1 E2切胶回收 16

3.2.4全基因克隆 17

3.2.5里氏木霉内切-1，4-β-D-葡聚糖酶II基因胶回收验 里氏木霉内切β-1，4-D-葡聚糖酶II基因的克隆:http://www.youerw.com/shengwu/lunwen_62287.html