中文摘要：

【目的】用分子改造的方法改造里氏木霉（Trichoderma reesei）的纤维二糖水解酶基因cbh1，提高其纤维素酶活力，为工业化生产纤维素酶提供参考。【方法】用DNase Ⅰ消化里氏木霉cbb1，回收100bp左右的片段后用T4DNA连接酶连接，以连接产物作为模板进行PCR扩增，将PCR改造的cbh1基因转化里氏木霉原生质体，使改造的cbh1基因与里氏木霉原有的cbh1基因发生同源重组。通过比较滤纸酶活力的方法筛选纤维素酶活力提高的突变菌株，在NCBI上比对分析突变菌株的cbh1序列。【结果】经克隆测序获得基因片段大小为637bp，与里氏木霉同属菌株cbh1基因的相似性在88％-98％，确定为里氏木霉cbh1的基因片段。经DNaseⅠ消化15min、T4DNA连接酶连接、经PCR扩增获得改造后的cbh1基因。将改造cbh1基因片段转化里氏木霉原生质体，得到cbh1基因突变体库。从cbh1基因突变体库中筛选获得1株纤维素酶滤纸酶活比出发菌株高1．7518倍的突变菌株E2-1。序列比对分析发现，与出发菌株相比，突变菌株E2-1的cbh1基因有14个碱基发生了突变，其中5个碱基为置换突变，8个碱基为缺失突变，1个碱基为插入突变，分布于cbh1基因的9个位置。【结论】改造后的cbhI基因能与里氏木霉的染色体DNA发生同源重组，进而提高突变菌株的纤维素滤纸酶活力。

英文摘要：

【Objective 】Molecular modification of cellobiohydrolase gene cbh1 from T. reesei was carried out to improve cellulase activity in order to provide scientific reference for commercial production of cellulase. 【Method 】Cellobiohydrolase gene cbh1 from T. reesei was digested with DNase I, and the recycled fragments about 100 bp were linked with T4 DNA ligase randomly. By using the connection product as templates, the cbh1 sequence was amplified by PCR method. The modified cbh1 gene was transferred into T. reesei protoplast to conduct homologous recombination with original cbh1. Mutant strains with high cellulase activity were screened based on filter paper enzyme activity.cbh1 gene sequence alignment was performed in the NCBI. 【Result 】The cloned gene fragment was 637 bp in length and shared over 88% of homology with the other isolates of T. reesei, so it was identified as cbh1 gene fragment of T. reesei. After digestion of DNase I for 15 min, linking with T4 DNA ligase and PCR amplification, the modified cbh1 gene was obtained and transferred into T. reesei protoplast to construct mutant library. According to filter paper enzyme activity, mutant strain named E2-1was screened from cbh1 mutant library. The filter paper activity of E2-1cellulase was 1.7518 times higher than original strain. Sequence alignment revealed that, compared with original strain,14 bases of cbh1 distributing on 9 loci from mutant strain E2-1were changed, which included 5 bases substitution mutation, 8 bases deletion mutation and 1 base insertional mutation. 【Conclusion 】The modified cbh1 gene could be homologously recombined with chromosomal DNA of T. reesei, which resulted in improving the filter paper enzyme activity of mutant strain cellulase.