论文总字数：19819字

摘 要

T.ressei Rut-C30 是到现在为止分泌胞外蛋白能力最强的菌株。虽然里氏木霉菌株酶系安全，但是β-葡萄糖苷酶分泌量严重不足，然而黑曲霉作为工业化生产机器，可以分泌葡萄糖苷酶。故将黑曲霉的β-葡萄糖苷酶基因（beta-glucosidase A，简称bglA）经PCR的方式获取并在里氏木霉进行重组和表达，采用诱导型启动子gla（来自葡萄糖淀粉酶基因），以pAN7-1为载体骨架构建重组质粒pAN7-1-bglA，并通过PEG介导原生质体转化的技术将重组质粒pAN7-1-bglA导入里氏木霉的原生质体中，从潮霉素平板中选出十个好的重组转化子，通过5次传代后，靶基因bglA可分别通过染色体DNA和PCR检测，表明该基因已整合到Trichoderma reesei基因组中。将原始里氏木霉和重组里氏木霉均以30g/L葡萄糖为主碳源进行摇瓶实验，以培养温度30℃，转速210rpm，培养72h，取发酵液上清在PH4.8的柠檬酸缓冲液中，50℃反应，测的纤维素酶滤纸酶活为：重组菌1.4U/mL，原始菌0.385U/mL，残糖：重组菌4.1g/L，原始菌0.4g/L。本实验证明了，黑曲霉β-葡萄糖苷酶基因可以实现在里氏木霉中的重组和表达，以及实现以葡萄糖为唯一碳源发酵产纤维素酶，相比于微晶纤维素生产里氏木霉的高成本来说，本实验能在工业化应用上取得巨大突破。

关键词：里氏木霉 β-葡糖糖苷酶 滤纸酶活 原生质体转化

Trichoderma reesei recombinant bacteria producing cellulase

ABSTRACT

T.ressei Rut-C30 is the strain that has the highest ability to secrete extracellular proteins until now. Although the enzyme strain of Trichoderma reesei is safe, the amount of β-glucanase is severely insufficient. However, Aspergillus niger can secrete glucosidase as an industrial production machine. Therefore, Aspergillus niger beta-glucosidas A (bglA) was obtained by PCR and recombined and expressed in Trichoderma reesei. The inducible promoter gla (from glucoamylase gene) was constructed using PAN7-1 as the vector backbone. Recombinant plasmid PAN7-1-bglA and the recombinant plasmid PAN7-1-bglA were introduced into protoplasts of Trichoderma reesei by PEG-mediated protoplast transformation techniques. Ten good recombinant transformations were selected from hygromycin plates. After 5 passages, the target gene bglA can be detected by chromosomal DNA and PCR, respectively, indicating that the gene has been integrated into the Trichoderma reesei genome. The original Trichoderma reesei and Trichoderma reesei were respectively shaken with 30g/L glucose as the main carbon source. The culture temperature was 30°C, the rotation speed was 210rpm, the culture period was 72h, and the fermentation supernatant was taken as the lemon at pH 4.8. In the acid buffer, at 50°C, the measured cellulase filter paper enzyme activity was: recombinant bacteria 1.4U/mL, original bacteria 0.385U/mL, residual sugar: recombinant bacteria 4.1g/L, original bacteria 0.4g/L. This experiment demonstrated that the recombination and expression in Trichoderma reesei can be achieved by the Aspergillus niger β-glucosidase gene and the production of cellulase by using glucose as the sole carbon source compared to the production of microcrystalline cellulose. For the high cost of Trichoderma, this experiment can make a huge breakthrough in the industrial application.

Keywords: Trichoderma reesei; Beta-glucosidase; Filter paper enzyme activity; Protoplast transformation

目 录

摘要 1

ABSTRACT 2

目 录 1

第一章 文献概述 1

1.1 引言 1

1.2 里氏木霉 1

1.3 纤维素酶简介、作用机制、生产方式及在不同领域的应用 2

1.3.1 纤维素酶的简介 2

1.3.2 纤维素酶的工作原理 2

1.3.3 纤维素酶的生产方式 3

1.3.4 纤维素酶在不同领域的应用 3

1.4 黑曲霉β-葡萄糖苷酶（beta-glucosidase A，bglA）基因 4

第二章 材料和方法 5

2.1 菌种 5

2.2 实验试剂 5

2.3 实验仪器 6

2.4 培养基 7

2.5 培养方法 8

2.5.1基因组提取 8

2.5.2黑曲霉β-葡萄糖苷酶基因的克隆 8

2.5.3酶切 9

2.5.4凝胶的回收处理 9

2.5.5大肠感受态制备 10

2.5.6一步克隆 11

2.5.7提质粒 11

2.5.8酶切验证 13

2.5.9黑曲霉原生质体转化 13

2.5.10还原糖含量的检验方法 15

2.5.11转化子的再次筛选 15

2.5.12测酶活 16

第三章 实验结果与讨论 17

3.1抗性摸索 17

3.1.1孢子抗性Hyg的选取 17

3.1.2 原生质体抗性浓度的选取 17

3.2诱导物摸索 18

3.3结果与讨论 18

第四章 展望 20

参考文献 21

致谢 24

第一章 文献概述

1.1 引言

以细菌、放线菌（如链霉菌、微粒体等）、古细菌（如GelyMyes、PygMyceta等）和真菌（如木霉、青霉等）、草食动物和昆虫为主的生物体也可用于生物量的利用[1]，生物量是主要被这些生物利用维生素酶分泌纤维转化成碳水化合物，为身体提供巨大能量。所以纤维素酶能被大量获取。

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