摘要
黑曲霉纤维素酶三类不同酶系中,纤维二糖水解酶基因表达处于很低水平,导致纤维素酶总体活力水平不高。为构建黑曲霉纤维素酶高产菌株,采用基因工程方法,全基因合成拼接黑曲霉高表达葡萄糖淀粉酶基因glaA的强启动子片段与纤维二糖水解酶基因cbhB编码区片段,然后将杂合基因克隆到二元载体pCAMBIA1301上,重组质粒通过农杆菌介导转化黑曲霉分生孢子,携带杂合基因的T-DNA片段插入到黑曲霉转化子的染色体上,共筛选到48个具有潮霉素抗性的转化子。纤维素酶活力水平测定结果显示,转化子A3-9的CMC酶活力最高,为野生型黑曲霉菌株的1.31倍;转化子B1-7与A3-6的滤纸酶活力最高,为野生型黑曲霉菌株2.51倍。另外,初步分析了杂合基因在黑曲霉中的表达所需的诱导条件。
Aspergillus niger produces three classes of cellulases for cellulose degradation and among these enzymes the expression level of cellobiohydrolases is very low,affecting the cellulose digestion efficiency by the cellulase system of Aspergillus niger.In order to construct an engineered strain with high cellulase enzyme activity,a hybrid gene containing strong promoter fragment of glucoamylase gene glaA and coding region of cellobiohydrolase gene cbhB was constructed via in vitro synthesis.The hybrid gene was subsequently cloned into the binary vector pCAMBIA1301 and the recombinant plasmid was introduced into conidiospores of Aspergillus niger by Agrobacterium tumefaciens-mediated genetic transformation.The transformants were hygromycin resistant with the hybrid gene inserted into chromosome DNA of Aspergillus niger via T-DNA illegitimate recombination.In total,48 transformants were seleted and their cellulase activities were tested.The results showed that transformant A3-9 had the highest CMC activity which was 1.31-fold as high as the wild type strain of Aspergillus niger,while both transformants B1-7 and A3-6 had the highest FPA activity which was 2.51-fold as high as wild type strain of Aspergillus niger.Furthermore,induction conditions for the hybrid gene expression were also analyzed.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第11期34-38,共5页
China Biotechnology
基金
天津市自然科学基金(08JCYBJC26600)
天津科技大学启动基金(20070436)资助项目
