| 张姝，胡蓉，黄健，吴建伟，国果，付萍，修江帆，尚小丽,2017,家蝇β-葡萄糖苷酶基因的克隆及重组表达[J].环境昆虫学报,(4):930-939 |
| 家蝇β-葡萄糖苷酶基因的克隆及重组表达 |
| Cloning and recombinant expression of β-glucosidase gene from Musca domestica |
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| DOI： |
| 中文关键词: 家蝇 β-葡萄糖苷酶 克隆 异源性表达 酶活性 |
| 英文关键词:Musca domestica β-glucosidase clone heterogenous express enzymatic activity. |
| 基金项目:贵州省教育厅培育项目[(2009)0137];国家科技支撑计划课题（2011BAC06B12）；国家自然科学基金（30960343）；国家教育部博士点专项基金[（2008）220] |
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| 中文摘要: |
| 克隆家蝇内源性β-葡萄糖苷酶（beta-glucosidase，BG）基因并建立原核表达体系，检测其表达产物的活性，了解家蝇内源性BG酶特点，为进一步解释家蝇极强环境适应能力和发现新的种群控制措施提供分子生物学基础。以草地贪夜蛾等结构信息相对完整且研究较为透彻的6种代表性昆虫的BG酶基因序列为参照对象，利用同源克隆结合RACE-PCR的方法，从家蝇cDNA中克隆得到BG酶基因的全长cDNA序列并对其进行生物信息学分析。分别采用原核表达载体pET-28a(+)构建原核表达体系并在大肠杆菌E.coli BL21(DE3) 中诱导表达BG酶基因的融合蛋白。采用七叶苷平板显色法和DNS法检测表达体系融合蛋白的酶活性，并对其性质进行初步的分析。家蝇体内克隆得到了长度为1933 bp的家蝇BG酶基因全长cDNA序列，推导其为562个氨基酸残基组成的蛋白多肽。重组原核质粒经诱导表达后，在65 kDa附近均出现特异性蛋白条带，证实重组质粒成功表达。重组表达的家蝇BG酶兼具内切β-1,4-葡聚糖酶、外切β-1,4-葡聚糖酶和BG的酶活性。家蝇BG酶是一种新的多功能纤维素酶，是昆虫纤维素酶研究的重要补充。 |
| 英文摘要: |
| Endogenous β-glycosidase (BG) gene of Musca domestica was cloned to construct a prokaryotic expression system, and the activities of expression products were detected. The characteristics of endogenous BG were explored to provide molecular and biological bases for further explaining the strong adaptive capacity of M.domestica to the environment and taking new measures for population control. Using the BG gene sequences of 6 representative, well-studied insects such as Spodoptera frugiperda as references, the whole-length cDNA sequence of BG gene that was cloned from cDNA of M.domestica was subjected to bioinformatics analysis by homology-based cloning in combination with RACE-PCR. Afterwards, pET-28(+) vector was used to construct a prokaryotic expression system that was transformed into E.coli BL21/DE to induce expressions of recombinant proteins. The BG activities of different recombinant proteins were detected by the aesculin plate method and DNS method, and their characteristics were preliminarily analyzed. Whole-length cDNA sequence (1933 bp) of BG gene was cloned from M.domestica, and deduced to be a polypeptide comprising 562 amino acid residues. After induced expression of recombinant prokaryotic plasmid, a specific band appeared at about 65 kDa, verifying that the recombinant plasmid was successfully expressed. The recombinant BG of M.domestica simultaneously had the activities of endo-β-1,4 glucanase (EC 3.2.1.4), exo-β-1,4-glucanase (EC 3.3.1.91) and BG (EC 3.2.1.21). BG from M.domestica is a novel multifunctional cellulase, as an important supplement for studies on inst cellulases. |
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