乙酰乳酸合成酶基因(budB)整合表达载体的构建  

The Construction of Acetolactate Synthase Gene Integration Expression Vector

作　　者：葛菁萍[1] 邓利廷 孙雯[1] 宋刚[1] 平文祥[1] Ge Jingping Deng Liting Sun Wen Song Gang Ping Wenxiang(Key Laboratory of Microbiology, College of Life Sciences, Heilongjiang University, Harbin 150080)

机构地区：[1]微生物黑龙江省高校重点实验室,黑龙江大学生命科学学院,哈尔滨150080

出　　处：《中国农学通报》2017年第8期25-30,共6页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金“从2,3-丁二醇代谢角度构建工程微生物群体及其生态学机制研究”(31570492)

摘　　要：为了进一步了解2,3-丁二醇合成路径中关键酶的表达量与产量之间的关系,本研究根据Gen Bank中乙酰乳酸合成酶基因(bud B)的基因序列设计引物,以产酸克雷伯氏菌(Klebsiella oxytoca)HD79基因组DNA为模板,通过PCR扩增技术得到目标酶基因,目的片段全长1680 bp。以表达载体p RSFDuet-1为骨架,利用基因工程手段构建整合表达载体p R1SW-bud B。电转化法将其转化至菌株HD79感受态细胞中,通过高浓度Kan筛选和PCR双重鉴定验证后测定ALS酶活力。结果表明,乙酰乳酸合成酶基因整合表达载体构建成功,酶活可达1.0627 U/mg,比原始菌株提高了4.35倍。在一定程度上为实现2,3-丁二醇的工业化生产奠定基础。To learn more about the relationship between expression levels and yield of the key enzymes in 2,3- BD synthesis route, the primers were designed according to the sequence of budB in GenBank. Taking genomic DNA of Klebsiella oxytoca HD79 as templates, the target acetolactate synthase gene （budB） was obtained by PCR. The target fragment was 1680 bp. The multicopying integrated expression vector pR1SW-budB was constructed using expression vector pRSFDuet- 1 as a framework by means of genetic engineering. The plasmid pR1SW-budB was transformed into Klebisella oxytoca HD79 by electric conversion, then screened through high concentration Kan, followed by PCR amplification before determination of enzyme activity. The results demonstrated successful construction of the integrated expression vector of acetolactate synthase gene, with high ALS activity of 1.0627 U/mg, which was 4.35 times of the original activity. These findings lay a foundation for the industrial production of 2,3-BD in some degree.

关 键 词：2 3-丁二醇 乙酰乳酸合成酶 产酸克雷伯氏菌 电转化法 酶活力 

分 类 号：Q933[生物学—微生物学]