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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (13): 67-70.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

含正确木聚糖酶基因转化子的活性筛选

刘亮伟 李运峰 郭晓丹   

  • 收稿日期:2010-01-14 修回日期:2010-03-01 出版日期:2010-07-05 发布日期:2010-07-05
  • 基金资助:

    国家自然科学基金项目

An activity-screening method for transformants containing accurate xylanase gene

Liu Liangwei, Li Yunfeng, Guo Xiaodan   

  • Received:2010-01-14 Revised:2010-03-01 Online:2010-07-05 Published:2010-07-05

PDF (PC)

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摘要：

基因工程中，人们很难有效地筛选到带有正确基因的转化子。本文将木聚糖酶基因与pET20b重组，转化BL21(DE3) 大肠杆菌，选取10个转化子，放入0.5ml LB培养基过夜培养，补加9.5ml培养基2.5h后诱导，收集并冻融裂解细胞，检测酶活性，7个转化子酶活性接近或高于阳性对照菌落，认定为阳性转化子，经DNA测序证明基因序列正确。与以往检测基因不同，本研究直接检测转化子酶活性，筛掉不能表达活性酶的突变基因，称为活性筛选方法；其次，用冻融裂解细胞方法简化裂解过程，便于自动化高通量操作；最后，比较初筛酶活性，筛选高酶活转化子。这个过程只要2-3天，具有快速简便特点。

关键词: 烤烟品系, 烤烟品系, 黑胫病, 抗病性, 病情指数, 人工接种

Abstract:

In enzyme engineering, screening for transformant containing accurate gene is labor-intensive and time-consuming. In this study, xylanase gene was cloned into pET20b to transform E. coli strain BL21(DE3) competent cells. 10 transformants were inoculated in 0.5ml LB for culturing overnight, 9.5ml LB was added and cultured for 2.5h. After induction for 5h, cells were harvested and lyzed by freezing-thawing procedure in a simplified lysis buffer. 7 transformants were regarded as positive for having activities closer to or higher than that of the positive control, thereafter, plasmid DNA was sequenced to confirm gene’s accuracy. Different from previous method screening transformant for gene, the present method screened transformant for enzyme activity, called activity-screening method. The preliminarily assayed activities were compared for finding out transformant having high activity. The procedure lasted for 2~3 days, having characteristic of high-throughput for assaying transformant’s enzyme activity in parallel.