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2025年8月8日 星期五
农业生物技术学报  2023, Vol. 31 Issue (2): 425-435    DOI: 10.3969/j.issn.1674-7968.2023.02.020
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
双向启动子探针载体的构建及其在谷氨酸棒杆菌中的应用
曹煜, 余心宇, 刘秀霞, 白仲虎*
江南大学 生物工程学院/工业生物技术教育部重点实验室/粮食发酵与食品生物制造国家工程研究中心,无锡 214122
Construction of Bidirectional Promoter Probe Vector and Its Application in Corynebacterium glutamicum
CAO Yu, YU Xin-Yu, LIU Xiu-Xia, BAI Zhong-Hu*
College of Biotechnology/Key Laboratory of Industrial Biotechnology/National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China
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摘要 双向启动子(bidirectional promoters, BDPs)为自然界常见表达元件之一,能够为遗传电路设计、代谢途径组装和优化提供新工具,但目前缺乏针对BDPs的高通量筛选手段。为了高通量筛选性能优越的BDPs,用于代谢工程中的多基因调控,本研究构建了一种BDPs活性探针载体p19BDP。该探针基于绿色荧光瞬时表征蛋白传感器蛋白1 (green fluorescent sensor for transiently expressed proteins 1, gSTEP1)和瞬时表征蛋白传感器蛋白质标签(sensor for transiently expressed proteins tag, STEPtag)相互作用后会产生荧光的原理,以“头对头”的方式将gSTEP1基因与STEPtag基因连入p19-0质粒(删除tac启动子的pXMJ19质粒改造获得)获得探针载体,在两个基因中间插入BDPs序列后,通过检测荧光信号初步表征BDPs活性。通过转录组数据分析筛选出了8个谷氨酸棒杆菌(Corynebacterium glutamicum)中的BDPs,利用探针载体p19BDP对其活性进行表征,其中两个诱导型启动子BDPcat和BDPicl的荧光值较诱导前分别提高了15.9倍和6.2倍,且在一定范围内荧光强度与诱导剂浓度呈正相关;其他6个内源BDPs (BDP1~BDP6)中,BDP6的荧光强度是对照启动子BDPcat (未诱导)的6.8倍。该探针载体不影响菌体生长,且gSTEP1/STEPtag复合物形成后其荧光值保持稳定。以上结果表明,探针载体p19BDP能够灵敏、有效地表征谷氨酸棒杆菌中BDPs强度。本研究为谷氨酸棒杆菌高通量挖掘天然BDPs提供了有效工具,同时为原核生物中BDPs筛选提供了新的思路。
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曹煜
余心宇
刘秀霞
白仲虎
关键词 谷氨酸棒杆菌探针载体双向启动子诱导型启动子    
Abstract:As one of the common expression elements in nature, the bidirectional promoters (BDPs) can provide new tools for genetic circuit design and metabolic pathway assembly and optimization. However, there is a lack of high-throughput screening and characterization methods for BDPs. In order to obtain the efficient BDPs for multi-gene regulation of metabolic pathways based on genome-level high-throughput screening, a BDPs probe vector p19BDP based on fluorescence generated by the interaction of the green fluorescent sensor for transiently expressed proteins 1 (gSTEP1) and the sensor for transiently expressed proteins tag (STEPtag) was constructed. The gSTEP1 and STEPtag gene were amplified by PCR, and ligated to the p19-0 vector in a "head-to-head" manner. After inserting the BDPs sequence between the two genes, the BDPs activity was assessed by detecting the fluorescence intensity. The p19BDP was used to characterize the activity of 8 BDPs screened by transcriptome data analysis in Corynebacterium glutamicum. The results showed that the fluorescence intensity of the BDPcat and BDPicl were 15.9 and 6.2 times higher than those before induction, respectively, the fluorescence intensity was positively correlated with the concentration of inducer in a certain range. For the other 6 endogenous BDPs (BDP1~BDP6), the fluorescence intensity of BDP6 was 6.8 times higher than that of the control promoter BDPcat (uninduced). The probe vecotr did not affect the growth of recombinant strain, and its fluorescence value remained stable after the formation of gSTEP1/STEPtag complex. The above results showed that the probe vector p19BDP could characterize sensitively and effectively the strength of BDPs in C. glutamicum. This study provides a powerful tool for high-throughput mining of natural BDPs in C. glutamicum and a new idea for characterizing BDPs in prokaryote.
Key wordsCorynebacterium glutamicum    Probe vector    Bidirectional promoters (BDPs)    Induce promoter
收稿日期: 2022-04-21     
ZTFLH:  S-3  
基金资助:国家自然科学基金(22078128; 21878124)
通讯作者: *baizhonghu@jiangnan.edu.cn   
引用本文:   
曹煜, 余心宇, 刘秀霞, 白仲虎. 双向启动子探针载体的构建及其在谷氨酸棒杆菌中的应用[J]. 农业生物技术学报, 2023, 31(2): 425-435.
CAO Yu, YU Xin-Yu, LIU Xiu-Xia, BAI Zhong-Hu. Construction of Bidirectional Promoter Probe Vector and Its Application in Corynebacterium glutamicum. 农业生物技术学报, 2023, 31(2): 425-435.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.02.020     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I2/425
 
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