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食品与发酵工业  2021, Vol. 47 Issue (15): 9-14    DOI: 10.13995/j.cnki.11-1802/ts.026047
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
植物乳杆菌CGMCC8198 β-羟脂酰-ACP脱水酶启动子的克隆及其冷激调节作用分析
周成慧1,2, 赵阳1,2, 王德行1,2, 席茂盛1,2, 罗学刚1,2*
1(天津科技大学 生物工程学院 工业发酵微生物教育部重点实验室暨天津市工业微生物重点实验室,天津,300457)
2(天津市微生物代谢与发酵过程控制技术工程中心,天津,300457)
Cloning of promoterregion of hydroxylizoyl β-ACP dehydrase gene from Lactobacillus plantarum CGMCC8198 and its cold shock response
ZHOU Chenghui1,2, ZHAO Yang1,2, WANG Dehang1,2, XI Maosheng1,2, LUO Xuegang1,2*
1(Key Lab of Industrial Fermentation Microbiology of the Ministry of Education,Tianjin Key Lab of Industrial Microbiology,College of Biotechnology,Tianjin University of Science & Technology,Tianjin,300457,China)
2(Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control,Tianjin,300457,China)
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摘要 温度对乳酸菌储存和应用有重要影响,为挖掘乳酸菌温控启动子以及探索冷激反应的分子机制,对植物乳杆菌CGMCC8198经4和37 ℃处理后的转录组测序数据进行了分析,并对其中的差异基因β-羟脂酰-ACP脱水酶编码的fabZ基因表达情况进行验证,进一步应用MEGA 7.0软件分析其进化亲缘关系。再通过BPROM、BDGP、IPSW以及WebLogo 3.0对基因上游区域进行预测,最后克隆启动子,构建绿色荧光蛋白启动子报告分析质粒,并转化大肠杆菌进行低温诱导分析。结果发现fabZ基因低温刺激后上调,生物信息学结果也显示此基因与低温应答有关系;在基因上游500 bp区域有强启动子,且启动子中的-10区(TGTAAC)和-35区(TTACCG)6个核苷酸的位置与经典的-10区、-35区相似;大肠杆菌低温诱导实验表明在14 ℃刺激20、40、60 min后,含fabZ启动子菌株的荧光相对强度分别是37 ℃时的1.30、2.38和1.42倍。该研究发现植物乳杆菌的fabZ基因具有低温上调特性,为探究乳酸菌冷激应答与fabZ基因功能机理,以及构建乳酸菌低温诱导表达载体奠定一定的基础。
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周成慧
赵阳
王德行
席茂盛
罗学刚
关键词:  乳酸菌  生物信息学分析  启动子  增强绿色荧光蛋白  冷激应答    
Abstract: Temperature has an important effect on the storage and application of lactic acid bacteria (LAB). In order to explore the temperature-regulated promoter with cold shock response of LAB, the transcriptome sequencing of Lactobacillus plantarum CGMCC8198 at 4 ℃ and 37 ℃ was carried out and analyzed. The upstream region of hydroxylizoyl β-ACP dehydrase gene fabZ was analyzed and the promoter was predicted. Finally, the reporter plasmid was constructed with enhanced green fluorescent protein and fabZ promoter in Escherichia coli. Real-time PCR and Bioinformatics analysis showed that fabZ was related to the hypothermia response and could up-regulated by low-temperature. A strong promoter sequence was predicted in the 500 bp upstream of fabZ. The nucleotide positions in the Pribnow box (TGTAAC) and the Sextama box (TTACCG) were similar to classical ones. The relative fluorescence intensity of E. coli mediated by fabZ promoter at low temperature were increased by 0.30, 1.38 and 0.42 times after incubation at 14℃ for 20, 40 and 60 min, respectively. The results showed that fabZ of L. plantarum was up-regulated at low temperature, made a certain foundation to explore the cold shock response of L. plantarum and the mechanism of fabZ, as well as to construct a low-temperature induced expression vector of L. plantarum.
Key words:  lactic acid bacteria    bioinformatics analysis    promoter    enhanced green fluorescent protein    cold shock response
收稿日期:  2020-10-30      修回日期:  2020-12-08           出版日期:  2021-08-15      发布日期:  2021-08-23      期的出版日期:  2021-08-15
基金资助: 国家重点研发计划项目(2017YFD0400303);天津市自然科学基金重点项目(18JCZDJC33800);工业微生物优良菌种选育与发酵技术公共服务平台项目(17PTGCCX00190);天津科技大学青年教师创新基金项目(2016LG06);天津市高等学校创新团队培养计划资助项目(TD13-5015)
作者简介:  硕士研究生(罗学刚教授为通讯作者,E-mail:luoxuegang@hotmail.com)
引用本文:    
周成慧,赵阳,王德行,等. 植物乳杆菌CGMCC8198 β-羟脂酰-ACP脱水酶启动子的克隆及其冷激调节作用分析[J]. 食品与发酵工业, 2021, 47(15): 9-14.
ZHOU Chenghui,ZHAO Yang,WANG Dehang,et al. Cloning of promoterregion of hydroxylizoyl β-ACP dehydrase gene from Lactobacillus plantarum CGMCC8198 and its cold shock response[J]. Food and Fermentation Industries, 2021, 47(15): 9-14.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.026047  或          http://sf1970.cnif.cn/CN/Y2021/V47/I15/9
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