腾冲嗜热厌氧杆菌cmr4基因的表达及生物信息学分析

Expression and Bioinformatics Analysis of cmr4 in Thermoanaerobacter tengcongensis

腾冲嗜热厌氧杆菌(Thermoanaerobacter tengcongensis)cmr4基因是CRISPR-CasⅣ-B亚型原核防御系统的一部分。为研究其在腾冲嗜热厌氧杆菌热适应中的作用,通过PCR技术扩增得到cmr4基因,构建原核表达载体pET-28a::cmr4并在大肠杆菌BL21中表达Cmr4蛋白;同时利用生物信息学软件分析腾冲嗜热厌氧杆菌、布氏酸菌以及肉毒杆菌中cmr4所编码氨基酸的理化性质。成功构建了原核表达载体pET-28a::cmr4,腾冲嗜热厌氧杆菌cmr4基因并在大肠杆菌BL21中得到表达,Cmr4蛋白其分子质量为40 kD,以可溶形式存在。实时荧光定量PCR结果表明腾冲嗜热厌氧杆菌cmr4 mRNA在75℃和80℃高表达;生物信息学分析显示腾冲嗜热厌氧杆菌cmr4基因编码349个氨基酸,Cmr4为亲水性蛋白,等电点为5.19,不存在糖基化位点,潜在的磷酸化位点有29个。本研究结果对Cmr4在嗜热菌中热适应作用机制的研究有一定的参考价值。

cmr4 of Thermoanaerobacter tengcongensis is a part of the CRISPR-CasⅣ-B subtype prokaryotic defense system.In order to study its function in thermal adaptation of Thermoanaerobacter tengcongensis,the cmr4was cloned using PCR.Then the prokaryotic expression vector pET-28a::cmr4 was constructed to express Cmr4 in Escherichia coli BL21;meanwhile,the basic physio-chemical properties of cmr4 encoded amino acids in Thermoanaerobacter tengcongensis,Acidianus brierleyi and Clostridium botulinum A str.Hall was analyzed by bioinformatic approaches.The results showed that the properties vector pET-28a::cmr4 was successfully constructed and expressed in E.coli BL21,the size of molecular weight of Cmr4 was 40 k D mainly existed in soluble form.RT-qPCR analysis showed that the mRNA expression of cmr4 was highly expressed in 75℃and 80℃.Bioinformatics analysis showed that the cmr4 of Thermoanaerobacter tengcongensis encoded 349 amino acids,and its encoding protein Cmr4 was hydrophilic protein whose isoelectric point was 5.19.There was no glycosylation site in Cmr4,but it had 29 potential phosphorylation sites.The results of this study have certain enlightenment to study thermophilic adaptation of Cmr4 in Thermoanaerobacter tengcongensis.