Bioinformatics analysis and heterologous expression of RfbC gene in marine bacterium P. a. Hao2018

Abstract

There is a special kind of prokaryotic single-celled organisms in the ocean, which are called marine bacteria. Marine bacteria are a kind of microorganisms widely distributed in the marine environment. The high complexity of the marine environment enables marine bacteria to secrete macromolecular bioactive substances such as extracellular polysaccharide. As one of the main metabolites, the research on extracellular polysaccharide has been deepened in recent years, and the research and development and utilization of marine bacteria and extracellular polysaccharide have also developed rapidly. In this study, Pseudoalteromonas agarivorans Hao 2018, a marine bacterium, was taken as the research object to explore the cloning and expression of dTDP-4- dehydrorhamnose 3,5 epimerase gene in marine bacterium P.a.Hao.2018. Firstly, the amino acid sequence of Rfbc enzyme (dTDP-4- dehydrorhamnose 3,5 epimerase) was found from NCBI database, and the sequence alignment and phylogenetic tree were carried out. At the same time, the secondary structure and tertiary structure of Rfbc enzyme were predicted. In this experiment, the genome of marine bacteria Pseudoalteromonas agarivorans Hao 2018 was extracted, the dTDP-4- dehydrorhamnose 3,5 epimerase gene was cloned by PCR amplification, and then the cloned fragment sequence was connected with pET-16b (plasmid) to further construct an expression vector. It was transformed into the expression host Escherichia coli BL21(DE3). After the expression vector was verified, the target enzyme protein was finally obtained by IPTG induction, cell fragmentation and protein purification. Marine bacteria have important biological functions. The results of this study are expected to provide a theoretical basis for further exploring the metabolic pathway of marine bacteria Hao 2018 and its application in the field of biotechnology.