Homologous Cloning, Prokaryotic Expression And Construction Of Knockout Vector Of Clostridium Hydrogenase Gene

Dissimilatory iron-reducing bacteria grown in soil play an important role inbioremediation of organics and heavy metal pollution. Research in recent years showed that,in the process of dissimilatory iron-reducing coupled with oxidation of organics and hydrogen,many microorganisms could store energy for growth.Acetic acid and hydrogen are important intermediate products generated from organicmatter degradation. Meanwhile, they are significant electron donor of many iron-reducingbacteria. Clostridium is the most studied fermentative hydrogen-producing bacterium.Hydrogenase catalyzes generation and consumption of hydrogen. Therefore, it is inferred thatClostridium could reduce ferric iron with hydrogen as its electron donor.The object of this research is a bacterial strain possessed high iron reducing abilityseparated from paddy soil. Primarily, its genomic DNA was extracted as the template of16SrDNA amplification. Based on16S rDNA sequencing and neighbor-joining tree, the phyleticrelationships of the strain was investigated. Using homologous cloning technique, a conservedfragment of hydrogenase gene was obtained. After sequencing, it is showed that the length is761bp, coding polypeptide containing253amino acid residues. Comparison result showedthat, this hydrogenase gene fragment is similar to several Clostridium strains' hydrogenasegene submitted. The similarity in nucleotide level with FeFe-hydrogenase gene of Clostridiumpasteurianum strain DSM525is82%, while the amino acid sequence was77%homologous tothe hydrogenase of Clostridium kluyveri DSM555.Bioinformatics analysis showed that,761bp fragment is the common conserved regionamong many hydrogen-producing bacteria. Thus, it is reasonable to believe that fragmentcontained the active centers of hydrogenase, and might be the major functional domain. Thegene fragment was used for the structure of prokaryotic expression vector pET47b-Hyd,which was transformed into E.coli for prokaryotic expression. Moreover, knockout vector(pMD-19-HTH) included tetracycline resistance gene was constructed by overlap PCRtechnique with the purpose of achieving mutant lacked hydrogenase function and lay the foundation for uncovering relationship between the iron-reducing ability andhydrogen-evolution.In addition, the change of hydrogenase gene expression level during iron-reducingprocess was studied via RT-PCR and Real-Time PCR technique. Results displayed that, theaddition of iron influenced hydrogenase gene expression level largely, indicated hydrogenaseis highly possible to participate in iron-reducing process directly or indirectly.