Studies On Clonging,Expression And Function Of RpoD In Azospirillum Brasilense

Transcriptional regulation is one of the most widely studied regulatory mechanisms in the field of molecular biology.RNA polymerase(RNAP)that consists of core-RNAP and sigma factor plays a major role in transcription,which can specifically bind to the promoter to begin transcription.Sigma factor can specifically recognize the promoter element and promote transcription initiation.Besides,sigma factor,a main regulatory factor regulates many biological metabolic processes,is not only a unique subunit of RNAP,but also an important part of the signal transduction system.The RpoD protein is a member of the sigma factor family and plays an important role in microbial growth and development,biological metabolism,and regulation of some stress responses.The RpoD gene conserved sequence was obtained by blasting the six genome sequenced Azospirillum strains,Azospirillum brasilense Az39,Azospirillum brasilense Sp7,Azospirillum lipoferum 4B,Azospirillum humicireducens SgZ-5,Azospirillum sp.B510,and Azospirillum thiophilum BV-S.And then,the RpoD gene was cloned by using the DNA of Azospirillum brasiliensis L25,and verified through the protein expression in Escherichia.coli BL21,the carbon source utilization function and some tolerances behavior.In addition,to construct RpoD-GFP fusion protein,the GFP gene was cloned with plasmid pCR2.1-TOPO-GFP,verified through the protein expression in Escherichia.coli BL21.And the below are main conclusions of this research:1.Sequence alignment revealed that this 1959 bp fragment,encoding a protein consisting of 653 amino acids,has a conserved domain of the sigma70 family and can be categorized into the first basic sigma factor of the sigma70 protein family.Phylogenetic analysis indicated that the similarity of RpoD gene between strain L25 and A.brasilense was closet.The pET-32a(+)expression vectors were constructed by inserting an RpoD gene into the EcoR I / Hind III.2.Exogenous RpoD gene was induced in E.coli BL21 and detected by SDS-PAGE.The bright band at about 100 KD indicated that the fusion protein of RpoD gene and pET-32a(+)vector partial sequence was obtained,which indicated that the exogenous RpoD gene was Successfully expressed in E.coli.3.The experiment of the carbon source utilization of exogenous RpoD gene in E.coli BL21 showed that the exogenous gene RpoD could significantly promote the bacteria to use more kinds of carbon sources.While,at the same time the ability to use a few types of carbon sources were weakened or even disappeared.4.The transgenic E.coli BL21 containing with exogenous RpoD gene could tolerant stress of high temperature,high osmotic pressure,oxidation,IPTG and acid stress conditions to growth compared with the none transgenic E.coli BL21.And the expression of exogenous RpoD gene promoted the stress tolerance of E.coli BL21 under high temperature and IPTG stress conditions,while it could promote or inhibit the bacteria growth under the stress of oxidation,acidity,and high osmotic pressure.Furthermore,the green fluorescent protein gene GFP was cloned,and the recombinant plasmid pET-32a-RpoD-GFP was successfully constructed.Besides,the light green strain containing the pET-32a-RpoD-GFP recombinant plasmid was obtained through adding inducer agent IPTG to induce GFP protein expression.Those result indicated that GFP gene was successfully expressed in E.coli BL21,which will be helpful for screening out whether RpoD gene has successfully transduced into nitrogen-fixing bacteria.Taken together,our results show that RpoD protein,as a class of global regulatory factor,plays multiple functional roles in metabolic regulation and environmental stress resistance,which has laid the foundation for further studies on some biological function and stress resistance of azotobacter bacteria.