The Molecular Mechanism Of EndoTT Function In Thermoanaerobacter Tencongensis

?Objective? Thermoanaerobacter tengcongensis,an anaerobic thermophile which was isolated from Tengchong hot spring in Yunnan province of China is the first strain sequenced complete genomein China..SSB1,SSB2 and SSB3 are three single stranded DNA binding proteins annotated in Thermoanaerobacter tengcongensis,the SSB1 are encoded by tte0829 gene.In vitro studies have shown that the SSB1 protein has a complex,in vitro biochemical function regulated by multiple environmental factors,which can be combined with ss DNA,ds DNA,and damaged ds DNA,and can also be used to cut off the ectopic sites on ss DNA.Because SSB1 has the double function of single stranded DNA binding activity and single strand DNA cleavage activity at the same time,it is called Endo TT.However,it is still not clear what the physiological function of Endo TT in Thermoanaerobacter tengcongensis and what role it plays in the thermal adaptation This study provides a theoretical basis for revealing the function of Endo TT at different temperatures and the effects of its interaction proteins.?Method?(1)The Endo TT bifunctional complement plasmids ?endo TT::endo TT,Single stranded DNA binding complement plasmids ?endo TT::endo TTB and Single stranded DAN cleavage of the complement plasmids ?endo TT::endo TTS were constructed by homologous recombination.The three complement plasmids were cultured through transferred into WT and ?endo TT at 60?,and transferred to the solid medium of Thermoanaerobacter tengcongensis.transformants selected and verified by genomic extraction and specific primer PCR.(2)The growth trend of WT,?endo TT::endo TT,?endo TT::endo TTB,?endo TT::endo TTS strain at 75 ? and 80 ? was observed by phenotypic observation.(3)The transcriptions of Thermoanaerobacter tengcongensis WT and ?endo TT grown at 50 ?,60 ?,75 ? and 80 ? were analyzed by RNA sequencing,The differential expression genes of ?endo TT at different temperatures were defined,selected the specific temperature of the transcribed Fengdu high gene.(4)Analyzed the m RNA expression of transcribed fengdu high gene at specific temperature in ?endo TT by real-time PCR.?Results?(1)The compensation strains of ?endo TT::endo TT,?endo TT::endo TTB,?endo TT::endo TTS was constructed successful under base of ?endo TT.(2)Growth curve results show that the growth rate of?endo TT::endo TTB is slower than ?endo TT :: endo TTS at 75 ? and 80 ?.(3)The transcriptions of WT and?endo TT grown at 50 ?,60 ?,75 ? and 80 ? were analyzed by RNA sequencing.The results showed that501 differentially expressed genes(including 296 genes expressed at specific temperatures)may be associated with Endo TT or even with heat adaptation,using GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)for differential expression Gene analysis,the signal transduction,fatty acid synthesis and ABC transport system in the Thermoanaerobacter tengcongensis heat adaptation process plays an important role.(4)The tte0270,cel A,arg H,arg C,opp F3 and pur R3 genes were detected by real time-PCR at the specific temperature.The expression of tte0270 and arg H were significantly expressed at 50? and 60?,Cel A,arg C was only expressed at 60?,pur R3 was significantly expressed at 75? and 80?,opp F3 was only expressed at 80?.?Conclusion? Different functional strains of Endo TT was constructed.The ?endo TT::endo TTS with single strand DNA cleavage activity grows fast at high temperature,it was presumed that Endo TT could play single-stranded cleavage at high temperature.?endo TT transcriptome analysis at 4 temperatures,found 501 differentially expressed genes involved in carbohydrate metabolism,amino acid metabolism,ABC transport system and other means,and found that tte0270,cel A,arg H,arg C,opp F3 and pur R3 genes differentially expressed at different temperatures,suggesting that these genes play protein and Endo TT encoding the existence the interaction and influence its function.