| Toxin-antitoxin systems are widely distributed in prokaryotes, They can mediatecell growth or inhibition by interacting of co-expression products of a pair of genes inTA system under environment stress conditions. But by now, only mazEF and relBETA systems of E.coli chromosome have been deeply researched and just a smallamount of experimental research for TA system in cyanobacteria chromosomes.Thehomologous genes of TA systems in the chromosome of Anabaena sp.PCC7120hadbeen studied for the first time in this thesis, based on the analysis by bioinformatics,we used the vector with two promoters to control the expression of toxic gene andantitoxic gene and identified the physiological characteristics of them; The deletionmutants of all3211/asl3212and asl4561/asl4562had been constructed for the furtherresearch. It includes:1) According to the genetic structure and conservative sequence of TA system inbacteria chromosomes, the characteristics of genetic structure, homology of protein,prediction of isoelectric point and domain, we found two pairs of genes in Anabaenasp.PCC7120: all3211/asl3212had homology with mazEF TA system,asl4561/asl4562was homologous to relBE TA system;2) Recombinant plasmid with two promoters was constructed, all3211gene wascloned into the downstream of PBAD, and asl3212gene was fused to Placpromoter, theexpression of these two genes were induced to analyze the toxin-antitoxin function toE.coli cells: after arabinose induction, all3211had the toxic effect to E.coli, and thetoxicty could be inhibited when inducing both all3211and asl3212by arabinose andIPTG, it showed all3211/asl3212were a pair of TA system gene, all3211was toxinand asl3212was antitoxin;3) Recombinant expression vectors were constructed to control the expression ofasl4561and asl4562in the E.coli, to identify the physiological function of this pair ofrelBE homologous genes; the expressing conditions of asl4561/asl4562gene hadbeen optimized. The result showed that the E.coli cells were inhibited after theexpression of asl4561induced by IPTG, it showed that the protein coded by asl4561function to E.coli regulation system, causing damage, when asl4561-asl4562werecoexpressed, the toxicty can be inhibited, indicating that the interaction between theproducts of this two genes inhibited the function of toxin. The optimal condition ofexpression of asl4561-asl4562gene as follows: adding0.8mM IPTG after cultivateBL21for1-1.5hours in LB with100μg/mL Kan, inducing at28℃for6h;4) The gene all3211/asl3212or asl4561/asl4562in the chromosome of Anabaena sp.PCC7120were replaced by Kan resistance fragments to constructdeletion mutants of Anabaena sp.PCC7120. There was no significant effect onphysiological function when the deletion mutants are cultivated under normalcondition. The construction of those mutants have established foundation for thefurther study on the mechanism of function and regulation of these genes;5) Based on the results in E.coli, recombination plasmids drived by petEpromoter were constructed. The all3211, all3211-asl3212, asl4561-asl4562andasl4561fragments were fused to PPetEwhich indeced by copper from Anabaenasp.PCC7120, then transfered into the PCC7120mutants of all3211-asl3212andasl4561-asl4562deletion. The products of all3211and asl4561induced by copper ionshowed toxic effect on the cells of PCC7120. However, the toxicity could be inhibitedwhen all3211/asl3212and asl4561/asl4562were coexpressed by copper inducing.The growth of mutants was decreased with the increase of the different concentrationof copper ion, it showed that toxin cause death of most algae cells and inhibition of apart of the algal cell growth. According to the results, all3211/asl3212andasl4561/asl4562were two TA systems in Anabaena sp.PCC7120chromosome,all3211and asl4561were toxic genes, the corresponding upstream genes asl3212and asl4562were antitoxic genes. |
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