| Cyanophyceae can be also called cyanobacteria,it is an ancient prokaryote,which is widespread with a strong adaptability that can make it survive in a variety of extreme environments.Overgrowth of cyanobacteria can lead to water bloom that will destroy the water ecological balance and pollute environment.Anabaena sp.PCC7120 is one of the the main species that lead to water bloom,cells of Anabaena sp.PCC7120 cotain lots of toxin-antitoxin system(TAS)genes,the TAS contains a toxin gene that encodes stable toxin protein while the antitoxin gene encodes unstable antitoxin protein.Some researches had showed that under some environmental stresses,the toxin protein acted on host cells and led to growth inhibition or death of cells.Gene pair asr0757/alr0758,located on the chromosome of Anabaena sp.PCC7120,was predicted to have a high homology with MazEF in E.coli with analysis on NCBI,and they both had the same conservative structure.Genes asr0757 and alr07587 were likely to constitute a TAS.To study whether asr0757 / alr0758 belonged to TAS and verified its biological functions,our experient was divided into two parts:1): Designed the specific primers of genes asr0757/alr0758,then amplified the gene fragments by Touch-Down PCR,built the TA clones,inserted the gene fragments into pET-30 a to construct the expression vectors pET-30a-asr0757 and pET-30a-alr0758 respectively,translated the vectors into E.coli BL21,then induced the expression of genes asr0757 and alr0758,purified the expression proteins and identified them with western blot.Result of SDS-PAGE showed that the purpose proteins had been expressed successfully when induced 6h at 37?,protein asr0757 was 12.5KD and alr0758 was 17.9KD,the asr0757 had a good expression quantity while alr0758 just expressed on a low level,so we optimized the induced conditions of toxin gene alr0758 by setting up the induction time gradient and IPTG concentration gradient,result of optimization showed that at 37?,when induced 10 h with 0.4m M IPTG,gene alr0758 had its largest expression quantity.Protein purification and western blot both revealed that the expressed proteins were just the purpose proteins.2): Built the vector with two promoters,which contained both asr0757 and alr0758 genes,then gene asr0757 and alr0758 were induced separately or together to express proteins for verifying the influences of selective expression products on thegrowth of cells.Results showed that the purpose proteins asr0757(13.5KD)and alr0758(17.9KD)were induced successfully,the growth of bacteria would not be affected when gene asr0757 was induced only,but the growth of host cells was obviously suppressed when gene alr0758 was induced separately,the growth of host cells returned to normal when genes asr0757 and alr0758 were induced at the same time.All above revealed that asr0757 and alr0758 constituted a gene pair of MazEF family with functions of anti-toxicity and toxicity.At present,only a handful of TAS genes located on the chromosome of bacteria were verified.Innovation points of this article are as follows: It is the first time to identify the likely TAS gene pair asr0757/alr0758 systematically,it is also the first time to construct vector with two promoters that can express toxin and antitoxin proteins at the same time,which aims to reveal impacts of the products on host cells.All above provide a foundation for researches of other predicted genes in Anabaena sp.PCC712,and the results in this paper create a good condition for its future study,help improve the TA system theory and has important theoretical significance on supplyment to the development of the molecular mechanism. |
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