A Study Of A Pair Of Toxin-antitoxin Gene Alr9029/asr9028 In The Plasmid Of Anabaena Sp.PCC7120

The type ?toxin-antitoxin systems(TAs)are widely distributed in the chromosome of cyanobacterium Anabaena sp.PCC7120,which typically comprise an auto-regulated operon encoding a functional and stable toxin and a labile antitoxin inhibitor.They are involved in mediating bacterial programmed death to keep plasmid replication stable,and in triggering environment stress responses to induce bacterial growth inhibition and death.In the TAs that have been identified,many characteristics of them are not clear including their functions,the modes of their interaction and the mechanism of regulation.At the same time in a large number of predicted TAs,a huge amount of work needs to be done about their validation and classification.As a model organism,the whole genome sequencing of Anabaena is an important achievement in the field of photosynthesis research in the past few decades.The most popular research is about PCC6803 and PCC7120.The genome sequences of PCC6803 are announced as early as 1996,while the sequences of PCC7120 are not finished until 2001.This essay used alr9029-asr9028 gene pair on plasmid of Anabaena sp.PCC7120 as the research project,through methods of expression vectors construction,protein expression experiments and deletion mutant construction to identify toxin-antitoxin system and expected to reveal their mechanism.It mainly involved the following aspects:(1)Analysis the sequences of gene pair alr9029-asr9028 according to the bioinformatics prediction.The genome of Anabaena sp.PCC7120 is about 10 Mb with 5368 protein-coding genes,45% of its predicted products similar to the identified function proteins.The gene pair alr9029-asr9028 in plasmid of Anabaena sp.PCC7120 is highly homologous with phd-doc family.alr9029-asr9028 is preliminarily identified as one of phd-doc TAs by the evolutionary tree analysis and protein structure prediction.(2)Expression vectors construction of gene alr9029 and asr9028.The specific primers were designed to amplified target genes alr9029 and asr9028 by PCR.Enzyme sites of BamH? and Hind ? were added to the ends of target genes,after double enzyme digestion and gel purification,the amplified fragments were ligated into the corresponding sites of vector pET30 a to get expression vectors pET-30a-alr9029 and pET-30a-asr9028 successfully.Colony coating experiments showed that the expression of protein Alr9029 can inhibit the growth of E.coli to some extent,which exhibited its toxic effects.All of those above proved that alr9029-asr9028 belongs to TAs and Alr9029 is toxin protein.The expression vectors from host were successfully induced by IPTG.SDS-PAGE analysis showed that the toxin protein Alr9029 and antitoxin protein Asr9028 were expressed normally and they were both soluble proteins.(3)Purification and identification of Alr9029 and Asr9028 proteins.His-tag purification column was used to purify toxin and antitoxin protein and Western blot was conducted for further verification study.The results showed that Ni column could effectively purify His tag fusion protein.(4)Constructing deletion mutants of gene pair alr9029-asr9028 by tri-mating conjugation.The purpose was to reveal the functions of the gene pair to the growth of Anabeana sp.PCC7120,the heterocyst formation and the pigment integrity.Failure of Mutants construction meant more effective methods were needed for further research.Nowadays,the study of type ?toxin-antitoxin system mainly focuses on the chromosome while less on the plasmid.This paper,gene pair of alr9029-asr9028 on ? plasmid is chosen as the research object,has a certain effect on the systematic classification of TAs in plasmid.Trying to construct gene mutation on plasmid by tri-parental mating for the first time,will have significant guidance to the related functional verification and the discovery of relevant regulatory mechanism.At the same time,as a regulatory mechanism under lacking nutrition situation,TAs is one of the important supplements on bacteria metabolic regulation,and hopefully provides a new way to deal with cyanobacterial blooms.