| Toxin- antitoxin system is composed of two genes co-expressed, one of which is the toxin protein with unstable coding, the other stable one. Through their products, TA system gets involved in physiological activities of the cell growth inhibition or cell death under the stress environment. Toxin-antitoxin system widely exists in prokaryotes. Through an analysis of bioinformatics, we found in sequenced prokaryotes there were probably 671 toxin-antitoxin loci, belonging to seven classic toxin- antitoxin gene families, which respectively are: ccdAB, rel BE, parDE, higBA, mazEF, phD/doc and vapBC/vag. Currently more researches have been conducted on relBE and mazEF family.Under the condition of normal growth, relBE and mazEF continue to synthesize toxin and excessive antitoxin and inhibits the toxicity of toxin proteins by forming the heterogeneous six polymers. However when cells are confronted with external stress, protease degrades antitoxin and releases toxin from complex. In this way, the growth of bacteria will be inhibited since toxin protein specifically cuts mRNA so as to block the synthesis of protein.This experiment studied on a pair of genes asl2401 and all2402 in Anabaena sp. PCC7120 chromosome by constructing a dual promoter expression vector. We made an analysis about influences from protein toxin and antitoxin protein on the cell growth respectively by individual induction and double induction to express and then determined its toxin-antitoxin effects. It mainly includes the following aspects:1)Through an analysis of gene asl2401 and all2402 sequence by using bioinformatics, and a prediction of their encoding products, the nature of the protein, and the senior structure of protein, we can further determine that their toxins-antitoxin gene family is higBA. Currently we have very few researches on TA system of this family and their toxin mechanism is not clear, so this study will play a role in enriching TA system.2) With genes asl2401 and all2402, we constructed cloning vector and expression vector, and then inducted them to express. After electrophoretic detection, we found gene asl2401 easy to be constructed successfully and able to be efficiently induced for expression, while the expression vector of toxin gene all2402 is difficult to construct, and only a few successfully built ones can induce protein expression but with quantity not high. For those successfully built expression vectors, we kept them for a period of time and sequence them, there were no results. So it can be speculated that the toxin gene expressed toxin protein and killed the cells, which stopped the experiments from going on. This was why we built double promoter and induced it to express, verify our prediction, and continued our experiment into the next phase so as to determine physiological regulatory function in Anabaena sp. PCC7120.3) We placeed the toxin gene all2402 under PBAD promoter, and antitoxin gene asl2401 under PLAC promoter, to build double promoter and then induce it to express vector. After this, we made an analysis of protein products about its toxic and anti-toxic effects: toxin gene all2402 can be induced by L-(+)- arabinose to produce toxin protein and had toxic effects on cells; Antitoxin gene asl2401 can be co-induced by L-(+)-arabinose and IPTG to express antitoxin protein which interacted with toxin protein and removed the toxicity of the latter. So we can determine the physiological regulation function of the target genes in Anabaena sp. PCC7120.4) TA system, a regulatory mechanism of prokaryotic cells dealing with the lack of nutrition, is an important complement of bacteria metabolism. At present, Lakes and reservoirs in China have been contamination by algae outbreak. So we expected that TA system research on cyanobacteria chromosome can also pave a way for the removal of algae in our environment. |
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