| Cyanobacteria are tiny photosynthetic prokaryotes,which are considered to be the earliest creatures to live in the land.Because cyanobacteria formed the strong ecological competitive ability and environmental adaptability during the long-time evolution,they were spread around all the water body and soil environment.They can cause a serious threaten on human and animals' health by producing cyanotoxins.Therefore,the health problem of soil and water caused by cyanotoxins became to an important environmental issue during the last decades.Microcystins(MCs)is one type of cyanotoxins,which is widely distributed and has the most toxicity.The MCs family had more than 90 chemical structures and every stable chemical property,which makes them hardly to be removed from environment.MC is synthesized by the non-ribosomal peptide synthetase system(NRPS),which controlled by a MC synthetase(mcy)gene cluster containing nine or ten genes.It has been reported repeatedly that the genetic basis of mcy gene cluster is frequently mutated leading to the inactivation of microcystin biosynthesis.Therefore,the amount and the distribution of mutated mcy gene controlled the MC concentration in environment,which became to an important scientific issue.Focused on this,our study based on the 800 single filaments(Planktothrix)isolated from eight water samples in 5 Apls lakes,established a cultivation independent method to detect the whole mcy gene cluster,searching for the mutation in nature environment;and we analyzed the phylogenetic divergence of those mutants when compared with genotypes still containing the original mcy gene cluster,exploring the revolution relationship between them;and also,we obtained quantitative information on the abundance of each of the discovered mcy mutants and relate the abundance both spatially and temporally to environmental conditions.All in all,this study established a cultivation independent method to detect the mcy gene cluster in Planktothrix filaments,which important had meaning theoretically and practically,based on this method the main results in this study shows as follows:1.The length of DNA fragments extracted from single filament by ultrasonic was stable around 1 kbp to 10 kbp,which was independent with the concentration of filament sample.The central peak was 6 kbp,and the fragments mainly concentrated between 4 kbp and 8 kbp.The optimum ultrasonic intensity of DNA extraction was 15%,and the optimum ultrasonic time was 1 s.The extracted DNA was degraded within 2 days under-20°C,but this storing time can be prolonged to more than 3 months with the addition of dilution buffer.2.There was 80 PCR reactions can be amplified clearly from one single filament.As the DNA polymerase Phusion amplified DNA fragments with the maximum length up to 7738 bp.The maximum amplicon length afforded by one single filament DNA was about 6.2 kbp.Based on these two restrictive conditions above,the major primer system of mcy gene cluster was designed,which consisted with 16 primer pairs.Under the condition of Phusion and the designed primer system,the detective sensitivity of PCR reaction reached up to 1-10 pg/?L DNA template.3.The jumping gene ISPlr1 insterted into mcy gene cluster within 3 regions,which were 4 times in the mcyD,60 times in the IGS region between mcyE and mcyG and 5 within mcyA gene respectively,in total of 69 times.There were two deletions detected within mcy gene cluster,one was in mcyD and the other one was located from mcyH to mcyA,the proportion of these two deletions were 6.9% and 2.6%,respectively.And also,there was a functional gene fragment inserted into the IGS region between mcyT and mcyD(20.6%),and recombination within mcyA(Dhb)was 59%.4.The repetitive regions(RRs)with special structure was found 7 times within mcy gene cluster,which was 44 bp with one hairpin structure inside,highly content of GC(75%).All the 7 RRs were in the special position of mcy genes cluster,3 of them contained the 5 insertion sites of ISPlr1,one RR existed within the inserted foreign functional genes(mcyTD insertion),one RR located within the shorter recombination gene in mcyA(Dhb),and the last two were located 622 bp upstream and 121 bp downstream of the mcy gene cluster respectively.5.It was hardly to find a second mutation within one filament,in which had an insertion mutation.But the deletion of mcyHA and mcyD had a high probability to site in the same filament(71.4%).The two types of mcyA gene(Mdha and Dhb)had a similar proportion in environment.The deletion of mcyHA was positively related to Dhb.Two phylogenetic gene site gene loci(rbcXL and cpcBA)were sequenced and analyzed,the results showed that most of the filaments belong to same gene phylogenetic sequencing,which indicates that the evolution of mcy gene had no relation with the phylogenetic evolution.6.The jumping gene ISPlr1 insterted into mcy gene cluster within 3 regions,which were 4 times in the mcyD,60 times in the IGS region between mcyE and mcyG and 5 within mcyA gene respectively,in total of 69 times.The 5 insertion sites of the ISPlr1 were corresponding to 5 different direct repeat sequences(DR).The same DR corresponded to the same direction of the insertion,DR1,DR3 and DR4 corresponded to the same direction,DR2 and DR5 corresponded to the opposite direction.7.The cell numbers of Planktothrix showed positively related with the length of filaments,so as the total DNA content.But there was no significant relationship between filament length(the total DNA content)and the PCR results.Large amount of mcy mutants was found in in Hallwilersee and Ammersee,but most of the mutants did not disturb the MC synthetase.Zürichsee had the lowest propotion of mcy mutants,and did not detect any ISPlr1 inserted within the functional genes.There were more mutants disturbed the MC synthetase in W?rthersee and Mondsee,and the proportion of mutants without interrupt the MC synthetase were relatively low compared with other three lakes.Mutants caused by ISPlr1 insertion was quite low in Mondsee,so there was no seasonal variation detected.mcyTD insertion,mcy D and mcyHA deletion increased from 5.7%,5.2% and 0% in Spring to 27.0%,19.4% and 7.3% in Autumn respectively.The shorter recombination gene in mcyA(Dhb)decreased from 81.1% in spring to 44.8% in Autumn.This study firstly developed the toolkit able to detect mutations in situ occurring anywhere within the large mcy synthesis gene cluster as amplified from one single filament.According to the culture-independent method,we analyzed the population for all mutations affecting MC synthesis in real-time independently,also analyzed the phylogenetic divergence of those mutants when compared with genotypes still containing the original mcy gene cluster,obtaind quantitative information on the abundance of each of the discovered mcy mutants and relate the abundance to environmental conditions both spatially and temporally.The mcyD deletion within mcy gene cluster was first discovered in our study,also the 5 insertion sites of transposons ISPlr1.This study also proved that the variation of mcy mutants was not related to the evolution of the conserved sequence. |
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