| Genetic engineering has been widely used in various fields such as agriculture,environment,medicine,and food.It has great potential in solving major problems in agriculture,health,and ecology over the world.Although creating huge economic,environmental,and social benefits,the safety of genetically modified organisms has also aroused widespread concern and discussion around the world.As an important vector of gene modification technology,plasmids are widely used in gene modification of plants,animals,and microorganisms.Therefore,exploring the impact of plasmid vectors on the host and the surrounding ecological environment is necessary for evaluating the safety of genetically modified organisms from the source of genetic engineering.This is also of great significance to the further promotion and commercial application of genetically modified products.Since the construction of plasmid vectors in the 1970 s,human research on the impact of plasmid vectors on the host has never stopped.Many studies have shown that plasmid vectors can have off-target effects on host cells while conferring various characteristics on host cells,such as cell growth,tolerance,mutation rate,chromosomes,protein expression,metabolism,etc.At present,the rapid development of omics technology,genomics,transcriptomics,proteomics,and metabolomics is making these techniques and method more important to explore the undesired effects of plasmid vectors and predict their potential ecological risks.However,the omics method is not very purposeful for data analysis.Furthermore,changes in the transcriptome may not necessarily lead to changes in proteomics and metabolomics or may not be able to accurately predict the potential ecological risks of plasmid vectors.Therefore,it is important to introduce more detection indicators to clarify the impact of plasmids on host non-target effects and their mechanisms.Using the bacterivorous nematode C.elegans to detect the off-target effects of plasmid on host bacteria has become a potential detection method.C.elegans was designated as a model organism in 1974 by Nobel Prize winner Sydney Brenner.C.elegans can grow and reproduce with a single bacterium as a food source and can survive in different bacteria.Hermaphrodite C.elegans can reproduce through selffertilization.The growth cycle of C.elegans is generally 5-7 days,and the body is transparent to facilitate observation of its cells and tissues under a microscope.Because of these characteristics,the phenotype of C.elegans,including reproduction,life span,body size,pumping rate,body bend,and other life-history traits,is easy to determine.Using the C.elegans to feed the host bacteria carrying the plasmid vector to explore the response of C.elegans phenotype can not only reflect the impact of the plasmid vector on the host bacteria but also reflect the potential of the plasmid vector on the predator of the host and even humans.Therefore,C.elegans is an ideal potential organism to detect the off-target effects of plasmid vectors and their mechanism.This paper consists of three main research contents:(1)Introduce different types of plasmid vectors into E.coli OP50,observe the response of C.elegans fecundity,life span,body size,pumping rate,and body bends,Test the feasibility of using C.elegans to explore the off-target effects of plasmid vectors and potential ecological risks.(2)To analyze the non-target effects of plasmids from the perspective of the plasmid structure,we used methods such as gene knockout,gene replacement,and gene expression to determine the elements function in the plasmid that regulates the fecundity of C.elegans.We applied a series of molecular methods to carry out the role elements of functional analysis for exploring how the action elements regulate the host.(3)To explore the non-target effects of plasmids from the metabolism of the host bacteria,we used enzymology,proteomics,and other molecular methods to find out the key metabolites that the plasmid regulates the host bacteria and combine the metabolites of the bacteria and the mutant strains to explore the regulatory pathway of bacteria.The main results are as follows:1.C.elegans can be used as an effective tool to detect off-target effects and potential ecological risks of plasmid vectors.The plasmid vectors pEX18 Gm,p BBR1MCS-2,p BBR1MCS-3,p BBR1MCS-5,p ACYC184,p SC101-TIMER,R6 KBOX,and p UC19 were respectively introduced into E.coli OP50 and fed with C.elegans.Compared to the fecundity of C.elegans feeding on the E.coli OP50,the lifetime fecundity of C.elegans feeding of E.coli OP50 carried the plasmid pEX18 Gm significantly increased by 20%.However,all plasmid vectors have no significant effect on the life span,body size,pumping rate,and body bends of C.elegans.2.The plasmid pEX18 Gm are not specific to the bacteria in promoting the reproduction of C.elegans.The plasmid pEX18 Gm was introduced into the indigenous Salmonella,Klebsiella,Escherichia,and laboratory bacteria E.coli SM10 to feed C.elegans,respectively.Compared to the fecundity of C.elegans that feed on bacteria with no plasmid,the introduction of plasmid pEX18 Gm into different types of bacteria can increase the fecundity of C.elegans,and its lifetime fecundity was increased by38.8%,15.3%,17.1%,16.5%,respectively.3.Gene knockout,gene replacement,and gene expression methods were used to eliminate the effects of the constituent elements of plasmid pEX18 Gm.The gentamicin acetyltransferase encoding gene(aac C1)and the levansucrase-encoding gene(sac B)were excluded.The results revealed that the replicon pMB1 was the functional element that increased the C.elegans fecundity.Taking advantage of the cut experiment on the DNA sequence of the replicon pMB1,this further confirmed that the reduced DNA fragments in the replicon pMB1 can still increase the fecundity of C.elegans but with no replication function.4.By measuring the plasmid copy number,the path of C.elegans increased due to the metabolic burden on the host during plasmid replication being eliminated.The results of the Northern Blot analysis indicated that the reduced DNA fragment in pMB1 did not transcribe any RNA,so the transcribing RNA or protein way is excluded.We speculated that the functional DNA fragment may directly act on the host to regulate bacterial metabolism in the form of DNA,and then increase the fecundity of C.elegans.We inserted the functional DNA fragment into the plasmid p BBR1MCS-5 forward and reverse,preliminarily confirming this hypothesis’ s possibility.5.We found that the sonication solution of the strain E.coli OP50 carrying the plasmid pEX18 Gm can increase the fecundity of C.elegans.By processing the cellsonicated solution with Proteinase K and RNase,we confirmed that the increased fecundity of the substance is related to these proteins.Using different specifications of centrifugal filter devices to treat the cell sonication solution,the results preliminarily confirmed that the size of the substance is between 10 k D-30 k D.6.Using the i TRAQ proteomics to compare the protein expression of strain E.coli OP50 carrying and not carrying the plasmid pEX18 Gm,22 candidate proteins were screened.The candidate proteins were expressed one by one in E.coli and fed to the C.elegans.We found that the dihydrofolate reductase(DHFR)expression can significantly increase the fecundity of C.elegans.Using q RT-PCR and exogenous addition of DHFR inhibitor trimethoprim further confirmed that DHFR is the key protein of plasmid pEX18Gm-induced C.elegans fecundity change.Feeding DHFR directly to C.elegans does not lead to an increase in the reproduction of C.elegans,these results indicated that DHFR regulates the reproduction of C.elegans indirectly.7.The plasmid pEX18 Gm regulates the reproduction of C.elegans by altering bacterial folate and methionine metabolism.The exogenous addition of DHFR substrate folic acid and DHFR product tetrahydrofolate experiment confirmed that DHFR affects the reproduction of C.elegans in an indirect way by regulating host bacterial folate metabolism.By adding 5-methyl-tetrahydrofolate and methionine exogenously,we found that 5-methyl-tetrahydrofolate and methionine all can increase the fecundity of C.elegans.We speculated that the methionine cycle may be the key metabolic pathway that pEX18 Gm which induces the increase of C.elegans fecundity.The plasmid pEX18 Gm was introduced into the E.coli BW25113 mutants of methionine synthase gene(met E,met H,mmu M)and fed to C.elegans.These findings indicated that the Met H(vitamin B12-dependent methionine synthase)was necessary for the plasmid pEX18 Gm to increase the fecundity of C.elegans.It was found by LC-MS/MS that the plasmid pEX18 Gm can increase the methionine content in E.coli BW25113 by 4.75 times.The above experimental results indicate that the plasmid pEX18 Gm can increase the content of methionine by affecting the bacterial folate and methionine metabolism,thereby promoting the reproduction of C.elegans. |
PDF Full Text Request