Construction Of Pseudomonas Donghuensis HYS Preliminary Study On The Toxicity Of Caenorhabditis Elegans

A strain of high-yield siderophore isolated from the East Lake waters was found in the preliminary work of the experiment.Genome-wide sequencing and Pseudomonas donghuensis HYS~T were identified.The interaction between Pseudomonas and the host has been extensively studied.Among them,Caenorhabditis elegan as a model organism,which is widely used in the study of the interaction-There are two types of toxic lethal effects of Pseudomonas sp.Of C.elegans,one is the chronic lethal model and the other one is the acute lethal model.Pseudomonas aeruginosa has a toxic effect on C.elegans,and it has been proved that the toxicity of Pseudomonas donghuensis HYS~T to C.elegans is more obvious in the chronic lethal model.In order to explore the mechanism of Pseudomonas donghuensis HYS~T on the toxicity of C.elegans,the whole genome transposon of the strain was inserted into the mutant library to screen the functional gene.The mutant strain with attenuated traits was screened by chronic lethal model,and the attenuated mutants with stable traits were obtained by screening.Then,Tail-PCR and semi-random primers PCR were used to detect the transposon flanking,finally to get the relevant functional genes.In the end,Fifteen genes were identified by two rounds of screening.In the selected genes,the gene enoly-CoA hydratase at the genome Scaffold 1532640-32641 has been screened 6 times and has the high homology compared with PA0745,which has been reported to be toxic to C.elegans in Pseudomonas aeruginosa,so,the gene causes attention.the gene knockout strain and the compensated strain is constructed to determine its toxicity to C.elegans.The gene function was analyzed by NCBI.It was also found that the gene had hydratase activity and was involved in the metabolism of fatty acids.The gene function was identified by enzyme activity reaction system.By analyzing the difference of the content of knockout and wild type fatty acids,The results showed that the protein encoded protein had hydrazine activity and was involved in fatty acid metabolism.The upstream and downstream sequences of the gene were analyzed.The relationship between the four genes was found to be close to one gene cluster and verified by co-transcription detection.In addition,the most upstream gene was knocked out and found that the knockout strain had an attenuated effect.In addition,two genes,NAD dependent epimerase and polyketide cyclase,were screened on the genome Scaffold 14 105154-105155,Scaffold 14 107680-107681,respectively,and the two genes were closely aligned and guessed on the same gene cluster.Gene upstream and downstream genes transferred out,observe the arrangement,the co-transcription detection.The results showed that these genes were co-transcribed and located on the same gene cluster.The toxicity of the strain NAD dependent epimerase was significantly decreased,indicating that the gene had toxic effect on C.elegans.But the effect of the gene cluster on C.elegans and the interrelationship between the two genes need to be further explored.In conclusion,the mechanism of Pseudomonas donghuensis HYS~T on the toxicity of C.elegans-was studied by constructing the mutant libra of the strain and using the chronic lethal model.The toxic effects of enoly-CoA hydratase,NAD dependent epimerase and its gene cluster on C.elegans were studied,and the causes of toxicity were deduced.