Identification Of The Novel Genes Involved In The Regulation Of Avoidance Behavior To PA14 Of C.elegans

Organisms must make a correct choice to respond to the changes of environment and then adapt to them when they live in a complex environment.Therefore,sensory modulation is critically essential for the organisms~’sensations,behaviors,responses and survival.Avoidance behavior is one of the most effective and economic defense mechanisms when facing with the dangerous and harmful environments.As a common neurobiological behavior,much remained unclear about the molecular mechanisms underlying avoidance though a few pathways or neurons have been reported.So in our current study using Caenorhabditis elegans,we focuses on the avoidance behavior induced by the pathogenic bacteria as well as the related mechanism,which is of great significance to understand how organisms effectively respond to the complex external environments.C.elegans is an important model in the field of biological research because of its short life cycle,simple structure,transparent whole body,easy to be cultured,suitable for high-throughput screening,and easy genetic manipulation.The causal genes that have virulence in many other animal models are usually proved to work in this worm model.Meanwhile,many human bacterial pathogens,such as Pseudomonas aeruginosa PA14,can also effectively infect C.elegans.Therefore,it is considered as a simple and economical model host for studying the pathogenic mechanism of microorganisms and the response of hosts.In WNT signaling pathway,Wnt ligand is a secreted lipid-modified glycoprotein that binds to its cell surface receptor Frizzled(Fz),in which a highly conserved serine residue on palmitic acid and cysteine residues of palmitic acid are covalently linked.If the Wnt proteins do not accumulateβ-catenin in the cell,they are therefore referred to as non-canonical WNT signaling pathways.The non-canonical WNT signaling pathway is often involved in the polarization of cells and intracellular calcium ion change,but whether the non-canonical WNT signaling pathway mediates the neurobiological behavior of avoidance in the Caenorhabditis elegans is still unclear.Based on those backgrounds,we observed the avoidance of C.elegans against P.aeruginosa.Combined the techniques of RNAi,pan-neuronal rescue,microinjection,fluorescent localization and so on,the different genes in non-classical WNT signaling pathways were investigated for the effects on the avoidance behavior,which laid the foundation for elucidating the molecular mechanisms of avoidance behavior.The main results obtained in this study include:1.The ligand cwn-1 and receptor mig-1 are involved in the avoidance of nematodes against P.aeruginosa PA14.In our study,we used the C.elegans as the research object,using a flat plate which was rich in Pseudomonas aeruginosa to do the avoidance experiment.By screening the ligands and receptors in the non classical WNT signal pathway,we found that the nematode of the non classical WNT signaling ligand cwn-1 and the receptor mig-1 mutation were added to the platelets of Pseudomonas aeruginosa,the avoidance index increased.This indicates that the ligand cwn-1 and receptor mig-1 of non classical WNT signaling pathway are involved in the avoidance of nematodes by Pseudomonas aeruginosa.2.It was found that plc-1、plc-3、egl-8 and itr-1 in PLC signaling pathway were involved in the avoidance of nematodes to pathogenic bacteria PA14.We found that plc-1,plc-3,egl-8 and itr-1 mutants in PLC pathway were added to the small plates of Pseudomonas aeruginosa and their avoidance index increased.Because the PLC pathway is located in the downstream of the non classical WNT pathway,this shows that the plc-1,plc-3,egl-8 and itr-1 in the downstream PLC pathway of the non classical WNT signaling pathway may escape the Pseudomonas aeruginosa.At the same time,we found that the plc-2 of phospholipase C family is not involved in the avoidance of nematodes to Pseudomonas aeruginosa.In addition,we found that the PKC pathway which is downstream of the non classical WNT pathway is not involved in the escape of nematodes to Pseudomonas aeruginosa.3.Study on the relationship between WNT signaling pathway and PLC signaling pathway.We found that the WNT signaling pathway ligand cwn-1 and the receptor mig-1 are consistent with PLC signaling pathway in the PA14 escaping phenotype,so we added the plc-1 agonist in the ligand cwn-1 of the non classical WNT signaling pathway,and statistics its avoidance index.We found that after cwn-1 was added to the phospholipase C family gene plc-1 agonists,the avoidance index was between the cwn-1 individual mutant and the N2escaping index treated with a single phospholipase C family gene plc-1 agonist.According to this,we speculate whether the effect of plc-1 agonists on the whole animal is complete,or the downstream of the non classical WNT pathway may be partially mediated by the PLC pathway or other pathways,which need to be further studied.4.Fluorescence co injection showed that the cwn-1 neurons were expressed in the head neurons.By constructing the recombinant plasmid vectors of cwn-1、mig-1 and egl-8 and red Mcherry fluorescent plasmids,the GFP nematode strain,tph-1::GFP,which contains the gene expression location gene,was injected with GFP by microinjection.The fluorescence injection experiments of cwn-1、mig-1 and egl-8 were completed.By querying WormBase,we found that TPH-1 gene was strongly expressed in ADF and NSM neurons.Only cwn-1 was found in the head neurons.Innovation of my current investigation includes as follows:For the first time,we identified the new gene cwn-1、mig-1、plc-1、plc-3、egl-8 and itr-1which was involved in the pathogen avoidance of Caenorhabditis elegans.Downstream of the non classical WNT pathway may be partly mediated by the PLC pathway.