| In order to survive in soil, which is a very complex environment. Caenorhabditis elegans developed a simple but fully functional system of neurons. This particularity makes it the model organism to study the development of neurons system and related behaviors. C. elegans can detect a wide range of chemicals and physical conditions in the environment, such as volatile and water-soluble compounds, as well as temperature, oxygen concentration, pH... which are often associated with food or dangerous factors. This ability mainly depends on the function of twelve pairs of amphid chemosensory neurons, with cilium structure, that are able to sense the signals in the environment. In response to modifications of the external conditions. C. elegans can adapt, and use different strategies such as chemotaxis. retreat or stress behaviors.Olfaction in C. elegans is a versatile and sensitive strategy to seek food and avoid dangers by sensing volatile chemicals emitted by the targets. Not only food-bacteria can produce volatile odor that attract nematodes. but a recent research showed that many pathogenic micro-organisms, including Pseudomonas aeruginosa and Serratia marcescen. can also produce these compound. Why the nematode gets attracted by theses pathogenic bacteria, is not known, though it is a significant scientific problem in the field of life science. Thus using C. elegans as model could allow us to better understand the molecular mechanism involved in the detection of chemical compounds and find new targets for drugs development.To unravel the molecular mechanism of this olfactive ability in C. elegans. we selected Bacillus nematocid B16. a bacteria known for its aptitude to produce volatile compounds attractive for nematodes. We identified molecules in partcular the2-heptanone and the signal transduction pathway underlying the olfactive phenomenon?So we were able to clarify the interaction between the pathogens and their hosts as well as the underlying molecular mechanism. These results laid to a theoretical foundation for the use of pheromones in pest biological control.We obtained the following results. 1. Determined the main neurons mediating the perception of the signal produced by B. nematocida B16.Through chemotaxis assays of odr-1and odr-1mutants, in which to function of AWC and AWA olfactory neurons is lost, respectively, we found that the phenomenon of chemotaxis was reduced in both mutants; furthermore the affection of AWC olfactory neurons was extremely obvious. So, in the process of sensing B. nematocida B16, AWC olfactory neurons play a primary role.2. Determined three main attractive signal molecules produced by B. nematocida B16and the olfactory neurons in charge of detecting them.In the signal molecules generated by B. nematocida B16, there are three chemicals, benzaldehyde.2-heptanone and2-ethyl hexanol, that have the remarkable ability to attract C. elegans. Benzaldehyde and2-heptanone were detected by AWC olfactory neurons, and2-ethyl alcohol was detected by AWA olfactory neurons.3. Determined G protein-coupled receptor STR-2expressed in AWC olfactory is the specific receptor of2-heptanone.In the research of C. elegans olfaction process, explicit evidence for an olfactory-role has been documented only for the ODR-10receptor, which is highly expressed in AWA olfactory neurons and necessary for a response to diacetyl. Chemotaxis assays were used to identify the response of mutants to2-heptanone, we found that animals with the str-2mutation were defective in their response to2-heptanone, but exhibited wild-type behavior in their response to benzadelhyde and2-ethyl alcohol. STR-2is supposed to be the specific receptor of2-heptanone.Gene str-2was expressed in mammalian cells. HEK293T. and immuno-fluorescent staining of cells indicated that gene str-2is expressed on the cell membrane. Assay of the Ca2+level reveals that STR-2mediates Ca2+level elevation in response to2-heptanone. These results are consistent with the previous experiment, and STR-2is the specific receptor of2-heptanone.4. Determined the olfactory signal transduction pathway of sensing of2-heptanone.We combined the methods of RNA interference and mutant to test the gene that is necessary to sense the2-heptanone molecule, including G protein a subunit (odr-3, gpa-3. egl-30), guanylate cyclase (odr-1, daf-11), phospholipase C (plc-1, egl-8). calcium channel (cmk-1, cal-1) and other elements. We found that PLC pathway and cGMP pathway affect the chemotaxis to2-heptanone. We expressed gene str-2in odr-1mutants and found that cGMP pathway is mainly involved in the expression of the receptor STR-2; we also expressed the gene egl-30in the AWC neurons of egl-30mutants, and identified that PLC pathway plays a ajor role in C. elegans sensing2-heptanone.Innovation of this paper:1、Determined2-ethyl hexanol is able to attract C.elegans for the first time, and idefitied the olfactory neurons of C. elegans detecting it.2、Determined the ligand of G protein coupled receptor STR-2is2-heptanone. and expressed it in heterologous system for the first time, and it is the second GPCR which have been known the ligand after the research of ODR-10in1997.3、Proposed a new olfactory signal transduction pathway without cGMPsignal in AWC neurons of C. elegans. |
PDF Full Text Request