Study The Biological Function Of Phospholipase D In C.elegans

Phospholipase D (PLD) is a kind of phospholipases, which was firstdiscovered in plant, then also in bacteria, fungus and mammal. There are stillthree other members in the phospholipase family, they are: PLA1, PLA2, andPLC. The activity of PLD is regulated by some hormone, growth factors andother extra cellular signal molecules. The most important substrate of PLD isphosphatidyl choline (PC), and the products are choline and phosphatidic acid(PA) which is transformed to other signal molecules, such as, diacylglycerol(DG) and lysophosphatidic acid (LPA). Since its important role in cellsignaling pathways, people are paying more and more attention on this field.PLD is associated with many diseases, for example, in the diabetesmellitus (DM), the activity of PLD is distinct among various tissues and cells.At present, GPI-PLD is the only enzyme that can hydrolyze GPI anchorprotein in human being. Recently, more and more evidences indicate theabnormal expression of GPI-PLD is related to many diseases, such ashepatopathy, malignant tumor, inflammatory reaction, atherosclerotic plaqueand hematopathy. However, the mechanism is still not clear.Now we choose C.elegans as a model system to further explore thefunction of PLD. Not only because the genome sequence of C.elegans istotally mapped, but also because it has many advantages, such as: shortlifespan, easy to incubate, transparent epidermis. Moreover, it is a eucaryoticmetazoan whose cells, molecular constructions, and signal pathways aresimilar to higher animals. All these unique advantages above make C.elegansto be an excellent model organism.PLD-1 is the homologous gene of PLD in C.elegans, it has two specificmotifs of HKD, coding 1427 amino acids, but now there is still no report onPLD-1 of C.elegans.The basic method of our research is "loss-of-function phenotype", whichusually includes gene knockdown (RNA interference) and gene knockout. Inthis research, we choose the latter. We called the strain of PLD-1 deletedworms RB1040, and we also have another strain named RB1040::GFP, whichcan be used to locate the protein of PLD-1 in C.elegans. Because we observedits expression around the nervous system in head and body wall, we designedsome corresponding experiments: egg-laying, lifespan, chemotaxsis and foodattraction.During the experiment, we found the mumber of eggs laid by deletiongroup was less than that of wild type group, but the lifespan, locomotion,pharynx pumping and defecation of the two groups had no difference. Itimplied that the PLD-1 mutation in worm was mild. Further more, wedesigned the chemotaxsis and food attraction experiment and chose 5 odorantswhich could attract C.elegans through AWA and AWC neuron in the head ofworm. Finally, we found that the PLD-1 deletion group was more sensitive tothe odorant compared to the wild type group. So the conclusion can be drawnlike this: PLD-1 gene is likely to be a negative regulator to AWA and AWCneuron.Our research established the method to exam the chemotaxsis of worm,and it was the first time that we demonstrated the relationship between PLD-1gene and head neuron in worm. This result wounld benefit the exploration ofthe role of PLD in cellular signaling, and it also helps us to screen the drugswhich targeted to the PLD.In our future work, firstly, we will detect the different expression ofPLD-1 between deletion group and wild type group by Western blotting,which can help us to understand the role of PLD in cell signaling;Secondly,we will confirm our results in the cell level;finally, we are going to comparePLD-1 to other genes that are related to neuron system to define the functionand mechanism of PLD.