Study On Characteristic Of Secretion And Protein Function In C. Elegans

The 1 mm large soil nematode Caenorhabditis elegans was first used by Sydney Brenner to study the genetics of development and behavior.C. elegans is an ideal genetic model system. Firstly, its complete genome, consisting of five pairs of autosomal and one pair of sex chromosomes, has been sequenced and annotated. Secondly, due to the short generation time, self-fertilization and small size of C. elegans, forward and reverse genetic screens are fast, easy, cheap and can be easily scaled up. Thirdly, C. elegans is susceptible to manipulation using a range of genetic techniques. Because the advantages of C. elegans are numerous, we attempt to establish a research stage for the culture and manipulation of C. elegans, and study on the characteristic of secretion and protein function related secretory vesicles and membrane.We described all the techiniques using in the project, including maintaining a culture of a worm strain. In the laboratory, C. elegans is cultured on Petri dishes containing buffered agar and OP50 that serves as food.The C. elegans hermaphrodite consists of 959 somatic cells. Of all cells, the complete embryonic lineage has been determined. Furthermore, the positions, morphologies, synapses and gap junctions of all 302 neurons have been described. The primary culture system that allows culture of C. elegans embryonic cells had been described. We observed several types of cells and compared their morphological characterization.And using tph-1::GFP transgenic strain GR1366, we cultured the NSM neurons located on the pharynx in C. elegans. The major neurotramitter of NSM neuron is 5-HT. We undertook a series of patch clamp studies to in vitro electrophysiological analysis. The cultured NSM neuron expressed slowly inactivating, outwardly rectifying currents. By using high time resolution measurements of membrane capacitance, flash photolysis of caged Ca2+ and Carbon-fiber amperometric measurement, we first characterize ~10 fF Cm increasing and the 5-HT release spike current in vitro neurons in C. elegans.In this dissertation, we also present the construction and screening of deletion mutant libraries to generate C. elegans knockouts. This gene knockout strategy used a random mutagen, TMP and UV to mutagenize a very large number of worms. If PCR primers flanking an area of the gene of our interest are used to amplify from the genomic DNA samples, deletions between the primes can be detected. Once a DNA sample containing a deletion is identified, one can work back to identify the subculture of worms. Individual live animals carrying the deletion mutation can be used to study the molecular and cellular basis of secretory, even in the relationship of gene and behavior.