Cloning and Functional Assay of pigment dispersing factor on the Circadian Clock in the German Cockroach, Blattella germanica (L.)
Date Issued
2009
Date
2009
Author(s)
Lee, Chi-Mei
Abstract
In Drosophila melanogaster, Leucophaea maderae, and Gryllus bimaculatus, the pigment-dispersing factor (PDF) functions as output and coupling signal of locomotor circadian clocks. In this study, the German cockroach, Blattella germanica, was investigated to reveal the functions of PDF. The pdf cDNA had been cloned and its sequence and structure showed higher similarity with Romalea microptera and Meimuna opalifera than that of D. melanogaster and Bombyx mori. The pdf gene in the head of the German cockroach did not express rhythmically which displayed the similar pattern as D. melanogaster. The PDF precursor protein of the German cockroach was composed of 87 amino acids and its sequence was also similar to that of R. microptera and M. opalifera. Once the pdf cDNA had been cloned, we explored the functions of PDF in the German cockroach with RNA interference technique. After pdf double-stranded RNA (dsRNA) injection, the amount of pdf mRNA decreased significantly since the second day and this knockdown effect could persist for at last 56 days. With immunostaining technique, the clock cells of pdf dsRNA-injected cockroaches could not be stained by anti-PDF antibody. In the behavioral study, the injection of pdf dsRNA caused rhythmic males to become arrhythmic in light-dark cycles or constant darkness, but had no obvious effect on the locomotion of female cockroaches. In addition, due to the nocturnal nature of the German cockroach, the locomotor activity increased after light off or entering subjective night. However, this activity peak gradually disappeared after pdf dsRNA injection. Based on these two lines of evidences, PDF serves as an output regulator of locomotor circadian rhythm in the German cockroach. Beside, the effect of pdf dsRNA injection on the reproduction of female cockroaches had also been studied. The results showed that PDF might not be involved in the regulation of reproduction in female cockroaches.
Subjects
locomotion
circadian rhythm
neuropeptides
RNA interference
gene silencing
Blattella germanica
Type
thesis
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