Unveiling the circadian clockwork of the German cockroach Blattella germanica

• Main Authors: Chi-Yuan Huang, 黃啟原
• Other Authors: How-Jing Lee
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/ymb87f

碩士 === 國立臺灣大學 === 昆蟲學研究所 === 105 === A circadian rhythm is controlled by an endogenous circadian clock which is driven by autoregulatory transcription feedback loop of clock genes. This behavioral or physiological phenomenon repeats itself approximately 24 hours and commonly expresses in almost all organisms from bacteria to human. The molecular mechanisms of insect circadian clock have been well studied in fruit fly (Drosophila melanogaster). However, the components of clockwork may be different in non-model insects since some differences have been reported in monarch butterfly (Danaus plexippus). In previous studies, both Drosophila-like cryptochrome (Bgcry1) and mammalian-like cryptochrome (Bgcry2) were found in the German cockroach form the next-generation sequencing data. By knocking down Bgcry2 gene with RNA interference shows arrhythmicity in locomotion and expression level of cycle (Bgcyc) (a transcription factor of the circadian feedback loop), but mRNA levels of period (Bgper) and cycle (Bgcyc) genes are increased. In this study, I investigated the protein-protein interaction of circadian clock by performing co-immunoprecipitation, GST-pulldown assay and proximity ligation assays. These results show that BgCRY2 binds with either BgTIM or BgPER in the cytoplasm of HEK293T cells, while BgTIM interacts with BgPER in the cytoplasm. In combination of previous studies and present results, I propose a model in which BgTIM and BgPER form a complex and accumulate in the cytoplasm, and then disassociates before entering the nucleus. But BgPER may be independently translocated into the nucleus to act as a repressor of clock genes. Another mode is also proposed that BgCRY2 is carried by BgTIM to enter the nucleus and BgCRY2 may be the repressor based on the alignment results. I also suggest that a photoreceptor BgCRY1 may be a photoreceptor, expressing in the cytoplasm, to bind to BgTIM for degrading BgTIM during photophase. Although the clockwork of the German cockroach is largely following the Drosophila model, the minor difference is BgTIM, BgCRY2 and BgPER involved in an autoregulatory feedback loop and at least two modes of feedback loop involved in the circadian clockwork of the German cockroach. The BgTIM/BgCRY2 may enter nucleus for repression of their own transcription is a new finding in this study. This new hypothesis requires further investigation to verify the completeness of the circadian clockwork.