The Molecular Mechamism Of Negative Circadian Feedback Loop In Silkworm, Bombyx Mori

Chronobiology is one of the three most important subjects in 21 st century, chose n by the US Science in 1999, and that is also an important breakthrough in studying the coevolution and its mechanism of living organisms and natural environment. Chronobiology pays attention to the endogenous circadian clock system, the mechanisms of biological rhythm, and periodic changes of physiological functions, especially concerning about the research of circadian rhythm with nearly 24 h in a cycle. A lot of fields of life sciences related to its application, such as medicine and agriculture, in which chronobiology is playing an increasingly important role.The circadian research of insects focused on Drosophila melanogaster, Blattodea and Danaus plexippus. Silkworm is a mass rearing economy insects, and also the only model insects of Lepidoptera. that means silkworm has significant economic value and unique significance of model insects in agricultural. In this paper, the connection between the silkworm circadian clock regulation mechanism and its incubation behavior was indicated, on the basis of core pathway which was in ongoing laboratory studies. Western blotting, immunofluorescence, CO-IP and immunohistochemistry and other experimental techniques were used. The main results were summarized as follows:1 Bm N cell regulation mechanism of the endogenous circadian system was analyzedAfter the subcellular localization experiment of core clock proteins C RY1, C RY2, PER and TIM in Bm N silkworm cells, it was founded that CRY1 only existed in the cytoplasm, while CRY2, PER and TIM were present both in the cytoplasm and the nucleus; Through the western blotting of separated nuclear and cytoplasm protein, and the investigation of interactions between them, which were detected by CO-IP, the results showd that the expression levels all had a typical circadian r hythm of the CRY1, C RY2, PER and TIM proteins. In more detailed said, heterodimer of C RY1, PER is detected in the cytoplasm and C RY2 combined with PER and TIM respectively in the cytoplasm and the nucleus. Referencing to the protein interaction mechanism o f Drosophila melanogaster and Danaus plexippus, we suggested that the Bm N cell made C RY1 as photoreceptors, and then the light signal passed to PER. After activate PER combined with CRY2, they entered into the nucleus, and combined with CLK / CYC dimers subsequently. At this time, activation effect of E-box was prevented, thereby the transcription of Per, Tim and Cry2 genes were inhibited. In the dark mode, the sensitive TIM which combined with C RY2, stopped the PER / CRY2 dimer formation, and kept PER / C RY2 from C LK / CYC for inhibiting the transcription repression activity. 2 Bm N cells rhythm mechanism response to the temperature zeitgeberSubcellular localization of C RY1, CRY2, PER and TIM protein showed that timing type in temperature cycling, 20 ℃: 30 ℃(12h: 12h)in constant dark, was similar to the mechanism light zeitgeber. Western blotting test of separated nuclear and cytoplasm protein showed that endogenous circadian clock in Bm N cell can be entrained by the temperature zeitgeber, but its oscillatio n rhythm was inconsistent. CRY1 didn’t show a similar oscillation to light zeitgeber. CRY2 was more sensitive to temperature than the light. Interestingly, PER and TIM protein levels didn’t have the obvious rhythmicity, but subcellular localization of PER and TIM are different. PER was mainly concentrated in the nucleus, and TIM was mainly in the cytoplasm. Presumably CRY2 was the temperature timing signal sensor more likely, and PER was a negative regulatory element while TIM was the way to transport. Eve n though, the specific transmission mechanism in response to temperature signal was still unclear. 3 There was a positive connection between the core circadian clock oscillation system in head and the incubation behavior in silkworm embryos.The results of constant temperature of 12L: 12 D and instantaneous light stimulation in 30 min both showed that, after the head cell of silkworm embryos which is in late period received the light signal, C RY1 and CRY2 expression levels raised gradually, especially CRY1. There was no obvious rhythmical change observedin PER protein, but it appeared that the protein transported from the cytoplasm to the nucleus after light stimulation..We speculated that, in the head of embryos, CRY1 as the photoreceptor, accepted the light rhythm(or light stimulation) to start the core circadian clock negative feedback signal path, and further regulate the embryos’ hatching behavior(rhythm).