Cloning And Functional Identification Of Cry Gene From Bombyx Mori

The circadian clock plays a vital role in rhythmic activity of insect by providing the timing component, such as light, tempetature, the season shifting and so on. In insect, the molecular mechanism had been the subject of intense investigation in Drosophila, while less attention has been directed at the clockwork in other, non-drosophilid insect. Bombyx mori is a model insect in Lepidoptera. It has many advantages as a model of circadian clock research. In our study ,we mainly evaluated the Bm-clock genes by focusing on the functions of a Drosophila-like Cryptochrome(Cry), designated Cry1, and a vertebrate-like Cry, designated Cry2, that both expressed in Bombyx mori as a clock gene. We firstly cloned Cry genes, and investigated the expression profile of Cry in different development stages and in different organs, then we investigated the circadian rhythm of Cry genes in different circadian light and temperature cycle in the adult of Bombyx mori. We also investigated the expression profile of Cry in diapause-inducing tempetature and light in order to study the relationship between Cry and dispause. At last, we prepared polyclonal antibody of CRY1 and CRY2, and localizated the tissue-specificated expression of CRY1 and CRY2 in different light and temperature. In our study, we had already drew the conclusion as following:There were two kinds of Cry gene in Bombyx mori , and the result of cloning Cry by 5'and 3'RACE showed the length of cDNA respectively were 2.166kb and 2.389kb, the blast results showed that the sequence were homogeneity with the database in NCBI. The sequence also had the characteristic of 5'-UTR and 3'-UTR. The expression profile in different development stages and in different organs showed that Cry had tissues and development stages specificity. It was mainly expression in the head of adult. The results of IHC showed that CRY1 and CRY2 mainly localizated in the forebrain of adult and it had tissue-specificated.The forebrain founctioned as vision-forming, so we concluded that CRY1 and CRY2 had functions as shift signal of the environment.We investigated the expression profile of Cry in classical light and temperature cycle by Real–time PCR. The results showed Cry had a circadian rhythm in adult of Bombyx mori, and at the same light cycle, the rhythmic phase shift of Cry1 and Cry2 had a consistency, the wave-shape was nearly similar, this indicated us that Cry1 and Cry2 maybe in the same loop of circadian rhythm, or it was the correlate gene in loop. In different temperature cycle, Cry1 and Cry2 also showed circadian rhythm, and had a distinct phase shift and wave-shape change. We can know Cry was an important clock gene in the regulation of temperature rhythm. But we can't find a relationship between Cry1 and Cry2 in different temperature cycle, so we concluded that Cry1 and Cry2 maybe in different loop in temperature circadian regulation.The expression profile of Cry1 and Cry2 in embryo of Bombyx mori in diapause-inducing environment showed that Cry1 and Cry2 had a relationship in the regulation of diapause. The transcriptional level of Cry1 in light environment was significantly higher than other tested genes in dark environment, while cycle phase of the Cry1 expression was changed by temperature environment. The expression of Cry2 up-regulated at 15℃and down-regulated at 25℃incubation temperature, the cycle phase changed at 15℃, while the incubation photoperiod had no influence on transcriptional level and cycle phase of Cry2. The results suggested that there was a CRY-centric circadian clock loop in which CRY1 functioned as a photoreceptor in Bombyx mori. It provided us a clue to study the relationship between the diapause and circadian clock.