【作者】 梁辉； 王文栋； 朱晓苏； 陶卉； 徐丽； 司马杨虎； 徐世清；

【Author】 LIANG Hui,WANG Wen-Dong,ZHU Xiao-Su,TAO Hui,XU Li,SIMA Yang-Hu,XU Shi-Qing~* (National Engineering Laboratory for Modern Silk,Department of Applied Biology,Medical College of Soochow University,Suzhou,Jiangsu 215123,China)

【机构】 苏州大学医学部应用生物学系现代丝绸国家工程实验室；

【摘要】 隐花色素基因(Cry)是已确认的主要生物钟基因之一。昆虫Cry基因分为Cry1和Cry2两类,果蝇只有Cry1,蜜蜂等膜翅目昆虫只有Cry2,而家蚕同时具有两类Cry基因。本文调查了Cry1和Cry2基因在家蚕的不同组织和发育时期特异的表达;并且调查了Cry1和Cry2基因在家蚕成虫期不同组织中不同光节律和温节律下的振荡表达;同时,在卵期采用不同的滞育诱发环境来调查Gry1和Cry2基因对环境因子的响应;最后通过制备多克隆抗体,对CRY1和CRY2在家蚕成虫脑部的组织特异表达进行组织定位;为CRY1和CRY2的功能研究提供了线索和证据。通过试验研究主要得到以下结果:家蚕不同发育时期和不同组织的芯片表达谱调查显示,Cry1和Cry2基因的表达具有组织和发育时期的特异性,Cry1和Cry2基因在家蚕蛾期的表达量高于其它发育时期,在家蚕蛾的头和触角中的表达量高于四肢和飞行肌。免疫组化结果表明,CRY1和CRY2蛋白质集中在家蚕蛾脑部的前脑叶高量表达,并且具有组织细胞分布的特异性;蚕蛾前脑叶的主要功能是与视觉形成和感受外界环境变化有关,因此推测CRY1和CRY2具有生物钟环路中转换环境授时因子变化的作用。在家蚕蛾期,采用经典的光制和温制的振荡节律处理,利用Real-time PCR的方法调查Cry1和Cry2基因的节律振荡表达,结果显示Cry1和Cry2基因在家蚕成虫期头部的表达呈现振荡节律变化,并且在相同光制中Cry1和Cry2基因的变化趋势非常的一致,变化波形几乎相同,但表达量有差别。推测Cry1和Cry2基因在家蚕生物钟环路中处于相同环路中,可能是相关联基因。在不同温制下,Cry1和Cry2基因的表达出现了显著的相位位移和波形变化,推知Cry基因在响应温度节律的机制中也发挥着重要的作用,但在定量分析结果中找不到Cry1基因和Cry2基因对温度节律的相关性变化,推测Cry1基因和Cry2基因在温度调节生物钟变化的机制中处于不同的环路中。通过采用常规诱导滞育发生的温度与光照条件处理胚胎发育后期蚕卵,调查家蚕胚胎生物钟信号环路主要基因Cry1和Cry2的表达对授时因子振荡变化的响应。结果显示,光照能够上调Cry1基因在家蚕胚胎发育后期的表达,温度则可改变该基因的振荡周期相位;温度在改变Cry2基因振荡表达相位的同时低温(15℃)还能够上调该基因在家蚕胚胎发育后期的表达,提示家蚕胚胎中可能存在与其他昆虫类似的以CRY1为光感受器的生物钟环路,为我们研究家蚕滞育发生与生物钟的关系提供了一定的线索。更多还原

【Abstract】 Cryptochrome gene(Cry) is one of the major biological clock genes which were widely distributed in bacteria and eukaryotes.Cry genes of insect species are clearly divided into two types,Cry1 and Cry2.Only Cry1 is expressed in Drosophila,while only Cry2 was expressed in bees and other hymenopteran insects.In this study,we mainly evaluated the Bm-clock genes by focusing on the functions of a Drosophila-like Cryptochrome(Cry),designated Cryl,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: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 Cryl 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.更多还原