温度与光照节律对家蚕成虫生物钟基因Cry1和Cry2表达的影响

Effects of Temperature and Light Rhythm on Expression of Clock Genes Cry1 and Cry2 in Bombyx mori Adult

为了深入研究家蚕昼夜节律授时作用机制,设置4种光制和2种温度振荡周期,调查家蚕成虫生物钟核心钟基因———隐花色素编码基因Cry1和Cry2的表达特点。采用定量PCR方法检测Cry1和Cry2在正常温度与光照环境下(25℃,LD 12 h∶12 h)的家蚕各发育阶段的表达谱,2个基因都在成虫期显著上调表达,而在卵期和5龄幼虫期只有痕迹量表达;2个基因在成虫组织的表达谱显示在头部的表达水平最高,此外Cry1在触角、Cry2在飞行肌的表达水平也相对较高。RT-PCR检测分析不同光制下成虫头部、触角和飞行肌中2个Cry基因的表达谱都呈现出振荡节律性,发现光制变化能够改变这2个基因表达的振荡相位,其中Cry1的振荡表达相位变化出现在光照来临时,并且从中枢神经所在头部组织和触角至外周组织飞行肌的响应呈现时间梯度;而Cry2基因表达产物则可能不是光制授时因子的第一信响应物质。2个Cry基因都对温度振荡节律表现出敏感性,在昼夜时长内改变温度振荡周期,Cry1的表达只是振荡相位发生改变,而Cry2的表达还出现振荡波形倒置,推测它们在生物钟环路中可能与温度信号的转换有一定关系,但作用有差异。

In order to elucidate the timing mechanism of circadian rhythm in silkworm（Bombyx mori）,expression patterns of two critical clock genes,Cry1 and Cry2 which encode the cryptochrome of silkworm adult,were investigated under treatment of 4 photoperiods as well as 2 periodic oscillations of temperature.Expression patterns of Cry1 and Cry2 were assayed by Real-time PCR at each developmental stage of silkworm under normal temperature and light condition（25 ℃,LD 12 h∶12 h）.It was found that both genes were remarkably up-regulated at the adult stage,while only trace expression could be detected at the embryonic stage and during the 5th instar larval stage.Expression profile of both genes in adult tissues displayed that head tissue had the highest expression level.Meanwhile,the expression level of Cry1 in antennae and Cry2 in flight muscle was also quite high.RT-PCR analyses revealed that oscillatory rhythm existed in the expression of both Cry genes in head,antenna and flight muscle of the adult under different photoperiods,indicating that change of photoperiod could alter expressional oscillatory phase of these two genes.Oscillatory phase of Cry1 expression was altered when the illumination started.In addition,the responses from the head and antenna where central nervous system was located to the flight muscle of peripheral tissue showed a time gradient pattern.However,expression product of Cry2 gene might not be the first substance responding to light zeitgeber.Both Cry genes were sensitive to the periodic oscillation of temperature.Change of oscillating cycle within the circadian period led to an alteration in the oscillatory phase of Cry1 expression and an inversion in the oscillatory wave form of Cry2 expression.It is suggested that both Cry1 and Cry2 are related to signal transformation of temperature in biological clock,but their functions may differ.