家蚕生物钟基因Bmtim2的克隆与序列分析

Cloning and Sequence Analysis of Biological Clock Gene Bmtim2 in Bombyx mori L.

[目的]为进一步研究家蚕生物钟基因的功能提供依据。[方法]从家蚕品种p50未受精卵提取总RNA,利用快速扩增cDNA末端(RACE)技术克隆家蚕生物钟基因Bmtim2cDNA序列。最后运用实时定量RT-PCR对该基因在家蚕不同发育时期的表达情况进行初步分析。[结果]利用RACE扩增,获得长度为1867bp、包括完整3'非翻译区的家蚕生物钟基因timeless2(Bmtim2)cDNA序列。经推导,获得该基因序列编码的584个氨基酸残基的蛋白质序列。该蛋白序列具有TIM蛋白的C末端保守区,与黑腹果蝇、尖音库蚊的TIM2基因具有近缘进化关系。Bmtim2基因在家蚕生活周期各时点均有表达。[结论]该结果对研究Bmtim2基因在家蚕发育过程的时空表达差异及其与日周节律的关系具有参考价值。

[Objective] The research aimed to provide basis for further research on the functions of biological clock gene in Bombyx mori L, [Method] Total RNA was extracted from unfertilized egg of silkworm variety p50. Bmtim2 eDNA sequence of the biological clock gene was cloned by using rapid amplication of eDNA ends （RACE） technology. Finally the expression of this gene in different growth stages of B. mori＇was preliminarily studied by using Real-time quantitative PCR. [Result] eDNA sequence of the biological clock gene timeless2 （Bmtim2）in B. mori with the length of 1 867 bp which included complete 3＇ untranslated region was obtained through RACE amplification. Through derivating, the protein sequence with 584 amino acid residues that was coded by this gene sequence was obtained. The protein sequence had C-terminal conservative region of TIM protein and it had closely -related evolution relationship with TIM 2 gent of Drosophila melonogaster and Culex pipiens, Bmtim 2 gene was expressed at each time point of the life cycle in B. mori. [Conclusion] These results had an important reference value for studying the time-space expression differences of Bmtim2 gene in the growth process orB. mori and its relationship with the circadian rhythm.