棉铃虫复眼中Clock生物钟基因的昼夜表达模式

Daily Expression of Clock Gene in Compound Eye of Helicoverpa armigera

【目的】克隆并分析棉铃虫(Helicoverpa armigera)复眼Clock(Clk)生物钟基因的c DNA序列,探讨棉铃虫复眼中Clk生物钟基因的昼夜表达模式及其表达水平的影响因子,以确认其在复眼中是否起着调节生物节律的功能,为理解复眼中生物钟基因网络提供理论参考。【方法】以2日龄棉铃虫复眼为试验材料,采用RT-PCR和RACE末端扩增技术克隆棉铃虫Clk生物钟基因。利用生物信息学软件对得到的棉铃虫CLK氨基酸序列进行生物信息学分析。采用实时荧光定量PCR(q RT-PCR)技术,检测棉铃虫成虫不同器官(头、胸、腹、足、翅、脑、触角、复眼)中Clk生物钟基因的表达水平;通过设置不同的光周期环境,检测复眼中Clk生物钟基因的昼夜表达模式;通过在暗期设置6 h不同波段光(UV、蓝光和绿光)照射,检测复眼中Clk生物钟基因的表达水平;通过设置棉铃虫雌雄蛾交配处理,检测交配结束0 h和3 h复眼中Clk生物钟基因的表达水平;通过饥饿处理棉铃虫雌雄蛾,检测复眼中Clk生物钟基因的表达水平。【结果】克隆得到棉铃虫Clk生物钟基因的c DNA序列,命名为He CLK(Gen Bank登录号为KM233158),开放读码框1 860 bp,编码619个氨基酸组成的多肽,理论推测分子量(Mw)为69.32 k D,等电点(p I)为5.71。推导得到的氨基酸序列具有3个跨膜拓扑结构,包含多个昆虫CLK蛋白的保守区域(PAS和HLH),其与甜菜夜蛾(Spodoptera exigua)和黑脉金斑蝶(Danaus plexippus)的同源性较高,分别为97%和74%。与点蜂缘蝽(Riptortus pedestris)和马铃薯甲虫(Leptinotarsa decemlineata)的同源性较低,分别为53%和52%。q RT-PCR结果表明在检测的成虫器官中,He CLK在复眼中表达水平最低,触角中表达水平最高。在14L﹕10D光周期下,复眼中He CLK的表达量在白天增高,夜晚下降。生物钟基因的昼夜表达模式在1 d黑暗下可以持续,而在持续黑暗下固有表达节律消失。复眼中He CLK的表达水平在6 h光照后上调,但不同波段光照射无显著性差异。复眼中He CLK的表达水平在交配后有下调趋势,在雄蛾交配后表达水平显著性下降。复眼中He CLK的表达水平在饥饿处理后无显著性变化。【结论】成功从夜蛾棉铃虫的复眼中克隆得到Clk生物钟基因,由Clk生物钟基因推导得到的氨基酸序列具有典型的CLK蛋白特征,且与昆虫CLK蛋白同源性较高。在检测的棉铃虫成虫器官中,He CLK在复眼中的表达水平最低。He CLK在外周组织复眼中的表达水平受蛾类自身节律、光照和蛾类生理状态的影响,证实棉铃虫复眼中He CLK在调节生物节律方面具有重要作用,但生物钟基因网络在复眼与中枢神经中是否类似有待进一步深入研究。

【Objective】The objective of this study is to clone and analyze a circadian clock gene, Clock（Clk） in the compound eyes of Helicoverpa armigera, examine the diurnal changes and determinants of Clk m RNA levels in compound eyes of cotton bollworm, and to determine whether Clk performed circadian functions in compound eyes, which will provide a theoretical reference for understanding the circadian clock machinery in compound eyes. 【Method】The total RNA was isolated from the compound eyes of 2-day-old H. armigera moths, and the Clk was cloned by reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of c DNA ends（RACE）. The amino acid sequence of CLK from cotton bollworm was analyzed using bioinformatics softwares. The relative m RNA levels of Clk were determined among various adult organs（head, thorax, abdomen, leg, wing, brain, antennae and compound eye） of H. armigera by quantitative real-time PCR（q RT-PCR）. The diurnal change of Clk m RNA levels in compound eyes was measured under different photoperiods. Moths were illuminated by UV, blue and green lights, respectively, for 6 h from the beginning of the scotophase, and the expression levels of Clk in compound eyes were determined after light exposure. Female and male moths were paired for mating, and the expression levels of Clk in compound eyes were determined from the moths that had completed mating 0 h and 3 h. The expression levels of Clk in compound eyes were determined from the moths that were not fed. 【Result】An open reading frame of 1 860 bp was cloned, encoding 619 amino acids, designed as ＂He CLK＂（Gen Bank accession number KM233158）. The molecular mass of the deduced protein was predicted to be 69.32 k D, and the calculated isoelectric point（p I） was 5.71. Deduced amino acids sequence contained three transmembrane topologies, and several conserved domains of insect CLK（PAS and HLH）. He CLK showed high homology with Spodoptera exigua（97% identity） and Danaus plexippus（74% identity）, and low homology with Riptortus pedestris（53% identity） and Leptinotarsa decemlineata（52% identity）. q RT-PCR revealed that He CLK showed the lowest m RNA levels in compound eyes, and the highest m RNA levels in antennas among tested adult organs. The m RNA levels of He CLK in compound eyes increased during the day, and decreased during the night under 14L﹕10D. The cycling of the circadian clock gene m RNA levels persisted for 1 d under dark condition, but did not persist further under constant darkness. He CLK was up-regulated in compound eyes after light exposure, but there was no significant difference in m RNA levels of He CLK among different wavelengths of light. The expression levels of He CLK were tended to be down-regulated in the compound eyes after copulation, and there was a significant difference in m RNA levels of He CLK between mated males and virgin males. The expression levels of He CLK in compound eyes were not influenced by starvation. 【Conclusion】He CLK from the compound eyes of a nocturnal moth, H. armigera, was cloned. Deduced amino acids sequence contained the conserved domains of CLK proteins, and shared high homology with insect CLK. He CLK showed the lowest m RNA levels in compound eyes among tested adult organs. The expression levels of He CLK in peripheral tissues（compound eyes） were regulated by the circadian rhythms, light condition and physiological status of moths, confirming that He CLK played an important role in circadian rhythms in compound eyes. Whether the circadian clock machinery is similar between compound eyes and the central nervous system need to be further studied.