棉铃虫生物钟基因HeDbt的克隆和表达模式分析

【目的】克隆并分析棉铃虫Helicoverpa armigera生物钟基因Double-time (Dbt),明确该基因的昼夜表达模式,探讨其表达水平的影响因子,为研究夜蛾科昆虫复眼中生物钟基因的作用机制奠定基础,为理解外周组织中生物钟基因功能提供参考。【方法】采用RT-PCR和RACE技术从2日龄棉铃虫雌成虫复眼中克隆生物钟基因Dbt,并利用在线网站和软件进行生物信息学分析。采用qPCR技术检测棉铃虫雌、雄成虫不同组织(头、脑、复眼、触角、胸、腹、足和翅)中Dbt的表达水平;检测光周期14L∶10D和持续黑暗(DD)下雌、雄成虫头和复眼中Dbt的昼夜表达模式;在暗期用棉铃虫敏感波段光(UV、蓝光和绿光)照射2日龄成虫6 h,检测复眼中Dbt表达水平的变化;在暗期进行雌、雄成虫交配,检测交配结束及3 h后复眼中Dbt表达水平的变化。【结果】成功克隆到棉铃虫生物钟基因Dbt的cDNA序列,命名为HeDbt(GenBank登录号: KM233159),开放阅读框长1 026 bp,编码314个氨基酸组成的多肽。HeDbt理论推测分子量为39.79 kD,等电点(pI)为9.55,不具有跨膜拓扑结构,包含典型的昆虫DBT蛋白保守区域,其与甜菜夜蛾Spodoptera exigua和柞蚕Antheraea pernyi DBT的同源性较高, 氨基酸序列一致性分别为99%和97%。qPCR结果表明,HeDbt在成虫各组织中均有表达,在头、脑和复眼中表达水平较低,在胸和腹中表达水平较高;在14L∶10D和DD下,头和复眼中HeDbt未呈现明显的昼夜表达节律。暗期光照和交配后,复眼中HeDbt的表达均显著下调,但雌、雄成虫间HeDbt表达水平整体相似。【结论】成功克隆得到棉铃虫生物钟基因HeDbt,其在棉铃虫成虫头和复眼中表达水平较低,且不具有昼夜规律性,但复眼中Dbt的表达受到光照和交配的影响。本研究为进一步探索夜蛾外周组织生物钟基因功能奠定了基础。     

Neurobiology of the fruit fly's circadian clock

Studying the fruit fly Drosophila melanogaster has revealed mechanisms underlying circadian clock function. Rhythmic behavior could be assessed to the function of several clock genes that generate circadian oscillations in certain brain neurons, which finally modulate behavior in a circadian manner. This review outlines how individual circadian pacemaker neurons in the fruit fly's brain control rhythm in locomotor activity and eclosion.     

Normal vision can compensate for the loss of the circadian clock

Circadian clocks are thought to be essential for timing the daily activity of animals, and consequently increase fitness. This view was recently challenged for clock-less fruit flies and mice that exhibited astonishingly normal activity rhythms under outdoor conditions. Compensatory mechanisms appear to enable even clock mutants to live a normal life in nature. Here, we show that gradual daily increases/decreases of light in the laboratory suffice to provoke normally timed sharp morning (M) and evening (E) activity peaks in clock-less flies. We also show that the compound eyes, but not Cryptochrome (CRY), mediate the precise timing of M and E peaks under natural-like conditions, as CRY-less flies do and eyeless flies do not show these sharp peaks independently of a functional clock. Nevertheless, the circadian clock appears critical for anticipating dusk, as well as for inhibiting sharp activity peaks during midnight. Clock-less flies only increase E activity after dusk and not before the beginning of dusk, and respond strongly to twilight exposure in the middle of the night. Furthermore, the circadian clock responds to natural-like light cycles, by slightly broadening Timeless (TIM) abundance in the clock neurons, and this effect is mediated by CRY.     

Human intestinal circadian clock: expression of clock genes in colonocytes lining the crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per-1, per-2, and clock mRNA were detected by real-time RT-PCR. The three-dimensional distributions of PER-1, PER-2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per-1, per-2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER-1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER-1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.     

昆虫钟基因研究进展

昆虫进化形成了内在的生物钟机制以协调行为、生理及代谢节律与外部环境信号同步,从而更有效地利用资源并获得适应性优势。行为、生理及代谢昼夜调控的协调对于昆虫有效应对可预见的生理上的挑战至关重要。生化过程和代谢变化与外部环境的昼夜节律同步性受基因表达的控制,钟基因在昆虫的重要生理过程如中枢及外围生物钟机制、光周期信号传导、光周期介导的外围组织调控、代谢以及免疫中发挥着重要作用。根据信号转导过程中的作用,昆虫钟基因分为3类——信号输入基因、信号震荡起搏器和信号输出基因,它们通过相互作用形成了复杂的转录-翻译反馈回路并参与调控昆虫昼夜节律和光周期事件。本文针对昆虫钟基因的鉴定、分类和功能,作用分子机制以及研究方法和挑战等方面作了总结,并展望了昆虫钟基因未来的研究方向,这将为昆虫钟基因的进一步功能研究及开发利用提供信息参考。     

Morphine withdrawal produces circadian rhythm alterations of clock genes in mesolimbic brain areas and peripheral blood mononuclear cells in rats

Previous studies have shown that clock genes are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus, other brain regions, and peripheral tissues. Various peripheral oscillators can run independently of the SCN. However, no published studies have reported changes in the expression of clock genes in the rat central nervous system and peripheral blood mononuclear cells (PBMCs) after withdrawal from chronic morphine treatment. Rats were administered with morphine twice daily at progressively increasing doses for 7 days; spontaneous withdrawal signs were recorded 14 h after the last morphine administration. Then, brain and blood samples were collected at each of eight time points (every 3 h: ZT 9; ZT 12; ZT 15; ZT 18; ZT 21; ZT 0; ZT 3; ZT 6) to examine expression of rPER1 and rPER2 and rCLOCK . Rats presented obvious morphine withdrawal signs, such as teeth chattering, shaking, exploring, ptosis, and weight loss. In morphine-treated rats, rPER1 and rPER2 expression in the SCN, basolateral amygdala, and nucleus accumbens shell showed robust circadian rhythms that were essentially identical to those in control rats. However, robust circadian rhythm in rPER1 expression in the ventral tegmental area was completely phase-reversed in morphine-treated rats. A blunting of circadian oscillations of rPER1 expression occurred in the central amygdala, hippocampus, nucleus accumbens core, and PBMCs and rPER2 expression occurred in the central amygdala, prefrontal cortex, nucleus accumbens core , and PBMCs in morphine-treated rats compared with controls. rCLOCK expression in morphine-treated rats showed no rhythmic change, identical to control rats. These findings indicate that withdrawal from chronic morphine treatment resulted in desynchronization from the SCN rhythm, with blunting of rPER1 and rPER2 expression in reward-related neurocircuits and PBMCs.     

棉铃虫蛾复眼光反应特性

用视网膜电位图(electroretinogram,ERG)技术研究了棉铃虫Helicoverpa armigera蛾暗适应过程中对单色光和白光刺激的光感受性变化。结果显示：(1)依ERG振幅大小(峰-峰值),在340～605 nm波谱内有3个大小不等的峰-主峰位于绿 黄光区562 nm,次峰在蓝光区483 nm,第3个峰在近紫外区400 nm,显示其至少有3种感受器;(2)性别、日龄及暗适应时间长短对其光谱敏感性有影响,低龄时雄蛾对单色光刺激较雌蛾敏感,高日龄时相反;1～5日龄内, 3日龄蛾的视网膜电位(ERP)值最高;随暗适应时间延长,其复眼对近紫外区400 nm敏感性明显增加;(3)一定光强度范围内,随单色光和白光光强度增强该蛾复眼的ERP值增大,初期增加较缓,中期较快,呈近似S型曲线,显示其复眼具有较强的光强度自调节和适应机制。     

Interactions of polymorphisms in different clock genes associated with circadian phenotypes in humans

Several studies have shown that mutations and polymorphisms in clock genes are associated with abnormal circadian parameters in humans and also with more subtle non-pathological phenotypes like chronotypes. However, there have been conflicting results, and none of these studies analyzed the combined effects of more than one clock gene. Up to date, association studies in humans have focused on the analysis of only one clock gene per study. Since these genes encode proteins that physically interact with each other, combinations of polymorphisms in different clock genes could have a synergistic or an inhibitory effect upon circadian phenotypes. In the present study, we analyzed the combined effects of four polymorphisms in four clock genes (Per2, Per3, Clock and Bmal1) in people with extreme diurnal preferences (morning or evening). We found that a specific combination of polymorphisms in these genes is more frequent in people who have a morning preference for activity and there is a different combination in individuals with an evening preference for activity. Taken together, these results show that it is possible to detect clock gene interactions associated with human circadian phenotypes and bring an innovative idea of building a clock gene variation map that may be applied to human circadian biology.     

昆虫生物钟分子调控研究进展

昆虫生物钟节律的研究是人类了解生物节律的重要途径。昆虫在生理和行为上具有广泛的节律活动,如运动、睡眠、学习记忆、交配、嗅觉等节律活动,其中昼夜活动行为节律的研究广泛而深入。昆虫乃至高等动物普遍具有保守的昼夜节律系统,昼夜生物钟节律主要包括输入系统:用于接受外界光和温度等环境信号并传入核心振荡器,使得生物时钟与环境同步;核心时钟系统:自我维持的昼夜振荡器;输出系统:将生物钟产生的信号传递出去而控制生物行为和生理的节律变化。早期分子和遗传学研究主要关注昼夜节律振荡器的分子机制及神经生物学,阐明了昼夜生物钟节律的主要分子机制及相关神经网络。最近更多的研究关注生物钟信号是如何输入和输出。本文以果蝇运动节律的相关研究为主要内容,围绕生物钟输入系统、振荡器、输出系统这3个组成部分对昆虫生物钟研究进展进行总结。     

Synchronization of the mammalian circadian timing system: Light can control peripheral clocks independently of the SCN clock

A vast network of cellular circadian clocks regulates 24‐hour rhythms of behavior and physiology in mammals. Complex environments are characterized by multiple, and often conflicting time signals demanding flexible mechanisms of adaptation of endogenous rhythms to external time. Traditionally this process of circadian entrainment has been conceptualized in a hierarchical scheme with a light‐reset master pacemaker residing in the hypothalamus that subsequently aligns subordinate peripheral clocks with each other and with external time. Here we review new experiments using conditional mouse genetics suggesting that resetting of the circadian system occurs in a more “federated” and tissue‐specific fashion, which allows for increased noise resistance and plasticity of circadian timekeeping under natural conditions.     

Temporal-spatial characterization of chicken clock genes: circadian expression in retina,pineal gland,and peripheral tissues

The molecular core of the vertebrate circadian clock is a set of clock genes, whose products interact to control circadian changes in physiology. These clock genes are expressed in all tissues known to possess an endogenous self-sustaining clock, and many are also found in peripheral tissues. In the present study, the expression patterns of two clock genes, cBmal1 and cMOP4, were examined in the chicken, a useful model for analysis of the avian circadian system. In two tissues which contain endogenous clocks--the pineal gland and retina--circadian fluctuations of both cBmal1 and cMOP4 mRNAs were observed to be synchronous; highest levels occurred at Zeitgeber time 12. Expression of these genes is also rhythmic in several peripheral tissues; however, the phases of these rhythms differ from those in the pineal gland and retina: in the liver the peaks of cMOP4 and cBmal1 mRNAs are delayed 4-8 h and in the heart they are advanced by 4 h, relative to those in the pineal gland and retina. These results provide the first temporal characterization of cBmal1 and cMOP4 mRNAs in avian tissues: their presence in avian peripheral tissues indicates they may influence temporal features of daily rhythms in biochemical, physiological, and behavioral functions at these sites.     

A spatial model of the plant circadian clock reveals design principles for coordinated timing

Individual plant cells possess a genetic network, the circadian clock, that times internal processes to the day‐night cycle. Mathematical models of the clock are typically either “whole‐plant” that ignore tissue or cell type‐specific clock behavior, or “phase‐only” that do not include molecular components. To address the complex spatial coordination observed in experiments, here we implemented a clock network model on a template of a seedling. In our model, the sensitivity to light varies across the plant, and cells communicate their timing via local or long‐distance sharing of clock components, causing their rhythms to couple. We found that both varied light sensitivity and long‐distance coupling could generate period differences between organs, while local coupling was required to generate the spatial waves of clock gene expression observed experimentally. We then examined our model under noisy light‐dark cycles and found that local coupling minimized timing errors caused by the noise while allowing each plant region to maintain a different clock phase. Thus, local sensitivity to environmental inputs combined with local coupling enables flexible yet robust circadian timing.     

Effects of iron status on expression of circadian clock genes and serum lipid metabolism in sucking piglets

The aim of the study is to determine the effects of iron on circadian clock gene expression and serum lipid metabolism in sucking piglets. Twenty-four neonatal piglets were selected and randomly assigned into three groups (A, B, and C) with eight replicates. Group A were received 1 mL physiological saline by intramuscular administration at d 3 and d 10; group B were received 1 mL iron dextran (100 mg) by intramuscular administration at d 3 and 1 mL physiological saline at d 10, respectively; group C were received 1 mL of iron dextran (100 mg) by intramuscular administration at both d 3 and d 10. Our results reveal that the relative expressions of Cry1, Cry2, Per1, Per2, and Bmal in liver were significantly different in the three groups (p < 0.05). Meanwhile, the content of triglyceride (TG) and high-density lipoprotein (HDL) in serum were also affected by the iron supplementation (p < 0.05). These results indicated that iron affected hepatic circadian clock genes significantly, meanwhile, it may possible association with lipid metabolism.     

The Tilapia collagen peptide mixture TY001 protects against LPS-induced inflammation,disruption of glucose metabolism,and aberrant expression of circadian clock genes in mice

The Tilapia collagen peptide mixture TY001 has been shown to accelerate wound healing in streptozotocin-induced diabetic mice and to protect against streptozotocin-induced inflammation and elevation in blood glucose. The goals of the present study are to further study TY001 effects on lipopolysaccharide (LPS)-induced inflammation and metabolic syndrome. LPS is known to disrupt circadian clock to produce toxic effects, the effects of TY001 on rhythmic alterations of serum cytokines and hepatic clock gene expressions were examined. Mice were given TY001 (30 g/L, ≈ 40 g/kg) through the drinking water for 30 days, and on the 21^st day of TY001 supplementation, LPS (0.25 mg/kg, ip, daily) was given for 9 days to establish the inflammation model. Repeated LPS injections produced inflammation, impaired glucose metabolism, and suppressed the expression of circadian clock core genes Bmal1 and Clock; clock feedback gene Cry1, Cry2, Per1, and Per2; clock target gene Rev-erbα and RORα. TY001 prevented LPS-induced elevations of TNFα, IL-1β, IL-6, and IL-10 in the liver, along with improved histopathology. TY001 reduced LPS-elevated fasting blood glucose and increased LPS-reduced serum insulin levels, probably via increased glucose transporter GLUT2, enhanced insulin signaling p-Akt and p-IRS-1^Try612. Importantly, LPS-induced circadian elevations of serum TNFα and IL-1β and aberrant expression of circadian clock genes in the liver were ameliorated by TY001. Immunohistochemistry revealed that the LPS decreased Bmal1 and Clock protein in the liver, which was recovered by TY001. Taken together, TY001 is effective against LPS-induced inflammation, disruption of glucose metabolism and disruption of circadian clock gene expressions.

Abbreviations: TY001: Tilapia collagen peptide mixture; LPS: Lipopolysaccharide; TNFα: Tumor necrosis factor-α; IL-1β: Interleukin-1β; GLUT2: Glucose transporter 2