棉铃虫生物钟基因HeDbt的克隆和表达模式分析

Cloning and expression profiling of circadian clock gene HeDbt in Helicoverpa armigera (Lepidoptera: Noctuidae)

【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。

【Aim】The aim of this study is to clone and analyze circadian clock gene Double-time(Dbt)in Helicoverpa armigera,and to examine the diurnal expression pattern of Dbt mRNA levels and its determinants,so as to provide a theoretical basis for studying the mechanisms of action of the circadian clock genes in the compound eyes and understanding the function of circadian clock genes in peripheral tissues in noctuid moths.【Methods】Dbt was cloned from compound eyes of 2-day-old female adults of H.armigera by RT-PCR and RACE,and its deduced amino acid sequence was analyzed using online sites and software.The expression levels of Dbt among different tissues(head,brain,compound eyes,antennae,thorax,abdomen,leg and wing)of female and male adults were determined and compared by qPCR.The diurnal changes of Dbt mRNA levels in compound eyes and head were measured under a photoperiod of 14L∶10D and constant darkness(DD).The 2-day-old adults were irradiated by sensitive wavelength of UV,blue and green lights,respectively,for 6 h from the beginning of the scotophase,and the changes in the expression levels of Dbt in compound eyes were determined after light exposure.Female and male adults were paired for mating during scotophase,and the changes in the expression levels of Dbt in compound eyes were determined at 0 h and 3 h after copulation,respectively.【Results】The full-length cDNA of Dbt gene was cloned from the compound eyes of H.armigera and designated as HeDbt(GenBank accession number:KM233159).It has a 1 026 bp open reading frame encoding a predicted protein of 314 amino acids,with a molecular mass of 39.79 kD and a calculated isoelectric point(pI)of 9.55.Its deduced amino acid sequence has no transmembrane topologies,and shares several typical conserved domains of insect DBT.HeDBT shows a high homology with DBT proteins from Spodoptera exigua(99%amino acid sequence identity)and Antheraea pernyi(97%amino acid sequence identity).qPCR results illustrated that HeDbt was expressed in all the tested adult tissues,and had low expression levels in the head,brain and compound eyes and high expression levels in thorax and abdomen.The expression of HeDbt showed no obvious circadian rhythm in both the head and compound eyes under 14L∶10D and DD.The expression of HeDbt was down-regulated in compound eyes after light exposure and copulation,but with similar HeDbt mRNA levels between female and male adults.【Conclusion】A circadian clock gene HeDbt was successfully cloned from H.armigera.HeDbt had low mRNA levels in the head and compound eyes of H.armigera adults.Its expression in the head and compound eyes showed no obvious circadian rythym,but was influenced by light exposure and copulation of adults.Our study provides a theoretical basis for further studying the function of circadian clock genes in peripheral tissues in noctuid moths.