Comparative analysis of reference genes in honey bees, <i>Apis cerana</i> and <i>Apis mellifera</i>

Honey bees are vital pollinators in agriculture and important model insects. To understand the genetic and molecular aspects in their development, a reverse transcription quantitative polymerase chain (RT-qPCR) is used to investigate the target genes. However, it is essential to use the appropriate reference genes as endogenous controls for accurate normalization of target genes. To identify stable reference genes in two honey bee species, [<i>Apis mellifera</i> (<i>Am</i>) and <i>Apis cerana</i> (<i>Ac</i>)], we evaluated eight candidate reference genes including, <i>actin</i>, <i>atub</i>, <i>ef1α</i>, <i>gapdh</i>, <i>rpl13a</i>, <i>rpl32</i>, <i>rps18</i> and <i>tif</i>. Worker bees belonging to the two species were collected at each developmental day during the embryonic and postembryonic developmental stages. The <i>tyrosine hydroxylase</i> (<i>th</i>) gene was used as the target gene to validate the selected reference genes. Our results revealed that <i>rpl13a</i> was the most stable reference gene at all developmental stages of <i>Am</i> and <i>Ac</i>. In addition, gene combinations, including <i>Amrpl13a</i> &amp; <i>Amrps18</i> &amp; <i>Amactin</i>, <i>Amrpl13a</i> &amp; <i>Amrpl32</i>, <i>Acrpl13a</i> &amp; <i>Acrpl32</i>, <i>Acrpl13a</i> &amp; <i>Acrpl32</i> &amp; <i>Acef1α</i> followed by other combinations effectively normalized the expression of the target genes during the embryonic and postembryonic developmental stages of <i>Am</i> and <i>Ac</i>, respectively. Our findings provide a foundation for standardized RT-qPCR analysis to improve the accuracy of genes normalization during the different developmental stages of honey bees.蜜蜂是农业生产中至关重要的传粉昆虫，同时也是重要的模式昆虫。为解析其发育过程中的遗传与分子调控机制，常采用反转录定量聚合酶链式反应（RT-qPCR）探究靶基因的表达情况。然而，要实现靶基因表达的精准归一化分析，必须选用合适的内参基因作为内参对照。为在两种蜜蜂——西方蜜蜂（Apis mellifera，简称Am）与东方蜜蜂（Apis cerana，简称Ac）——中筛选稳定的内参基因，我们评估了8个候选内参基因，分别为肌动蛋白（actin）、α微管蛋白（atub）、延伸因子1α（ef1α）、甘油醛-3-磷酸脱氢酶（gapdh）、核糖体蛋白L13a（rpl13a）、核糖体蛋白L32（rpl32）、核糖体蛋白S18（rps18）以及翻译起始因子（tif）。我们于胚胎发育与胚后发育阶段的每日采集两种蜜蜂的工蜂样本。以酪氨酸羟化酶（tyrosine hydroxylase，th）基因作为靶基因，对筛选得到的内参基因进行有效性验证。研究结果显示，在西方蜜蜂与东方蜜蜂的所有发育阶段中，核糖体蛋白L13a（rpl13a）均为最稳定的内参基因。此外，针对西方蜜蜂的胚胎发育与胚后发育阶段，采用Amrpl13a与Amrps18、Amactin的组合，以及Amrpl13a与Amrpl32的组合，可有效实现靶基因表达的归一化分析；针对东方蜜蜂的对应发育阶段，则可采用Acrpl13a与Acrpl32的组合、Acrpl13a、Acrpl32与Acef1α的组合，以及其他组合方案达成有效归一化。本研究结果可为蜜蜂不同发育阶段的标准化RT-qPCR分析提供理论基础，有助于提升靶基因归一化分析的准确性。