Table_3_Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions.DOCX

Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (β-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, β-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, β-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.

六齿小蠹（Ips sexdentatus）隶属于鞘翅目（Coleoptera）象甲科（Curculionidae）小蠹亚科（Scolytinae），是极具破坏性且经济重要性的森林害虫之一。深入解析其适应寄主有毒化合物的分子机制，可为未来该害虫的防控工作提供潜在突破口。基因表达研究正是实现此类研究目标的关键实验手段之一，而合适的内参基因筛选，是开展六齿小蠹（Ips sexdentatus）定量基因表达分析与功能基因组学研究的基础。
本研究针对鞘翅目昆虫常用的12种内参基因，在不同实验条件下开展筛选，以实现基因表达数据的精准可靠归一化。12种内参基因中的绝大多数，在发育阶段、组织特异性及性别特异性样本中均表现出相对稳定的表达模式，但在不同温度培养条件下则存在一定表达差异。
在发育条件下，微管蛋白β-1链（Tubulin beta-1 chain，β-Tubulin）为稳定性最优的内参基因，其次为翻译延伸因子（translation elongation factor，eEF2）与核糖体蛋白S3（ribosomal protein S3，RPS3）。在性别特异性条件下，RPS3、β-Tubulin与eEF2的表达稳定性位居前列。与之相对，组织特异性条件下稳定性较高的基因组合存在差异：头部组织中为RPS3与eEF2，脂肪体（fat body）中为液泡型ATP酶亚基A（V-ATPase-A）与eEF2，肠道（gut）中则为液泡型ATP酶亚基A（V-ATPase-A）与eEF2。在不同温度条件下，β-Tubulin与V-ATPase-A稳定性最优；而经保幼激素III（Juvenile Hormone III）处理后，泛素（ubiquitin，UbiQ）与V-ATPase-A的表达稳定性最高。
本研究通过基于实时定量反转录PCR（real-time quantitative reverse transcription PCR，RT-qPCR）的靶基因表达分析，对上述结果进行了进一步验证。综上，本研究为鞘翅目森林害虫六齿小蠹（Ips sexdentatus）在多样实验条件下梳理出了适用内参基因参考清单，并为该物种后续的基因表达与功能基因组学研究奠定了坚实基础。