Bassan Meire Menezes, Angelotti-Mendonc A Je Ssika, Alves Gustavo Rodrigues, Yamamoto Pedro Takao, Moura O Filho Francisco de Assis Alves
Departamento de Produc¸a~o Vegetal, Universidade de Sa~o Paulo, Escola Superior de Agricultura "Luiz de Queiroz", Brazil.
Departamento de Entomologia e Acarologia, Universidade de Sa~o Paulo, Escola Superior de Agricultura "Luiz de Queiroz", Brazil.
J Econ Entomol. 2017 Dec 5;110(6):2623-2629. doi: 10.1093/jee/tox253.
The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is considered the main vector of the bacteria associated with huanglongbing, a very serious disease that has threatened the world citrus industry. The absence of efficient control management protocols, including a lack of resistant cultivars, has led to the development of different approaches to study this pathosystem. The production of resistant genotypes relies on D. citri gene expression analyses by RT-qPCR to assess control of the vector population. High-quality, reliable RT-qPCR analyses depend upon proper reference gene selection and validation. However, adequate D. citri reference genes have not yet been identified. In the present study, we evaluated the genes EF 1-α, ACT, GAPDH, RPL7, RPL17, and TUB as candidate reference genes for this insect. Gene expression stability was evaluated using the mathematical algorithms deltaCt, NormFinder, BestKeeper, and geNorm, at five insect developmental stages, grown on two different plant hosts [Citrus sinensis (L.) Osbeck (Sapindales: Rutaceae) and Murraya paniculata (L.) Jack (Sapindales: Rutaceae)]. The final gene ranking was calculated using RefFinder software, and the V-ATPase-A gene was selected for validation. According to our results, two reference genes are recommended when different plant hosts and developmental stages are considered. Considering gene expression studies in D. citri grown on M. paniculata, regardless of the insect developmental stage, GAPDH and RPL7 have the best fit as reference genes in RT-qPCR analyses, whereas GAPDH and EF 1-α are recommended as reference genes in insect studies using C. sinensis.
亚洲柑橘木虱，Diaphorina citri Kuwayama（半翅目：木虱科），被认为是与黄龙病相关细菌的主要传播媒介，黄龙病是一种非常严重的疾病，已经威胁到全球柑橘产业。由于缺乏有效的防治管理方案，包括缺乏抗性品种，导致了研究这种病理系统的不同方法的发展。抗性基因型的产生依赖于通过RT-qPCR对柑橘木虱基因表达进行分析，以评估对媒介种群的控制。高质量、可靠的RT-qPCR分析取决于合适的内参基因的选择和验证。然而，尚未鉴定出合适的柑橘木虱内参基因。在本研究中，我们评估了EF 1-α、ACT、GAPDH、RPL7、RPL17和TUB基因作为这种昆虫的候选内参基因。使用数学算法deltaCt、NormFinder、BestKeeper和geNorm，在五个昆虫发育阶段，在两种不同的植物寄主[甜橙（Citrus sinensis (L.) Osbeck，无患子目：芸香科）和九里香（Murraya paniculata (L.) Jack，无患子目：芸香科）]上生长时，评估基因表达稳定性。使用RefFinder软件计算最终的基因排名，并选择V-ATPase-A基因进行验证。根据我们的结果，当考虑不同的植物寄主和发育阶段时，推荐使用两个内参基因。考虑在九里香上生长的柑橘木虱的基因表达研究，无论昆虫发育阶段如何，GAPDH和RPL7最适合作为RT-qPCR分析中的内参基因，而在使用甜橙的昆虫研究中，推荐使用GAPDH和EF 1-α作为内参基因。
