Choudhury Aparajita, Verma Shubham, Muthamilarasan Mehanathan, Rajam Manchikatla Venkat
Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Telangana, 500046, India.
Mol Biol Rep. 2021 Nov;48(11):7477-7485. doi: 10.1007/s11033-021-06766-3. Epub 2021 Oct 12.
Maruca vitrata is one of the potential insect pests that cause devastating losses to legume cultivation worldwide. Gene functional studies facilitate dissecting the molecular mechanisms underlying the infection process and enable devising appropriate molecular strategies to control this insect pest. Expression profiling using quantitative real-time PCR (qRT-PCR) provides insights into the functional characterization of target genes; however, ideal reference genes should be deployed in such studies to nullify the background variation and improve the accuracy of target gene expression. An ideal reference gene should have a stable expression across developmental stages, biological conditions, tissues, or experimental conditions.
Given this, the stability of eight candidate reference genes was evaluated in M. vitrata at different developmental stages, diets, and sexes by qRT-PCR method, and the data was analyzed using four independent algorithms, namely GeNorm, NormFinder, BestKeeper, and ΔCt, and one comprehensive algorithm, RefFinder.
The analysis showed that RP49 and RPL13 were the best suitable reference genes for studying target gene expression at different developmental stages. Further, the study identified RP49 and RPL24, and GAPDH and RPL24 as the ideal reference genes in M. vitrata fed with different diets and sexes, respectively. The reference genes reported in the present study will ensure the accuracy of target gene expression, and thus, will serve as an important resource for gene functional studies in M. vitrata.
豆野螟是一种潜在的害虫，在全球范围内给豆类种植造成毁灭性损失。基因功能研究有助于剖析感染过程背后的分子机制，并有助于制定合适的分子策略来控制这种害虫。使用定量实时PCR（qRT-PCR）进行表达谱分析可深入了解靶基因的功能特征；然而，在此类研究中应使用理想的内参基因来消除背景差异并提高靶基因表达的准确性。理想的内参基因应在不同发育阶段、生物学条件、组织或实验条件下具有稳定的表达。
鉴于此，通过qRT-PCR方法评估了8个候选内参基因在豆野螟不同发育阶段、食物和性别中的稳定性，并使用四种独立算法（GeNorm、NormFinder、BestKeeper和ΔCt）以及一种综合算法RefFinder对数据进行了分析。
分析表明，RP49和RPL13是研究不同发育阶段靶基因表达的最合适内参基因。此外，该研究分别确定RP49和RPL24以及GAPDH和RPL24为取食不同食物和不同性别的豆野螟的理想内参基因。本研究报道的内参基因将确保靶基因表达的准确性，因此将成为豆野螟基因功能研究的重要资源。
